ABSTRACT
Bordetella pertussis toxin (PT) and Clostridium botulinum C2 toxin are ADP-ribosylating toxins causing severe diseases in humans and animals. They share a common translocation mechanism requiring the cellular chaperones Hsp90 and Hsp70, cyclophilins, and FK506-binding proteins to transport the toxins' enzyme subunits into the cytosol. Inhibitors of chaperone activities have been shown to reduce the amount of transported enzyme subunits into the cytosol of cells, thus protecting cells from intoxication by these toxins. Recently, domperidone, an approved dopamine receptor antagonist drug, was found to inhibit Hsp70 activity. Since Hsp70 is required for cellular toxin uptake, we hypothesized that domperidone also protects cells from intoxication with PT and C2. The inhibition of intoxication by domperidone was demonstrated by analyzing the ADP-ribosylation status of the toxins' specific substrates. Domperidone had no inhibitory effect on the receptor-binding or enzyme activity of the toxins, but it inhibited the pH-driven membrane translocation of the enzyme subunit of the C2 toxin and reduced the amount of PTS1 in cells. Taken together, our results indicate that domperidone is a potent inhibitor of PT and C2 toxins in cells and therefore might have therapeutic potential by repurposing domperidone to treat diseases caused by bacterial toxins that require Hsp70 for their cellular uptake.
Subject(s)
Bacterial Toxins , Botulinum Toxins , Animals , Humans , Bordetella pertussis/metabolism , Domperidone/pharmacology , Botulinum Toxins/toxicity , Bacterial Toxins/metabolism , Pertussis Toxin , ADP Ribose Transferases/metabolismABSTRACT
Clostridioides difficile infections cause severe symptoms ranging from diarrhea to pseudomembranous colitis due to the secretion of AB-toxins, TcdA and TcdB. Both toxins are taken up into cells through receptor-mediated endocytosis, autoproteolytic processing and translocation of their enzyme domains from acidified endosomes into the cytosol. The enzyme domains glucosylate small GTPases such as Rac1, thereby inhibiting processes such as actin cytoskeleton regulation. Here, we demonstrate that specific pharmacological inhibition of Hsp70 activity protected cells from TcdB intoxication. In particular, the established inhibitor VER-155008 and the antiemetic drug domperidone, which was found to be an Hsp70 inhibitor, reduced the number of cells with TcdB-induced intoxication morphology in HeLa, Vero and intestinal CaCo-2 cells. These drugs also decreased the intracellular glucosylation of Rac1 by TcdB. Domperidone did not inhibit TcdB binding to cells or enzymatic activity but did prevent membrane translocation of TcdB's glucosyltransferase domain into the cytosol. Domperidone also protected cells from intoxication with TcdA as well as CDT toxin produced by hypervirulent strains of Clostridioides difficile. Our results reveal Hsp70 requirement as a new aspect of the cellular uptake mechanism of TcdB and identified Hsp70 as a novel drug target for potential therapeutic strategies required to combat severe Clostridioides difficile infections.
Subject(s)
Bacterial Toxins , Clostridioides difficile , Clostridium Infections , Humans , Bacterial Toxins/metabolism , Clostridioides difficile/metabolism , Domperidone/pharmacology , Domperidone/metabolism , Caco-2 Cells , Bacterial Proteins/metabolism , Enterotoxins/toxicity , Enterotoxins/metabolismABSTRACT
First derivative synchronous fluorescence spectroscopy (FDSFS) was applied to detect and quantify either ciprofloxacin (CIP) or levofloxacin (LEV) simultaneously with their photodegradation products, where the photolytic pathway for each analyte was found to be pH dependent. Under the guidance of early published articles, the structure of the produced photolytic products could be concluded, and further related to their resultant fluorescence spectra. The proposed method was subjected to full validation procedure which enables its application in investigating the photodegradation kinetics for both drugs. The obtained kinetic parameters were in accordance with previous reports and could be linked to predict the antibacterial activity of the resultant photodegradation products. These facts prove the suitability of the suggested FDSFS to serve as a stability-indicating assay method and to trace the photofate of CIP and LEV in the ecosystem as potential contaminants. Furthermore, the greenness of the suggested analytical methodology was evaluated via 'Green Analytical Procedure Index' (GAPI), which classifies it as an eco-friendly assay. Eventually, no extraction, treatment or preparation steps were needed during all analysis steps, which renders the proposed assay an appealing tool in environmental analysis.
ABSTRACT
Bakeries add extra potassium bromate to the dough to make homogeneous, elastic, fluffy bread. Bromate causes renal damage and cancer. FAO/WHO stated that bromate residues shouldn't be in baked products. A potentiometric sensor's membrane recipe was optimized for sensitive and selective bromate assay. We planned a custom experimental design of 21 sensors that included numerical and categorical factors (NPPE: PVC, matrix%, membrane thickness, and ionophore type). We defined sensor performance outcomes (Nernstian slope, quantification limit, correlation coefficient, response time and selectivity), and each sensor's outcome was determined. The computer software developed a predictive model for each outcome and the desirability function suggested the optimum sensor recipe. The sensor achieved a slope of -63.54 mV/decade and detection limit of 2 × 10-6 mol/L. The greenness profile was evaluated by the National Environmental Approach Index protocol. The developed sensor represents a reliable, fast, in-site tool for the assay of bromate in bakery products.
Subject(s)
Bread , Bromates , Potentiometry/methods , Ionophores , Bread/analysis , Research DesignABSTRACT
A novel potentiometric sensor was developed and optimized for the quantitative analysis of ephedrine in non-prescribed herbal supplements used as adjunctive therapy for weight loss. An initial optimization study aimed to reach the optimum membrane composition, sensor assembly, and experimental conditions. The study evaluated the effect of several factors on the sensor performance including different ion-exchangers, plasticizers, ionophores, membrane thicknesses, soaking solution concentrations, soaking time intervals, and pH. The optimized polyvinyl chloride membrane included tungstophosphoric acid hydrate as a cation exchanger, tricresyl phosphate as a plasticizer, and calix[8]arene as an ionophore to enhance the sensitivity and selectivity of the developed sensor. The polyvinyl chloride membrane was drop-casted over a polyaniline modified glassy carbon electrode surface to form a solid-state sensor. The proposed membrane succeeded to quantify ephedrine over a linear range of 6 × 10-6 to 1 × 10-2 M with a LOD of 3.60 × 10-6 M, acceptable selectivity, and fast response time. The IUPAC characterization of sensor response and International Conference on Harmonization validation parameters were calculated. The method successfully determined ephedrine concentration in spiked herbal mixtures and determined labeled and undeclared ephedrine content of weight loss herbal preparations.
ABSTRACT
A hybrid micelle based mobile phase was used to develop and validate a liquid chromatographic method for the separation and quantification of two local anesthetics namely; lidocaine hydrochloride (LID), and bupivacaine hydrochloride (BPV) in presence of the frequently co administered vasopressors phenyl ephrine (PHR) and ephedrine (EPH). Optimization of chromatographic separation conditions was performed applying experimental one factor at a time tool, and design of experiment, where the retention behavior of all analytes using both optimization protocols was in accordance. Chromatographic separation was carried on a C8 column operating at 40 °C at a flow rate of 1.5 mL/min. using a mobile phase consisting of 0.18 M sodium dodecyl sulphate, 10% acetonitrile, containing 0.3% triethyl amine and adjusted to pH 7 using 2 M ortho phosphoric acid, adopting UV detection at 230 nm. The proposed method was fully validated and applied to both in vitro and in vivo analysis of rat blood samples. The pharmacokinetics of both LID and BPV was followed when they were solitary injected or when co administered with either PHR or EPH. Moreover, the in vitro spiked experiment was also subjected to documented bio-analytical validation procedures.
Subject(s)
Anesthetics, Local , Chromatography, Liquid/methods , Drug Monitoring/methods , Vasoconstrictor Agents , Anesthetics, Local/blood , Anesthetics, Local/chemistry , Anesthetics, Local/pharmacokinetics , Animals , Bupivacaine/blood , Bupivacaine/chemistry , Bupivacaine/pharmacokinetics , Drug Interactions , Ephedrine/blood , Ephedrine/chemistry , Ephedrine/pharmacokinetics , Lidocaine/blood , Lidocaine/chemistry , Lidocaine/pharmacokinetics , Micelles , Rats , Vasoconstrictor Agents/blood , Vasoconstrictor Agents/chemistry , Vasoconstrictor Agents/pharmacokineticsABSTRACT
Micellar mobile phase was utilized for the separation and quantification of two antimicrobial mixtures with some analgesics that were found to augment their antimicrobial activity, namely; cefotaxime sodium (CFX) with ibuprofen (IBU) and naproxen (NPX) (mixture Ι), and azithromycin dihydrate (AZT) with diclofenac sodium (DIC) (mixture ΙΙ). Both mixtures were analyzed using a C18 column operated at 50⯰C using a mobile phase composed of sodium dodecyl sulphate (SDS), an organic modifier; (1-propanol or acetonitrile), and 0.3% triethylamine (TEA), adjusting pH to the required value with 2â¯M orthro-phosphoric acid, accompanied with UV detection. The proposed method was subjected to full validation procedure and was applied to determine the concentration of the studied antibiotics in homogenized liver, kidney and heart rat tissue samples, with or without the co-administered non steroidal anti-inflammatory drugs.
Subject(s)
Anti-Infective Agents/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Chromatography, Liquid/methods , Drug Interactions , Animals , Anti-Infective Agents/analysis , Anti-Inflammatory Agents, Non-Steroidal/analysis , Kidney/chemistry , Kidney/metabolism , Limit of Detection , Linear Models , Liver/chemistry , Liver/metabolism , Models, Chemical , Rats , Reproducibility of ResultsABSTRACT
An accurate and selective method using micellar liquid chromatography was developed to determine metformin hydrochloride both in its pharmaceutical dosage forms and human plasma. Separation was conducted using a Zorbax SB-Phenyl (250 × 4.6 mm id) stainless steel column at ambient temperature after pre-column derivatization with 9,10-phenanthraquinone. A mobile phase composed of 0.1 M sodium dodecyl sulfate, 10% 1-propanol and triethylamine (0.3%) in 0.02 M phosphoric acid, adjusted to pH 2.5, was used at a flow rate of 1 ml/min with fluorimetric detection at 450 nm after excitation at 306 nm. The proposed method showed high sensitivity with limit of quantification of 0.35 µg/ml and limit of detection of 0.23 µg/ml, being linear from 0.5 to 3.0 µg/ml. Being highly sensitive, the method could be applied to spiked human plasma, and also to follow the pharmacokinetic parameters of the studied drug in healthy volunteers after administration of both its immediate and sustained release tablet formulations. Such procedures were carried out without any extraction steps, which improves the accuracy and precision of the proposed method when applied to human plasma. Detailed validation procedures were also carried out giving results in accordance with the comparison method. The proposed method has also the advantage of being environmentally safe, where the use of organic solvents is highly limited in comparison with other traditional chromatographic separation methods that depend mainly on a high proportion of organic modifiers. This concept, in turn, emphasizes the application of green chemistry in the analysis of pharmaceutical products. The simplicity, relatively low cost and short analysis time of the suggested method makes it a candidate for routine quality control work.
Subject(s)
Delayed-Action Preparations , Fluorometry/methods , Metformin/analysis , Metformin/pharmacokinetics , Micelles , Chromatography, Liquid , Drug Compounding , Molecular StructureABSTRACT
A new method orthogonal projection to latent structures (O-PLS) combined with artificial neural networks is investigated for non-destructive determination of ebastine powder via near-infrared (NIR) spectroscopy. The modern NIR spectroscopy is efficient, simple and non-destructive technique, which has been used in chemical analysis in diverse fields. Being a preprocessing method, O-PLS provides a way to remove systematic variation from an input data set X not correlated to the response set Y, and does not disturb the correlation between X and Y. In this paper, O-PLS pretreated spectral data was applied to establish the ANN model of ebastine powder, in this model, the concentration of ebastine as the active component was determined. The degree of approximation was employed as the selective criterion of the optimum network parameters. In order to compare the OPLS-ANN model, the calibration models that use first-derivative and second-derivative preprocessing spectra were also designed. Experimental results showed that the OPLS-ANN model was the best.
Subject(s)
Butyrophenones/analysis , Butyrophenones/chemistry , Neural Networks, Computer , Piperidines/analysis , Piperidines/chemistry , Spectrophotometry, Infrared/methods , PowdersABSTRACT
An accurate, simple, sensitive and selective micellar liquid chromatographic method has been developed for the simultaneous determination of caffeine (CAF) and two non-steroidal anti-inflammatory drugs, namely ibuprofen (IBU) and ketoprofen (KET). The chemometric approach was applied to the optimization of separation of the studied drugs. To optimize their separation, the effect of six experimental parameters on retention was investigated by means of multivariate analysis. Separation was conducted using an ODS C18 (150 × 4.6 mm i.d.) stainless steel column at ambient temperature with UV detection at 260 nm. A mobile phase composed of 40 mM sodium dodecyl sulphate (SDS), 10% 1-propanol and 0.3% tri-ethylamine in 0.02 M phosphoric acid adjusted to pH 3 has been used at a flow rate of 1 mL/min. Regression models were characterized by both descriptive and predictive ability (R(2) ≥ 97.9% and R(cv)(2) ≥ 96.2%) and allowed the chromatographic separation of the drugs with a good resolution and a total analysis time within 15 min. The calibration curves were rectilinear over the concentration ranges of 2.0-25.0, 1.5-15.0 and 1.0-10.0 µg/mL for IBU, KET and CAF, respectively, with detection limits of 1.2, 1.0 and 0.6 µg/mL, and quantification limits of 1.6, 1.2 and 0.8 µg/mL, respectively. The results obtained were in good agreement with those obtained by the comparison method.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/analysis , Caffeine/analysis , Chromatography, Liquid/methods , Micelles , Reproducibility of ResultsABSTRACT
A validated simple, rapid, and selective spectrofluorimetric method was developed for the determination of some antihistaminic H(1) receptor antagonist drugs namely ebastine (EBS), cetirizine dihydrochloride (CTZ), and fexofenadine hydrochloride (FXD). The method is based on the reaction of the cited drugs with some Π acceptors namely p-chloranilic acid (CLA), tetracyanoethylene (TCNE), and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) to give highly fluorescent derivatives. The fluorescence intensity-concentration plots were rectilinear over the concentration ranges of 0.2-3.0, 0.2-2.5 and 0.15-2.0 µg/ml for EBS with CLA, DDQ, and TCNE respectively; 0.5-7.0, 0.5-6.0, and 0.2-4.0 µg/ml for CTZ with the previously mentioned reagents, and 0.2-3.5, 0.5-6.0, and 0.2-3.5 µg/ml for FXD. The factors affecting the formation of the reaction products were carefully studied and optimized. The method was applied for the determination of the studied drugs in their dosage forms. The results obtained were in good agreement with those obtained by the comparison methods. Reactions Stoichiometries of the complexes formed between the studied drugs and Π acceptors were defined by the Job's method of the continuous variation and found in 1:1 in all cases.
Subject(s)
Histamine H1 Antagonists/analysis , Histamine H1 Antagonists/chemistry , Receptors, Histamine H1/metabolism , Spectrometry, Fluorescence/methods , Capsules , Electron Transport , Fluorescent Dyes/chemistry , Solvents/chemistry , Tablets , Temperature , Time FactorsABSTRACT
An accurate, simple, sensitive and selective reversed phase liquid chromatographic method has been developed for the determination of ebastine in its pharmaceutical preparations. The proposed method depends on the complexation ability of the studied drug with Zn2+ ions. Reversed phase chromatography was conducted using an ODS C18 (150 × 4.6 mm id) stainless steel column at ambient temperature with UV-detection at 260 nm. A mobile phase containing 0.025%w/v Zn2+ in a mixture of (acetonitril/methanol; 1/4) and Britton Robinson buffer (65:35, v/v) adjusted to pH 4.2, has been used for the determination of ebastine at a flow rate of 1 ml/min. The calibration curve was rectilinear over the concentration range of 0.3 - 6.0 µg/ml with a detection limit (LOD) of 0.13 µg/ml, and quantification limit (LOQ) of 0.26 µg/ml. The proposed method was successfully applied for the analysis of ebastine in its dosage forms, the obtained results were favorably compared with those obtained by a comparison method. Furthermore, content uniformity testing of the studied pharmaceutical formulations was also conducted. The composition of the complex as well as its stability constant was also investigated. Moreover, the proposed method was found to be a stability indicating one and was utilized to investigate the kinetics of alkaline and ultraviolet induced degradation of the drug. The first-order rate constant and half life of the degradation products were calculated.
ABSTRACT
Two sensitive, selective, economic, and validated spectrofluorimetric methods were developed for the determination of ebastine (EBS) in pharmaceutical preparations depending on reaction with its tertiary amino group. Method I involves condensation of the drug with mixed anhydrides (citric and acetic anhydrides) producing a product with intense fluorescence, which was measured at 496 nm after excitation at 388 nm.Method (IIA) describes quantitative fluorescence quenching of eosin upon addition of the studied drug where the decrease in the fluorescence intensity was directly proportional to the concentration of ebastine; the fluorescence quenching was measured at 553 nm after excitation at 457 nm. This method was extended to (Method IIB) to apply first and second derivative synchronous spectrofluorimetric method (FDSFS & SDSFS) for the simultaneous analysis of EBS in presence of its alkaline, acidic, and UV degradation products.The proposed methods were successfully applied for the determination of the studied compound in its dosage forms. The results obtained were in good agreement with those obtained by a comparison method. Both methods were utilized to investigate the kinetics of the degradation of the drug.
ABSTRACT
Two rapid, simple, sensitive, selective and economic derivative spectrophotometric (first [D1] and second [D2]) and synchronous spectrofluorimetric (FDSFS and SDSFS) methods have been developed for the analysis of fexofenadine hydrochloride (FXD) in the presence of its different degradation products. Derivative spectrophotometry (D1) was used to measure FXD at 223 nm in the presence of its alkaline or acidic degradation products, and at 211 nm in the presence of its oxidative degradation product. Derivative spectrophotometry (D2) was used to determine FXD at 217 nm in the presence of its alkaline or acidic degradation products, and at 215 nm in the presence of its oxidative degradation product; the UV degradation product was measured at 211 nm. Synchronous spectrofluorimetry (FDSFS) was used to measure FXD in the presence of its alkaline or acidic degradation products at 406 nm, and at 367 nm in the presence of its oxidative or UV degradation products. Synchronous spectrofluorimetry (SDSFS) was applied to determine the drug at 225 nm in the presence of its alkaline, acidic, oxidative or UV degradation products. The proposed methods were successfully applied for the determination of the studied compound in its commercial tablets. The results obtained were in good agreement with those obtained by the comparison method.