Biochemical evaluation and ligand binding studies on glycerophosphodiester phosphodiesterase from Staphylococcus aureus using STD-NMR spectroscopy and molecular docking analysis.

Glycerophosphodiester phosphodiesterase (GDPD) is a highly conserved enzyme in both prokaryotic and eukaryotic organisms. It catalyses the hydrolysis of various glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, which serve as building blocks in several biosynthetic pathways. This enzyme is a well-known virulence factor in many pathogenic bacteria, including Staphylococcus aureus, and is thus considered a potential drug target. In this study, competent E. coli BL21(DE3)pLysS expression cells were used to express the GDPD enzyme from vancomycin-resistant Staphylococcus aureus (VRSA), which was then purified using size exclusion and anion exchange chromatography. The hydrolytic activity of GDPD was evaluated on the non-physiological substrate bis(p-nitrophenyl) phosphate (BpNPP), which indicated functional activity of the enzyme. 79 drugs were evaluated for their inhibitory potential against GDPD enzyme by the colorimetric assay. Out of 79 drugs, 13 drugs, including tenofovir (1), adenosine (2), clioquinol (11), bromazepam (12), lamotrigine (13), sulfadiazine (14), azathioprine (15), nicotine (16), sitagliptin PO4 (17), doxofylline (18), clindamycin phosphate (19), gentamycin sulphate (20), and ceftriaxone sodium (21) revealed varying degrees of inhibitory potential with IC50 values in the range of 400 ± 0.007-951 ± 0.016 µM. All drugs were also evaluated for their binding interactions with the target enzyme by saturation transfer difference (STD-NMR) spectroscopy. 10 drugs demonstrated STD interactions and hence, showed binding affinity with the enzyme. Exceptionally, tenofovir (1) was identified to be a better inhibitor with an IC50 value of 400 ± 0.007 µM, as compared to the standard EDTA (ethylenediaminetetraacetic acid) (IC50 = 470 ± 0.008 µM). Moreover, molecular docking studies have identified key interactions of the ligand (tenofovir) with the binding site residues of the enzyme.

Development and validation of a stability indicating RP-HPLC-DAD method for the determination of bromazepam.

A reliable, selective and sensitive stability-indicating RP-HPLC assay was established for the quantitation of bromazepam (BMZ) and one of the degradant and stated potential impurities; 2-(2-amino-5-bromobenzoyl) pyridine (ABP). The assay was accomplished on a C18 column (250 mm × 4.6 mm i.d., 5 µm particle size), and utilizing methanol-water (70: 30, v/v) as the mobile phase, at a flow rate of 1.0 ml min-1. HPLC detection of elute was obtained by a photodiode array detector (DAD) which was set at 230 nm. ICH guidelines were adhered for validation of proposed method regarding specificity, sensitivity, precision, linearity, accuracy, system suitability and robustness. Calibration curves of BMZ and ABP were created in the range of 1-16 µg mL-1 with mean recovery percentage of 100.02 ± 1.245 and 99.74 ± 1.124, and detection limit of 0.20 µg mL-1 and 0.24 µg mL-1 respectively. BMZ stability was inspected under various ICH forced degradation conditions and it was found to be easily degraded in acidic and alkaline conditions. The results revealed the suitability of the described methodology for the quantitation of the impurity (ABP) in a BMZ pure sample. The determination of BMZ in pharmaceutical dosage forms was conducted with the described method and showed mean percentage recovery of 99.39 ± 1.401 and 98.72 ± 1.795 (n = 6), respectively. When comparing the described procedure to a reference HPLC method statistically, no significant differences between the two methods in regard to both accuracy and precision were found.

Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water.

The study aims the development and validation of a LC-MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1-2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC-MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Environmental occurrence, fate and transformation of benzodiazepines in water treatment.

Benzodiazepine derivatives are prescribed in large quantities globally and are potentially new emerging environmental contaminants. Unfortunately, a dearth of data exists concerning occurrence, persistence and fate in the environment. This paper redresses this by reviewing existing literature, assessing the occurrence of selected benzodiazepine anxiolytics (diazepam, oxazepam and bromazepam) in wastewater influent and effluent and surface water from Slovenia, evaluating their removal during water treatment and identifying the transformation products formed during water treatment. Their occurrence was monitored in hospital effluent, river water and in wastewater treatment plant influent and effluent. The study reveals the presence of benzodiazepine derivatives in all samples with the highest amounts in hospital effluents: 111 ng L(-1), 158 ng L(-1) and 72 ng L(-1) for diazepam, bromazepam and oxazepam, respectively. Removal efficiencies with respect to biological treatment of diazepam were 16-18% (oxic), 18-32% (anoxic→oxic), 53-76% (oxic→anoxic) and 83% (oxic→anoxic→oxic→anoxic cascade bioreactors), while the removal oxazepam was 20-24% under anoxic conditions. Coupled biological and photochemical treatment followed by the adsorption to activated carbon resulted in a removal efficiency of 99.99%. Results reveal the recalcitrant nature of benzodiazepine derivatives and suggest that only combinational treatment is sufficient to remove them. In addition, eight novel diazepam and four novel oxazepam transformation products are reported.

Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction.

Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.

Comparison of CZE, MEKC, MEEKC and non-aqueous capillary electrophoresis for the determination of impurities in bromazepam.

The purpose of the present investigation was to develop a test for related substances in the benzodiazepine drug substance bromazepam based on capillary electrophoresis (CE). A final method for the determination of impurities in bromazepam is based on non-aqueous capillary electrophoresis (NACE). Five modes of capillary electrophoresis were investigated and compared for the said purpose. All the CE systems investigated make use of running buffers at low pH in order to protonate the analytes. A low pH of the running buffers was needed as the pK(a) values of benzodiazepines in general are in the range from 1.3 to 4.6. Dynamically coated capillaries were used to overcome the low electro-osmotic flow at low pH in the aqueous buffers investigated. CZE with and without dynamical coating of the internal surface of the fused capillaries was compared and also micellar electrokinetic chromatography (MEKC) as well as microemulsion electrokinetic chromatography (MEEKC) performed in dynamically coated capillaries were investigated. The NACE was chosen as the best technique as the low solubility of the benzodiazepines in water is easily overcome. The NACE system showed good selectivity and detectability for the substances investigated and the limit of quantitation for the impurities corresponded to 0.05% of the drug substance. Linearity was good.

Radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of 4 cases.

BACKGROUND: The radiopacity of ingested substances may serve as a clue to the presence of particular compounds, as this characteristic varies considerably among medications and household products. Tablet conglomerations are also variably radiopaque. We report 4 cases of clomipramine poisoning associated with formation of radiopaque masses, believed to be clomipramine, in the area of the stomach. CASE REPORTS: Four patients were admitted to the Toxicological Intensive Care Unit after ingestions of, respectively, 8.5 g (180 tablets of mixed strength), 7.5 g (100 tablets), 10.5 g (140 tablets), and 4.5 g (60 tablets) of clomipramine, along with other sedatives and antipsychotics. In each case, a rounded density was observed in the gastric area on plain chest radiograph. The hospital courses of each patient were marked by tachycardia, hypotension, QRS and QT prolongation, seizures, and decreased mental status. Three of 4 patients underwent unsuccessful endoscopy to remove tablet fragments and subsequently suffered gastrointestinal hemorrhage requiring transfusion. All patients were discharged recovered from the hospital. DISCUSSION: Clomipramine, a potent tricyclic antidepressant, has been previously reported to be nonradiopaque, and has not been reported to induce formation of concretions. These cases suggest that massive ingestions of clomipramine may form bezoars which are radiopaque and may be associated with serious toxicity. Careful consideration should be given prior to the use of gastric endoscopy for the retrieval of tablet fragments since significant hemorrhage, attributed to the procedure itself rather than to clomipramine toxicity, may ensue.

Acidic hydrolysis of bromazepam studied by high performance liquid chromatography. Isolation and identification of its degradation products.

A kinetic study on the acidic hydrolysis of bromazepam was carried out in 0.01 M hydrochloric acid solution at 25 and 95 degrees C. A reversed-phase HPLC method was developed and validated for the determination of bromazepam and its degradation products. Bromazepam degraded by a consecutive reaction with a reversible first step. Two degradation products were isolated and identified by infrared, 1H and 13C nuclear magnetic resonance and mass spectroscopy. Spectroscopic data indicated that N-(4-bromo-2-(2-pyridylcarbonyl)phenyl)-2-aminoacetamide was the intermediate degradation product of this acid hydrolysis, whereas 2-amino-5-bromophenyl-2-pyridylmethanone was the final one. Therefore, the mechanism of this acid-catalysed hydrolysis involved initial cleavage of the 4,5-azomethine bond, followed by slow breakage of the 1,2-amide bond. Statistical evaluation of the HPLC method revealed its good linearity and reproducibility. Detection limits were 3.8 x 10(-7) M for bromazepam, 6.25 x 10(-7) M for the intermediate and 8.16 x 10(-7) M for the benzophenone derivative.

Kinetics of the acid hydrolysis of diazepam, bromazepam, and flunitrazepam in aqueous and micellar systems.

A kinetic study of the acid hydrolysis of aqueous diazepam, bromazepam, and flunitrazepam was carried out at 25 degrees C using a spectrophotometric method. For diazepam and flunitrazepam, the experimental pseudo first-order rate constant decreased as the acid concentration was increased. The contrary behavior was found in the case of bromazepam. A kinetic scheme that includes the hydrolysis reaction of both protonated and nonprotonated species of the drug can account for these results. Also, the kinetics of the acid hydrolysis of the same drugs in the presence of micellar aggregates [nonionic polyoxyethylene-23-dodecanol (Brij 35); cationic cetyl trymethyl ammonium bromide (CTAB); and anionic sodium decyl (SdeS), dodecyl (SDS), and tetradecyl (STS) sulfate] was studied at 25 degrees C. Negligible effects were observed in the cases of nonionic and cationic micelles. Anionic micelles produced an inhibitory effect in the reaction velocity. This effect increased as the hydrophobic nature of the surfactant increased. All these facts are interpreted quantitatively by means of a pseudophase model.