Elucidation of Degradation Behavior of Nitrazepam and Other Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (II).

The degradation behavior of eight benzodiazepines (BZPs): alprazolam, etizolam, diazepam, triazolam, nitrazepam (NZP), flunitrazepam (FNZ), bromazepam, and lorazepam, in artificial gastric juice was monitored by a LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. For drugs that were degradable, such physicochemical parameters as reaction rate constant were measured to evaluate the effect of storage conditions on drug degradability, such as whether the degradation proceeds faster by increasing storage temperature, or whether the degradation reaction is reversible by adjusting pH. As a result, it was confirmed that although the eight BZPs degraded in artificial gastric juice, most of them could be restored when pH was increased, and the restoration rates differed depending on the pH and the type of BZP. As for NZP, an Arrhenius plot was drawn to obtain the physicochemical parameters, such as activation energy and activation entropy involved in the degradation reaction, and the reaction kinetics was discussed. In addition, two substances were confirmed as the degradation products of NZP in artificial gastric juice: one was a reversible degradation product (A) (intermediate) and the other was an irreversible degradation product (B) (final degradation product). The intermediate was identified as 2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide, and the final degradation product was 2-amino-5-nitrobenzophenone. Therefore, when detecting NZP in human stomach contents, such as during judicial dissection, it would be prudent to target NZP as well as the intermediate (A) and the final degradation product (B).

A molecularly imprinted polymer based chemiluminescence array sensor for one-step determination of phenothiazines and benzodiazepines in pig urine.

The residues of phenothiazines and benzodiazepines in foods of animal origin are dangerous to consumers. For inspection of their abuses, this study for the first time reported on the use of a chemiluminescence array sensor for the simultaneous determination of four phenothiazines and five benzodiazepines in pig urine. Two molecularly imprinted polymers were coated in different wells of a conventional 96-well microtiter plate as the recognition reagents. After sample loading, the absorbed analytes were initiated directly by using an imidazole enhanced bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system to emit light. The assay process consisted of only one sample-loading step prior to data acquisition, so one test was finished within 10 min. The limits of detection for the nine drugs in the pig urine were in a range of 0.1 to 0.6 pg/mL, and the recoveries from the fortified blank urine samples were in a range of 80.3 to 95%. Furthermore, the sensor could be reused six times. Therefore, this sensor could be used as a simple, rapid, sensitive and reusable tool for routine screening for residues of phenothiazines and benzodiazepines in pig urine.

Structure-based design and synthesis of imidazo[1,2-a]pyridine derivatives as novel and potent Nek2 inhibitors with in vitro and in vivo antitumor activities.

We present herein the discovery and development of novel and potent Nek2 inhibitors with distinctive in vitro and in vivo antitumor activity based on an imidazo[1,2-a]pyridine scaffold. Our studies identified a nonlinear SAR for activity against both Nek2 and cancer cells. Bioisostere and structure-based design techniques were employed to identify compounds 42c (MBM-17, IC50 = 3.0 nM) and 42g (MBM-55, IC50 = 1.0 nM), which displayed low nanomolar activity and excellent selectivity for Nek2. Both compounds effectively inhibited the proliferation of cancer cells by inducing cell cycle arrest and apoptosis. Importantly, the salts form of these two compounds (MBM-17S and MBM-55S) significantly suppressed tumor growth in vivo without apparent toxicity based on appearance and changes in body weight. In summary, MBM-17 and MBM-55 displayed the potential for substantial therapeutic application in cancer treatment.

Parts per trillion detection of 7-aminonitrazepam by nano-enhanced ELISA.

It is challenging to detect 7-aminonitrazepam (7-ANZP) residue in animal tissues simply and sensitively by the enzyme-linked sorbent immunoassay (ELISA) method. This paper demonstrates that utilizing a bioconjugate of gold nanoparticles and enzyme-labeled antibody as a signal probe increases the sensitivity of a traditional ELISA for 7-ANZP by nearly 20 times. The sensitivity of this ELISA for 7-ANZP was 5.6 pg/mL in buffer, and the limit of detection (LOD) of 0.18 µg/kg for 7-ANZP in urine could be achieved after the urine samples were simply hydrolyzed and diluted by buffer. This simple and sensitive method has potential application for improving the sensitivity of ELISA methods against various small molecules.

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography-tandem mass spectrometry.

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.

Mixed micelles of 7,12-dioxolithocholic acid and selected hydrophobic bile acids: interaction parameter, partition coefficient of nitrazepam and mixed micelles haemolytic potential.

The formation of mixed micelles built of 7,12-dioxolithocholic and the following hydrophobic bile acids was examined by conductometric method: cholic (C), deoxycholic (D), chenodeoxycholic (CD), 12-oxolithocholic (12-oxoL), 7-oxolithocholic (7-oxoL), ursodeoxycholic (UD) and hiodeoxycholic (HD). Interaction parameter (ß) in the studied binary mixed micelles had negative value, suggesting synergism between micelle building units. Based on ß value, the hydrophobic bile acids formed two groups: group I (C, D and CD) and group II (12-oxoL, 7-oxoL, UD and HD). Bile acids from group II had more negative ß values than bile acids from group I. Also, bile acids from group II formed intermolecular hydrogen bonds in aggregates with both smaller (2) and higher (4) aggregation numbers, according to the analysis of their stereochemical (conformational) structures and possible structures of mixed micelles built of these bile acids and 7,12-dioxolithocholic acid. Haemolytic potential and partition coefficient of nitrazepam were higher in mixed micelles built of the more hydrophobic bile acids (C, D, CD) and 7,12-dioxolithocholic acid than in micelles built only of 7,12-dioxolithocholic acid. On the other hand, these mixed micelles still had lower values of haemolytic potential than micelles built of C, D or CD. The mixed micelles that included bile acids: 12-oxoL, 7-oxoL, UD or HD did not significantly differ from the micelles of 7,12-dioxolithocholic acid, observing the values of their haemolytic potential.

Determination and importance of temperature dependence of retention coefficient (RPHPLC) in QSAR model of nitrazepams' partition coefficient in bile acid micelles.

Linear dependence between temperature (t) and retention coefficient (k, reversed phase HPLC) of bile acids is obtained. Parameters (a, intercept and b, slope) of the linear function k=f(t) highly correlate with bile acids' structures. Investigated bile acids form linear congeneric groups on a principal component (calculated from k=f(t)) score plot that are in accordance with conformations of the hydroxyl and oxo groups in a bile acid steroid skeleton. Partition coefficient (K(p)) of nitrazepam in bile acids' micelles is investigated. Nitrazepam molecules incorporated in micelles show modified bioavailability (depo effect, higher permeability, etc.). Using multiple linear regression method QSAR models of nitrazepams' partition coefficient, K(p) are derived on the temperatures of 25°C and 37°C. For deriving linear regression models on both temperatures experimentally obtained lipophilicity parameters are included (PC1 from data k=f(t)) and in silico descriptors of the shape of a molecule while on the higher temperature molecular polarisation is introduced. This indicates the fact that the incorporation mechanism of nitrazepam in BA micelles changes on the higher temperatures. QSAR models are derived using partial least squares method as well. Experimental parameters k=f(t) are shown to be significant predictive variables. Both QSAR models are validated using cross validation and internal validation method. PLS models have slightly higher predictive capability than MLR models.

Determination of critical micellar concentrations of two monoketo derivatives of cholic acid.

Critical micellear concentrations (CMC) were determined for two novel promoters of membrane permeability-7-monoketocholic acid (7-MKC) and 12-monoketocholic acid (12-MKC), using two non-invasive ((1)H NMR relaxation experiment and conductometry) and two invasive (spectral shift and partition coefficient of the probe molecule) methods. Studies by the former methods suggest the different aggregation abilities of the investigated bile acid derivatives. In an aqueous solution, 7-MKC has a somewhat lower CMC value (43 mM) than 12-MKC (50 mM). Further, it was found that, in addition to primary micelles, 7-MKC forms also secondary micelles. In the experiments with probe (hydrophobic) molecules, the aggregation properties of investigated bile acids did not differ in water, whereas the presence of urea altered the aggregation of 7-MKC. Based on the CMC value, 7-MKC is more hydrophobic than 12-MKC. The apparent hydrophobicity of 7-MKC is a consequence of the formation of secondary micelles, shifting the monomer equilibrium to the direction of primary micelles, which is manifested as a decrease in the CMC value.

Electroencephalographic properties of zaleplon, a non-benzodiazepine sedative/hypnotic, in rats.

The encephalographic (EEG) properties of zaleplon were investigated in comparison with those of other sedative hypnotics in conscious rats with chronically implanted electrodes. The oral administration of zaleplon (0.25-1.0 mg/kg), triazolam (0.0625-0.25 mg/kg), zopiclone (1.0-4.0 mg/kg), brotizolam (0.0625-0.25 mg/kg), and nitrazepam (0.125-0.5 mg/kg) lengthened the total sleep in a dose-dependent manner. On distribution of sleep-wakefulness stages, zaleplon, in particular, increased the slow wave deep sleep (SWDS), whereas triazolam, brotizolam, and nitrazepam increased the slow wave light sleep (SWLS) in a dose-dependent manner. Zopiclone significantly increased the SWDS at a dose of 2 mg/kg and both the SWLS and the SWDS at a dose of 4 mg/kg. All tested hypnotics caused no influence on fast wave sleep (FWS) at doses tested. The appearance of the sleep-inducing activity of zaleplon was more rapid than those of any compounds tested, and zaleplon significantly increased the relative EEG power density in the delta frequency band over that of triazolam at 20 and 30 min after the administration in the spectral analysis. Therefore, the present findings suggest that the non-benzodiazepine zaleplon can be expected to exhibit high practical potential as a hypnotic and is characterized by an increase in SWDS with rapid onset of hypnotic action.

An evaluation of a four-channel multiplexed electrospray tandem mass spectrometry for higher throughput quantitative analysis.

A four-channel multiplexed electrospray inlet system (MUX) coupled to a triple quadrupole mass spectrometer was investigated as a higher throughput approach to quantitative analysis. Four discrete samples may be simultaneously analyzed by virtue of a rotating sampler with a concomitant 4-fold increase in analytical throughput. Although absolute sensitivity was reduced using the MUX interface compared with analysis using traditional single electrospray interface, reproducibility of response was shown to be comparable. Source robustness was established for the analysis of both aqueous drug standards and drugs in biological media, and linearity of response for a test compound, diazepam, was demonstrated over 2 orders of magnitude. Analyte-dependent response differences were exhibited between the four channels of the interface, and this led to the overall conclusion that samples to be compared quantitatively must be analyzed through the same sprayer. In addition, each channel must be independently calibrated to afford true quantification. Should a deuterated internal standard be employed, however, quantitative comparisons can be made across channels. An HPLC pumping system providing individual back-pressure regulation to each channel was shown to provide adequate chromatography even in the event of a channel blockage. Furthermore, following multiple injections of biological samples onto the MUX interface, an eluent flow diversion was integrated into the first part of each analytical run. This served to prevent source fouling, and thus, no detrimental effects to response reproducibility or sensitivity were observed.

Study of partition of nitrazepam in bile salt micelles and the role of lecithin.

The effect of trihydroxy (sodium cholate and sodium glycocholate) and dihydroxy (sodium deoxycholate and sodium glycodeoxycholate) bile salt micelles on the spectrophotometric properties and on the solubility of nitrazepam in aqueous solution, at 25.0 degrees C and at ionic strength 0.1 M in sodium chloride, has been assessed. From the results obtained it was possible to calculate the partition coefficients (Kp) of nitrazepam between aqueous and micellar phases. The partition coefficients of nitrazepam have also been determined in mixed micelles of cholate or deoxycholate with lecithin (egg yolk phosphatidylcholine), which were used as a model of the gastrointestinal tract. Drug partition was found to depend on the bile acid (number of hydroxyl groups and conjugation with glycine), and our data indicate further that addition of lecithin to bile salt micelles decreases the values of the partition coefficients in the mixed micelles at physiological pH.

Simple preparation of the major urinary metabolites of flunitrazepam and nitrazepam.

An alarming increase in the misuse/abuse of nitrobenzodiazepine derivatives, especially flunitrazepam, prompted us to establish reliable analytical protocols for their routine detection. Whilst the parent drugs are readily available from a number of commercial sources, it was found difficult to obtain samples of the corresponding amino metabolites which were required as analytical standards. This lead us to develop the straightforward synthetic protocol described here, to convert the readily available parent drugs, namely flunitrazepam and nitrazepam, to their respective 7-amino derivatives. The method requires minimum laboratory facilities. It involves the reduction of the nitro functionality in the parent drug to an amino group using tin (II) chloride under mild conditions, using ultrasonication at room temperature. The method is simple and should give toxicology laboratories better access to these much needed compounds.

Deaggregation during the dissolution of benzodiazepines in interactive mixtures.

The purpose of this research was to investigate the influence of surfactants on the dissolution of benzodiazepines in interactive mixtures. The dissolution of ternary interactive mixtures consisting of micronized drugs (oxazepam, nitrazepam, and flunitrazepam) and micronized surfactants (sodium lauryl sulfate and cetrimide) adhered onto the surface of a lactose carrier (250-355 microm) was studied using the USP/NF paddle method. Dissolution was considered to occur from dispersed particle and aggregate fractions of the drugs, and data were modeled using multiexponential equations. The initial concentrations of the aggregates and dissolution rate constants were estimated using a Marquardt-Levenberg nonlinear least squares algorithm. The marked increase in dissolution rate which occurred with increasing concentrations of sodium lauryl sulfate and cetrimide resulted both from deaggregation of the benzodiazepine particles and from increases in the dissolution rate constants of the dispersed particle and aggregate fractions probably associated with an increased intrinsic dissolution rate. The presence of 5% sodium lauryl sulfate in the interactive mixture reduced the initial percent of aggregates from about 85% in a binary mixture to less than 10% and about doubled the dispersed particle dissolution rate constant. The presence of the surfactant in the surface particulate matrix of the interactive mixture was essential for its deaggregation effect. Sodium lauryl sulfate was more effective than cetrimide in achieving drug deaggregation.

On the partition of ampholytes: application to blood-brain distribution.

Partition coefficients in the water-octanol, -cyclohexane, and -dichloromethane systems were determined as a function of pH for three non-zwitterionic ampholytes (nitrazepam, albendazole sulfoxide, and sulfadimidine) and three zwitterionic ampholytes (morphine, difloxacin, and niflumic acid). From known macro- and microprotonation constants in water, the concentration of cation, anion, neutral form, and zwitterion can be found, and partition coefficients can then be calculated for the partition of the neutral form in water to the neutral form in the organic solvent for all six compounds. These micropartition coefficients were then used to obtain descriptors for the neutral form in the general linear free energy (LFER) equation of Abraham. Knowledge of the descriptors enables a number of physicochemical and biochemical properties of the neutral form to be estimated; a detailed analysis is given of the estimation of the blood-brain distribution ratio for the process of going from the neutral form in blood to the neutral form in brain. A related procedure leads to an estimation of the distribution ratio for the process of going from the zwitterion in blood to the neutral form in brain.

Effect of dimethyl-beta-cyclodextrin on nitrazepam stability.

Nitrazepam was found to form an inclusion complex with heptakis; 2,6 di-O-methyl-beta-cyclodextrin (DM-beta-CD) in solution. The phase solubility diagram was found to be AL type with no precipitation of the inclusion complex formed. The kinetic studies of nitrazepam hydrolysis in DM-beta-CD solution at 30, 40 and 50 degrees C was followed spectrophotometrically, as the degradation products of the drug did not interfere with such assay. It was found that the degradation of nitrazepam in acidic media followed the first order reaction kinetics at the temperatures studied. Inclusion complexation of nitrazepam in DM-beta-CD resulted in a relatively improved stability of the drug in solution at 30 degrees C. On the other hand, no appreciable stabilization was achieved at higher temperatures (40 and 50 degrees C).

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Preparation and investigation of inclusion complexes containing nitrazepam.

Nitrazepam is a valuable drug, being one of the often used minor tranquillizers in psychotherapy. However, its solubility characteristics are very unfavourable. Products were prepared with dimethyl-beta-cyclodextrin in different combinations (8.58%, 19.48% and 27.29%) and by several methods such as mixing, kneading, precipitation and spray-drying.

Involvement of the intestinal microflora in nitrazepam-induced teratogenicity in rats and its relationship to nitroreduction.

A study was undertaken to investigate the relationship between nitroreduction of nitrazepam and its teratogenic effects and the involvement of the intestinal microflora in Sprague-Dawley rats. Incubation of bacterial suspensions from rat cecal contents with nitrazepam resulted in extensive reduction to 7-aminonitrazepam. Rat liver homogenates also reduced nitrazepam but only under anaerobic conditions. Following oral administration of 300 mg/kg nitrazepam to pregnant rats, total excretion of reduced metabolites (7-aminonitrazepam and 7-acetylaminonitrazepam) in urine and feces accounted for approximately 30% of the administered dose. When antibiotics were administered to dams to deplete their intestinal microflora prior to administration to nitrazepam, the total excretion of the reduced metabolites in the urine and feces decreased to 2% of the dose. Nitroreductase activity of cecal contents was almost completely suppressed by antibiotic pretreatment, but the activity of liver homogenates was not significantly altered by the same treatment. The incidence of nitrazepam-induced malformations was markedly decreased by antibiotic pretreatment. These results suggest that the intestinal microflora plays an important role in the reductive metabolism of nitrazepam and that the teratogenicity of nitrazepam may be related to its nitroreduction by the microflora.

[Screening and detection of nimetazepam and its chief metabolites]. / Screening und Nachweis von Nimetazepam und seinen Hauptmetaboliten.

The article describes analytical data for the screening and detection of nimetazepam and its major metabolites. The methods comprise thin-layer chromatography, gas chromatography, UV-spectrophotometry, infrared- and mass spectrometry.