Sándor Nagy

Optimization of conditions for culture of the test bacteria used for direct bioautographic detection. 1. The gram-positive test bacterium Bacillus subtilis

The main purpose of this study was to determine optimum conditions for culture of a test microbe Bacillus subtilis (ATCC 6633) which enabled us to establish its use for direct bioautography. The viability of the bacteria on TLC plates was measured on the basis of their adenosine-5’-triphosphate (ATP) content as determined by bioluminescent luciferin/luciferase assay, the data being referred to values for total bacterial protein. In the first experiments, we used a ‘20-h’ culture of B. subtilis prepared by dilution of an optical density (OD) ≫ 0.4 culture to furnish a culture of OD = 0.4 (Method A). Later, on the basis of our optimization experiments we found that a ‘5–9-h’ broth culture of B. subtilis was suitable. Under these conditions the bacteria remained in the log phase (OD = 0.2–0.4) for 5–9 h (Method B) in immersion bacterial suspension. Because the test bacteria were in the log phase a much shorter incubation time (4–8 h) was sufficient for TLC plates instead of the original 18 h in a previous study. One advantage of this method, in addition to the shorter incubation time, is that we can use TLC plates coated with adsorbents other than silica.

Technological and biopharmaceutical optimization of nystatin release from a multiparticulate based bioadhesive drug delivery system

Formulation considerations of a new drug delivery system include controlling the site of release of the active ingredient, maintaining drug level for a suitable time and decreasing dosage frequency. In research and development practice, these therapeutic benefits can be attained by selecting suitable active ingredients and optimizing procedure parameters, determining the composition of the medicine, and dissolution properties. The aim of our study was to design a pharmaceutical preparation with increased local therapeutic effect in the therapy of gastrointestinal candidiasis. The polyene antibiotic nystatin may be an optimal choice for active agent, incorporated in a bioadhesive multiparticulate system. Choosing the proper excipients in the proper dosage form and ensuring prolonged residence time may further improve the optimal treatment. Using an experimental design, the micropellets were prepared with 5% nystatin content, taking the factors average pellet size (~200 to ~800 μm) and the amount of applied carbomer and hydroxyethylcellulose (0-5%) into consideration. Dissolution of the active ingredient was detected by UV spectrophotometric and microbiological assay. The bioadhesive character of the multiparticulate dosage form was examined by ex vivo wash-off test. The only factor which significantly influenced the examined parameters was average pellet size. The proportion of applied bioadhesive excipients had significance mostly in interactions with average pellet size. Eventually, optimized drug release (5-10 min mean dissolution time, 50-55% bioadhesion retention) could be achieved with 550 μm pellet size, containing carbomer and hydroxyethylcellulose in 85:15 ratio.

Preformulation studies and optimization of sodium alginate based floating drug delivery system for eradication of Helicobacter pylori.

The aim of this study was to design a local, floating, mucoadhesive drug delivery system containing metronidazole for Helicobacter pylori eradication. Face-centered central composite design (with three factors, in three levels) was used for evaluation and optimization of in vitro floating and dissolution studies. Sodium alginate (X1), low substituted hydroxypropyl cellulose (L-HPC B1, X2) and sodium bicarbonate (X3) concentrations were the independent variables in the development of effervescent floating tablets. All tablets showed acceptable physicochemical properties. Statistical analysis revealed that tablets with 5.00% sodium alginate, 38.63% L-HPC B1 and 8.45% sodium bicarbonate content showed promising in vitro floating and dissolution properties for further examinations. Optimized floating tablets expressed remarkable floating force. Their in vitro dissolution studies were compared with two commercially available non-floating metronidazole products and then microbiologically detected dissolution, ex vivo detachment force, rheological mucoadhesion studies and compatibility studies were carried out. Remarkable similarity (f1, f2) between in vitro spectrophotometrically and microbiologically detected dissolutions was found. Studies revealed significant ex vivo mucoadhesion of optimized tablets, which was considerably increased by L-HPC. In vivo X-ray CT studies of optimized tablets showed 8h gastroretention in rats represented by an animation prepared by special CT technique.

Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation.

INTRODUCTION In cellular viability assays the sole determination of a single parameter might not give precise information on the extent of toxicity. In our study we worked out a multiparametric microplate assay based on bioluminescent ATP quantification, esterase activity-related fluorescence, nucleic acid staining and total intracellular protein measurement from the same sample in MDCK and HepG2 tissue cultures. METHODS Dose-response analyses were done after ATP depletion by metabolic poisons (NaF, NaN3) and by ochratoxin A (OTA) mycotoxin treatments. A novel perchloric acid fixation/extraction technique was applied in order to obtain intracellular ATP levels, esterase activity, DNA content and protein data simultaneously. Esterase activity was assessed by a fluorogenic staining. Estimation of cell number was done by DAPI fluorescence. Our results were expressed as ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein and ATP/DAPI ratios. Apoptosis/necrosis rates were measured by Annexin V-propidium iodide and 7-aminoactinomycin D flow cytometric assays and effects of OTA on actin cytoskeleton were also studied by using labeled phalloidin for visualization of actin. RESULTS We could verify that the esterase assay was not an energy driven (true viability) process. ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein ratios, DAPI fluorescence and protein levels together with morphological and apoptosis/necrosis parameters deciphered subtle changes in cell viability with good between-run precision. Dose dependent loss in cell number and decreased protein levels were observed in all cases, while disorganization of actin microfilaments was seen in OTA treated cells. The two cell lines did not respond uniformly to the same treatments. DISCUSSION ATP/protein ratio proved to be a useful viability parameter however, the suppression and/or loss of intracellular protein could cause difficulty in interpreting ATP/protein data. We conclude that correct assessment of cellular viability should be done by measuring multiple parameters related to the specific mode of action of the tested toxic compound.

Optimization of conditions for culture of test bacteria used for direct bioautographic TLC detection. 2. Gram-negative test bacterium: Escherichia coli

Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a complex mixture. In this process the developed TLC plate is dipped into a broth culture of a test bacterium and the bacterium will grow directly on the plate. Optimum experimental conditions must, however, be used for each test bacterium. The main purpose of this study was to find optimum culture conditions for a Gram-negative test bacterium, Escherichia coli (ATCC 25922) enabling us to establish a direct bioautographic method with the shortest possible performance time. Because the intracellular adenosine-5’-triphosphate (ATP) level is a direct and sensitive measure of bacterial well-being, ATPassay was used for this purpose. As far as we know this is the first report of the use of an ATP method for optimization of direct bioautography with E. coli. Our optimizing experiments on E. coli culture showed that the bacteria had to be in the log phase (optical density, OD600nm = 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the logphase culture needed a shorter incubation time for bacterial growth on the TLC plate (3 h) than for the original ‘overnight’ culturing suggested in studies by others. In this paper we will show that: – ATP assay is a valid method for optimizing E. coli direct bioautography. – Bacterial ATP level oscillates during the growth phase in culture media. – TLC plates should be immersed in E. coli dipping suspension with OD600nm = 0.1–0.4. – Dipping a developed TLC plate for 10 s gave acceptable results. – Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum. – An ATP/protein ratio of 10–15 nmol mg−1 in dipping culture and ~5 nmol mg−1 on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction. – With our optimized coditions the total performance time of E. coli direct bioautography is 9.6 h instead of the originally reported 11.5 h. – Our procedure results in much sharper contrast of the inhibition zone than that without optimization. – ATP assay is a valid method for optimizing E. coli direct bioautography. – Bacterial ATP level oscillates during the growth phase in culture media. – TLC plates should be immersed in E. coli dipping suspension with OD600nm = 0.1–0.4. – Dipping a developed TLC plate for 10 s gave acceptable results. – Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum. – An ATP/protein ratio of 10–15 nmol mg−1 in dipping culture and ~5 nmol mg−1 on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction. – With our optimized coditions the total performance time of E. coli direct bioautography is 9.6 h instead of the originally reported 11.5 h. – Our procedure results in much sharper contrast of the inhibition zone than that without optimization.

Influence of different types of low substituted hydroxypropyl cellulose on tableting, disintegration, and floating behaviour of floating drug delivery systems

The object of the present study is to evaluate the effect of application of low-substituted hydroxypropyl cellulose (L-HPC) 11 and B1 as excipients promoting floating in gastroretentive tablets. Directly compressed tablets were formed based on experimental design. Face-centred central composite design was applied with two factors and 3 levels, where amount of sodium alginate (X1) and L-HPC (X2) were the numerical factors. Applied types of L-HPCs and their 1:1 mixture were included in a categorical factor (X3). Studied parameters were floating lag time, floating time, floating force, swelling behaviour of tablets and dissolution of paracetamol, which was used as a model active substance. Due to their physical character, L-HPCs had different water uptake and flowability. Lower flowability and lower water uptake was observed after 60 min at L-HPC 11 compared to L-HPC B1. Shorter floating times were detected at L-HPC 11 and L-HPC mixtures with 0.5% content of sodium alginate, whereas alginate was the only significant factor. Evaluating results of drug release and swelling studies on floating tablets revealed correlation, which can serve to help to understand the mechanism of action of L-HPCs in the field development of gastroretentive dosage forms.

Optimization of Growth Conditions for Test Fungus Cultures used in Direct Bioautographic TLC Detection. 3. Test Fungus: Candida albicans

Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones in direct bioautographic TLC. Bioluminescent ATP assay is a highly sensitive method for optimizing the viability of Candida albicans test fungus in bioautographic TLC detection. A suspension of microbes (OD600nm = 0.5-0.7, in Mueller-Hinton broth with 5% glucose) in the log phase of growth can be used for dipping TLC plates. On the basis of our results with Candida albicans, we can differentiate between microbiostatic (bacteriostatic or fungistatic) and microbio-cidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioauto-grams. This technique leads to new possibilities in studies of the interactions between microbes and antimicrobial compounds on bioautographic silica gel TLC plates by using scanning electron microscopy. In this paper we describe an optimization procedure for bioautographic TLC detection.

Influence of barium sulfate X-ray imaging contrast material on properties of floating drug delivery tablets

The objective of the study was to reveal the influence of necessarily added barium sulfate (BaSO4) X-ray contrast material on floating drug delivery tablets. Based on literature survey, a chosen floating tablet composition was determined containing HPMC and carbopol 943P as matrix polymers. One-factor factorial design with five levels was created for evaluation of BaSO4 (X1) effects on experimental parameters of tablets including: floating lag time, total floating time, swelling-, erosion-, dissolution-, release kinetics parameters and X-ray detected volume changes of tablets. Applied concentrations of BaSO4 were between 0 and 20.0% resulting in remarkable alteration of experimental parameters related especially to flotation. Drastic deterioration of floating lag time and total floating time could be observed above 15.0% BaSO4. Furthermore, BaSO4 showed to increase the integrity of tablet matrix by reducing eroding properties. A novel evaluation of dissolutions from floating drug delivery systems was introduced, which could assess the quantity of drug dissolved from dosage form in floating state. In the cases of tablets containing 20.0% BaSO4, only the 40% of total API amount could be dissolved in floating state. In vitro fine resolution X-ray CT imagings were performed to study the volume change and the voxel distributions as a function of HU attenuations by histogram analysis of the images. X-ray detected relative volume change results did not show significant difference between samples. After 24h, all tablets containing BaSO4 could be segmented, which highlighted the fact that enough BaSO4 remained in the tablets for their identification.