Preparation method of penicillin V potassium ß-cyclodextrin inclusion.

In order to conceal the unpleasant smell of pharmaceutical raw materials of Penicillin V potassium, we use ß-cyclodextrin (ß-CD) as a drug carrier. The Penicillin V potassium ß-CD inclusion complex was prepared by saturated aqueous solution method; the characterization of penicillin V potassium ß-CD inclusion complex is determined by scanning electron microscopy, Differential Scanning Calorimeter (DSC) and X-ray Diffraction (XRD), the formation constants of the complexes were determined by UV spectrophotometry; based on the results obtained from the orthogonal experimental design, the optimum preparation process are summarized as follow: ß-CD: Penicillin V potassium = 4: 1 (molar ratio), stirring temperature 50 centigrade, stirring time 12h, the encapsulation efficiency is 29.40%.

Molecular recognition and binding of beta-lactamase II from Bacillus cereus with penicillin V and sulbactam by spectroscopic analysis in combination with docking simulation.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 106 and 3.98 × 105 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics.

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization.

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.

Development of a method of analysis for profiling of the impurities in phenoxymethylpenicillin potassium based on the analytical quality by design concept combined with the degradation mechanism of penicillins.

Accurate analysis of all of the impurities present in a substance is critical for controlling the impurity profiles of drugs. Penicillins can easily yield a formidable array of degradation-related impurities (DRIs) with significantly different polarities and charge properties, which renders identifying each one a complicated matter. In this work, phenoxymethylpenicillin potassium (Pen V) was selected to find a way to quickly establish a robust analysis method for the impurity profiling of penicillin. Based on the analytical quality by design (AQbD) concept and the degradation mechanism of the drug, structures of all of the DRIs were first proposed. Then Pen V and its detected DRIs were separated and identified by liquid chromatography-tandem mass spectrometry method (LC-MS). Characteristic fragment ions and mass fragmentation process of Pen V and its detected DRIs were summarized. In addition, a quantitative structure-retention relationship (QSRR) model was constructed to predict the retention times of undetected impurities and to evaluate whether the chromatographic system can separate them. Finally, a stability-indicating high-performance liquid chromatography (HPLC) method was developed that can separate all of the DRIs of Pen V.

Tunable antimicrobial polypropylene surfaces: simultaneous attachment of penicillin (Gram +) and gentamicin (Gram -).

Surface reactions were performed on polypropylene (PP) surfaces to retard the simultaneous growth of Staphylococcus aureus (S. aureus) and Pseudomonas putida (P. putida) bacteria. Microwave plasma reactions in the presence of maleic anhydride (MA) resulted in the formation of acid groups on the surface of PP. Such surfaces were further modified by conducting two parallel reactions: (1) poly(ethylene glycol) (PEG) was attached to COOH groups of the PP surface, followed by penicillin V (PEN) reactions to target S. aureus destruction and (2) diglycidyl PEG was attached, followed by gentamicin (GEN) reactions, to create antimicrobial surfaces targeted at P. putida . Simultaneous gram "+" and gram "-" resistance was obtained by varying the PEN/GEN ratios on such modified PP surfaces, thus providing the controllable degree of gram "+" and gram "-" antimicrobial strength. While spectroscopic analyses revealed chemical attachments of PEN and GEN, the effectiveness against proliferation of S. aureus (Gram +) and P. putida (Gram -) bacteria was determined using liquid culture tests. These studies show for the first time the formation of tunable antimicrobial polypropylene surfaces with controllable strength.

Quantitative determination of penicillin V and amoxicillin in feed samples by pressurised liquid extraction and liquid chromatography with ultraviolet detection.

A rapid and simple method is proposed for the routine determination of amoxicillin (AMOX) and penicillin V (PENV) in swine feedingstuffs. The method is based on pressurised liquid extraction (PLE) followed by high performance liquid chromatography with ultraviolet detection (PLE-HPLC-UV) for antibiotic analysis. Parameters affecting PLE procedure, such as temperature, solvent composition, number of extraction cycles and sample cell size, were evaluated in order to achieve the highest extraction efficiency. The optimised method employed 11mL extraction cells, acetonitrile-water mixtures (25:75, v/v) for AMOX and (50:50, v/v) for PENV, as extraction solvent, 102.07atm of extraction pressure, 50 degrees C of extraction temperature, 5min of static time and 60% flush volume of the cell size. Extracts were filtered and directly analysed by HPLC-DAD/UV without further clean-up. Mean recovery rates for feed samples fortified with 200-500mgkg(-1) of both antibiotics were 86% for AMOX (RSD< or =6%) and 95% for PENV (RSD< or =3%). The method was successfully applied to the analysis of a commercial medicated swine feedingstuff, and the results were in good agreement with those obtained using mechanical shaking or ultrasonic extraction combined with solid phase extraction (UE-SPE), previously applied in the literature for feed analysis. The extraction efficiencies were evaluated by statistical comparison (analysis of variance, ANOVA-single factor) of the results obtained using the different extraction methods. Compared to the alternative techniques, PLE offers several practical advantages: easy to perform, fast, savings in solvent volume and in time, all steps are fully automated and further clean-up is not necessary for penicillin analysis.

Effect of Overencapsulation on the Disintegration and Dissolution of Licensed Formulations for Blinding in Randomized Controlled Trials.

Overencapsulation is a technique used to conceal tablet products for blinding in randomized controlled trials. A tablet is inserted in an opaque capsule shell with backfill excipient to prevent rattling. Regulatory authorities require evidence that such modification does not materially alter drug release to approve their use in trials. The objective of this study was to assess impact of overencapsulation on disintegration and dissolution of 4 immediate-release drug products (penicillin V, gemfibrozil, ciprofloxacin, and furosemide). Each unmodified tablet was compared to 3 overencapsulated tablets with differing backfill excipient (colloidal silica, lactose monohydrate, or microcrystalline cellulose). All 12 overencapsulated tablets met disintegration and dissolution acceptance criteria. Dissolution acceptance was dependent on apparatus as only 4/12 formulations met specifications using the rotating basket compared to 12/12 using the rotating paddle. Significant differences in release were observed at early time points (T5-T15). No correlation was observed between aqueous solubility and release, although dissolution of the lipophilic drug gemfibrozil was least impacted by overencapsulation. There was evidence that type/quantity of backfill delays release at early time points. These findings indicate that under the specified conditions, overencapsulated formulations of 4 drugs, 1 from each class of the Biopharmaceutics Classification System, met compendial requirements for release testing.

Photolysis of four ß­lactam antibiotics under simulated environmental conditions: Degradation, transformation products and antibacterial activity.

߭Lactam antibiotics are among the most widely used antibiotics in human medicine and their effects on the aquatic environment - concerning bacterial resistance - are controversially discussed. This study focused on the photolysis of the four ߭lactam antibiotics - amoxicillin, ampicillin, penicillin V and piperacillin - under simulated environmental conditions. It was observed that all investigated ߭lactam antibiotics are photolytically degradable by simulated sunlight (1 kW/m2) with half-lives between 3.2 and 7.0 h. Structure elucidation of transformation products performed with liquid chromatography coupled to high resolution mass spectrometry showed that the hydrolysis of the ߭lactam ring is the primary transformation reaction, followed by the elimination of carboxylic and dimethyl thiazolidine carboxylic acid. Growth inhibition tests on Bacillus subtilis showed the loss of bactericide activity of irradiated solutions of amoxicillin, ampicillin and piperacillin, suggesting the transformation of the ߭lactam ring is responsible for the antibiotic effect. In contrast, the solutions of penicillin V did not show any decline of the antibacterial activity after photolytic degradation, probably due to the formation of still active epimers.

Development of a Minimally Invasive Microneedle-Based Sensor for Continuous Monitoring of ß-Lactam Antibiotic Concentrations in Vivo.

Antimicrobial resistance poses a global threat to patient health. Improving the use and effectiveness of antimicrobials is critical in addressing this issue. This includes optimizing the dose of antibiotic delivered to each individual. New sensing approaches that track antimicrobial concentration for each patient in real time could allow individualized drug dosing. This work presents a potentiometric microneedle-based biosensor to detect levels of ß-lactam antibiotics in vivo in a healthy human volunteer. The biosensor is coated with a pH-sensitive iridium oxide layer, which detects changes in local pH as a result of ß-lactam hydrolysis by ß-lactamase immobilized on the electrode surface. Development and optimization of the biosensor coatings are presented, giving a limit of detection of 6.8 µM in 10 mM PBS solution. Biosensors were found to be stable for up to 2 weeks at -20 °C and to withstand sterilization. Sensitivity was retained after application for 6 h in vivo. Proof-of-concept results are presented showing that penicillin concentrations measured using the microneedle-based biosensor track those measured using both discrete blood and microdialysis sampling in vivo. These preliminary results show the potential of this microneedle-based biosensor to provide a minimally invasive means to measure real-time ß-lactam concentrations in vivo, representing an important first step toward a closed-loop therapeutic drug monitoring system.

Electrical conductances of dilute aqueous solutions of sodium penicillin G, potassium penicillin G, and potassium penicillin V in the 278.15-313.15 K temperature range.

Systematic determinations of electrical conductivities of sodium penicillin G, potassium penicillin G, and potassium penicillin V in the 278.15-313.15 K temperature range are reported. These conductivities are examined by applying the Quint-Viallard conductivity equations and the Debye-Hückel equations for activity coefficients. Determined dissociation constants and the limiting conductances of penicillin anions are based on the assumption that in dilute aqueous solutions, penicillin salts behave as acidic salts of dibasic acids, which are the final products of degradation reactions in acidic media.

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin-binding protein PonA1 reveal potential mechanisms of antibiotic resistance.

UNLABELLED: Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic-resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin-binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor-free form and in complex with penicillin V are reported. We used site-directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of ß-lactams. Structural comparison of the PonA1 apo-form and the antibiotic-bound form shows that binding of penicillin V induces conformational changes in the position of the loop ß4'-α3 surrounding the penicillin-binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested ß-lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall-targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. DATABASE: Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

Optimization of culture medium and conditions for penicillin acylase production by Streptomyces lavendulae ATCC 13664.

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylase. This extracellular enzyme recently has been reported to be a penicillin K acylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 microg/mL of CuSO4 x 5H2O was found to be best for acylase production. In such optimized culture medium, fermentation of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.

Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

Randomized, double blind comparison of brand and generic antibiotic suspensions: I. A study of taste in adults.

BACKGROUND: A belief that brand oral liquid medications taste better than their generic counterparts may influence prescribing habits among pediatricians. METHODS: We undertook a prospective, randomized, double blinded, comparative evaluation of the taste of brand and generic erythromycin ethylsuccinate, cephalexin monohydrate, erythromycin ethylsuccinate/sulfisoxazole, penicillin V potassium and trimethoprim-sulfamethoxazole in 42 adult volunteers. Subjects tasted one class of brand and generic antibiotics and rated them according to smell, texture, taste and aftertaste. RESULTS: At least one generic preparation of cephalexin, erythromycin ethylsuccinate/sulfisoxazole and penicillin V potassium was rated equal in taste to the respective brand name products. However, brand erythromycin estolate and trimethoprim-sulfamethoxazole name brand suspensions rated significantly higher than the other products tested. CONCLUSIONS: Based on our results brand name oral antibiotic formulations do not necessarily taste better than their generic counterparts.

13C and 15N-chemical shift anisotropy of ampicillin and penicillin-V studied by 2D-PASS and CP/MAS NMR.

The principal values of the chemical shift tensors of all 13C and 15N sites in two antibiotics, ampicillin and penicillin-V, were determined by 2-dimensional phase adjusted spinning sideband (2D-PASS) and conventional CP/MAS experiments. The 13C and 15N chemical shift anisotropies (CSA), and their confidence limits, were evaluated using a Mathematica program. The CSA values suggest a revised assignment of the 2-methyl 13C sites in the case of ampicillin. We speculate on a relationship between the chemical shift principal values of many of the 13C and 15N sites and the beta-lactam ring conformation.

Biopharmaceutical characterization of oral immediate release drug products. In vitro/in vivo comparison of phenoxymethylpenicillin potassium, glimepiride and levofloxacin.

The development of in vitro dissolution tests using the paddle and basket apparatus is described with respect to the qualification/validation of the testing procedure. Three examples of immediate release products containing phenoxymethylpenicillin potassium, glimepiride, and levofloxacin providing different solubility characteristics are evaluated. The solubility was high in the case of phenoxymethylpenicillin potassium and levofloxacin and low for glimepiride according to the biopharmaceutics classification system. The permeability is studied using the human colorectal carcinoma cell line CaCo-2. The permeability (10(-6) cm/s) of phenoxymethylpenicillin potassium, glimepiride, and levofloxacin was high. The determined permeability data are confirmed by absorption data obtained by means of numerical deconvolution of plasma concentrations. Recommendations are given for the biopharmaceutical characterization of the three immediate release drug products, taking into account in vitro and in vivo comparison as well as the biopharmaceutics drug classification system. The evaluated acceptance criteria are the following: phenoxymethylpenicillin potassium (80% in 30 min), glimepiride (80% in 15 min) and levofloxacin (80% in 30 min). Typically, for immediate release formulations, one limit is specified for the dissolution to ensure the release of the active ingredient within the present time period. Since phenoxymethylpenicillin potassium and levofloxacin belong to Case 1, no in vitro/in vivo correlation is expected, absorption may be gastric emptying dependent. Glimepiride is categorized to Case 2. Nevertheless, a correlation with the in vivo dissolution profile does not exist, because of the pH-dependent low solubility of the drug. Finally, recommendations are made for the batch control of drug products in accordance with the four Cases.

Evaluation of LC methods for the separation of phenoxymethylpenicillin and its related substances.

Five isocratic reversed-phase liquid chromatography (LC) methods have been examined for the separation of phenoxymethylpenicillin and its related substances. A method previously selected for the analysis of benzylpenicillin gave the best selectivity. The mobile phase of this method is composed of 0.5 M phosphate buffer (pH 3.5)-water-methanol (10:50:40), the amount of organic modifier was slightly adapted when it was used with different columns. Similar selectivity is obtained not only on different C18 materials but also on C8 packings. The selectivity on poly(styrenedivinylbenzene) is less good. As method performance test a resolution test with benzylpenicillin was used. The robustness of this method was examined by applying a full factorial design to test the influence of the content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the temperature of the column. The results show that the method is robust.

Voltammetric determination of penicillin V in pharmaceutical formulations and human urine using a boron-doped diamond electrode.

Simple, sensitive and selective differential pulse voltammetric method for determination of penicillin V on a bare (unmodified) boron-doped diamond electrode has been developed. Penicillin V provided highly reproducible and well-defined irreversible oxidation peak at very positive potential of +1.6V (vs. Ag/AgCl). The optimum experimental conditions for oxidation of penicillin V were achieved in acetate buffer solution (pH 4.0). The modulation amplitude of 0.1V, modulation time of 0.05s and scan rate of 0.05Vs(-1) were selected as optimum instrumental parameters for differential pulse voltammetry. Linear response of peak current on the concentration in the range from 0.5 to 40µM with coefficient of determination of 0.999, good repeatability (RSD of 1.5%) and detection limit of 0.25µM were observed without any chemical modifications and electrochemical surface pretreatment. The effect of possible interferents such as stearic acid, glucose, urea, uric acid and ascorbic acid appeared to be negligible which evidently proved the good selectivity of method. The practical analytical utility of proposed method was demonstrated by determination of penicillin V in pharmaceutical formulations (tablets) and human urine samples with satisfactory recoveries (from 98 to 101% for tablets and 97 to 103% for human urine).

Determination of ß-lactam antibiotics in milk based on magnetic molecularly imprinted polymer extraction coupled with liquid chromatography-tandem mass spectrometry.

In this work, a rapid and selective method was successfully developed using the magnetic molecularly imprinted polymer (MMIP) as sorbent for the extraction of ß-lactam antibiotics (BLAs) from milk samples. The MMIP has been prepared using penicillin V potassium (PENV) as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and Fe(3)O(4) magnetite as magnetic component. The experimental results showed that the MMIP had high affinity and selectivity toward PENV and other structurally related BLAs. The extraction process was carried out in a single step by mixing the extraction solvent, MMIPs and milk samples under ultrasonic action. When the extraction was completed, the MMIPs adsorbing the analytes were separated from the sample matrix by an external magnet. The analytes eluted from the MMIP were analyzed by liquid chromatography-tandem mass spectrometry. For achieving optimal preconcentration and reducing non-specific interactions, various parameters affecting the extraction efficiency such as extraction mode, extraction solvent, the amount of MMIPs, extraction time, washing solution and eluting solution were comprehensively evaluated. Under the optimal conditions, the detection limits of BLAs are in the range of 1.6-2.8 ng mL(-1). The relative standard deviations of intra- and inter-day ranging from 3.2% to 8.3% and from 3.6% to 9.8% are obtained, respectively. The method was applied to determine BLAs including PENV, amoxicillin and oxacillin in five milk samples from different provenances. The recoveries of BLAs in these samples from 71.6% to 90.7% are obtained.

Quantum chemical studies on the inhibition potentials of some Penicillin compounds for the corrosion of mild steel in 0.1 M HCl.

Inhibitive and adsorption properties of Penicillin G, Amoxicillin and Penicillin V potassium were studied using gravimetric, gasometric and quantum chemical methods. The results obtained indicate that these compounds are good adsorption inhibitors for the corrosion of mild steel in HCl solution. The adsorption of the inhibitors on mild steel surface is spontaneous, exothermic and supports the mechanism of physical adsorption. From DFT results, the sites for nucleophilic attacks in the inhibitors are the carboxylic acid functional group while the sites for electrophilic attacks are in the phenyl ring. There was a strong correlation between theoretical and experimental inhibition efficiencies.