Evaluation of hyaluronic acid nanoparticle embedded chitosan-gelatin hydrogels for antibiotic release.

The development of chitosan-gelatin (CS-G) hydrogels embedded with ampicillin-loaded hyaluronic acid nanoparticles (HA-NPs) for wound dressing is proposed. It was aimed to provide controlled ampicillin delivery by incorporation of HA-NPs into biocompatible CS-G hydrogel structure. According to in vitro ampicillin release studies, 55% of ampicillin was released from CS-G/HA-NPs hydrogels after 5 days. Antibacterial performance of CS-G/HA-NPs hydrogels was proven with agar disc diffusion test. For cytotoxicity assay, fibroblast cell viability increased in CS-G/HA-NPs hydrogels compared with CS-G group after 24 hr incubation. Consequently, the potential ability of CS-G/HA-NPs hydrogels as a controlled drug delivery system has been verified.

Ampicillin-mediated functionalized gold nanoparticles against ampicillin-resistant bacteria: strategy, preparation and interaction studies.

Antibiotic resistance is a highly challenging concern of infectious diseases, and it requires a rational approach to overcome. Through this work, we have synthesized ampicillin-capped gold nanoparticles (Amp-Au NPs) and studied its interaction with bacterial cells. In this process of synthesis, the primary amine group of ampicillin acts as both reducing as well as capping agent. In addition to synthesized gold nanoparticles, the ß-lactam ring remains free to interact with bacteria. This approach not only utilizes the maximum efficiency of nanoparticles and antibiotics towards ampicillin sensitive bacterial cells but also proves to be effective against ampicillin resistance bacteria. Our results illustrate that the optimized system of Amp-Au NPs was formulated by taking 1.25 mM ampicillin and 10-2 of gold ions concentration. UV-vis spectrum of gold nanoparticles and the presence of ampicillin were recorded at around 540 nm and 259 nm, respectively. Microscopic images indicate that particles are nearly spherical and are in size range between 25 and 50 nm. Moreover, formulated Amp-Au NPs show successful accumulation onto the surface of the bacterial cell as a result of which pores were formed into the bacterial membrane. The entry of nanoparticles into bacterial cells was validated through both atomic force microscopy and fluorescent microscopy. The adhesive properties of this coating material and its stability in various pH, i.e. pH 3, pH 7 and pH 10 conditions, could make them a good candidate in the prevention of biofilm formation. Amp-Au NPs show promising antimicrobial activity against ampicillin resistance Escherichia coli bacteria. Furthermore, antimicrobial studies indicate that the efficacy of Amp-Au NPs increased against both ampicillin sensitive and ampicillin resistance bacteria up to sixteen folds and four folds respectively.

One-step synthesis of carbon dots for selective bacterial inactivation and bacterial differentiation.

Novel carbon dots (CDs) were synthesized by a one-pot hydrothermal approach using ampicillin as a precursor, and the as-prepared CDs exhibited a high quantum yield (23%). The CDs were found to possess abundant surface functional groups, thus providing good permeability to the cell, and the antibacterial activity of CDs was evaluated. S. aureus and L. monocytogenes were selected as model bacteria, and our results showed that the CDs exhibited antibacterial activity against S. aureus and L. monocytogenes under visible light illumination, even at low concentrations. The antibacterial mechanism is believed to be the production of reactive oxygen species (ROS) from CDs under visible light irradiation, which attacked the bacterial cell membranes, resulting in the death of the bacteria. In addition, because of the multicolor fluorescence properties of CDs, staining of S. aureus and L. monocytogenes obtained multicolor fluorescence images at different excitation wavelengths. Based on these results, CDs are a promising candidate material for biological applications. Graphical abstract.

Development of an aptamer-ampicillin conjugate for treating biofilms.

Biofilm formation involves the development of extracellular matrix and initially depends on adherence and tropism by flagellar movement. With the widespread development of antibiotic resistance and tolerance of biofilms, there is a growing need for novel anti-infective strategies. No currently approved medications specifically target biofilms. Aptamers are single-stranded nucleic acid molecules that may bind to their targets with high affinity and affect the target functions. We developed a bifunctional conjugate by linking an aptamer targeting bacterial flagella with ampicillin. We investigated its influence on biofilm prevention and dissolution by ultraviolet-visible spectrophotometry, inverted microscopy, and atomic force microscopy. This conjugate had distinctive antibacterial activity. Notably, the conjugate was more active than either component, and thus had a synergistic effect against biofilms.

Design, synthesis and microbiological evaluation of ampicillin-tetramic acid hybrid antibiotics.

Exploiting iron-uptake pathways by conjugating ß-lactam antibiotics with iron-chelators, such as catechol and hydroxamic acid is a proven strategy to overcome permeability-related resistance in Gram-negative bacteria. As naturally occurring iron-chelating tetramic acids have not been previously examined for this purpose, an exploratory series of novel ampicillin-tetramic acid hybrids that structurally resemble ureidopenicillins was designed and synthesized. The new analogs were evaluated for the ability to chelate iron and their MIC activities determined against a representative panel of clinically significant bacterial pathogens. The tetramic acid ß-lactam hybrids demonstrated a high affinity to iron in the order of 10-30 M3. The hybrids were less active against Gram-positive bacteria. However, against Gram-negative bacteria, their activity was species dependent with several hybrids displaying improved activity over ampicillin against wild-type Pseudomonas aeruginosa. The anti-Gram-negative activities of the hybrids improved in the presence of clavulanic acid revealing that the tetramic acid moiety did not provide added protection against ß-lactamases. In addition, the hybrids were found to be efflux pump substrates as their activities markedly improved against pump-inactivated strains. Unlike the catechol and hydroxamic acid siderophore ß-lactam conjugates, the activities of the hybrids did not improve under iron-deficient conditions. These results suggest that the tetramic acid hybrids gain permeability via different membrane receptors, or they are outcompeted by native bacterial siderophores with stronger affinities for iron. This study provides a foundation for the further exploitation of the tetramic acid moiety to achieve novel ß-lactam anti-Gram-negative agents, providing that efflux and ß-lactamase mediated resistance is addressed.

Synthesis of 2-oxoquinoline-3-carboxamide of ampicillin and amoxicillin as inhibitors of penicillin binding protein 1A of pseudomonas aeruginosa.

A series of ampicillin and amoxicillin derivatives were prepared by N-acylation with N1-substituted 1, 2-dihydro-2-oxoquinoline carboxylic acids by Schotten-Baumann procedure and evaluated as inhibitors of Pseudomonas aeruginosa using molecular docking study. Most of the derivatives showed remarkableactivity against Gram positive and Gram negative bacteria in vitro.

Iron transport-mediated drug delivery: practical syntheses and in vitro antibacterial studies of tris-catecholate siderophore-aminopenicillin conjugates reveals selectively potent antipseudomonal activity.

An artificial tris-catecolate siderophore with a tripodal backbone and its conjugates with ampicillin and amoxicillin were synthesized. Both conjugates exhibited significantly enhanced in vitro antibacterial activities against Gram-negative species compared to the parent drugs, especially against Pseudomonas aeruginosa . The conjugates appeared to be assimilated by an induced bacterial iron transport process as their activities were inversely related to iron concentration. The easily synthesized tris-catecolate siderophore has great potential for future development of various drug conjugates to target antibiotic-resistant Gram-negative bacteria.

Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates.

Semi-synthetic ß-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the ß24 position. ßPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, ßPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. ßPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

A simple method for simultaneous determination of some organic liquids in in-process materials and effluents produced in the manufacture of amoxicillin and ampicillin.

A simple GC method for simultaneous determination of pivaloylchloride, methylacetoacetate, ethylacetoacetate and 2-ethylhexanoic acid in the presence of each other has been developed using glass column packed with 1% Silicone OV-17 on Gaschrom Q 100-120 mesh with temperature programming. Various performance parameters including precision, linearity and limit of detection have been evaluated. The method was found to be suitable for the analysis of these chemicals in in-process materials and effluents associated with the manufacture of amoxicillin and ampicillin. The run time was less than 15 min. The method has been successfully applied to determine the level of these hazardous organic liquids in real time samples.

Influence of mass transfer limitations on the enzymatic synthesis of beta-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose.

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.

Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation.

Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.).

Synthesis, structural analysis and antibacterial activity of a butyl ester derivative of ampicillin.

BACKGROUND: Ester groups placed on medicinal agents have been found to increase lipophilicity (more positive log p value). Alterations in a partition coefficient may affect medicinal activity. Formation of an ester group such as -C(O)OCH(2)CH(2)CH(2)CH(3) from a carboxyl group [-C(O)OH] can be efficiently accomplished utilizing diazoalkanes and produces a prodrug-like structure of ampicillin (a bipartate drug carrier). Formation of an ester group may produce beneficial changes in medicinal parameters such as log P, log BB, and molecular polar surface area (TPSA). METHODS: An ester of ampicillin was synthesized by replacing the carboxyl group with a butyl ester group. The ester formed was synthesized utilizing diazobutane, which is a gas at room temperature and reacts with the acidic hydrogen of the carboxylic acid. The ampicillin ester was dissolved into LB agarose media at levels of 0.228 and 0.180 mg/ml, and its antibacterial activity was compared to that of normal ampicillin. Medicinal parameters such as log P, solubility, polar surface area, log BB, molecular dipole, Clog P, intestinal absorption and the 'Rule of 5' were determined. RESULTS: The ampicillin ester consisted of yellow crystals that were stable for >12 weeks at 0 degrees C. The ester has reduced hydrogen bonding capacity and increased lipophilic character (by log P) compared to the parent structure. Each concentration of the ester tested induced 100% growth inhibition of ampicillin-susceptible but streptomycin-resistant Escherichia coli bacteria, and >30% growth inhibition of an ampicillin-resistant E. COLI. This ester compound of ampicillin showed zero violations of the 'Rule of 5', which indicates good bioavailability and good bioactivity. Intestinal absorption indicated by TPSA value showed moderate absorption activity for both parent and ester compounds. The lipophilic substituent constant for the butyl ester is +0.941, which indicates increased lipophilicity. The ester group improves log BB and dermal permeability of ampicillin. CONCLUSION: The butyl ester of ampicillin inhibited the growth of susceptible and resistant E. COLI. Favorable values for parameters such as TPSA, log P, log BB, and zero violations of the 'Rule of 5' suggest good bioavailability and good bioactivity.

Synthesis and biological activity of tris- and tetrakiscatecholate siderophores based on poly-aza alkanoic acids or alkylbenzoic acids and their conjugates with beta-lactam antibiotics.

New linear and tripodal tri-aza- and tetra-aza alkanoic acids or alkylbenzoic acids were prepared as basic structures for siderophore mimetics from polyamines and oxocarbonic acids or formylbenzoic acids by catalytic hydrogenation. From these acids acetylated tris- and tetrakiscatecholates or 8-acyloxy-2,4-dioxo-benzoxazine derivatives as well as compounds with spacer groups were synthesized. These derivatives were coupled with ampicillin, amoxicillin, bacampicillin or cefaclor to new siderophore antibiotic conjugates. Most of the catecholate derivatives showed high siderophore activities in strains of Pseudomonas aeruginosa and Escherichia coli in a growth promotion assay under iron limitation conditions. The beta-lactam conjugates were highly active in vitro against Gram-negative bacteria correlating to the siderophore activity of the catecholate moiety and depending on the beta-lactam part. One ampicillin conjugate based on 5-(aminoethyl)-2,5,8-triazaalkylbenzoic acid was highly active against Gram-negative and Gram-positive bacteria. It was shown that conjugates with enhanced activity against Gram-negative bacteria use active iron uptake routes to penetrate the bacterial outer membrane barrier. Correlations between structure and biological activity were studied.

Analysis of an ampicillin propyl ester prodrug which inhibits the growth of Escherichia coli.

An ampicillin prodrug was synthesized by utilizing the chemical reaction of ampicillin with diazopropane (CH(3)CH(2)CHN(2)) in an organic solvent. The result is esterification of the carboxylic acid functional group. The ampicillin prodrug is a solid that forms yellow crystals which are soluble in water and LB agarose media. The ampicillin prodrug was stable for more than 10 weeks when stored at < or = 0.0 degrees C. The prodrug has reduced hydrogen-bonding capability compared with the unmodified structure of ampicillin. Evaluation of the logP parameter (the octanol/water partition coefficient) indicates that the ampicillin prodrug (logP=1.773) has increased lipophilic characteristics relative to the unmodified ampicillin structure (logP=1.06). The lipophilic substituent constant for the esterification of the carboxylic acid is 0.713, a positive value which indicates that the substituent has a lipophilic nature. The ampicillin prodrug was solubilized into LB agarose media at a concentration of 0.228 mg/ml, and was found to induce 100% growth inhibition of an ampicillin-susceptible and streptomycin-resistant Escherichia coli strain (designated DH1), and induced greater than 30% growth inhibition of an ampicillin-resistant E. coli strain (designated PKK). Synthesis of this prodrug utilizing a diazoalkane was highly efficient, with no undesirable by-products being formed.

Penicillin acylase-catalyzed ampicillin synthesis using a pH gradient: a new approach to optimization.

The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-(-)-phenylglycine amide (D-PGA) to 6-aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (V(S)/V(H)) is pH-dependent and exhibits a maximum at pH 6.5-7.0 for a saturated solution of 6-APA; 2) at a fixed 6-APA concentration below saturation, V(S)/V(H) increases with decreasing pH. Optimum synthetic efficiency could, therefore, be achieved by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing the pH to 6.3 in the course of 6-APA consumption. A conversion of 96% of 6-APA and 71% of D-PGA into ampicillin was accomplished in an optimized procedure, which significantly exceeds the efficiency of enzymatic synthesis performed at a constant pH of either 7.0 or 6.3.

Synthesis and antimicrobial activity of new pyrrolecarboxylic acid derivatives of ampicillin and amoxicillin.

Twelve new penicillin derivatives were prepared for microbiological evaluation by N-acylation of ampicillin (CAS 69-53-4) and amoxicillin (CAS 26787-78-0) with activated pyrrolecarbocylic acids via mixed anhydrides following the Schotten-Baumann procedure. An alternative synthetic approach via chloroanhydrides was checked and rejected because of the instability established for these intermediates. NMR and IR spectral data together with TLC confirmed the structure and the purity of the new products. Antimicrobial tests in vitro indicated a reduction of the antibacterial activity compared with that of ampicillin and amoxicillin as reference antibiotics, but their minimal inhibitory concentrations (MIC) were still in the range of 0.62-16 micrograms/ml against standard and clinical Gram positive strains. Preliminary toxicological evaluations showed low toxicity.

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation.

Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.

Synthesis and properties of dextran-linked ampicillin.

Ampicillin was coupled to dextran of average molecular weight 9,000 or 81,200 via the cyanogen bromide method. The degree of drug substituted per glucose unit (DSG) was varied from 0.104 to 0.028 (by weight bases: 22.1-5.9%) depending on the ratio of the reactants. Water solubility of dextran-linked ampicillin increased compared with free ampicillin, and the solubility decreased as the amount of ampicillin substituted increased. Plasma concentration of ampicillin, which appeared after intravenous administration of dextran-linked ampicillin in rats, was higher than when free ampicillin was administered, and the more so, the higher the molecular weight of dextran. Plasma half-life of dextran-linked ampicillin was two times longer than that of free ampicillin in rats. Antibacterial activities of dextran-linked ampicillin were evaluated against Staphylococcus aureus, Bacillus substillis, and Escherichia coli at two concentration levels according to the cup-plate method by measuring the diameter of inhibition zone, which was comparable to that of free ampicillin.

Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase.

[reaction--see text] Penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) was immobilized in a simple and effective way by physical aggregation of the enzyme, using a precipitant, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). These had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less. Penicillin G acylase CLEAs also catalyzed the synthesis of ampicillin in a broad range of organic solvents.