DNA binding studies of antibiotic drug cephalexin using spectroscopic and molecular docking techniques

The purpose of this study was to explore an accurate characterization of the binding interaction of antibiotic drug cephalexin with calf thymus DNA (CT-DNA) as a relevant biological target by using UV absorption, fluorescence spectroscopy and circular dichroism (CD) in vitro under simulated physiological conditions (pH = 7.4) and also through a molecular modeling study. The results showed that the drug interacts with the DNA helix via a minor groove binding mode. The thermodynamic parameters were calculated and showed that the reaction between the drug and CT-DNA was exothermic. In addition, the drug enforced traceable changes in the viscosity of DNA. The molecular modeling results indicated that cephalexin forcefully binds to the minor groove of DNA with a relative binding energy of ?21.02?kJ mol^?1. The obtained theoretical results were in good agreement with those obtained from experimental studies.     

Rapid and sensitive high-performance liquid chromatographic determination of four cephalosporin antibiotics in pharmaceuticals and body fluids

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250 x 4-mm, 5-microm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)-CH(3)OH 78-22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV-Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-microl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/microl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/microl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1-8 ng/microl.     

Kinetically controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase

Advantages of performing penicillin G amidase catalysed synthesis of ampicillin and cephalexin by enzymatic acyl transfer to the β-lactam antibiotic nuclei in a highly condensed system using mainly undissolved substrates, with no apparent aqueous liquid phase, were demonstrated. It was shown that synthesis can be performed in the absence of a liquid phase formed by water or an organic co-solvent. This highly condensed system is formed by a liquid phase given by one of the reactant, the phenylglycine methyl ester (PGM), that remains liquid in these operative conditions and the partially dissolved β-lactam nucleus. Operating in such highly condensed system, the water that causes the hydrolysis of PGM is limited to the water hydrating the support on which the enzyme is covalently immobilised. In this way the reaction system is maintained at a controlled degree of hydration.

In the present work the reaction system was modulated by eliminating the solvent (aqueous or aqueous/organic), reducing the amount of water to the minimum for the biocatalytic activity and using PGM as solvent and reagent at the same time. The synthesis was conducted with equimolar amounts of PGM and the β-lactam nucleus, with a reduced hydrolysis of the activated acyl donor. We have also studied a simple and efficient method for the workup of the reaction where the unreacted reagents can be recovered after selective filtration and precipitation.     

A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties

The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95–100% and a recovered activity of 50–65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g_{dry weight}) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.     

Non-isothermal cephalexin hydrolysis by penicillin G acylase immobilized on grafted nylon membranes

A new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer. Penicillin G acylase (PGA) and cephalexin were employed as catalyst and substrate, respectively. Cephalexin hydrolysis was studied in bioreactors operated under isothermal and non-isothermal conditions. A hydrolysis increase was found when the temperature of the warm membrane surface was kept constant and the temperature of the other membrane surface was kept at a lower value. The hydrolysis increase was linearly proportional to the applied temperature difference. Cephalexin hydrolysis increased to about 10% when a temperature difference of 1°C was applied across the catalytic membrane. These results have been attributed to the non-isothermal cephalexin transport across the membrane, i.e., to the process of thermodialysis. In this way, the enzyme immobilized on and into the membrane reacts with a substrate concentration higher than that produced by simple diffusion under isothermal conditions.     

Antifungal Cobalt(II), Copper(II), Nickel(II) and Zinc(II) Complexes of Furanyl-,Thiophenyl-, Pyrrolyl-, Salicylyl- and Pyridyl-derived Cephalexins

Some novel cephalexin-derived furanyl, thiophenyl, pyrrolyl, salicylyl and pyridyl Schiff's bases and their cobalt (II), copper (II), nickel (II) and zinc (II) complexes have been synthesized and studied for their antifungal properties against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. The presence of metal ions in the investigated Schiff's base complexes reported here lead to significant antifungal activity, whereas the parent ligands were generally less active.     

Characterization of the activity of penicillin G acylase immobilized onto nylon membranes grafted with different acrylic monomers by means of γ-radiation

Penicillin G acylase (PGA) has been immobilized onto nylon membranes grafted with methylmethacrylate (MMA) or diethyleneglycoldimethacrylate (DGDA) monomers by means of γ-radiation. Hexamethylenediamine (HMDA) has been used as spacer between the grafted membranes and the enzyme. Glutaraldehyde (GA) was used as crosslinking to couple the enzyme to the HMDA. The catalytic membranes so prepared were studied as a function of pH and temperature of the solution containing the substrate. The membranes showing the best characteristics were the ones grafted with DGDA. The dependence of the behavior of these membranes on several experimental conditions was studied, i.e., the temperature and duration of the aminoalkylation process, spacer concentration, the glutaraldehyde concentration and the enzyme concentration. The experimental conditions giving the best performance of the catalytic membranes have been deduced. The time requested to obtain 50% of substrate conversion, i.e., hydrolysis of cephalexin, has been studied as a function of its initial concentration.     

Chronopharmacological Study of Cephalexin in Dogs

Recent studies have identified a 24 h rhythm in the expression and function of PEPT1 in rats, with significantly higher levels during the nighttime than daytime. Similarly, temporal variations have been described in glomerular filtration rate and renal blood flow, both being maximal during the activity phase and minimal during the rest phase in laboratory rodents. The aim of this study was to assess the hypothesis that the absorption of the first‐generation cephalosporin antibiotic cephalexin by dogs would be less and the elimination would be slower after evening (rest span) compared to morning (activity span) administration, and whether such administration‐time changes could impair the medication's predicted clinical efficacy. Six (3 male, 3 female; age 4.83±3.12 years) healthy beagle dogs were studied. Each dog received a single dose of 25 mg/kg of cephalexin monohydrate per os at 10∶00 and 22∶00 h, with a two‐week interval of time between the two clock‐time experiments. Plasma cephalexin concentrations were determined by microbiological assay. Cephalexin peak plasma concentration was significantly reduced to almost 77% of its value after the evening compared to morning (14.52±2.7 vs. 18.77±2.8 µg/mL) administration. The elimination half‐life was prolonged 1.5‐fold after the 22∶00 h compared to the 10∶00 h administration (2.69±0.9 vs. 1.79±0.2 h). The area under the curve and time to reach peak plasma concentration did not show significant administration‐time differences. The duration of time that cephalexin concentrations remained above the minimal inhibitory concentrations (MIC) for staphylococci susceptiblity (MIC=0.5 µg/mL) was>70% of each of the 12 h dosing intervals (i.e., 10∶00 and 22∶00 h). It can be concluded that cephalexin pharmacokinetics vary with time of day administration. The findings of this acute single‐dose study require confirmation by future steady‐state, multiple‐dose studies. If such studies are confirmatory, no administration‐time dose adjustment is required to ensure drug efficacy in dogs receiving an oral suspension of cephalexin in a dosage of 25 mg/kg at 12 h intervals.     

固定化巨大芽孢杆菌青霉素G酰化酶酶促合成头孢氨苄的研究

β—内酰胺系列抗菌素抗菌谱广、疗效高、毒副作用小,国际上研究与应用日渐广泛深入。头孢氨苄(Cephalexin)是重要的半合成抗菌素之一,由头孢霉素母核7—氨基脱乙酰氧基头孢烷酸(简称7-ADCA)和侧链结构物苯甘氨酸或其甲酯(PGME)经酰化而生成。酰化有化学法和酶法两种。采用青霉素G酰化酶或a—氨基酸酯酶或,a—氨酰转移酶的酶法,具有工艺操作简单、无需基团保护、环境污染轻等优点。继日本人于70年代初试验成功酶法之后,80年代初我们开展了这方面研究。制备方面,胞外酶优于胞内酶;使用方面,固定化酶优于固定化细胞。在用具有青霉素G酰化酶活性的固定化大肠杆菌(Escherichia coli)细胞合成头孢氨苄的基础上,又研究了用固定化巨大芽孢杆菌(Bacillus megaterium)BP931胞外青霉素G酰化酶酰化合成头孢氨苄的条件。本文报道这一研究结果。     

Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation

The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase^®) and for the free enzyme were used. For Assemblase^®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed.

The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase^® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane.

The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.