Kinetics of the reactions of amoxycillin sodium and potassium clavulanate alone and in combination were investigated in the liquid and frozen states at selected pH values of 2.0, 4.6 and 7.0. A stability indicating HPLC assay was developed to perform simultaneous quantification of these compounds validated under stressed conditions.Amoxycillin and clavulanate degradation obeyed first-order kinetics under all conditions of this study. The effect of temperature, buffer, concentrations and complexation were investigated. Both compounds showed acceleration in rates due to general acid catalysis from buffer species. The buffer catalysis rate constants due to total phosphate and total acetate at 55 degrees celsius were 5.84x10(subscript)-1 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and 1.53 X10(subscript)-1 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 for amoxycillin, 2.33 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and 4.4x10(subscript)-1 mol dm(subscript)-3)(subscript)-1 h(subscript)-1 for clavulanate respectively. The buffer independent rate constant values were obtained and interpreted according to the available literature data. Increase in the initial concentration of amoxycillin or clavulanate did not change the first-order rate constant values of these antibiotics significantly at liquid state temperatures. However in the buffer systems, the rate of hydrolysis of amoxycillin in the combination was significantly subject to clavulanate catalysis. This novel finding was influenced by phosphate buffer concentration. A kinetic model was proposed and the second-order catalytic rate constant values at pH 7.0 and 55 degrees celsius were estimated for clavulanate catalysis of amoxycillin (k(subscript)cvc) to be k(subscript)cvc = 1.75 X10(subscript)2 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and for phosphate catalyzed of clavulanate catalysis of ++ / amoxycillin (k(subscript)phccv) as k(subscript)phccv = 2.87 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1.The temperature dependence of the rate of amoxycillin sodium degradation over the pH range evaluated did not change significantly. However the E(subscript)a values of potassium clavulanate decreased slightly with increase in pH. Both the compounds showed similar E(subscript)a values at pH 4.6 in acetate system. Hence 71.2 kJ mol(subscript)-1 for amoxycillin and 75.1 kJ mol(subscript)-1 for clavulanate.The investigation on complexation effects by HPbetaCD on the rate of hydrolysis of amoxycillin and clavulanate indicated no significant change in the rate of reaction of amoxycillin in the acetate buffer system. But the rate of clavulanate hydrolysis in combination was decreased by approximately 10%. The rate constant within the cyclodextrin complex and the stability constant of the complex obtained for clavulanate at pH 4.6 and 55 degrees celsius were k(subscript)c = 1.54x 10(subscript)-1 h(subscript)-1 and K(subscript)c = 74.2 (mol dm(subscript)-3)(subscript)-1.Extrapolation of the rate constant values to the frozen state from the liquid state data indicated marked acceleration of the rate of amoxycillin and clavulanate in all the pH values investigated. The highest acceleration in rate recorded was 15.0 fold for clavulanate in the hydrochloric acid system and the lowest value was 4.4 fold for amoxycillin at -7.3 degrees celsius. The rate constant values obtained were interpreted in terms of the concentration model (Pincock and Kiovsky 1966), phase-temperature relationship of the solutes, buffer catalysis, pH change and polymerization reactions.In the hydrochloric acid system a kinetic model was deduced providing adequate explanation of the experimental results. The stabilizing effect of sodium chloride used for maintaining constant ionic strength (mu=0.5) was ++ / enormous in this system. The shelf-life of amoxycillin was increased from 2.2 h to 58.3 h at -7.3 degrees celsius when sodium chloride was included in the system. It also stabilized the rates of the reactions significantly in the buffer systems.The buffer systems used in this study stabilized the rates of the reaction of both the drug compounds considerably. The shelf-life of amoxycillin in phosphate buffer was 621.3 h at -13.5 degrees celsius and in acetate buffer the shelf-life of clavulanate was 71.9 h at the same temperature. These are the highest shelf-life values recorded so far in the literature for amoxycillin and clavulanate at this frozen temperature.

Biofilms are surface-bound bacterial colonies that are held together by a self-produced extracellular polymeric matrix. They are highly resistant to antibiotics and host defence mechanisms, and are known to be the cause of persistent infections. Biofilm-degrading enzymes have been shown to prevent biofilm formation, remove mature biofilm, and enhance the efficacy of antibiotics. This study investigated the antibacterial and antibiofilm actions of the commercial enzyme Pectinex Ultra SP-L (Pectinex), alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. The cytotoxicity of Pectinex was determined on human cell cultures in vitro. Pectinex (7.42 – 950 PGU/ml) was not bactericidal, and had no effect on the antibacterial efficacy of amoxicillin-clavulanate and ciprofloxacin in cultures of S. aureus (ATCC 12600) and P. aeruginosa (ATCC 9027), respectively. However, in clinical cultures of P. aeruginosa, Pectinex caused an 89.0% (from 1.0 to 1.89 μg/ml) and 92.8% (from 1.67 to 3.22 μg/ml) increase in the MIC and MBC of ciprofloxacin, respectively. In clinical cultures of S. aureus, both bactericidal indices of amoxicillin-clavulanate were increased by 28.0% (from 2.0 to 2.56 μg/ml). In all bacterial cultures, low concentrations of Pectinex (≤ 118.75 PGU/ml) and prolonged incubation periods (≥ 6 h) were both associated with increased viability and biofilm biomass. Over a short incubation period (≤ 6 h), higher concentrations of Pectinex (237.5 – 950 PGU/ml) effectively inhibited biofilm formation in P. aeruginosa ATCC (237.5 – 950 PGU/ml) and clinical (950 PGU/ml) strains but not in S. aureus cultures. Pectinex (237.5 – 950 PGU/ml) was cytotoxic to HeLa cells, lymphocytes and neutrophils, and induced morphological features that included shrunken rounded cells, blebs, apoptotic bodies, cytoplasmic vacuoles and cell debris. The effects at 475 and 950 PGU/ml were comparable to mitomycin C 10 μg/ml and staurosporine 1 μg/ml. Pectinex was shown to either enhance or reduce biofilm biomass and cell viability in cultures of S. aureus and P. aeruginosa. The manifested effects depended on the concentration of the enzyme, the specific bacterial species and strain, and the maturity of the biofilms. Further studies are still needed in order to determine the actions of Pectinex on other clinical pathogens. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Pharmacology / PhD / Unrestricted

Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaC. / Mycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC.

Compostos farmacêuticos são identificados em matrizes ambientais em ordens de grandeza de ng.L-1 a μg.L-1. Dentre os fármacos, os antibióticos têm recebido atenção especial devido aos problemas que podem causar à biota aquática. O objetivo do presente estudo foi avaliar a citotoxicidade de Amoxicilina e Clavulanato de Potássio isolados e em associação para mexilhões Perna perna utilizando o ensaio do tempo de retenção do corante vermelho neutro, que avalia a estabilidade da membrana lisossômica de hemócitos dos organismos teste. A Amoxicilina causou citotoxicidade aos mexilhões nas concentrações de: CEO: 1 ng.L-1, CI25-24h: 0,44 ng.L-1, CI25-48h: 1,19 ng.L-1 e CI25-72h: 0,85 ng.L-1, o Clavulanato de Potássio foi citotóxico nas concentrações de: 10 ng.L-1 em 24h e 50 ng.L-1 e 100 ng.L-1 em 48h e 72h. Os valores de concentração inibitória foram de CI25-24h: 3,11 ng.L-1, CI25-48h: 3,45 ng.L-1 e CI25-72h: 3,43 ng.L-1. No ensaio realizado com a associação dos fármacos todas as concentrações foram citotóxicas aos mexilhões em 48h e em 72h apenas 40 ng.L-1 de Amoxicilina + 10 ng.L-1 de Clavulanato de Potássio e 200 ng.L-1 de Amoxicilina + 50 ng.L-1 de Clavulanato de Potássio. As concentrações inibitórias foram: CI25-48h: 1,67 ng.L-1 e CI25-72h: 1,36 ng.L-1 a partir dos dados de Amoxicilina e CI25-48h: 0,42 ng.L-1 e CI25-72h: 0,34 ng.L-1 a partir dos dados de Clavulanato de Potássio. / Pharmaceutical compounds are identified in environmental matrices in orders of magnitude from ng.L-1 to μg.L-1. Among the drugs, antibiotics have received special attention due to the problems that can cause to aquatic biota. The aim of this study was to evaluate the cytotoxicity of Amoxicillin and Potassium Clavulanate in isolated and associated forms to marine mussels Perna perna through the neutral red retention time assay which assesses the stability of hemocytes lisossomal membrane of test organisms. Amoxicillin caused cytotoxicity to the mussels in concentrations of: OEC: 1 ng.L-1, IC25-24h: 0.44 ng.L-1, IC25- 48h: 1.19 ng.L-1 and IC25-72h: 0.85 ng.L-1, Potassium Clavulanate was cytotoxic at concentrations of 10 ng.L-1 in 24h; 50 ng.L-1 and 100 ng.L-1 at 48h and 72h. The inhibitory concentration values were IC25-24h: 3.11 ng.L-1, IC25-48h: 3.45 ng.L-1 and IC25-72h: 3.43 ng.L- 1. The test conducted with the combination of drugs all concentrations were cytotoxic to mussels in 48h and 72h only 40 ng.L-1 Amoxicillin + 10 ng.L-1 and Potassium Clavulanate 200 ng.L-1 Amoxicillin + 50 ng.L-1 Potassium Clavulanate. The inhibitory concentrations were IC25-48h: 1.67ng.L-1 and IC25-72h: 1.36 ng.L-1 from the data of Amoxicillin and IC25- 48h: 0.42 ng.L-1 and IC25-72h: 0.34 ng.L-1 from the Potassium Clavulanate data.

Compostos farmacêuticos são identificados em matrizes ambientais em ordens de grandeza de ng.L-1 a μg.L-1. Dentre os fármacos, os antibióticos têm recebido atenção especial devido aos problemas que podem causar à biota aquática. O objetivo do presente estudo foi avaliar a citotoxicidade de Amoxicilina e Clavulanato de Potássio isolados e em associação para mexilhões Perna perna utilizando o ensaio do tempo de retenção do corante vermelho neutro, que avalia a estabilidade da membrana lisossômica de hemócitos dos organismos teste. A Amoxicilina causou citotoxicidade aos mexilhões nas concentrações de: CEO: 1 ng.L-1, CI25-24h: 0,44 ng.L-1, CI25-48h: 1,19 ng.L-1 e CI25-72h: 0,85 ng.L-1, o Clavulanato de Potássio foi citotóxico nas concentrações de: 10 ng.L-1 em 24h e 50 ng.L-1 e 100 ng.L-1 em 48h e 72h. Os valores de concentração inibitória foram de CI25-24h: 3,11 ng.L-1, CI25-48h: 3,45 ng.L-1 e CI25-72h: 3,43 ng.L-1. No ensaio realizado com a associação dos fármacos todas as concentrações foram citotóxicas aos mexilhões em 48h e em 72h apenas 40 ng.L-1 de Amoxicilina + 10 ng.L-1 de Clavulanato de Potássio e 200 ng.L-1 de Amoxicilina + 50 ng.L-1 de Clavulanato de Potássio. As concentrações inibitórias foram: CI25-48h: 1,67 ng.L-1 e CI25-72h: 1,36 ng.L-1 a partir dos dados de Amoxicilina e CI25-48h: 0,42 ng.L-1 e CI25-72h: 0,34 ng.L-1 a partir dos dados de Clavulanato de Potássio. / Pharmaceutical compounds are identified in environmental matrices in orders of magnitude from ng.L-1 to μg.L-1. Among the drugs, antibiotics have received special attention due to the problems that can cause to aquatic biota. The aim of this study was to evaluate the cytotoxicity of Amoxicillin and Potassium Clavulanate in isolated and associated forms to marine mussels Perna perna through the neutral red retention time assay which assesses the stability of hemocytes lisossomal membrane of test organisms. Amoxicillin caused cytotoxicity to the mussels in concentrations of: OEC: 1 ng.L-1, IC25-24h: 0.44 ng.L-1, IC25- 48h: 1.19 ng.L-1 and IC25-72h: 0.85 ng.L-1, Potassium Clavulanate was cytotoxic at concentrations of 10 ng.L-1 in 24h; 50 ng.L-1 and 100 ng.L-1 at 48h and 72h. The inhibitory concentration values were IC25-24h: 3.11 ng.L-1, IC25-48h: 3.45 ng.L-1 and IC25-72h: 3.43 ng.L- 1. The test conducted with the combination of drugs all concentrations were cytotoxic to mussels in 48h and 72h only 40 ng.L-1 Amoxicillin + 10 ng.L-1 and Potassium Clavulanate 200 ng.L-1 Amoxicillin + 50 ng.L-1 Potassium Clavulanate. The inhibitory concentrations were IC25-48h: 1.67ng.L-1 and IC25-72h: 1.36 ng.L-1 from the data of Amoxicillin and IC25- 48h: 0.42 ng.L-1 and IC25-72h: 0.34 ng.L-1 from the Potassium Clavulanate data.

No description available.