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In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the μM to sub-μM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several β-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.

Ampicillin belongs to the class of beta-lactam antibiotics, which corresponds the most prescribed antibiotics in medicine. This justifies the importance of elucidating the effect of several variables for the synthesis process (pH, temperature, co-solvent) on the behavior of the compounds involved in the enzymatic reaction. This paper aims to present new ampicillin solubility data in aqueous medium and the characterization of the solid phase of the antibiotic. In the phase equilibrium experiments it was considered the effect of pH (3 up to 7.5), temperature (283.15 K up to 298.15 K) and the ethanol concentration in the aqueous solution (varying between 0 and 70 wt% ethanol). The solubility curves deviate from U-shape solubility curves with respect to pH in the range of 0e70 wt%. This is due to ampicillin anhydrous becomes trihydrated molecule in the solid phase at alkaline pH region, changing the antibiotic properties significantly. This change in crystalline structure was confirmed by characterization of antibiotic solid phase carried out from thermogravimetric (TGA), Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) analyses. Dissociation constants have also been measured at the studied values of temperature and ethanol composition, using potentiometric titration. Finally, a thermodynamic model considering the ideal solution was applied to describe mathematically the solubility curves measured close to experimental uncertainties

El creciente aumento en las importaciones de bienes e insumos en Colombia, especialmente en el caso de la materia prima amoxicilina, donde se evidencia un incremento en un 26.82% en el periodo de 2002 al 2012 y teniendo en cuenta las directrices del Plan Nacional de Desarrollo 2010 – 2014, que promueve la innovación y el mejoramiento de la productividad, se plantea una sustitución gradual de las importaciones de este insumo, por materia prima producida a nivel nacional. Para llevar a cabo tal planteamiento surge la necesidad de realizar un estudio de Vigilancia Tecnológica e Inteligencia Competitiva, teniendo como factores críticos de vigilancia la producción del antibiótico, análisis del mercado en el contexto nacional e internacional y características de la materia prima, con el fin de obtener resultados que permitan analizar las ventajas y desventajas de crear una planta de producción en Colombia y evaluar su factibilidad económica, siempre y cuando se integre la academia tanto nacional como extranjera con la industria farmacéutica. Las estrategias recomendadas para el desarrollo de esta investigación a partir de los resultados obtenidos, demostraron que para obtener la amoxicilina por vía enzimática no se requiere partir del intermediario 6-APA, sino de la sal potásica de penicilina G (PGK) directamente, usando la enzima penicilin acilasa (PA) de Kluyvera citrophila, inmovilizada en hidrogel de sílice amino modificado, precipitada con iones de zinc y en el medio de reacción tert-pentanol anhidro, con el fin de obtener un mayor rendimento de la síntesis. Abstract. The increasing growth of Colombia’s imports on goods and supplies, specially in the case of raw material amoxiciline, when an increasing of 26.82% is noticed, from years 2002 to 2012 and taking into account the development national plan (plan nacional de desarrollo) 2010-2014 guidelines which promotes innovation and producity improvement; it pases a gradual imported raw material substitution of these supplies by raw material supply produced all over the country. To carry out such approach, there’s a need to perform a technological surveillance and a competitive intelligence research, having a critical factors at surveillance the antibiotic production market analysis in national and international context and raw material characteristics. So that results can be obtained and analyse adventages and disadventages of bulding and amoxiciline production plant in Colombia and asses its economical feasibility whenever it involves both national and international academy and pharmaceutical industry. Recomended strategies for this research development from results obtained showed that to produce amoxiciline by means of enzymatic away, it is not required to start from G- APA intermediate but directly from penicilin salt G potassium (PGK). Usign enzyme penicilin acilase (PA) Kluyvera citrophyla inmobilized in hidrogel of sílice amino modified precipitated with zinc ions and a tert-pentanol-anhidro source reaction in order to get a better synthesis performance. Maestría

Objetivos: Avaliar a adequabilidade do modelo farmacocinético-farmacodinâmico (PK-PD) (NOLTING et al., 1996b) para modelar o efeito bactericida da piperacilina (PIP) em ratos Wistar infectados experimentalmente com Escherichia coli ATCC 25922. Metodologia: Experimentos de Farmacocinética: Determinou-se as concentrações plasmáticas totais e livres teciduais de PIP, através de microdiálise (MD), após administração de 240 mg/kg i.v. bolus a ratos Wistar granulocitopênicos (ciclofosfamida) infectados no músculo esquelético (105 UFC/mL) com E. coli. As amostras de plasma e de MD foram analisadas por CLAE. As sondas de MD foram calibradas por retrodiálise. Experimentos de Farmacodinâmica: Os animais imunodeprimidos e infectados foram tratados com PIP nas doses de 120 ou 240 mg/kg, em intervalos de 4/4, 6/6 e 8/8 horas por 24 h. Em tempos pré-determinados, os animais foram sacrificados (n = 3/tempo), o músculo infectado foi retirado, homogeneizado e o número de UFC/mL foi determinado em placas de ágar-sangue, após diluições sucessivas. Um grupo não tratado foi utilizado como controle. Modelagem PK-PD: A partir dos dados farmacocinéticos e farmacodinâmicos obtidos, avaliou-se efeito de morte bacteriana em função do tempo com o auxílio do programa de regressão não-linear SCIENTIST® v.2.0. Resultados e Discussão: Os parâmetros farmacocinéticos após a administração de PIP (240 mg/kg) foram t½ de 40 ± 8 min; CL de 0,46 ± 0,021 (L/h/kg) e um Vdss de 0,30 ± 0,06 L/kg. O perfil de PIP livre tecidual foi previsto a partir dos parâmetros plasmáticos utilizando ajuste simultâneo dos dados de plasma e tecido e um fator de proporcionalidade de 0,342 ± 0,101. Os parâmetros do modelo PK-PD obtidos foram: EC50 de 1,31 ± 0,27 μg/mL e kmax 1,39 ± 0,20 h-1. Os valores dos parâmetros da modelagem PK-PD obtidos in vivo diferiram dos descritos na literatura para o mesmo antibiótico e bactéria quando simulados in vitro. Conclusões: O modelo Emax-modificado descreveu os perfis de crescimento e morte bacteriana em função do tempo obtidos nas diferentes posologias testadas sendo adequado para modelagem PK-PD da piperacilina nas condições experimentais investigadas. Purpose: The objective of this study was to model the killing effect of a β-lactam antibiotic, piperacillin (PIP), in neutropenic and E. coli ATCC 25922 infected rats after different dosing regimens using a modified Emax PK-PD model. Methodology: Pharmacokinetic studies: Total plasma and free tissue concentrations of PIP, determined by microdialysis, were investigated after i.v. bolus of 240 mg/kg of the drug to immunecompromised (cyclophosphamide) and E. coli infected (107 CFU) Wistar rats. Microdialysis probes recoveries were determined by retrodialysis. Plasma and tissue samples were analyzed by HPLC. Pharmacodynamic studies: The infected rats were treated with iv bolus PIP 120 mg/kg or 240 mg/kg q8h, q6h, q4h. Three animals were sacrificed at predetermined times up to 24 hours. The infected muscle was removed, homogenized and the number of CFU/mL was determined by plate counting after 24 hours of incubation at 37ºC. A control group without treatment was used. PK-PD modeling: PIP killing effect as a function of time was fitted using the Emax-modified model with the aid of a non-linear regression computer program SCIENTIST® v.2.0. Results and Discussion: The pharmacokinetic parameters determined for PIP 240 mg/kg iv bolus were: t½ of 40 ± 8 min; CL of 0.46 ± 0.021 (L/h/kg) and Vdss of 0.30 ± 0.06 L/kg. Piperacillin free tissue levels were predicted using plasma data ina a simultaneous fitting with a proportionality factor of 0.342 ± 0.101. The parameters derived from PK-PD modeling were: bacterial killing rate (kmax) of 1.39 ± 0.20 h-1 concentration to produce 50% of de maximum effect (EC50) of 1.31 ± 0.27 μg/mL. The PK-PD parameters determined in vivo were different from those reported for the same bacteria and drug in vitro. Conclusions: The Emax model adequately described PIP antibacterial effect in animals for the different dosing regimens investigated.