RESUMO
OBJECTIVES: The combination of aztreonam and avibactam has been proposed for the treatment of infections caused by metallo-ß-lactamase-producing Gram-negative organisms, given the stability of aztreonam against metallo-ß-lactamases plus the broad coverage of avibactam against AmpC ß-lactamases and ESBLs. This study aimed to evaluate the efficacy of the combination against four clinical isolates with defined but diverse ß-lactamase profiles. METHODS: The MICs of aztreonam were determined without and with avibactam (1, 2, 4, 8 and 16 mg/L). Using the MIC values, the static time-kill kinetic studies were designed to encompass aztreonam concentrations of 0.25, 0.5, 1, 2 and 4 times the MIC at the respective avibactam concentrations from 0 to 8 mg/L. Aztreonam and avibactam concentrations were determined by LC-MS/MS during the course of the time-kill kinetic studies to evaluate whether avibactam protects aztreonam from degradation. RESULTS: Three of the four isolates had aztreonam MICs ≥128 mg/L in monotherapy. Dramatically increasing susceptibility associated with a decrease in aztreonam MIC was observed with increasing avibactam concentration. Against all isolates, the combinations resulted in greater killing with a much lower dose requirement for aztreonam. The resulting changes in base-10 logarithm of cfu/mL at both the 10 h and 24 h references (versus 0 h) were synergistic. In contrast, a significantly higher concentration of aztreonam in the monotherapy was required to produce the same kill as that in the combination therapy, due to rapid aztreonam degradation in two isolates. CONCLUSIONS: The aztreonam/avibactam combination protects aztreonam from hydrolysis and provides synergy in antimicrobial activity against multiple ß-lactamase-expressing strains with a wide MIC range.
Assuntos
Antibacterianos/análise , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Aztreonam/análise , Aztreonam/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , Cromatografia Líquida , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Teóricos , Espectrometria de Massas em Tandem , Inibidores de beta-Lactamases/farmacocinéticaRESUMO
The existence of the blood-brain barrier (BBB) complicates the treatment of many central nervous system (CNS) disorders, including the copper storage disease, Wilson's disease. Its CNS symptoms represent a serious problem, since therapeutics for Wilson's disease do not cross the BBB. One strategy to overcome this obstacle is the transfer of drugs across the BBB with colloidal carrier systems like liposomes. The aim of the present study was to encapsulate triethylenetetramine (TETA), a copper chelating agent, into surface modified liposomes and to investigate their permeation across the BBB. Liposomes were modified with cationized bovine serum albumin or penetratin, a cell penetrating peptide. Liposomes were characterized regarding size, PDI, zeta potential and encapsulation efficiency. Size was between 139.4±1.9nm to 171.1±3.5nm with PDI's below 0.2. Zeta potentials of vectorized liposomes were at least 6.9mV higher than those of standard liposomes. Cryo-TEM micrographs displayed liposomal structure, integrity and the similarity of structure and size between loaded, unloaded, vectorized and non- vectorized liposomes. In vivo experiments in rats showed an up to 16-fold higher brain uptake of TETA in vectorized liposomes compared to free TETA or TETA in non-vectorized liposomes, proving successful brain delivery using target seeking surface modifications. Tissue analysis indicated TETA concentrations in the brain being high enough to treat Wilson's disease.
Assuntos
Encéfalo/metabolismo , Quelantes/administração & dosagem , Quelantes/farmacocinética , Lipossomos/administração & dosagem , Lipossomos/química , Trientina/administração & dosagem , Trientina/farmacocinética , Animais , Disponibilidade Biológica , Proteínas de Transporte/química , Proteínas de Transporte/farmacocinética , Peptídeos Penetradores de Células , Lipossomos/farmacocinética , Lipossomos/ultraestrutura , Masculino , Tamanho da Partícula , Ratos , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacocinética , Distribuição TecidualRESUMO
The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappAâB), and the Papp from basolateral to apical (PappBâA) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappAâB of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappAâB and the PappBâA were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent.