Drug Delivery and Adjunct Strategies in the Treatment of Pseudomonas aeruginosa Infections.

摘要

Pseudomonas aeruginosa infections are the main cause of morbidity and mortality in patients suffering from cystic fibrosis (CF). I hypothesized that the entrapment of antibiotics and antioxidants in liposomes may increase their delivery and retention in the lung thus reducing the formation of P. aeruginosa biofilms and inflammation-induced oxidative damage respectively, and consequently alleviate the injury to the lungs. Initial experiments were carried out with CF sputum, a thick viscoelastic mixture of entangled host-derived mucins, actins, DNA, and bacterial components, to show that the components of the CF sputum individually and also expectorated CF sputum containing bacteria (expelled routinely by the patient to clear the lungs) can inactivate tobramycin, the most commonly prescribed antibiotic to CF patients. The entrapment of tobramycin in liposomes reduced the inactivation and increased the killing of P. aeruginosa; however, both conventional and liposomal tobramycin were greatly inhibited by intact CF sputum.;Since the activity of tobramycin was inhibited by components of the intact sputum, I examined if the co-incubation of the antibiotic formulations with sputum 'liquefiers' would increase the activity of tobramycin. Both DNase (a DNA-cleaving enzyme) and alginate lyase (a polysaccharide-cleaving enzyme) were proven to be effective with alginate lyase proving a better setting to the conventional and liposomal tobramycin to completely eradicate P. aeruginosa in expectorated CF sputum. The mucolytic antioxidant N-acetylcysteine (NAC) was also used but did not increase tobramycin activity.;Another strategy for improving the effectiveness of antibiotics against P. aeruginosa is the co-administration of antibiotics with different mechanisms of action. In a series of experiments, I explored the effectiveness of bismuth (attached to a lipophilic thiol), a proven antimicrobial compound, either alone or co-entrapped with tobramycin against P. aeruginosa . Entrapment of both compounds reduced cell signaling, virulence factors of P. aeruginosa at lower concentrations compared to the conventional tobramycin-bismuth combination. Liposomal tobramycin-bismuth did not inhibit biofilm formation; however it did penetrate and killed cells in the center of biofilms.;Since sputum 'liquefiers' increased the activity of tobramycin, the antimicrobial properties of the improved antibiotic formulation containing tobramycin and bismuth against mucoid P. aeruginosa strains in the presence or absence of alginate lyase was also assessed. The interaction and uptake of the tobramycin-bismuth formulation by P. aeruginosa through electron microscopy, and visualization of biofilms by confocal microscopy was also conducted in order to assess the degree of alginate production. The formulation reduced alginate levels at a lower concentration compared to the conventional form, and increased the uptake of tobramycin by P. aeruginosa . Compared to alginate lyase which detached and reduced mucoid P. aeruginosa biofilms, the liposomal formulation did not detach adhered cells but reduced alginate production.;Effective treatment in CF patients should not only involve the use of antibiotics and/or mucolytics but strategies that also modulate the immune and inflammatory processes that also play a role in the lung injuries. Accordingly, the extracts of the North American Panax quinquefolius (ginseng), with proven immune-modulating but nontoxic properties was chosen. Interestingly, I was able to show in both in vitro and an animal P. aeruginosa lung infection model that North American ginseng contains antipseudomonal and antibiofllm properties. Liposomal or conventional form of tobramycin, or ginseng equally reduced bacterial counts in the lungs, while co-administration of tobramycin and ginseng proved to be synergistic. Results from in vivo studies also showed that treatment of animals infected with P. aeruginosa reduced inflammatory markers.;Activation of inflammatory cells such as neutrophils and macrophages in CF are known to contribute to organ injury via the generation of reactive oxygen species. Antioxidants are also known to ameliorate oxidant-induced injuries. Thus, the protective effects of the antioxidant NAC in a liposomal formulation against an oxidant-induced damage were assessed in both in vitro and in vivo models. Hydrogen peroxide induced oxidative damage in HepG2 hepatocytes, while liposomal NAC greatly attenuated the production and or effects of reactive oxygen species, and increased cellular glutathione levels compared to conventional NAC. Lipopolysaccharide (LPS) administration to animals resulted in an inflammatory response while the prior administration of conventional NAC or liposomal NAC dampened the inflammatory response and protected against the LPS-induced hepatic injuries, a treatment effect being better for the group of animals treated with the liposomal NAC formulation. (Abstract shortened by UMI.)