Nanoparticle-based delivery of efflux pump inhibitors and antibiotics to treat mycobacterial infections. Reducing thioridazine toxicity to potentiate antituberculosis therapy

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has been treated with the same arsenal of antibiotics since the 1970 s; and with an estimated one third of the world s population latently infected at the moment, new regimens are sorely needed. Treatment remains lengthy and difficult, and inappropriate dosing or administration of drugs as well as patient non-compliance is contributing to the rising threat of multi-, extensively- and now totally- drug resistant TB. Should further expansion of resistant strains become a reality it would render our current antibiotics useless. To address the need for new therapies, this thesis explores both in vitro and in vivo approaches using a novel nanoparticle-based efflux pump inhibitor-antibiotic combination treatment. Bacterial efflux pumps (EPs) are inducible membrane-based transporters that can reduce intracellular concentrations of drugs, which leaves open a window for the development of permanent genetic drug resistance. By blocking these EPs with EP inhibitors such as thioridazine (TZ), while simultaneously treating with one of the most efficient anti-TB drugs, rifampicin (RIF), we predicted a synergistic effect likely caused by an increased intracellular concentration of both drugs in the bacteria. The first aim of this study was to develop a protocol for the encapsulation of TZ in polymeric poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs). Our group has previously established that PLGA NPs with RIF localize in the same cells as the bacteria, and improve therapy both in vitro and in vivo relative to free drug. Slow release from these particles and facilitated macrophage uptake could potentially lead to treatment that can be administered less frequently in a more targeted and consequently less toxic manner. This may be especially beneficial in the case of TZ, which is associated with serious dose-dependent cardiotoxicity. The second aim of the study was therefore to determine if the encapsulated form of TZ was less toxic than the free form. The third aim of this study was to explore the possible role of TZ as a potentiator of traditional anti-TB therapy. We found that encapsulating TZ in PLGA NPs reduced TZ toxicity, and had a synergistic effect when administered together with RIF. This was the case both in vitro, in primary murine macrophages infected with Mycobacterium bovis BCG, and in vivo, in the zebrafish model infected with Mycobacterium marinum