Efflux pump inhibition or cell membrane destabilization in Mycobacterium tuberculosis to enhance the activity of TB drugs and prevent drug resistance

BACKGROUND The current treatment for tuberculosis (TB) is complicated by its length and complexity, which has lead to inadequate response and emergence of multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). As a result, there is a very urgent need for new powerful TB regimens and novel strategies. Unfortunately, only few new TB drugs are being developed. Newer generation fluoroquinolones, especially moxifloxacin (MXF), are the most promising new TB drugs in susceptible TB and MDR-TB. In addition, oxazolidinones (OXAZO) are already available for MDR/XDR-TB treatment (linezolid, LZD) and very active derivatives are in development (PNU-100480). Furthermore, accumulating evidence suggests an important role for mycobacterial efflux pumps in the extrusion of TB drugs and emergence of resistance to both fluoroquinolones as well as LZD. Clearly, inactivation of mycobacterial efflux pump systems by efflux pump inhibitors (EPI) may be an advantageous strategy to increase intrabacterial concentrations of MXF and OXAZO resulting in enhanced anti-mycobacterial effect of these drugs and prevention of drug resistance. Another approach to increase intrabacterial drug concentrations is the addition of colistin (COL), which may result in enhanced permeability of the bacterial outer membrane. Recent in vitro findings from our consortium on the EPI thioridazine (THZ) and COL, and promising clinical results in XDR-TB patients who used MXF, LZD and THZ confirm the role of EPI and the potential of a regimen based on these drugs. AIMS The overall aim of this proposal is 1.to develop and bring a novel TB therapeutic regimen based on MXF plus an OXAZO to the phase of testing in humans 2.to maximize the potential of this novel regimen and limit its adverse effects by co-administration of mycobacterial EPIs and/or COL resulting in increased intrabacterial drug concentrations. APPROACH AND METHODS Four work packages are representing a comprehensive and application-oriented approach to bring the new TB treatment regimen and concept up to the phase of testing in humans. In WP1 (in vitro pharmacological studies), we will identify the efflux pumps used by Mtb to extrude MXF or OXAZO. In addition, an in vitro system will be developed that enables for screening of the most suitable inhibitors of these pumps, which are subsequently examined in WP2-4. In WP2 (in vitro pharmacodynamic studies), we will assess the susceptibility (MIC and time-kill kinetics) of two clinically relevant Mtb strains of the Beijing and East-African-Indian genotypes towards MXF, OXAZO, EPI and COL alone or in combinations. In WP3 (in vivo pharmacokinetic studies in mice) we will investigate the pharmacokinetics of MXF, OXAZO, EPI and COL in mice to develop drug dosage schedules that mimic drug exposures in humans. In WP3 and WP4 (below) an innovative blood sampling and bio-analytical ‘dried blood spots’ technique will be validated, requiring only a drop of blood for drug concentration measurement. In WP4 (in vivo therapeutic efficacy study in mice), the novel therapeutic regimens will be evaluated in our mouse model of TB caused by a Beijing genotype strain, by assessing elimination of Mtb in infected organs, prevention of emergence of drug resistance and relapse, and tolerability of the drug regimens. RELEVANCE This project is extremely relevant, as it prioritizes the development of new treatment regimens and concepts in TB, a disease confronted with rapid spread of multi-drug resistance. In addition, the current proposal is strongly application-oriented, quickly allowing for follow-up studies in the human setting. Furthermore, these studies by an academic consortium warrant specific attention, considering the limited involvement of pharmaceutical companies in TB drug development. This proposal is also highly innovative from a scientific point of view as it will provide valuable insights into the potentially groundbreaking concept of increasing intrabacterial TB drug concentrations by the addition of EPI or COL. The project also develops a novel in vitro screening system for EPIs, useful for future research. The emphasis on clinically relevant Mtb strains and pharmacokinetic/pharmacodynamic principles is innovative in the TB field. Finally, application of the novel ‘dried blood spots’ technique may provide a substantial reduction in animals required for this type of research. KNOWLEDGE TRANSFER AND IMPLEMENTATION To transfer knowledge from this project, a large number of target groups at a national and international level have been designated. The experienced multidisciplinary project team for this proposal involves all key preclinical and clinical TB pharmacological researchers from The Netherlands. They are very well embedded in these national and international gremia to enable optimal knowledge transfer and follow-up implementation.