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In vitro and in vivo evaluations of an innovative drug combination for pulmonary administration against tuberculosis

par Ravon, Faustine
Président du jury Amighi, Karim
Promoteur Fontaine, Véronique
Co-Promoteur Wauthoz, Nathalie
Publication Non publié, 2024-08-23

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Résumé :

Tuberculosis (TB) is a mycobacterial infection affecting mainly the lungs, caused by the pathogen Mycobacterium tuberculosis (Mtb). Before Coronavirus disease 2019 (COVID-19) pandemic, TB was the world’s first leading cause of death from a single infectious agent. Since COVID-19, TB represents the second cause of death by an infectious agent (in the period 2020-2022) 1. Based on the data reported by the World Health Organization (WHO), in 2022, over 10.6 new million people suffered from TB. Moreover, TB caused 1.3 million deaths worldwide 1. Current treatment recommended by the WHO still requires the use of the all-oral 6-month rifampicin-based regimen (2 months of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol followed by 4 months of INH and RIF) 1. This long multi-drug regimen affects patient adherence and persistence to medication, leading to treatment failure, relapse of infection, antibiotic resistance with the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) 1. Moreover, the Bacillus Calmette-Guerin (BCG) vaccine does not provide a complete protection, especially for adolescents and adults 2. Hence, a new treatment strategy is needed to comply with WHO’s goal to end the global tuberculosis epidemic by 2035 3. Drug repurposing strategy uses existing drug for a new treatment that was not indicated before. This strategy is very attractive to develop new anti-TB treatment. In this context, it was discovered that depleted phthiocerol dimycocerosates (PDIM, complex lipids of the mycobacteria cell envelope) mycobacteria strains were susceptible to glycopeptides (such as vancomycin (VAN)) usually used to combat Gram-positive bacteria 4. Rens and colleagues used this susceptibility to glycopeptides antibiotics to identify drugs able to target mycobacterial cell wall lipids. Among Food and Drug Administration (FDA)-approved lipid lowering drugs, tetrahydrolipstatin (THL) was identified as able to synergize with VAN to inhibit Mycobacterium bovis BCG and Mtb 5. The poor oral bioavailability of VAN and THL, and the predominant tropism of Mtb infection to the lungs and to alveolar macrophages (AM), make their pulmonary administration very attractive. Therefore, the research project aims to enhance the therapeutic potential of the VAN/THL combination by ensuring the synergistic action of the VAN/THL combination against different strains of mycobacteria, evaluating the possibility of administering the VAN/THL combination directly into the lungs, developing inhalable formulations with appropriate lung deposition, and verifying the efficacy of these inhalable formulations in cellular and animal models.The drug combination, VAN/THL in weight ratio 10:1, has demonstrated an effective synergistic action in vitro against Mtb, but also an inhibition action against Mycobacterium abscessus and Mycobacterium marinum. This first study confirmed previous results obtain by Rens and colleagues and reinforced the possibility of developing an anti-TB therapy based on the VAN/THL combination. To evaluate lung local tolerability of VAN, THL, and the VAN/THL combination, bronchial cells, alveolar cells, and monocytes were exposed to concentrations around and above their minimal inhibitory concentration (MIC). VAN had no inhibitory activity on the tested human cell lines, even at a concentration 125 times higher than its MIC, while the THL, alone or in combination with VAN presented a cytostatic action. Monolayer epithelium showed no significant irreversible damage at concentrations up to 100 times the combination MIC. BALB/cAnNRj mice exposed to concentration of 50 times the combination MIC delivered endotracheally 3 times a week for 3 weeks, demonstrated no clinical signs or significant weight loss. The increase of pro-inflammatory biomarkers (i.e., interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, proportion of inflammatory cells) and cytotoxicity in bronchoalveolar lavage fluid (BALF) were non-significant. Lung histopathology did not demonstrate significant tissue damage. VAN/THL combination at doses up to 50 times the combination MIC was found to be thus well tolerated by pulmonary route. This second study was a promising result and encouraged the development of the VAN/THL combination in dry powder for pulmonary administration.Inhalable microparticles containing VAN/THL combination at the weight ratio 10:1 were produced using the spray-drying technique according to a drug-drug co-formulation strategy. This permitted to promote the co-deposition and co-dissolution of both drugs into the lung. To develop VAN/THL formulations for pulmonary administration, it was necessary to firstly produce spray-dried VAN microparticles of 2 µm then recovered by THL (10:1 w/w) with or without hydrogenated castor oil (HCO) to increase their macrophage uptake. Dry powders for inhalation were characterized in terms of physicochemical as well as their in vitro aerodynamic and dissolution properties. VAN/THL with 25 % HCO presented the best properties, combining a microparticle size suitable for delivery to the lungs, optimal deposition, and co-dissolution of its both drug components. Furthermore, X-ray powder diffraction analyses demonstrated stable patterns (over 9 months). This third study proved the possibility to formulate spray-dried powders for pulmonary administration composed of VAN and THL.To evaluate the efficacy of the different spray-dried formulations in infected macrophages and mice, a new macrophage infection model using M. bovis BCG expression ESX-1 of M. marinum (BCG::ESX-1Mmar) was developed and has demonstrated drastic reduction of 62.5 to 99.9 % of the cytoplasmic mycobacterium for dry powder with and without HCO, respectively. Another macrophages model using Listeria monocytogenes was also used to confirm results and ensure the optimal intracellular activity of the VAN/THL formulations. The reduction of 92.6 to 94.8 % of the cytoplasmic Listeria monocytogenes was observed for dry powder with and without HCO, respectively. In vivo efficacy studies were performed but unfortunately did not demonstrate any effect of the formulations. Moreover, an in vitro synergistic activity of THL and RIF against Mtb was previously discovered in the laboratory. The spray-dried formulations were then tested in vivo in association with RIF to test a potential synergy of action between THL and RIF in animal model. VAN/THL with HCO associated with RIF demonstrated a statistical inhibition action against Mtb in comparison with the group treated only with RIF. However, VAN/THL without HCO demonstrated no inhibition against Mtb. This result demonstrated the importance to bring both drugs in the synergistic ratio (10:1 w/w) in the microparticles to obtain promising efficacy results for the treatment of TB. This fourth study provided very interesting information on the activity of the spray-dried formulations against Mtb. Further investigations are required to continue the pharmaceutical development of this VAN/THL combination and reach TB patients.