Date of Award
PhD (Doctor of Philosophy)
Professor Sally-Ann Cryan
Dr Mary O'Sullivan
Professor Joseph Keane
Health Research Board
Tuberculosis, Inhaled, PLGA, Retinoic Acid, Linezolid
Tuberculosis (TB) is a serious public health issue that requires attention. According to the World Health Organisation (WHO), millions of new cases of TB are recorded each year making it the one of the top causes of death worldwide, yet the availability of new treatments are limited. This issue is compounded by the rise of multi-drug resistant TB (MDR-TB) whereby patients becomes resistant to first-line therapy. MDR-TB is often related to non-compliance, due to lengthy regimens and intolerable side effects. The hypothesis of this project is that a convenient, inhaled treatment for tuberculosis, using a targeted drug delivery system, have the potential to reduce dosage regimens, toxicity and enhance efficacy of anti-tuberculosis therapies. This could increase patient adherence leading to a more positive prognosis for patients and reduce the incidence of MDT-TB worldwide.
Previous work carried out by our group demonstrated that unloaded- and antitubercular drug (ATD)-loaded microparticles (MPs) manufactured by double emulsion, solvent evaporation (DESE), could reduce bacterial viability in an in vitro TB infection model. This project sought to optimise the manufacture of these delivery systems and explore the use of novel therapeutic cargos in preparation for efficacy testing in an in vivo model of TB infection. The first-line anti-tubercular antibiotic rifampicin was selected alongside a group 5 anti-tubercular antibiotic used in MDRTB, linezolid. All-trans-Retinoic acid (ATRA) was also selected as a cargo for its hostdirected and antibacterial properties. Rifampicin-loaded and ATRA-loaded poly (D,Llactide- co-glycolide) (PLGA) MPs were first manufactured by DESE resulting in particles suitable for alveolar deposition (1-5 μm). The manufacture of Inhalable ATDloaded MPs was subsequently scaled-up using spray drying which coincided with the introduction of the third cargo, linezolid. ATD-loaded MP formulations maintained the physico-chemical properties suitable for inhalation (unloaded-MPs (UNL-MP):1.91 ± 0.4 μm, rifampicin-loaded MPs (RIF-MP):1.77 ± 0.1 μm, linezolid-loaded MPs (LINMP): 1.79 ± 0.04 μm, ATRA-loaded MPs (ATRA-MP): 2.07 ± 0.5 μm) and had an average encapsulation efficiency > 55 %. The spray dried PLGA MPs also controlled release of the cargos whilst the batch size increased approximately 10-fold when 24 compared to DESE MP manufacture. Antibiotic-loaded MPs were further modified by covalently attaching the pro-inflammatory cytokine interferon-ɣ (IFN-ɣ) to the surface. PLGA RIF-MPs were also manufactured using an electrohydrodynamic (EHA) method known as electrospraying producing spherical, homogenous MP batches (1.94 ± 0.46 μm) with an encapsulation efficiency of 93.2 ± 3.6 %.
Following process optimisation, each of the MP formulations were tested in vitro and in vivo alongside the active ingredients in solution. Rifampicin formulations proved most successful in reducing Mycobacterium tuberculosis (Mtb) (H37Ra) viability in vitro initially, however, after tailoring the studies specifically for host-directed treatments, the significant antibacterial properties of ATRA formulations became apparent. These results translated in vivo, with pulmonary administration of both rifampicin and ATRA formulations significantly reducing Mtb (H37Rv) viability in a murine model of infection. mRNA expression of pro-inflammatory cytokine genes from the lungs of mice treated with the ATDs in solution and encapsulated in MPs indicated two distinct mechanisms of immunomodulation. In addition, rifampicin treatments produced a more inflammatory mRNA expression profile in the lung when compared to ATRA treatment. Nonetheless, rifampicin solution plus IFN-ɣ, RIF-MPs, ATRA solution plus rifampicin and ATRA-MPs were all comparable in their ability to reduce the pulmonary pathology in the lungs following Mtb infection.
Overall the results of this project are promising for future research into inhaled treatments for TB. Firstly, the use of rifampicin as a control treatment and cargo in this project enabled the successful development of three protocols for the manufacture of ATD-loaded MPs. Secondly, the data generated on the effect of ATRA-MPs in TB infection provides a foundation on which further pre-clinical testing can be built. ATRA-MPs delivered intratracheally in a mouse model of TB infection can reduce bacterial load and importantly the pulmonary pathology, making this a potentially patient friendly, inhaled treatment. Although an improvement in the formulation stability and aerosol properties is required, the results here support the idea of combining MPs with HDTs as a method of potentially reducing the incidence of resistance.
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O'Connor G. Development of Inhaled Anti-Tubercular Therapies Using Bioactive Carriers [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2018.
Available for download on Saturday, May 30, 2020