An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance. Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Inhalable poly(lactic-co-glycolic acid) (PLGA) microparticles encapsulating all-trans-Retinoic acid (ATRA) as a host-directed, adjunctive treatment for Mycobacterium tuberculosis infection.

Ending the tuberculosis (TB) epidemic by 2030 was recently listed in the United Nations (UN) Sustainable Development Goals alongside HIV/AIDS and malaria as it continues to be a major cause of death worldwide. With a significant proportion of TB cases caused by resistant strains of Mycobacterium tuberculosis (Mtb), there is an urgent need to develop new and innovative approaches to treatment. Since 1989, researchers have been assessing the anti-bacterial effects of the active metabolite of vitamin A, all trans-Retinoic acid (ATRA) solution, in Mtb models. More recently the antibacterial effect of ATRA has been shown to regulate the immune response to infection via critical gene expression, monocyte activation and the induction of autophagy leading to its application as a host-directed therapy (HDT). Inhalation is an attractive route for targeted treatment of TB, and therefore we have developed ATRA-loaded microparticles (ATRA-MP) within the inhalable size range (2.07 ±â€¯0.5 µm) offering targeted delivery of the encapsulated cargo (70.5 ±â€¯2.3%) to the site of action within the alveolar macrophage, which was confirmed by confocal microscopy. Efficient cellular delivery of ATRA was followed by a reduction in Mtb growth (H37Ra) in THP-1 derived macrophages evaluated by both the BACT/ALERT® system and enumeration of colony forming units (CFU). The antibacterial effect of ATRA-MP treatment was further assessed in BALB/c mice infected with the virulent strain of Mtb (H37Rv). ATRA-MP treatments significantly decreased the bacterial burden in the lungs alongside a reduction in pulmonary pathology following just three doses administered intratracheally. The immunomodulatory effects of targeted ATRA treatment in the lungs indicate a distinct yet effective mechanism of action amongst the formulations. This is the first study to-date of a controlled release ATRA treatment for TB suitable for inhalation that offers improved targeting of a HDT, retains antibacterial efficacy and improves pulmonary pathology compared to ATRA solution.

Anti-GD2-ch14.18/CHO coated nanoparticles mediate glioblastoma (GBM)-specific delivery of the aromatase inhibitor, Letrozole, reducing proliferation, migration and chemoresistance in patient-derived GBM tumor cells.

Aromatase is a critical enzyme in the irreversible conversion of androgens to oestrogens, with inhibition used clinically in hormone-dependent malignancies. We tested the hypothesis that targeted aromatase inhibition in an aggressive brain cancer called glioblastoma (GBM) may represent a new treatment strategy. In this study, aromatase inhibition was achieved using third generation inhibitor, Letrozole, encapsulated within the core of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). PLGA-NPs were conjugated to human/mouse chimeric anti-GD2 antibody ch14.18/CHO, enabling specific targeting of GD2-positive GBM cells. Treatment of primary and recurrent patient-derived GBM cells with free-Letrozole (0.1 µM) led to significant decrease in cell proliferation and migration; in addition to reduced spheroid formation. Anti-GD2-ch14.18/CHO-NPs displayed specific targeting of GBM cells in colorectal-glioblastoma co-culture, with subsequent reduction in GBM cell numbers when treated with anti-GD2-ch14.18-PLGA-Let-NPs in combination with temozolomide. As miR-191 is an estrogen responsive microRNA, its expression, fluctuation and role in Letrozole treated GBM cells was evaluated, where treatment with premiR-191 was capable of rescuing the reduced proliferative phenotype induced by aromatase inhibitor. The repurposing and targeted delivery of Letrozole for the treatment of GBM, with the potential role of miR-191 identified, provides novel avenues for target assessment in this aggressive brain cancer.

Sharpening nature's tools for efficient tuberculosis control: A review of the potential role and development of host-directed therapies and strategies for targeted respiratory delivery.

Centuries since it was first described, tuberculosis (TB) remains a significant global public health issue. Despite ongoing holistic measures implemented by health authorities and a number of new oral treatments reaching the market, there is still a need for an advanced, efficient TB treatment. An adjunctive, host-directed therapy designed to enhance endogenous pathways and hence compliment current regimens could be the answer. The integration of drug repurposing, including synthetic and naturally occurring compounds, with a targeted drug delivery platform is an attractive development option. In order for a new anti-tubercular treatment to be produced in a timely manner, a multidisciplinary approach should be taken from the outset including stakeholders from academia, the pharmaceutical industry, and regulatory bodies keeping the patient as the key focus. Pre-clinical considerations for the development of a targeted host-directed therapy are discussed here.

Bronchodilator effects, pharmacokinetics and safety of PSX1002-GB, a novel glycopyrronium bromide formulation, in COPD patients; a randomised crossover study.

AIMS: To establish the dose-response relationship for pharmacodynamics (bronchodilatation), safety and pharmacokinetics of a novel particle engineered formulation of Glycopyrronium bromide (PSX1002-GB) in patients with chronic obstructive pulmonary disease (COPD). METHODS: Patients with moderate to severe COPD with bronchodilator reversible lung function were enrolled into this randomized, placebo-controlled, double-blind, dose-ranging, single dose, five way cross-over study (n = 37). Patients received single doses of PSX1002-GB (12.5-100 µg) via pMDI with one-week washouts between treatments. RESULTS: PSX1002-GB caused a bronchodilator response observed at 5 min post-dose at all doses. Significant improvements in mean change from baseline FEV1 at 24 h were seen at all doses compared with placebo; Mean changes were 0.071L (95% CI 0.041-0.101), 0.087L (95% CI 0.056-0.118), 0.102L (95% CI 0.072-0.133) and 0.120L (95% CI 0.089-0.150) for 12.5, 25, 50 and 100 µg respectively. PSX1002-GB 50 and 100 mcg caused rapid bronchodilation at 5 min after dosing. PSX1002-GB was well tolerated with similar adverse event rates reported compared to placebo. There were no clinically relevant changes in heart rate, blood pressure or ECG parameters (including QTc interval). CONCLUSION: Single doses of PSX1002-GB (12.5-100 µg) were well tolerated. PSX1002-GB 50 and 100 mcg delivered by pMDI produced rapid onset bronchodilation that was sustained over a 24 h period.

Treatment of Mycobacterium tuberculosis-Infected Macrophages with Poly(Lactic-Co-Glycolic Acid) Microparticles Drives NFκB and Autophagy Dependent Bacillary Killing.

The emergence of multiple-drug-resistant tuberculosis (MDR-TB) has pushed our available repertoire of anti-TB therapies to the limit of effectiveness. This has increased the urgency to develop novel treatment modalities, and inhalable microparticle (MP) formulations are a promising option to target the site of infection. We have engineered poly(lactic-co-glycolic acid) (PLGA) MPs which can carry a payload of anti-TB agents, and are successfully taken up by human alveolar macrophages. Even without a drug cargo, MPs can be potent immunogens; yet little is known about how they influence macrophage function in the setting of Mycobacterium tuberculosis (Mtb) infection. To address this issue we infected THP-1 macrophages with Mtb H37Ra or H37Rv and treated with MPs. In controlled experiments we saw a reproducible reduction in bacillary viability when THP-1 macrophages were treated with drug-free MPs. NFκB activity was increased in MP-treated macrophages, although cytokine secretion was unaltered. Confocal microscopy of immortalized murine bone marrow-derived macrophages expressing GFP-tagged LC3 demonstrated induction of autophagy. Inhibition of caspases did not influence the MP-induced restriction of bacillary growth, however, blockade of NFκB or autophagy with pharmacological inhibitors reversed this MP effect on macrophage function. These data support harnessing inhaled PLGA MP-drug delivery systems as an immunotherapeutic in addition to serving as a vehicle for targeted drug delivery. Such "added value" could be exploited in the generation of inhaled vaccines as well as inhaled MDR-TB therapeutics when used as an adjunct to existing treatments.