Tuberculosis (TB) is a global infectious disease caused by Mycobacterium tuberculosis (M.tb) and ranks as the second leading cause of death from infectious diseases worldwide (Chakaya et al., 2021). TB spreads through M.tb-containing aerosol droplets, transmitted from an infected patient to an uninfected individual. Upon inhalation, M.tb invades alveolar macrophages, forming granulomas, which are lesions consisting of various immune cells encapsulating the bacteria. This containment slows down bacterial metabolism, leading to a dormant state called latent TB (Luies and du Preez, 2020). Latent TB affects approximately 25 % of the population, presenting a significant global health concern (Haddad et al., 2018). However, some granulomas lose their integrity over time, releasing contagious bacteria and creating cavities in the airway walls, resulting in lung damage for TB patients. Targeting and eliminating the dormant bacteria within granulomas or actively replicating M.tb in cavities pose significant challenges for TB treatment (Chee et al., 2018).

The traditional treatment regimen for TB involves a lengthy course of 6–12 months, comprising first-line drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) and second-line drugs (ofloxacin, amikacin, ciprofloxacin, etc.) administered orally or intravenously (Jnawali et al., 2013). However, the repeated use of these decades-old anti-TB drugs has led to the emergence of drug resistance, such as multi-drug resistance (MDR-TB) and extremely drug resistance (XDR-TB), where TB patients become unresponsive to first and second-line drugs (Allué-Guardia et al., 2021). Fortunately, advancements in TB treatment led to the approval of three anti-TB drugs (bedaquiline, delamanid, and pretomanid), particularly for the management of MDR-TB and XDR-TB patients (Fong, 2023).

Combination therapy is commonly employed for TB treatment to minimize the risk of drug resistance, shorten treatment duration, and decrease the likelihood of disease relapse (Kerantzas and Jacobs, 2017, Larkins-Ford et al., 2022). Ongoing clinical trials are exploring different treatment regimens that involve a combination of first- and second-line drugs, aiming to replace existing protocols and address MDR/XDR-TB cases (Global Alliance for TB Drug Development, 2023, Global Alliance for TB Drug Development, 2020, Global Alliance for TB Drug Development, 2019). In this project, we explored the combination of bedaquiline (BDQ) and pretomanid (PTD) for TB treatment owing to complementary mechanisms targeting different sites of M.tb for inhibiting replication (Fong, 2023). BDQ, a diarylquinoline, operates by inhibiting the proton pump of ATP synthase and was approved for TB treatment in 2012 by US Food and Drug Administration (FDA) (Koul et al., 2007, Treatment of Multidrug-Resistant Tuberculosis, 2022). PTD, a bicyclic nitroimidazole approved by FDA in 2019, operates by inhibiting mycolic acid biosynthesis, which is crucial for cell wall production, leading to the eradication of actively replicating M.tb. Moreover, it demonstrates efficacy in eliminating non-replicating M.tb (latent TB) through the release of nitric oxide (Gils et al., 2022, Manjunatha et al., 2009). However, the low aqueous solubility of BDQ and PTD limits the bioavailability (reported bioavailability <40 % in animal models; not determined in humans) (Jaw-Tsai et al., 2023, Lyons, 2018) and further facilitates the need for solubility enhancement to increase therapeutic outcomes.

Various drug delivery strategies are used to improve the aqueous solubility and overcome the poor bioavailability of antibiotics, such as salt formation (Gupta et al., 2018), prodrug formation (Rautio et al., 2018), self-emulsifying drug delivery systems (SEDDS) (Neslihan Gursoy and Benita, 2004), cyclodextrin complexation (Parvathaneni et al., 2021, Patil et al., 2023b), and solid dispersion (Nguyen et al., 2023, Pardhi and Jain, 2021). However, nanocarriers have been widely investigated as one of the solubility enhancement techniques and polymeric nanoparticles (NPs) offer high drug loading, sustained or controlled drug release, target specificity by surface modification, and safety and biocompatibility for human use (Castro et al., 2022). Biodegradable poly(lactic-co-glycolic acid) (PLGA) is an extensively investigated polymer for drug delivery applications and is already reported for delivery of rifampicin (Andreu et al., 2019, Vibe et al., 2016) and ethionamide (Kumar et al., 2011a, Kumar et al., 2011b), and co-delivery of amikacin and moxifloxacin (Abdelghany et al., 2019). Further, the interest in local administration using dry powder inhalers (DPIs) of antibiotics to increase local drug deposition, reduce drug resistance, and lower systemic exposure is growing, as the lungs are the primary organ of M.tb infection. The successful delivery of the inhaled dose depends on powder properties and compatibility with the inhalation device. Consistency and predictability of the delivered dose are vital, and existing literature demonstrates the intricate interplay of powder and device factors influencing aerosolization performance (Ding et al., 2021). This complexity necessitates a Quality-by-Design (QbD) approach for developing and manufacturing of new DPI products (EMA, 2018a). QbD is a modern, regulatory-based quality management system that emphasizes the design phase of developing new pharmaceutical products. The principles of QbD were introduced in international guidelines, including the Q8(R2) (EMA, 2018b), Q9 (EMA, 2018c), and Q10 (EMA, 2018d) guidelines by the International Council for Harmonization (ICH) for human use. The most crucial element of QbD is risk assessment (RA), which identifies factors with the highest impact on the final drug product's quality. Here, implementing QbD methodology will enhance the practicality and effectiveness of powder preparation using spray drying, leading to more optimized and efficient DPI therapeutic.

Previous studies predominantly concentrated on optimizing PLGA NPs (Kunda et al., 2015, Vanza et al., 2023), employing a design of experiments (DoE) for the spray drying of NPs (Karas et al., 2023, Tse et al., 2021), and formulating DPI preparations through a QbD approach (Mukhtar et al., 2020, Pallagi et al., 2016). This investigation, however, takes a novel perspective by exploring the QbD strategic approach in the preparation of DPI for PLGA NPs using the spray drying technique. We propose the formulation of a combination of BDQ-PTD (BPa) drugs co-loaded into polymeric nanoparticles (BPa PLGA NPs) and administered as BPa PLGA NPs spray-dried (BPaD) powder for inhalation. The rationale behind this approach is to utilize combination therapy to prevent the development of drug resistance, while delivering the drugs through inhalation to ensure localized drug deposition and enhance patient compliance by minimizing systemic exposure and associated toxicities. To achieve these goals, we implemented a QbD approach to optimize resource utilization and develop an efficient design space based on our extensive prior knowledge of spray drying. Additionally, the Box-Behnken design, as part of the DoE, was used to optimize the process parameters for preparing the dry powder formulation. The optimized DPI formulation was evaluated for drug content, solid state characteristics, morphology, aerosolization performance, drug release, stability, and in vitro antibacterial activity to ensure the efficacy in TB patients.