Design of Experiment (DoE) Approach for Developing Inhalable PLGA Microparticles Loaded with Clofazimine for Tuberculosis Treatment

Abstract

Tuberculosis (TB) is an airborne bacterial infection caused by Mycobacterium tuberculosis (M. tb), resulting in approximately 1.3 million deaths in 2022 worldwide. Oral therapy with anti-TB drugs often fails to achieve therapeutic concentrations at the primary infection site (lungs). In this study, we developed a dry powder inhalable formulation (DPI) of clofazimine (CFZ) to provide localized drug delivery and minimize systemic adverse effects. Poly (lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) containing CFZ were developed through a single emulsion solvent evaporation technique. Clofazimine microparticles (CFZ MPs) displayed entrapment efficiency and drug loading of 66.40 ± 2.22 %w/w and 33.06 ± 1.45 µg/mg, respectively. To facilitate pulmonary administration, MPs suspension was spray-dried to yield a dry powder formulation (CFZ SD MPs). Spray drying had no influence on particle size (~1 µm), zeta potential (−31.42 mV), and entrapment efficiency. Solid state analysis (PXRD and DSC) of CFZ SD MPs studies demonstrated encapsulation of the drug in the polymer. The drug release studies showed a sustained drug release. The optimized formulation exhibited excellent aerosolization properties, suggesting effective deposition in the deeper lung region. The in vitro antibacterial studies against H37Ra revealed improved (eight-fold) efficacy of spray-dried formulation in comparison to free drug. Hence, clofazimine dry powder formulation presents immense potential for the treatment of tuberculosis with localized pulmonary delivery and improved patient compliance.

Introduction

Tuberculosis (TB) is a chronic infectious bacterial disease that resulted in approximately 1.3 million deaths worldwide in 2022 alone [1]. TB is caused by Mycobacterium tuberculosis (M. tb), a pulmonary pathogen that enters the human body through the lungs via inhalation and causes pulmonary TB [1,2,3,4,5]. Clinically, TB is classified as latent TB infection (LTBI) and active TB infection [6]. Current therapy for active TB infection involves Directly Observed Treatment Short course (DOTS) with a combination of drugs that include rifampicin, isoniazid, ethambutol, and pyrazinamide with high intravenous doses of streptomycin for 6 to 9 months to cure the infection and prevent the spread [7]. However, with conventional therapy, a very minimal amount of drug reaches the lungs, requiring long-term treatment with high doses to maintain effective drug concentration in the lungs. In most cases, long-term treatment often leads to non-compliance that eventually causes the development of multi-drug resistant tuberculosis (MDR-TB). Because current treatment options are limited, reformulating an existing highly active antibacterial drug, such as clofazimine, in a new drug delivery system could be an intriguing approach to overcoming TB, specifically by targeting the infection site.

Clofazimine (CFZ) is a poorly water-soluble antibiotic and anti-inflammatory drug belonging to the riminophenazine class. CFZ is highly active against various mycobacteria, such as Mycobacterium leprae, Mycobacterium avium complex (MAC), and M. tuberculosis. CFZ is clinically approved for the treatment of leprosy and is also recommended as a second-line agent for MDR-TB treatment [8]. Though the mechanism of action of CFZ is not fully understood, a few studies suggest that its antibacterial effect is caused by the release of toxic lysophospholipid enzymes [9]. CFZ has also been used in combination with other anti-TB drugs, and there is an ongoing Phase-II clinical study of CFZ with a combination of delamanid, bedaquiline, and fluoroquinolones (norfloxacin and levofloxacin) to evaluate its therapeutic effectiveness in MDR-TB [10]. However, current clinical treatment of MDR-TB requires a 100 mg oral dose of CFZ [8]. Importantly, a minimum of 30 days of oral dosing is required to reach the steady-state concentration, thereby emphasizing the use of large loading doses and delay in anti-tuberculosis action [11]. The high oral doses often lead to severe adverse effects such as discoloration of the skin and conjunctiva, bioaccumulation in body fat tissue, cardiotoxicity, and severe GI distress like abdominal pain, nausea, diarrhea, and vomiting [12]. Additionally, the CFZ oral capsule is characterized by poor aqueous solubility and variable oral bioavailability (45 to 62%).

Pulmonary drug delivery could be an effective alternative as it allows for direct delivery of CFZ into the lungs and achieves effective lung concentrations [13,14]. In comparison to conventional oral drug delivery, it requires a lower dose, shorter treatment duration, and lower dosing frequency. Moreover, a large pulmonary surface area with thin alveolar epithelium offers the advantage of faster onset of drug action. Though several types of pulmonary delivery formulations and devices are available, dry powder inhalers have distinctive advantages of high stability, ease of handling, and portability over pressurized metered-dose inhalers, soft-mist inhalers, and nebulizers. Additionally, inhalable dry powder formulations containing microparticles can also take advantage of alveolar macrophage phagocytosis and target the mycobacteria residing in them. Previously, Brunaugh et al. explored the use of air jet milling to produce inhalable CFZ powder. However, their study lacked data pertaining to the stability and in vitro efficacy of CFZ formulation [8]. This is important as air jet milling often induces surface defects in the particles, leading to alterations in their crystalline structure, subsequently affecting stability and aerodynamic characteristics [15]. Here, we propose to develop a polymer-based inhalable CFZ powder via spray drying that is stable and efficacious against TB.

Polymer-based drug delivery systems have been explored extensively to improve solubility and increase the bioavailability of lipophilic drugs [16,17,18]. Poly (lactic acid-co-glycolic acid) (PLGA) is a biodegradable polymer that is safe for pulmonary delivery [19,20]. In addition, PLGA microparticles (MPs) offer improved drug loading and sustained drug release and allow for the passive targeting of alveolar macrophages [14,21]. Further, formulating PLGA MPs into dry powders will enhance stability and prevent drug leakage from MPs in suspension [22]. In this study, we have used a Design of Experiment (DoE) approach to systematically identify and optimize the spray drying process parameters for the successful production of CFZ-loaded PLGA MPs (CFZ SD MPs). Moreover, the resultant CFZ SD MPs were characterized for encapsulation efficiency, drug loading, and in vitro aerosolization performance. Additionally, the in vitro antibacterial activity of CFZ SD MPs was evaluated.

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Materials

Clofazimine was procured from Acros Organics (Morris Plains, NJ, USA). Poly (lactic-co-glycolic acid) (MW: 7000 to 15,000; 50:50) acid terminated was purchased from PolySciTech (West Lafayette, IN, USA). Polyvinyl alcohol (PVA) and L-leucine were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Phosphate buffered saline (PBS) was obtained from Corning Inc. (Corning, NY, USA). Middlebrook broth 7H9 and Tween® 80 were purchased from VWR International (Radnor, PA, USA). Dichloromethane (DCM, HPLC grade), Acetonitrile (ACN, HPLC grade), Water (HPLC grade), and Resazurin were procured from ThermoFisher Scientific (Waltham, MA, USA). Avirulent Mycobacterium tuberculosis strain (H37Ra) was procured from the American Type Culture Collection (ATCC 25177) (Manassas, VA, USA).

Rongala, D.S.; Patil, S.M.; Kunda, N.K. Design of Experiment (DoE) Approach for Developing Inhalable PLGA Microparticles Loaded with Clofazimine for Tuberculosis Treatment. Pharmaceuticals 2024, 17, 754. https://doi.org/10.3390/ph17060754

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