Comprehensive Evaluation of Storage Stability and Cytotoxicity of Co-Spray-Dried Theophylline Dry Powders for Inhalation: Follow-Up Study

Abstract

Background/Objectives: The stability and biological safety of newly developed formulations must be established to support their therapeutic efficacy and clinical translation in pulmonary drug delivery. Therefore, this follow-up study comprehensively evaluated the short- and long-term stability and the in vitro cytotoxicity of optimized, co-spray-dried theophylline (THN) dry powders for inhalation against A549 lung epithelial cells.

Methods: Two established formulations were selected: THN-RAF (raffinose–leucine–glycine based) and THN-TRE (trehalose–leucine based). Stability was assessed under accelerated conditions (40 °C/75% RH, 3 months) and long-term desiccator storage (25 °C, 1 year) using laser diffraction, SEM, XRPD, FTIR, DSC, TGA, and Andersen Cascade Impaction. As THN-TRE had been previously confirmed to be cytocompatible, only THN-RAF and its components were evaluated against A549 human alveolar epithelial cells using the MTT assay.

Results: Under accelerated conditions, both formulations exhibited pronounced recrystallization (Xc up to 89.9%), agglomeration (D [0.9] up to 217.08 µm for THN-TRE), and deterioration in aerodynamic performance (FPF as low as 11.55%, MMAD up to 6.68 µm). By contrast, long-term desiccator storage induced substantial recrystallization (Xc up to 80.7%) while preserving thermal, chemical, and aerodynamic performance (FPF ≈ 40%; MMAD 4.99–5.21 µm). THN-RAF was more resistant to stress-induced agglomeration than THN-TRE. Cytotoxicity assessment confirmed cytocompatibility of THN-RAF, with cell viability exceeding 70.99% at all tested concentrations (up to 500 µg/mL).

Conclusions: These findings reveal a marked discrepancy between the outcomes of ICH accelerated testing and long-term desiccator storage. They underscore the importance of considering moisture-protective packaging configurations when designing stability protocols for amorphous inhalable formulations.

Introduction

Theophylline (THN) is a methylxanthine bronchodilator that has long been used in the management of asthma and chronic obstructive pulmonary disease (COPD) due to its bronchodilator and anti-inflammatory properties [1]. Its pharmacological effects are mediated through multiple mechanisms, including non-selective phosphodiesterase inhibition, adenosine receptor antagonism, and restoration of histone deacetylase-2 activity at lower concentrations, which may enhance corticosteroid responsiveness [1,2]. Low-dose theophylline has also been investigated as an adjunctive anti-inflammatory therapy, although recent clinical studies have reported variable therapeutic benefits [3]. Consequently, pulmonary delivery remains an attractive strategy to maximize local drug concentrations while minimizing systemic exposure and associated adverse effects [4]. However, despite its therapeutic potential, no inhaled theophylline product is currently available for routine clinical use [4].

Pulmonary drug delivery is constrained by several anatomical, physiological, and immunological barriers [5,6,7]. Nevertheless, inhalation offers a non-invasive, targeted route with the potential for rapid absorption. Other challenges include low pulmonary deposition efficiency, inadequate drug retention, poor patient adherence, and incorrect inhaler technique [5,8]. Formulation-related concerns include drug stability, particle size distribution, and excipient safety [9]. Several devices are available for direct pulmonary administration, including dry powder inhalers (DPIs).

DPIs can provide high physicochemical stability and efficiently deliver relatively high drug doses to the lungs [10]. They are also environmentally preferable [11], and offer a compact, portable, and coordination-free mode of administration; however, their performance depends on the patient generating sufficient inspiratory flow [12].

DPIs are generally easy to use and are associated with high patient adherence [13]. They are compatible with diverse active ingredients, including small molecules and biologics [7,14]. Recent technological advances have sought to improve DPI efficacy, dose uniformity, and physical stability [15], including through spray drying [16]. Co-spray-drying is an advanced particle-engineering approach that can improve the aerosolization and physicochemical stability of DPI formulations compared with conventional systems [17]. Co-spray-dried fine excipients have also been reported to improve dispersibility by filling carrier macropores [18]. When integrated with quality-by-design principles, spray drying can generate stable powders with optimized particle attributes for efficient pulmonary delivery and controlled release [19].

Nevertheless, the stability of newly developed co-spray-dried DPIs remains insufficiently characterized. Regulatory guidance, particularly the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q1A–Q1F guidelines, defines protocols for pharmaceutical stability testing [20,21]. The physical stability of inhalable dry powders is critical because solid-state changes may impair aerosolization performance [22]. Amorphous saccharide-based powders are particularly challenging because they may recrystallize during storage, thereby reducing aerodynamic performance [23]. These risks can be mitigated through moisture-protective packaging, hydrophobic excipient coatings such as leucine or stearates, and formulations engineered to have high glass-transition temperatures (Tg) [23]. Appropriate analytical methods are likewise essential for detecting stability-related changes [24].

For capsule-based DPIs, capsule composition (e.g., gelatin or hydroxypropyl methylcellulose [HPMC]) affects moisture interactions and electrostatic charging and therefore influences powder emission, retention, aerosolization efficiency, puncture behavior, and formulation stability [25,26]. Our group previously found that HPMC capsules provided the most favorable conditions for powder stability. By maintaining an appropriate residual moisture content and structural integrity while minimizing fragmentation after activation, these capsules supported superior structural and aerodynamic stability [27]. We have also investigated the physicochemical and aerodynamic stability of several novel spray-dried inhalation powders [28,29,30].

As pulmonary administration requires a favorable safety profile, in vitro cytotoxicity assessment is an essential step in predicting respiratory toxicity and identifying potential safety concerns before animal and clinical studies [31,32]. The A549 human alveolar epithelial adenocarcinoma cell line is widely accepted for evaluating the cytotoxicity of DPI formulations and has demonstrated good predictive value in previous studies [33,34,35].

Consequently, the present work builds upon our previously developed and optimized DPI formulations [33,36]. Co-spray-drying THN with saccharide–amino acid carriers—trehalose dihydrate (TRE) or raffinose pentahydrate (RAF) combined with leucine (LEU) or a leucine–glycine mixture (LEU–GLY)—yielded partially amorphous microparticles with markedly improved solubility, rapid THN dissolution, fine particle fractions (FPFs) of 43–48%, and predicted deep lung deposition of 36–40%. These carrier systems may enhance therapeutic efficacy while enabling dose reduction.

Although our previous studies established the formulation design and initial characterization of THN-TRE and THN-RAF DPI systems, their storage stability remained unexplored. The present study extends this earlier work by systematically evaluating the long-term and accelerated stability of both optimized formulations, with emphasis on physicochemical, solid-state, thermal, and aerodynamic performance after storage. Furthermore, the in vitro cytocompatibility of THN-RAF was evaluated using A549 cells to establish its preliminary pulmonary safety profile, complementing the previously reported safety assessment of THN-TRE [33]. This follow-up investigation provides the stability evidence required to assess their suitability for further pharmaceutical development. To the best of our knowledge, this is the first report presenting up to 1 year of stability data for these specific saccharide-based DPI formulations in combination with cytotoxicity profiling, thereby addressing an important gap in the literature.

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Materials

Anhydrous theophylline (THN), used as the active ingredient, was supplied by Hungaropharma Ltd. (Budapest, Hungary). The excipients were raffinose pentahydrate (RAF; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), trehalose dihydrate (TRE; Sigma-Aldrich Chemie GmbH, Steinheim, Germany), L-leucine (LEU; Molar Chemicals Kft., Budapest, Hungary), and glycine (GLY; VWR International LLC, Leuven, Belgium). Ezeeflo™ size 3 hydroxypropyl methylcellulose (HPMC) capsules (ACG-Associated Capsules Pvt. Ltd., Mumbai, India) were used to contain the powders during storage. Soft multilayer polyethylene bags (40–100 µm thick) were purchased from Patika Pack Kft. (Budapest, Hungary). Potassium bromide (J&K Scientific Limited, Beijing, China) was used for FTIR background measurements. Distilled water was obtained using an in-house Millipore Direct-Q 5 UV water-purification system (Millipore, Molsheim, France). A mixture of sorbitan monooleate (Span® 80; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and cyclohexane (VWR BDH Chemicals, Paris, France) was used to coat the aerodynamic-impactor collection plates. Aliquots were filtered through 0.45 µm Millex-HV syringe filters (Millipore Corporation, Bedford, MA, USA). [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and sodium dodecyl sulfate were supplied by (Sigma, St. Louis, MO, USA).

Soliman, L.; Paróczai, D.; Burián, K.; Ambrus, R. Comprehensive Evaluation of Storage Stability and Cytotoxicity of Co-Spray-Dried Theophylline Dry Powders for Inhalation: Follow-Up Study. Pharmaceutics 2026, 18, 1027. https://doi.org/10.3390/pharmaceutics18081027

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