Abstract
Background: A number of viral and bacterial pathogens are responsible for multiple respiratory diseases in humans. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most recent example of the significant global health burden related to these infectious diseases. Although numerous antibacterial and antiviral treatments are available, not all of them are effective at rapidly achieving the desired therapeutic outcomes when administered in their conventional dosage forms such as tablets, capsules, or injections. The key limitations include numerous side effects due to the administration of high doses and insufficient drug concentration in the respiratory tract, the primary target site of many of these infections. Alternatively, inhaled treatment is considered an efficient approach for treating infections like SARS-CoV-2, by supporting the administration of lower doses and ensuring higher concentrations of the drug(s) in the lung. The inhaled treatment combining two or more anti-infective agents can increase potency and reduce the possibility of drug resistance. Repurposed drugs are often chosen against these respiratory pathogens and during pandemic situations as they are readily available and require less time to develop. Subsequently, this thesis aimed to develop both single and combinational inhalable dry powders containing suitable repurposed agents having anti-infective properties, mainly targeting viral respiratory pathogens, such as SARS-CoV-2, while also evaluating their effectiveness against selected bacterial respiratory pathogens (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Klebsiella pneumoniae).
Methods: Early in the COVID-19 pandemic when limited options were available to target SARS-CoV-2, four commercially available and repurposed drugs having anti-infective properties, namely ivermectin, remdesivir, disulfiram, and ebselen were selected to develop as inhalable dry powders. Ivermectin and ebselen were developed as a single drug containing dry powder. On the other hand, remdesivir was developed as a combination with disulfiram or ebselen due to their reported synergistic activity. The inhalable dry powders were prepared by spray drying technique and in the presence/absence of different amino acids such as L-leucine, L-methionine, and L-tryptophan. The prepared dry powders were characterized using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to assess the dry powder morphology and particle size, residual solvent amount, crystallinity, and drug-drug/drug-excipient interactions. The stability of ivermectin and remdesivir-disulfiram combinational dry powders was assessed at 25 °C/<15% RH and 25 °C/53% RH conditions for one month. The cytotoxicity and anti-SARS-CoV-2 activity of ivermectin dry powder and combinational dry powders of remdesivir with disulfiram/ebselen were assessed in vitro using cultured human airway epithelial cells (Calu-3 cells). In addition, the cytotoxicity and antimicrobial activity of ebselen dry powders were assessed using an alveolar epithelial cell line (A549 cells) and against Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae.
Results: All the prepared dry powders were within the size range of 1–5 μm indicating their suitability for inhalation. The used raw materials were crystalline in nature. However, the spray-dried ivermectin dry powder was amorphous whereas the other formulations such as ebselen dry powders, remdesivir-disulfiram, and remdesivir-ebselen combinational dry powders remained crystalline. The residual solvent amount was ~1% (w/w) for all the dry powders, and no drug-drug/drug-excipient interactions were observed. Different drugs showed distinct morphological features after spray drying. The optimized ivermectin and ebselen dry powders were wrinkled whereas remdesivir-disulfiram and remdesivir-ebselen combinational dry powders were spherical. The average fine particle fraction of optimized ivermectin, remdesivir-disulfiram combination, remdesivir-ebselen combination, and ebselen dry powders were 83%, 61%, 65%, and 68%, respectively. All these optimized dry powders contained L-leucine as an excipient. The stability study conducted for ivermectin and remdesivir-disulfiram combinational dry powders revealed no significant difference in the physicochemical and in vitro aerosolization properties of the respective dry powders. The 50% cytotoxic concentration (CC50) values of the optimized ivermectin, remdesivir-disulfiram combination, and remdesivir-ebselen combinational dry powders were 39.1 μM, 41.51 μM and >100 μM, respectively. The half maximal inhibitory concentration (IC50) of optimized ebselen dry powder was 225 μg/mL tested in A549 cells. All the dry powders showed comparable anti-infective and antimicrobial activity to the raw drugs. The half maximal effective concentration (EC50) of the ivermectin, remdesivir-disulfiram combination, and remdesivir-ebselen dry powders against SARS-CoV-2 were 15.8 μM, 4.43 μM, and 8.04 μM, respectively. In addition, the ebselen dry powders showed potent antimicrobial effects against Gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae with a low minimum inhibitory concentration (MIC) of 0.31 μg/mL and 0.16 μg/mL, respectively. However, the dry powders lacked potent antimicrobial activity against the Gram-negative pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae. It is important to highlight that all the cellular toxicity (CC50 and IC50) and anti-infective as well as the antimicrobial (EC50 and MIC) values for the dry inhalable formulations were comparable to those obtained with respective non-formulated drugs.
Conclusions: The stable inhalable dry powders containing single and combinational antimicrobial agents were successfully developed by spray drying technique. L-leucine-containing dry powders showed better aerosolization properties compared to the L-leucine-free formulations or other amino acids. More importantly, the potency of all the prepared dry powders remained comparable to the raw active agents, with the dry powders showing limited cell toxicity in the respiratory cell lines used in this study. Further preclinical studies in animal models (e.g., humanized mice, African green monkeys) will test the suitability of the prepared dry powders to inhibit SARS-CoV-2, Staphylococcus aureus and Streptococcus pneumoniae.