Jorge Alves-Silva

ALVEOLUS - Inhaled Plant Volatiles for Lung Diseases

Chronic respiratory diseases present high mortality and disability rates, particularly in high income countries, where the presence of associated risk factors is highly prevalent. Among respiratory diseases, aspergillosis and cystic fibrosis have been in the spotlight due to increasing resistance and lack of effective treatments, respectively. Indeed, Aspergillus fumigatus, one of the causative agents for aspergillosis, was included in the recent WHO fungal priority pathogens list, highlighting its relevance and urgent need for mitigation. In addition, the same list reports very high mortality rates in immunocompromised patients with invasive aspergillosis caused by azole-resistant strains. Furthermore, aspergillosis is often prevented resorting to prophylaxis that also contributes to the emergence of resistant strains. Importantly, respiratory diseases are often associated with an inflammatory response that often leads to other complications such as increased mucus production and tissue remodeling. For example, in cystic fibrosis, the mucus clogs the airways and traps germs leading to infections, inflammation, respiratory failure, and other complications. Validating the role of inflammation in lung diseases, it has been shown that oral anti-inflammatory drugs delay cystic fibrosis. Nevertheless, anti-inflammatory drugs may present toxic effects and their use is limited in some patients with other underlying pathologies, such as asthma. Therefore, the development of less toxic and more amenable anti-inflammatory agents is paramount. Aromatic and medicinal plants stand out as a potential source of new anti-inflammatory drugs with many traditional uses supporting these effects. Moreover, their widely known antimicrobial effects, confirmed by their widespread use in cleaning agents, is also relevant in the context of lung infections.

Over the years our research group has validated the anti-inflammatory and antimicrobial potential of many plant volatiles and pointed out possible mechanisms of action. Despite the undeniable potential, these compounds present several drawbacks that continue to limit their clinical translation, such as volatility, lipophilicity and oxidation. Therefore, to achieve therapeutic efficacy, frequent administrations are required which jeopardizes patients’ adherence to therapy. In addition, due to their liquid form and high volatility, conventional techniques used to increase lung deposition, such as the addition of carrier systems, cannot be efficiently applied. To overcome these limitations the present proposal aims to develop an innovative inhalatory strategy to improve their stability, lung retention and sustained release, by resorting to solid matrix-based lipid nanoparticles. Overall, the ALVEOLUS proposal will boost the clinical translation of plant volatiles by paving the way for the development of effective inhalatory preventive and therapeutic strategies for lung diseases.

Scientific research is one of my main passions, with natural products drawing my attention since early days. In the last years, I have directed my research to the potential of these products, namely plant extracts and their volatile compounds as health promoting agents. This field of research has a direct impact on population welfare and can improve their quality of life. In this context, disruptive and impactful ideas are a must in order to fill knowledge gaps and truly impact patient’s health.
The foundation for my career plan was established during my PhD in Pharmaceutical Sciences, concluded in 2022 at the University of Coimbra (UC). During my PhD studies I had the opportunity to develop a breakthrough study that pointed out a cardioprotective potential for a volatile natural compound (doi: 10.1016/j.phrs.2022.106151). In addition, I collaborated in several projects showing the potential of essential oils and isolated volatile compounds as anti-inflammatory and antimicrobial agents (doi: 10.3389/fphar.2019.00446; 10.1016/j.jep.2018.06.025; 10.1016/j.indcrop.2018.01.024; 10.1016/j.jep.2019.112120). These studies demonstrated that volatile extracts (essential oils) obtained from aromatic and medicinal plants present strong effects against several pathogenic fungi, including Aspergillus spp. highly relevant in lung diseases. Furthermore, we demonstrated that several volatile extracts present a strong anti-inflammatory effect by modulating the canonical NF-κB signaling pathway. More recently, I also collaborated in studies showing the anti-aging potential of essential oils (doi: 10.3390/nu15081930; 10.3390/antibiotics12010179), which is highly relevant in the context of lung diseases, due to the high susceptibility of this organ to senescence and “inflammageing”. At this stage of my career, I would like to establish an independent research path and I´m truly interested in developing non-invasive routes of administration for plant volatiles, being the inhalation route one of my main goals. Indeed, lung diseases such as cystic fibrosis, aspergillosis or chronic obstructive pulmonary disease greatly impact patient’s quality of life and present very high mortality and disability rates. Plant extracts and compounds could be an excellent preventive/therapeutic strategy due to their widely reported anti-inflammatory and antimicrobial potential that could alleviate the inflammatory response and infection progress in these pathologies, thus improving patient’s quality of life.

Despite their huge potential, the translation of plant volatiles to a clinical setting is very challenging due to their high volatility, lipophilicity, and proneness to oxidation, which decreases their stability and bioavailability. Importantly, these compounds are highly suitable for inhalation, but some features such as their small size limits their use due to poor lung retention, being easily and readily removed upon exhalation. Built upon my previous work and skills acquired during my academic studies, including motivation and resilience to achieve the proposed goals, I designed the current proposal that gathers my main research interests and reflects the scientific path I intend to build.

The ALVEOLUS proposal includes several tasks that spawn from the development of inhalable particles in a dry powder to the in vivo validation of the therapeutic potential of this inhalatory system. The DDL Career Development Grant from the Aerosol Society would be of utmost importance to carry out the first task, namely the development of the solid lipid nanoparticles (SLN) containing the volatile compound(s) of interest. In this stage, relevant parameters for nanoparticle formulation need to be optimized such as particle size, polydispersity coefficient and zeta potential assessed by dynamic light scattering, Fourier transform infrared spectroscopy and differential scanning calorimetry (methodologies and equipment available at my institution). The encapsulation and loading efficiency will also be determined to assess the capacity of the SLN to incorporate the compound(s). In vitro release of the compound(s) will be determined using the dialysis bag method. Particle morphology will be assessed by transmission electron microscopy. Stability assays of the compound-containing SLN in solution for short- and long-term storage will be assessed by monitoring particle size and zeta potential. The following tasks aim to improve mucoadhesion and lung retention, by adsorbing SLNs to a mucoadhesive compound. A dry powder will be obtained by submitting the SLNs to spray-drying. Alternatively, bulking systems will be used to increase particle size, thus improving the aerodynamic properties of the SLNs, which allows for a deep lung deposition and increased retention. Particle size, density, flow behavior and surface characteristics of the obtained powders will be assessed according to the European Medicines Agency guidelines (EMEA/CHMP/QWP/49313/2005 Corr). Lung distribution of the powder will be disclosed using an Andersen Cascade Impactor, according to the European Pharmacopoeia. The final stages of this proposal resort to a pre-clinical validation using animal models of lung diseases, such as cystic fibrosis, chronic obstructive pulmonary disease, thus paving the way for the clinical translation of these compounds. The results obtained during the DDL Career Development Grant will be of the utmost importance, since they will leverage my applications to other funding opportunities, in order to fully accomplish the present proposal.

ALVEOLUS project brings novelty to my previous studies as it resorts to pharmaceutical technology to develop advanced drug delivery systems and is a step forward in the clinical translation of plant volatiles. The proposal also enriches my knowledge on drug formulation and routes of administration, namely the inhalatory one supporting my scientific maturity and will enable me to leverage of my own research line, thus consolidating an independent research career.
Having in mind that multidisciplinary teams are the cornerstone for a successful proposal, scientific collaborations will be key to bring to the project the skills and experience I lack. This proposal counts on previously established collaborations with researchers of the Faculty of Pharmacy and Chemistry Department of the UC and clinicians from Coimbra Hospital and University Centre. Moreover, new collaborations have been established namely with the Drug Sciences Department of University of Porto. Clinical collaborations are quite relevant, and I aim to reinforce them to bring clinical relevance and the patient need perspective to future projects. I also intend to collaborate with the pharmaceutical industry to further optimize the delivery support device, resorting to the wide network of industrial sponsors of the DDL Conference. These collaborations are critical as they will facilitate the clinical translation of the drug delivery system developed during the project. In addition, the direct collaboration with the pharmaceutical industry, will grant me a product-focused approach, which will provide the necessary skills to create a drug delivery system that could generate value and reach the market, thus improving patient’s quality of life. Furthermore, being awarded the DDL Career Development Grant, will open up my network by allowing the contact with several members of the DDL committee that have extensive experience in several fields of research relevant for my career plan.
Furthermore, I believe that mentorship, leadership, and communicational skills are an essential part of the skillset required for a successful independent research career. Therefore, this research project will allow me to establish my research line, which will allow me to mentor master and PhD students, thus contributing to their growth as resilient and motivated scientists. In addition, I plan to participate in several dissemination initiatives, both for academic, such as attending the DDL Conference 2023, Global Conference on Pharmaceutics and Novel Drug Delivery Systems or International Summit on Pharmaceutics and Drug Delivery Systems, and non-academic audiences.

Finally, the line of research I intend to create, based on the current proposal, is highly relevant to my institution as a specialized and trained researcher in inhalable encapsulated nanoparticles will be available. This skillset is currently missing at the institution and could greatly contribute to the research in disease-related strategic areas. Indeed, this research project will allow to install a workflow that can be adapted and applied to different research areas