2023-12 JAMPDD Vol 36, Issue 6 - Abstracts - ISAM International Society for Aerosols in Medicine e.V.

My first research work was on the structure‐function correlation of the lung in the laboratory of the late Dr. Ewald R. Weibel at the University of Bern [1]. Later, I began studies of particle‐lung interaction, including particle‐cell interaction and cell motility, in the laboratory of Dr. Joseph D. Brain at the Harvard School of Public Health [2]. This work evolved into my most important research work, which I pursued for many years with a wonderful group of young investigators at the University of Bern.

Over the years, there was a gradual transition from structure‐function correlation to structure‐particle interaction, including studies on the effects of nanoparticles on our health. I conducted these studies in close collaboration with Dr. Joachim Heyder at the Helmholtz Center Munich, and Dr. Barbara Rothen‐Rutishauser at the University of Fribourg [3].

To explore the mechanisms of how particles, especially nanoparticles, access our innermost being, I endeavored to look as deeply and as closely as possible into the lungs to learn what path inhaled aerosols take in the lungs. This led me on a wonderful journey around the world to learn from and collaborate with scientist friends.

References

[1] Gehr, P., Bachofen, M. and Weibel, E.R. (1978). Respir. Physiol. 32, 121‐140.

[2] Gehr, P., Brain, J.D., Bloom, S.B. et al. (1983). Nature 302, 336‐338.

[3] Gehr, P. (2018). Colloids Surf. B, 172, 395‐399.

B. Viral Infections and COVID

B. 01 SARS‐CoV 2 Transmission by Breathing in Children and Adults

Gerhard Scheuch

GS BIO‐INHALATION, Gemünden, Germany.

In the last years a new corona virus has had a major impact on our lives. At the very beginning the mechanism of transmission was not completely clear. Evidence was accumulating that aerosols play an important role.

There are different mechanisms by which aerosols can be generated in the human respiratory tract. It has long been known that normal quiet breathing can produce aerosol particles that are released into the ambient air.

In a study at the University of Frankfurt, Germany, we showed that in Corona patients admitted to the hospital, about 16% exhaled more than 5000 aerosol particles/L of breathing air during normal breathing, while healthy adults exhaled only about 200 P/L, we called these patients superemitters.

In children and adolescents, two studies failed to identify superemitters. And in addition we found that children and adolescents exhaled significantly fewer aerosol particles than adults even in the absence of an infection. In one study, we found on average only about 78 P/L exhaled by 6 – 11 year old children and 99 P/L in 12 – 17 year old adolescents.

We can therefore assume that adults are significantly more contagious than children and adolescents when infection occurs via exhaled aerosols.

B. 02 Review of Protective Measures against Direct and Indirect Infections

Christof Asbach

Gesellschaft für Aerosolforschung e.V, Köln, Germany. Institut für Umwelt & Energie, Technik & Analytik e. V. (IUTA), Duisburg, Germany.

The COVID‐19 pandemic has bestowed multiple challenges onto all of us. It had been known since the early phase of the pandemic that the highest infection risk is indoors, where the concentration of airborne pathogens (as any other aerosol particles) may accumulate. Proper care must therefore be taken in all indoor environments to reduce the virus concentration and therefore to minimize infection risks. Various different measures had been proposed, including frequent window airing, the use of room air cleaners and the wearing of protective face masks. Even though there were repeated attempts to play individual measures off against each other, the combination of measures turned out to be the most effective way of prevention. It also turned out to be crucial to distinguish between direct and indirect infections. Indirect infections can only occur indoors, when the virus concentration accumulates over time. The risk of indirect infections can significantly be reduced by cleaning or replacing the indoor air. This method is, however, ineffective against direct infections, which occur if a person directly inhales an infectious person's exhaled breath. Such direct infections can best be avoided by properly wearing efficient face masks.

The presentation will review the different methods for prevention of infections and evaluate them in terms of their efficacy against direct and indirect infections.

B. 03 Non‐Clinical Models of SARS‐CoV‐2 Transmission by Aerosol – What Are the Models and What Can We Learn From Them?

Philip J Kuehl,¹Jason Cox,¹Hammad Irshad,¹Edward G. Barrett,¹Sean N. Tucker,²and Stephanie N. Langel³

¹Lovelace Biomedical, Albuquerque, USA.

²Vaxart, Inc., South San Francisco, USA.

³Case Western Reserve University School of Medicine, Cleveland, USA.

It is well accepted that one of the primary modes of transmission for obligate respiratory pathogens, such as SARS‐CoV‐2, is through aerosols. Evaluating questions around viral transmission in the clinical setting has multiple concerns making it difficult to conduct well‐controlled studies. Multiple groups have developed non‐clinical models of SARS‐CoV‐2 transmission with the majority of them utilizing the Syrian golden hamster. Several key factors that must be considered in the evaluation of these models include: 1) the animal provides a model of viral burden but does not have many of the clinical phenotypes, 2) the characterization of the dose in naïve animals requires uses fit for purpose methods that are often lacking or not present, 3) the quantification of particle size is difficult based on the non‐specific models used and the low concentration of viral aerosols and 4) the control systems often aren't really control systems. Based on this our teams developed a hamster transmission model that allows for quantification of dose with fit for purpose methods using liquid impingers with PCR for viral aerosol concentration and controlled flow to calculate dose. We used up to four naïve hamsters that allowed sufficient animal numbers to conduct drug/vaccine efficacy and attempted to address the gap in particle size. Our model addresses many of the key factors mentioned yet highlights remaining gaps related to the non‐clinical disease phenotype.

B. 04 3D Human Airway Epithelial Models to Study SARS‐CoV‐2 Pathogenesis and to Discover Antivirals

Guy Barbin,Bernadett Boda, Rosy Bonfante, Samuel Constant, and Song Huang

Epithelix, Plan‐Les‐Ouates, Switzerland.

The respiratory system is the main entry portal of SARS‐CoV‐2 which infects initially and principally the airway epithelia. Since the first step of SARS‐CoV‐2 infection is taking place in airway epithelial cells, it is logic to use 3D airway epithelial model as drug testing platform.

Epithelix has developed and is offering standardized air‐liquid interface 3D human airway epithelial cultures from nasal or bronchial (MucilAir™) and small‐airway (SmallAir™) origins. These epithelial models closely mimic the morphology and function of the native tissues: such as cilia formation and beating, mucus production and secretion, mucociliary clearance, and secretion of antiviral molecules. These models have been successfully used for the development of antivirals against influenza, rhinoviruses, respiratory syncytial virus, amongst others.

This talk will highlight how these reconstituted human airway epithelial models can be used to characterize viral infection kinetics, tissue‐level tropism and transcriptional immune signatures induced by SARS‐CoV‐2. Relevance of these models for the preclinical evaluation of antiviral candidates will also be addressed in the context of repositioning of marketed drugs or evaluation of novel therapies and combinations delivered systematically or through aerosol therapy. Case study study will be presented highlighting that Molnupiravir combined with different repurposed drugs further inhibits SARS‐CoV‐2 infection in human nasal epithelium in vitro.

B. 05 From Medication to Intubation: Therapy of COVID 19 in Hospital Patients

Thomas Voshaar

Bethanien Hospital, Moers, Germany.

The SARS‐CoV‐2 pandemic presented our generation with challenges we had not previously encountered. Yet not everything was unknown, as is now often read. It has long been clear that respiratory viruses spread rapidly and highly effectively via aerosols. Thus, the respiratory system, especially the lungs, is also the primary target organ and, similar to influenza, is primarily affected and respiratory failure is the primary concern in severe disease progression.

Especially since any therapy for viral infections is less successful than for bacterial infections, the prevention of severe courses is a priority. Since the course of the disease also depends on the initial virus dose, protective measures such as effective masks and also room ventilation and air filtration are of great importance in a pandemic caused by respiratory viruses. However, vaccination has the greatest importance. In the case of respiratory viruses, it has no significant impact on breaking chains of infection; rather, its goal is to prevent very severe illness and reduce mortality.

After an infection has occurred, many drugs have been used without prior studies. The list ranges from vitamin D to zinc to colchicine, hydroxychloroquine, and ivermectin.

In the living guidelines, antiviral agents such as remdesivir (Veklury), molnupiravir (Lagevrio), and nirmatrelvir (Paxlovid) are recommended for early use, as is the monoclonal antibody sotrovimab (Xevudy).

In advanced disease, the focus is on more nonspecific inhibition of inflammation. In this situation, dexamethasone and the anti‐IL‐6 antibody tocilizumab may be used.

The most difficult and particularly controversial treatment is for severe, extensive bilateral pneumonia with respiratory failure. This condition is most commonly referred to as ARDS, although this is not an entity and the situation in COVID is different from other ARDS presentations. Treatment options range from simple oxygen administration, to high flow oxygen administration, to CPAP and non‐invasive ventilation (NIV), to invasive ventilation and ECMO. At the onset of the pandemic, a strategy of early intubation was recommended, resulting in mortality of 60 to 90%. Under invasive ventilation to date mortality is about 40 to 60%, strongly dependent on the age of the patient. The mortality with ECMO in Germany is about 80%.

Supported by known pathophysiological mechanisms, we favor the avoidance of invasive ventilation and use all forms of noninvasive treatment. The discussion on this approach focuses on the importance of hypoxemia, which is not identical with (tissue) hypoxia. The latter is mainly dependent on oxygen content, which is determined by oxygen saturation and hemoglobin content.

B. 06 Upper Airway Dehydration, Rehydration and the Management of Respiratory Disease

David A. Edwards

Harvard School of Engineering, Boston, USA.

Respiratory disease and breathing abnormalities worsen with dehydration of the upper airways. We find that humidification of inhaled air results in an osmotic pressure drop over airway mucus owing to very slight ion concentration differences above and below airway mucus. These osmolarity differences increase on the breathing of dry air, and are heightened by mouth breathing and exercise. A net osmotic force pressing mucus onto underlying cilia can develop with mouth breathing, and high minute volume as accompanies strenuous exercise, delivering a compressive force of up to around 100 cm H2O, promoting ATP secretion and activating neural pathways. We predict evolution of upper airway fluid structure, and airway dysfunction consequent to the breathing of dry air, including upper‐airway ATP and inflammatory cytokine levels, and cough incidence in normal and laryngeal hypersensitive human airways. We show that deposition of divalent hypertonic salts targeted with 8‐15 μm MMAD droplets rehydrates the upper airways significantly longer than monotonic salts, reducing inflammation and propensity to cough. These findings are compared with human clinical data.

B. 07 Does Pre‐existing Severe Asthma Increase Risk of Morbidity and Mortality from COVID‐19?

Rajiv Dhand,Paul Terry, and Eric Heidel

University of Tennessee College of Medicine, KNOXVILLE, USA.

Background:People with severe asthma perceive that they have an increased risk of poor outcomes after acquiring COVID‐19. However, the association of severe asthma with severity of COVID‐19 remains unclear.

Methods:We searched the PubMed (MEDLINE) database, using the search‐terms “asthma,” “SARS‐Cov‐2,” and “COVID‐19,” and cross‐referenced citations in identified studies (print or online) before December 1, 2022. We performed meta‐analyses using a common‐effect inverse‐variance model and established pooled effects based on adjusted odds ratios and 95% confidence intervals from studies where confounding variables were considered in multivariate models. Forest plots were used to visually depict the findings.

Results:We found 9 studies that categorized severe asthma based on the number and/or type of medicine used to control asthma, and/or the GINA severity level, recent history of severe asthma exacerbation, or an Asthma Control Test (ACT) score <19. The studies yielded a pooled OR = 1.50 (1.36 – 1.64), p < 0.001, and OR = 1.10 (1.00 – 1.20), p = 0.041 with COVID‐19 severity and mortality, respectively. There was no statistical evidence of heterogeneity for either meta‐analysis.

Conclusion:Increased risk of mortality and severe COVID‐19 in patients with pre‐existing severe asthma is possible. Due to several methodological limitations in reported studies, additional evidence is needed to adequately inform patients with severe asthma about risks due to COVID‐19.

B. 08 RNA Interference against SARS‐CoV‐2

Beatrice Tolksdorf,¹Daniela Niemeyer,^2,3Julian Heinze,³Johanna Berg,¹Christian Drosten,^2,3and Jens Kurreck¹

¹Technische Universität Berlin, Berlin, Germany.

²German Centre for Infection Research (DZIF), Berlin, Germany.

³Charité‐Universitätsmedizin Berlin, Berlin, Germany.

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) has so far globally infected more than 762.7 million people and led to more than 6.8 million deaths as of March 2023. Despite successful vaccine development, treatment options are still limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed a small interfering RNA (siRNA) targeting the highly conserved leader sequence in the 5’‐untranslated region (5’‐UTR) of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS‐CoV‐2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS‐CoV‐2 alpha and omicron variant. It was even highly active in virus replication assays with the SARS‐CoV‐1 family member. Introduction of 2’‐methoxy (2’‐OMe) and 2’‐fluoro (2’‐F) modifications sustained the interfering activity and increased resistance to degradation by nucleases. We therefore envision the delivery of this siRNA to the lung by inhalation as a plausible administration route for the treatment of COVID‐19. We will test the proposed route of delivery in our advanced bioprinted 3D lung model consisting of a base of endothelial cells, primary lung fibroblasts and macrophage‐like cells that are overlayed with an alveolar epithelial cell line.

B. 09 Combating SARS‐CoV‐2 by an Inhalable Nanostructured Dry Powder Formulation

Justin Stella,and Marc Schneider

Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken, Germany.

The severity of COVID‐19 is related to the penetration of the virus into the lung epithelium and its replication, which is facilitated by angiotensin‐converting enzyme 2 (ACE2) and the serine protease TMPRSS2 [1]. The goal of this project is to produce inhalable nanostructured microparticles containing camostat mesilate, a proven inhibitor of TMPRSS2, to prevent SARS‐CoV‐2 uptake [2]. To improve the dissolution and mucolytic properties of camostat mesilate, it is spray‐dried with N‐acetylcysteine. The resulting spherical microparticles are well soluble in water, between 2‐5 μm in size, and show promising aerodynamic properties. In a second approach, nanoparticles with a size of around 250 nm are embedded in the microparticle matrix [3]. The large surface area of the nanoparticles should allow the virus particles to bind and disrupt virus incorporation. The aerodynamic properties of the resulting nanostructured microparticles were also suitable, and the redispersibility of the embedded nanoparticles was confirmed. Both mechanisms are expected to reduce virus entry into epithelial cells, and modification of the nanoparticle surface with antibodies could improve binding strength.

References

[1] Kawase, M. et al. (2012). Journal of Virology 86, 6537–6545. doi: 10.1128/JVI.00094‐12

[2] Hoffmann, M. (2020). Cell, 181(2): 271‐280. doi: 10.1016/j.cell.2020.02.052

[3] Torge, A. (2019). Aerosol Science and Technology, 53(4): 1‐28. doi: 10.1080/02786826.2018.1542484

B. 10 Biopharmaceutical Characterization ofMorus albaRoot Bark Compounds and Extracts in Search of Inhalable Natural Product Formulations against Acute Respiratory Infections

Jacqueline Schwarzinger,Sigrid Adelsberger, Judith Rollinger, Ulrike Grienke, Lea Ann Dailey, and Gabriela Hädrich

University of Vienna, Vienna, Austria.

High mortality rates of acute respiratory infections (ARIs), particularly bacterial‐viral co‐infections, demonstrate the need for new anti‐ARI agents (1). Prenylated flavonoids fromMorus albaroot bark (known as mulberry Diels‐Alder adducts (MDAAs)) inhibit both viral and bacterial neuraminidases (2). MDAAs show low oral bioavailability, thus pulmonary administration could provide a therapeutic advantage (3). This study will investigate if MDAA extracts and their active constituents, sanggenon C and D, show suitable pharmacokinetic (PK) properties for lung delivery. Furthermore, the presence of PK synergies in terms of solubility and permeability are investigated. In vitro cytotoxicity and permeability were measured in Calu‐3 cells, while pH‐dependent thermodynamic solubility was measured by a miniaturized shake flask method. UPLC‐ESI‐MS method was used for quantitative analysis. Extract cytotoxicity (IC50: 141 μg/mL) was primarily due to the sanggenon C content (IC50: 41 μg/mL). Sanggenon C solubility was 194 μg/ml at pH 7.4. These preliminary findings will now be taken forward for further assessment of the biopharmaceutical characterization of MDAA for pulmonary delivery.

References

[1] Bloom, DE. and Cadarette, D. (2019). Front Immunol. 10, 549. doi: 10.3389/fimmu.2019.00549.

[2] Grienke, U., Richter, M., Walther, E et al. (2016). Sci Rep 6, 27156. DOI: 10.1038/srep27156.

[3] Thilakarathna, SH. and Rupasinghe, HP. (2013). Nutrients 5, 3367‐3387. doi:10.3390/nu5093367.

B. 11 Development and Characterization of Nintedanib Dry Powder Formulation as a Potential Post COVID‐19 Pulmonary Fibrosis Treatment

Valentina Ruggiero,Francesca Mariano, Rita Patrizia Aquino, and Paola Russo

University of Salerno, Fisciano, Italy.

Recently, Nintedanib, an orally administered intracellular tyrosine kinase inhibitor approved for idiopathic pulmonary fibrosis, was also proposed for post‐COVID‐19 pulmonary fibrosis [1]. In this context, the present study explores the possibility of developing dry powder of Nintedanib suitable for inhalation therapy to reduce doses and systemic side effects. Particles were prepared by spray drying the drug alone (3‐5% w/v) or with appropriate excipients such as leucine (2.5‐10% w/w), from different hydroalcoholic solutions i.e., water/ethanol (7/3 v/v) or water/2‐Propanol (from 7/3 v/v to 5/5 v/v) mixtures. After production, the powders obtained were characterized in terms of particle size distribution, morphology, density and respirable fraction. The results indicated that leucine improved the aerosol performance of the powders, increasing the value of the fine particle fraction (FPF) from 47.9% in the powder‐only batches to 73.1% using 5% w/w leucine. This improvement was attributed to the ability of the amino acid to change both the surface area of the particles and the fluidity of the powder. Therefore, this study demonstrated the possibility of developing dry powder of Nintedanib for local administration in the lungs, confirming their potential application for the treatment of post‐COVID‐19 pulmonary fibrosis.

References

[1] Mohammadi, A., Balan, I., Yadav, S. et al. (2022). Cureus 14(3): e22770. doi:10.7759/cureus.22770.

B. 12 Intranasal Aerosol Deposition in 3D Models using the Aptar Pharma BiVax Atomizer

Beth Laube,¹Jana Kesavan,²Gonçalo Farias,³Nektaria Karavas,⁴Mathilde Blondel,³and Julie Suman⁴

¹Johns Hopkins University, Baltimore, USA.

²U.S. Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, USA.

³Aptar Pharma, Le Vaudreuil, France.

⁴Aptar Pharma, Congers, USA.

We studied the effect of head position (upright [Up], tilted backwards at 45° [45] and supine [Su]), angle of insertion (30° and 45°) and distance of insertion (6mm and 9mm) on intranasal deposition of fluorescein aerosol in 3D models of 18 and 5 year old (yo) humans. Aerosol was generated by the Aptar Pharma BiVax 200 μL intranasal vaccination atomizer. Deposition was quantified in the anterior nose, the main nasal cavity, which was divided into upper, middle and lower horizontal thirds, and on an exit filter. The lower third included the nasopharynx, a target for nasally administered vaccines. A design of experiments was utilized to test 6 conditions (n = 3) for each model. Mean percent deposition was minimal in the upper third and on the exit filter for all conditions. A multivariate analysis showed that anterior nose deposition was significantly higher in the 18 yo, whereas middle third deposition was significantly higher in the 5 yo. Head position, but not angle or distance of insertion, impacted middle third deposition in both models combined, with Up <45

B. 13 OM‐85 is Boosting Immune Responses and Inhibiting Viral Replication in Murine Rhinovirus‐Infection Models

Helena Obernolte,¹Philippe Vollmer Barbosa,¹Olga Danov,¹Sabine Wronski,¹Katherina Sewald,¹Armin Braun,¹Anne Vaslin Chessex,²Claire Abadie,²and Christian Pasquali²

¹Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany.

²OM Pharma, Preclinical Research Department, Meyrin, Switzerland.

Rhinovirus (RV) is the main cause of the common cold and major risk factor of exacerbations in chronic lung disease. OM‐85, a soluble bacterial lysate, acts via its anti‐microbial and immunomodulatory properties. We tested efficacy against viral infection using murine in vivo and ex vivo models.

Balb/c mice (i.n.) or murine precision‐cut lung slices (PCLS) were treated with OM‐85 and infected with RV1b. Clinical parameters, viral load, histopathology, cytokine release and gene expression were analyzed 24h to 48h after infection.

OM‐85 significantly reduced viral load and stimulated immune reactions and cell migration in lungs from RV‐infected mice. BAL cytokine analysis showed an inhibition of RV‐induced soluble immune mediators as IFN‐γ, IP‐10, IL‐1β and MIP‐1α by OM‐85. In infected PCLS transcriptomic analysis confirmed the in vivo found immune changes induced by OM‐85 per se and/or by viral infection. E.g. chemokine‐mediated signaling pathway was shown to be of significant importance in OM‐85 and RV1b infected PCLS identified by GO biological processes.

In conclusion, OM‐85 is able to boost lung immune mechanisms and to modulate RV induced immune responses in murine ex vivo and in vivo models that could explain its anti‐viral efficacy.

B. 14 Maximizing Intranasal Drug Delivery by Optimizing Traditional Spray Pumps Using GentleMist Technology^TM: A multicenter Study

Mari Tesch,¹Fernando Valerio‐Pascua,²Armando Cabrera,³Abir Malakar,⁴Mohammad Mehedi Hasan Akash,⁴Saikat Basu,⁴and Franck Rahaghi⁵

¹Dr. Ferrer Biopharma, Hallandale Beach, USA.

²Hospital CEMESA, San Pedro Sula, Honduras.

³Aventura Hospital, Aventura, USA.

⁴South Dakota State University, Brookings, USA.

⁵Cleveland Clinic, Weston, USA.

Intranasal (IN) drug delivery presents an avenue for delivering targeted and effective treatment for respiratory viral illnesses. We conducted three different studies utilizing GentleMistTM nasal spray (GM) to evaluate the feasibility of enhancing IN therapeutic penetration and optimizing drug delivery through traditional sprays. These studies included a in vitro physical spray test, a human factor (HF) simulated, and an HF usability study. Physical spray test evaluated drug penetration for two different nozzle orientations (a) Improved Protocol:12‐15° angle to the horizontal axes (b) Traditional OTC protocol:‐67.5° to the horizontal axes which are the instructions for OTC nasal sprays. Results showed that the orientation at an optimal angle of 12‐15° allowed for improved drug delivery to targeted infection and inflammation sites. HF studies determine whether the application instructions of the nasal spray at the optimal angle can be followed. Results showed that the instructions were followed correctly, safely, and effectively without patterns of use errors. The optimization of the GM spray angle and microparticle uniformity array likely impacted the aerodynamic particle size distribution rendering a better drug target delivery without triggering cough or gag reflex. Findings suggest that the application instructions of the nasal spray at the optimal angle can enhance intranasal therapeutic penetration used to maximize intranasal drug delivery for respiratory viral illnesses.

C. Best Oral Presentation Session

C. 01 Development of LNP Formulations for RNAi Inhalation

Philippe Vollmer Barbosa,^1,2Sebastian Hook,³Josi Steinke,¹Helena Obernolte,¹Valerie Beneke,¹Axel Schambach,¹Katherina Sewald,¹Katharina Schwarz,¹and Armin Braun¹

¹Fraunhofer ITEM, Hannover, Germany.

²Hannover Medical School, Hannover, Germany.

³Twincore Hannover, Hannover, Germany.

RNA interference is a powerful and versatile tool for various lung diseases. However, delivery of functional RNA into the lung remains challenging. Here, we report the setting up of an in vitro/ex vivo screening for suitable lipid nanoparticle (LNP) formulations for inhalation.

We used mCherry mRNA for assessment of functional RNA delivery by fluorescent marker expression. We tested hydrodynamic diameter and encapsulation efficiency of the formulated LNPs before and after nebulization with an Aeroneb nebulizer and droplet size of the aerosole.

Stable formulations were further assessed in an isolated perfused rat lung (IPL) model. For this, an explanted perfused rat lung was subjected to the nebulized LNP carrying mCherry‐mRNA successively cultured for up to 48 hours. mCherry expression was assessed using confocal microscopy.

Prescreening of LNP formulations of 15 potential candidates demonstrated nebulization stability for four candidates and resulted in droplet sizes of 3.9 to 4.9 μm. One candidate was excluded because of highly reduced encapsulation efficiency. Three candidates were tested in the IPL and yielded distinct mCherry expression in the airway epithelial cells after 24 and 48 hours. During the maximum four‐hour exposure time in the IPL, no severe side effects could be observed, and the tissue was viable for 48 hours.

Using this screening setup allows higher predictivity for translation towards in vivo and later clinical applications.

C. 02 Exploring the Role of Electrostatic Effects on Inhaled Aerosol Deposition in Alveolatedin vitroAirway Models

Ron Bessler,Josue Sznitman, Rami Fishler, and Saurabh Bhardwaj

Technion ‐ Israel institute of technology, Haifa, Israel.

It is well known that large amounts of net electrical charge are accumulated on inhaled aerosols during their generation using commonly available inhalers. This often leads to undesired increased deposition in the extra‐thoracic airways. Since the electrostatic force is inversely proportional to the square of the distance (i.e. particle to the lumen wall), its role has long been recognized as potentially significant in the deep lungs. However, due to the complexity of establishing in vitro models of the acinar region at scale, experiments have been largely limited to the upper airways. Here, we devise a microfluidic alveolated airway channel coated with conductive material to quantify the importance of electrostatic effects on inhaled aerosol deposition. Specifically, our aerosol exposure assays focus on inhaled spherical particles of 0.2, 0.5, and 1.1 μm that are recognized to reach the acinar regions and underline the roles of diffusion and sedimentation. Our experiments show that deposition is overwhelmingly biased to the inter‐alveolar septal spaces relative to the alveolar cavities even when aerosols are neutralized; this phenomenon is most pronounced when aerosols are intrinsically charged. Our observations give new insight into the role of electrostatic deposition mechanisms in the acinar region and emphasize how charge overshadows the traditionally accepted dominance of diffusion or sedimentation when considering deposition phenomena in the deep lungs.

C. 03 A Robust Lung‐on‐Chip Inhalation Platform for Toxicity and Therapy Assessment

Arunima Sengupta,¹Nuria Roldan,²Lea Lara De Maddalena,²Andreas Hugi,²Janick Stucki,²Oliver Wisser,³Tobias Krebs,³Nina Hobi,²and Olivier. T Guenat¹

¹ARTORG Organs‐on‐Chip Technologies, University of Bern, Bern, Switzerland.

²AlveoliX AG, Swiss Organs‐on‐Chip Innovation, Bern, Switzerland.

³Vitrocell Systems GmbH, Waldkirch, Germany.

Exposure to toxic inhalants accelerate the development of chronic lung conditions like COPD (emphysema) and fibrosis. Innovative new‐approach‐methodologies that use human‐derived cells and tissue have the potential to overcome the limitations of animal studies in clinical translation. We have employed here the new Cloud α AX12 [1] platform which integrates the cutting‐edge^AXlung‐on‐chip technology (AX12) [2] with a cloud‐based exposure system (Cloud α) to mimic realistic inhalation exposure in the distal lung. A triple co‐culture system was established on‐chip with human alveolar epithelial cells (^AXiAECs) [3], macrophages and lung endothelial cells. Exposure to nanoparticles (TiO2 and ZnO) and aerosolized chemical (PHMG) resulted in significant cytotoxic effects, including barrier breakdown and elevated gene expression of inflammatory cytokines. Nebulized corticosteroids, fluticasone propionate, effectively alleviated inflammation induced by aerosolized PHMG. The Cloud α AX12 platform offers reproducible conditions and ease of use for inhaled medicine development and hazard assessment as an alternative to animal models in inhalation toxicity and drug efficacy testing, particularly in pre‐clinical and precision medicine studies.

References

[1] Sengupta, A. (2023). Frontiers in Pharmacology, 14. doi:10.3389/fphar.2023.1114739

[2] Stucki, J. D. (2018). Scientific Reports, 8(1). doi:10.1038/s41598‐018‐32523

[3] Sengupta, A. (2022). Frontiers Tox, 4, doi:10.3389/ftox.2022.840606

C. 04 3D‐Bioprinting of Bacterial Biofilms on Human Lung Epithelial Cells for Building Complexin vitroInfection Models

Samy Aliyazdi,^1,2Sarah Frisch,¹Alberto Hidalgo,³Nicolas Frank,¹Daniel Krug,¹Rolf Müller,^1,2Ulrich F. Schäfer,²Thomas Vogt,⁴Brigitta Loretz,¹and Claus‐Michael Lehr^1,2

¹Helmholtz‐Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany.

²Saarland University, Saarbrücken, Germany.

³Charite‐University Hospital Berlin, Berlin, Germany.

⁴University Clinic Saarland, Homburg, Germany.

Chronic, biofilm‐associated lung infections cause a major health burden due to imparted antibiotic resistance mechanisms, raising the requirement for novel aerosolized therapeutic approaches. The development of such is hampered by a lack of reliable models allowing their testing on bacterial biofilms, human cells and their interactions simultaneously. Biofilm‐infectedin vitromodels in air‐interface culture would be desirable but challenging due to rapid cell death caused by bacterial overgrowth and released virulence factors.

This problem was approached by 3D bioprinting ofE.coliMG1655 biofilms grown in a bioink. A mix of 3% gelatin ‐ 1% alginate was found to be optimum in terms of rheology, printability and bacterial growth. Printed biofilm showed very similar properties to native biofilm according to imaging, antibiotic susceptibility testing and metabolic profiling. Bioprinting enabled controlled deposition of biofilm on air‐liquid interface cultivated Calu‐3 cells without severe cytotoxicity after 24h. Shape retention of printed biofilm on cell layers was confirmed even after dissolution of non‐crosslinked bioink, indicating that printed bacteria continued to produce their own matrix of extracellular polymers.

Bioprinted bacterial biofilms may work as reproduciblein vitromodel for chronic lung infections, and fill a demand to test aerosolized therapeutics designed to act against biofilms, thus complementing animal experiments with human relevant aspects.

C. 05 Spray‐Drying of Anti‐Tuberculosis Phage Fionnbharth

Isobel Tetreau,¹Ilaria Rubino,¹Melissa Harrison,¹Mani Ordoubadi,¹Sheila Co,¹Sasha E. Larsen,²Carlos A. Guerrero,³Rhea N. Coler,²Graham F. Hatfull,³Dominic Sauvageau,¹and Reinhard Vehring¹

¹University of Alberta, Edmonton, Canada.

²Seattle Children's Research Institute, Seattle, USA.

³University of Pittsburgh, Pittsburgh, USA.

An estimated 10 million people contract tuberculosis (TB) each year, and 1.6 million die from the disease (World Health Organization [2022]. Global Tuberculosis Report 2022. ISBN 978‐92‐4‐006172‐9). With cases of multi‐drug resistant TB increasing, mycobacteriophages have the potential to revolutionize complimentary combination TB treatment. A stable, dry mycobacteriophage powder could allow global distribution without need for cold chain infrastructure. This work provides proof‐of‐concept for production of a powder containing anti‐M. tuberculosisphage Fionnbharth that retains phage activity following spray drying. Several phage buffer and excipient combinations were evaluated. Phages were dried in solutions of 50 mg/mL trehalose dissolved in either water, 6 mM sodium citrate solution, or standard phage buffer. Activity level was determined via plaque titering againstM. smegmatis, performed in triplicate on pre‐drying feedstock and on solutions of produced powders reconstituted in water or the initial base buffer diluted to 50%. Overall titer reduction due to spray drying and reconstitution was no more than 1.0 ± 0.1 log (PFU/mL) (sodium citrate formulation, reconstituted in diluted buffer). The best‐performing formulation, 50 mg/mL trehalose in standard phage buffer, had a titer loss of 0.2 ± 0.1 log (PFU/mL) when reconstituted in diluted buffer. This minimal manufacturing loss shows impressive potential for a stable phage powder suitable for inhalation.

D. Barriers to Pulmonary Drug Delivery

D. 01 Microbial Biofilms and the Failure of Antibiotic Therapy in Chronic Infections

Tom Coenye

Ghent University, Ghent, Belgium.

In this talk I will present an introduction to microbial biofilms and their involvement in (chronic) infections. I will focus on three specific aspects. First I will present an overview of the state‐of‐the‐art concerning mechanisms contributing to the lack of antimicrobial susceptibility in microbial biofilms. Secondly, I will present examples of innovative approaches that are being developed for the treatment of biofilm‐related infections. Finally, I will discuss how basic science can help clinical microbiology labs to deal with biofilms, specifically in the context of diagnosis and antimicrobial susceptibility testing.

D. 02 A Closer Look at the Alveolar Epithelium of the Lung

Matthias Ochs

Institute of Functional Anatomy, Charité, Berlin, Germany. Core Facility Electron Microscopy, Charité,Berlin, Germany. German Center for Lung Research (DZL), Berlin, Germany

The cellular nature of the alveolar epithelium remained enigmatic until the invention of electron microscopy and its application to the lung in the early 1950s. It is continuous and consists of two cell types, type I cells with thin cell extensions and cuboidal type II cells. While type I cells cover about 95% of the alveolar surface, thus providing the vast majority of the epithelial surface for gas exchange, type II cells have two main functions: they produce surfactant and constitute the progenitor cell population for renewal of the alveolar epithelium. In addition to these two cell types, important components of the alveolar surface can be found on top of the epithelium: an alveolar lining layer containing intraalveolar surfactant subtypes and the alveolar epithelial glycocalyx. The interactions between these components remain to be investigated.

A new dimension to electron microscopy of the lung was added by 3D techniques. These methods are complementary regarding sample size and resolution and provide 3D datasets of the lung much more efficiently and at higher quality than conventional serial section transmission electron microscopy. We investigated the topological complexity of type I cells by 3D‐EM and revealed their high morphological diversity and topological complexity. These characteristics have implications for cellular plasticity during alveolar development, maintenance and repair. They should also be taken into consideration when in vitro models are developed.

D. 03 Extracellular Matrix‐Modulating Nano‐Structured Microparticles for Pulmonary Inhalation as Potential Delivery System for Lung Cancer

Salma M. Abdel‐Hafez,^1,2and Marc Schneider¹

¹Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Germany.²Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Exploiting the pulmonary route for delivery of anticancer drugs provides direct access to the lungs and decreases the systemic toxicity. In this regard, nanoparticles (NPs) could offer several advantages such as improved local bioavailability, reduced phagocytosis and enhanced cellular uptake. However, penetration of NPs into solid tumors, such as lung adenocarcinoma, is physically hindered by the tumor extracellular matrix (ECM)⁽¹⁾. Thus, nano‐structured microparticles (MPs) for inhalation were developed embedding acid‐degradable NPs⁽²⁾in a water‐soluble, collagenase‐containing matrix, via spray drying. Released collagenase would modulate the dense ECM, improving the penetration of anticancer drug loaded NPs into the tumor. The nano‐structured MPs were characterized for: aerodynamic properties, sufficient redispersibility upon hydration and maintaining the collagenase activity after spray drying. We established a three‐dimensional spheroid model composed of human lung adenocarcinoma (A549) cells and lung fibroblasts (MRC‐5) to test NP penetration and hence their potential for cancer treatment.

References

[1] Dolor A. et al. (2018) Digesting a Path Forward: The Utility of Collagenase Tumor Treatment for Improved Drug Delivery, 15, 2069‐2083. 10.1021/acs.molpharmaceut.8b00319

[2] Abdel‐Hafez S.M. et al. (2022). Formulation attributes, acid tunable degradability and cellular interaction of acetalated maltodextrin nanoparticles, 288 ,119378. 10.1016/j.carbpol.2022.119378

D. 04 Repurposing Gallium for Local Treatment of Bacterial Pneumonia through Inhalable Nano‐Embedded Microparticles

Gabriella Costabile,¹Emma Mitidieri,¹Daniela Visaggio,²Giulia Ferri,³Ivana d'Angelo,⁴Emanuela Frangipani,⁵Roberta d'Emmanuele di Villa Bianca,¹Antonio Recchiuti,³Paolo Visca,^2,6Raffaella Sorrentino,⁷and Francesca Ungaro¹

¹Dept. of Pharmacy, University of Napoli “Federico II”, Napoli, Italy.

²Dept. of Science, Roma Tre University, Roma, Italy.

³Dept. of Medical, Oral and Biotechnological Sciences, Laboratory of Molecular Medicine, University "G. d'Annunzio", Chieti, Italy.

⁴Di.S.T.A.Bi.F., University of Campania "Luigi Vanvitelli", Caserta, Italy.

⁵Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy.

⁶Fondazione Santa Lucia IRCCS, Roma, Italy.

⁷Dept. of Molecular Medicine and Medical Biotechnology, University of Napoli Federico II, Napoli, Italy.

The ability of iron‐mimetic Ga(III) to alleviate Pseudomonas aeruginosa (Pa) pneumonia was recently documented after IV administration in mice and in cystic fibrosis (CF) patients.¹Moreover, the added value of lung delivery of Ga(III) was shown in rats.²To improve Ga(III) permanence in the lungs, the design of a drug delivery system specifically engineered for Ga(III) inhalation is advisable. To this purpose, we developed Ga(III) inhalable nano‐embedded microparticles (NEM) for the treatment of Pa infections, specifically tuning their features to overcome mucus and biofilm surrounding bacteria.³NEM showed high Ga(III) content, optimal aerosol performance, and sustained release in lung lining fluids. NEM displayed promising in vitro anti‐Pa activity, and enhanced Pa phagocytosis by human macrophages. Upon NEM intratracheal insufflation in rats, improved Ga(III) persistence in lungs and reduced levels in kidneys were achieved as compared to both IV and intratracheal Ga(III) solutions. Finally, NEM significantly protected mice from lethal Pa pneumonia, opening the path to future clinical application of inhaled Ga(III) in CF patients.

The Italian Cystic Fibrosis Research Foundation is gratefully acknowledged.

References

[1] Goss C.H. et al. (2018) Sci Transl Med. 10, eaat7520. doi:10.1126/scitranslmed.aat7520.

[2] Mitidieri E. et al (2021). Pharmacol Res. 170, 105698. doi: 10.1016/j.phrs.2021.105698.

[3] Costabile G. et al. (2022) Int J Pharm. 629,122400. doi: 10.1016/j.ijpharm.2022.122400.

D. 05 Inhaler Technique Mastery: Train, Train, Train again or Is there a Better Way?

Mark Sanders, and Darragh Murnane

University of Hertfordshire, Hatfield, United Kingdom.

Introduction:Guidelines call for inhaler technique (InT) training, at first and whenever possible (1). There are no consensus checklists (2). Patients continue to make errors and the frequency of mistakes hasn't changed (3).

Aim:Review InT frameworks, apply learning theory

• Steps: industry strives to require fewest usage steps to portray simplicity

• Patients don't always read instructions (PIL), illiteracy is a concern

• Clinician demonstration: incentivised in some countries, helps short‐term only

• Tools: limited availability

Academic theory: how people learn: Fleming & Mills VARK theory describes learning modes; Visual (V), Aural (A), Reading (R), Kinesthetic (K) and multimodal.

InT training can be; periodic (P) or continuous (C); real‐time guidance (RTG) or retrospective (R) feedback.

Applying VARK to InT tools:

PIL = R, P, ‐:PIL illustrated = R+V, P, ‐;Placebo = K, P, RTG;Whistle Trainer (WT) = A+R, P, RTG;Online media = V, P, R;Digital inhalers = V+K, P, R;WT+companion app = V+A+K, P, RTG;Retrofit WT+app = V+A+K, C, RTG.

A better way forward: The paradigm must change, using multiple learning styles engages more people. Tools that combine multimodal VARK learning styles and have C and RTG should be developed to improve InT and clinical outcomes.

References

[1] Reddel, Helen K., et al. Am. J. Respir. Crit. Care Med 205.1 (2022): 17‐35

[2] Dekhuijzen, PN Richard, et al J. Allergy Clin. Immunol: In Practice (2022)

[3] Sanchis, Joaquin, et al. Chest 150.2 (2016): 394‐406

D. 06 Effect of Turning off Heated Humidification on Nebulized Drug Delivery, Temperature, and Absolute Humidity during Mechanical Ventilation

Hui‐Ling Lin,¹James Fink,^2,3and Jie Li³

¹Chang Gung University, Taoyuan, Taiwan.

²Aerogen Pharma, San Mateo, USA.³Rush University, Chicago, USA.

Background:The study aimed to investigate the effect of turning off humidity during nebulization on inhaled dose and humidification. .

Methods:In an intubated adult model of mechanical ventilation with heated humidifier, salbutamol (5.0 mg/2.5 mL) was nebulized with jet (JN) and mesh nebulizers (VMN), placed pre humidifier and proximal to patient Y with: (1) dry circuit/humidifier off, (2) heated humidified circuit /humidifier on for 1 and 2 hours (3) heater turned off (T = 0 and 30 min after; n = 5). Drug on filter distal to ETT was eluted and analyzed UV Spec (276 nm). Temperature, absolute humidity (AH), and relative humidity (RH) were measured at humidifier outlet and proximal to Y‐adaptor with custom hygrometer.

Results:With the VMN at humidifier inlet, drug deposition was lower with the dry circuit and humidifier off than through a heated circuit at 1 h and 2 h (p < 0.001). With the VMN placed at the Y and JN placed at humidifier inlet, drug dose was lower with humidification than dry circuit, but did not improve when humidifier was turned off (p < 0.001). With JN at the Y, drug dose was similar with humidifier on and off (p = 0.712). In dry circuit with VMN at humidifier inlet, RH increased to >90 %, with 8°C temp drop with AH at Y < 20 mg/L. Both VMN and JN proximal to patient, AH was below 10 mg/L.

Conclusion:During mechanical ventilation, turning off heated humidifier did not improve drug delivery and nebulization alone does not provide safe levels of AH to inspired gas.

D. 07 Nanomedicines via the Pulmonary Route: a Promising Strategy to Reach the Target?

Mélina Guérin,and Elise Lepeltier

MINT, Univ Angers, Inserm, CNRS, SFR ICAT, ANGERS, France.

Whether it be infectiology or oncology, research on nanomedicines as new tools to fight complex pathologies has increased tenfold in many disciplines in decades. This process has further accelerated since the introduction of the Covid19 vaccines. According to the International Organization of Standardization, a nanoparticle is a “nano‐object with all external dimensions in the nanoscale where the lengths of the longest and the shortest axes of the nano‐object do not differ significantly”.¹When it comes to human health, nano‐objects are designed to protect, transport and improve the solubility of compounds in order to allow the delivery of an active ingredient with biological activity on its target. In this case, the nano‐objects are called nanodrugs and can be administered by different routes, such as oral, intravenous or pulmonary. In the latter route, nanodrugs can be aerosolized or nebulized to reach the deep lung.²This poster summarizes the existing nanomedicines that can be administered via this route, from their synthesis to medical interest, including the organs they can reach and the pathologies ‐ infections, genetic diseases, cancer ‐ that they could treat in the near future.

References

[1] ISO/TR 18401:2017(en), Nanotechnologies — Plain language explanation of selected terms from the ISO/IEC 80004 series

[2] Tiwari, G. et al. (2012). Drug Delivery Systems: An Updated Review. International Journal of Pharmaceutical Investigation, 2, 2–11. DOI : 10.4103/2230‐973X.96920

D. 08 Clinically Relevant Target Product Profiles from Physiologically Based Pharmacokinetic Modelling

William Ganley,¹Jonathan Tournaire,²Reanne Beaird,¹Cristina Rey Blanes,¹andIrene Rossi¹

¹Nanopharm Ltd, an Aptar Pharma Company, Cwmbran, United Kingdom.

²Aptar Pharma, Le Vaudreuil, France.

The reformulation of drugs into inhaled dosage forms can offer several benefits such as targeted delivery to the site of action, reduced systemic exposure, and avoidance of first pass metabolism. However, developing a well‐performing reformulated inhaled product presents a number of challenges starting with identifying an appropriate dose and aerosol characteristics to administer that dose.

In this study, a whole body physiologically‐based pharmacokinetic model was built to simulate intravenous administration of itraconozole and validated against clinical data. The model was then used to predict the proportion of the intravenous dose that was delivered to the lung tissue, which was found to be only 12 mg from a 200 mg 60‐minute intravenous infusion.

Using this target, a formulation design space was defined in terms of dissolution profile and aerodynamic particle size distribution to develop a dry powder inhaler formulation that could achieve the same local exposure. This design space provides a quantitative target product profile for the reformulated itraconazole product.

This study demonstrated a model‐informed quality by design process that can accelerate the development of inhaled formulations by targeting the desired clinical outcomes from the outset. Overall, the approach used in this study can help overcome the challenges associated with developing inhalation formulations and improve the success rate of drug development in this field.

D. 09In vitroComparison of Aerosol Delivery Using Symmetric and Asymmetric Nasal Cannula during Nasal High Flow Therapy (NHFT)

Maria Cabrera,^1,2Pierre‐François Dequin^1,3,4, Nathalie Heuzé‐Vourc'h,^1,2and Stephan Ehrmann^1,3,4

¹INSERM, Research Center for Respiratory Diseases, U1100, Tours, France.

²University of Tours, Tours, France.

³CHRU de Tours, Médecine Intensive Réanimation, Tours, France.

⁴INSERM CIC 1415, CRICS‐Triggersep F‐CRIN research network, Tours, France.

Feasibility of delivering inhaled medication through nasal high flow (NHF) is established and significant bronchodilation has been demonstrated clinically. NHF systems constantly evolve. Recently asymmetric cannulas were developed to improve efficacy. Our objective was to compare symmetric and asymmetric cannula in term of aerosol delivery.

NHF was set at 60L/min 37°C using the AirVo2 system. To determine the inhalable mass aerosol was administered through symmetric and asymmetric (Optiflow‐Duet) nasal cannula (3 sizes) in an artificial nares system connected to a filter. To determine the respirable mass an aerosol was administered through symmetric and asymmetric cannula (1 size) in a nasal cast connected to a lung model and a ventilator simulating adult breathing. A filter between the cast and the lung model represented aerosol lung deposition. The mass of NaF collected on filters (inhalable and respirable mass) was quantified by an electroanalytical method.

About 20‐25% of aerosol reached the outlet of nasal cannula regardless the type and size of nasal cannula. About 3% of aerosol reached the lungs regardless the type of nasal cannula. Statistical analysis showed no significant difference between symmetric and asymmetric nasal cannula in inhalable and respirable mass.

Aerosol delivery seems not to be quantitatively affected by the asymmetric design of cannulas. The delivery of bronchodilator through asymmetric cannulas is expected to induce significant bronchodilation.

D. 10 Design ofin vitroAirway Mucus Simulants for Use in Biopharmaceutics Studies

Lucy Goodacre,¹Irene Rossi,²Cecile Dreiss,¹William Ganley,²and Ben Forbes¹

¹King's College London, London, United Kingdom.

²Nanopharm, An Aptar Pharma Company, Cwmbran, United Kingdom.

Nasal mucus sits at the air‐mucosa interface in the nasal cavity, occupying a central position when delivering intranasal medications. Most currentin vitromodels lack biorelevance due to the absence of readily available, well‐characterised nasal mucus simulants. Artificial, synthetic and native mucus simulants were designed based on gelling polymer networks, commercially available mucins, and porcine gastric mucus, respectively.

Protocols for preparing each simulant were developed to control critical processing parameters and simulants were manufactured robustly to a consistent quality. The simulants were designed by benchmarking to the physicochemical properties of respiratory mucus: solids content 2.8% ± 2.6% (w/w) [1], pH 6.2‐6.5 [2], and rheological properties G’ 7.3 ± 2.9 Pa; G’’ 2.1 ± 0.7 Pa [3]. The native simulant was manufactured to possess solids content 3% (w/w), pH 6.1‐6.5. All simulants produced gels that were characterised in terms of their viscoelasticity using rheology.

The next step will be to evaluate these mucus simulants for their functionality in studies of nasal formulation deposition, dissolution and transport.

References

[1] Rubin, B., Druce, H., Ramirez, O et al. (1997). Am J Respir Crit Care Med 155, 2018‐23. doi:10.1164/ajrccm.155.6.9196110

[2] Washington, N., Steele, R., Jackson, S et al. (2000). Int J Pharm 198, 139‐46. doi:10.1016/s0378‐5173(99)00442‐1

[3] Radiom, M., Hénault, R., Mani, S., et al. (2021). Soft Matter 17, 7585‐95. doi:10.1039/D1SM00512J

D. 11 Drug Solubility in the Context of an Inhalation‐Based Biopharmaceutics Classification System (iBCS)

Andreea Floroiu,^1,2Brigitta Loretz,³Johannes Krämer,⁴and Claus‐Michael Lehr^3,1

¹Saarland University, Saarbrücken, Germany.

²Eurofins PHAST Development, Konstanz, Germany.

³Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany.

⁴DISSO GmbH, Homburg, Germany.

Dissolution testing is an important tool for predicting thein vivoperformance of orally inhaled drug products (OIDPs). Standardized methods together with an inhalation‐based Biopharmaceutical Classification System for pulmonary drugs have the potential to allow prediction of differences inin vivoperformance[1].

Solubility of 9 drug substances was measured in media representative for the oral and pulmonary routes of administration to confirm the need for a classification system for inhaled drugs. The media complexity was reduced stepwise for identifying suitable media for dissolution testing.

Drug substances were classified as having either critical or non‐critical pulmonary solubility according to their dose number (Do)[2][3].

Solubility of drug substances in simulated lung lining fluids was proven to be dependent on the physicochemical properties of the drug and composition of the media. Our results indicate that case‐by‐case studies are required for identifying suitable dissolution media, and for developingin vitrotests capable of predicting differences in thein vivoperformace of OIDPs.

References

[1] Hastedt, J.E., Bäckman, P., Cabal, A. et al. (2022). Mol. Pharm. 19, 7, 2032–2039 doi:10.1021/acs.molpharmaceut.2c00113.

[2] Amidon, G.L., Lennernäs, H., Shah, V.P. et al. (1995) Pharm. Res. An Off. J. Am. Assoc. Pharm. Sci. 12, 413–420. doi:10.1023/A:1016212804288.

[3] Hastedt, J.E., Bäckman, P., Clark, A.R. et al. (2016). AAPS Open. 2, 1–20. doi:10.1186/s41120‐015‐0002‐x.

D. 12 Assessing Pulmonary Absorption and the Role of ABC Transporters in Cell Models of the Human Lung Epithelium

Sina Simon,^1,2Carina Cantrill,¹and Claus‐Michael Lehr²

¹F. Hoffmann‐La Roche, Basel, Switzerland.

²Saarland University, Saarbrücken, Germany.

Until now, the active mechanisms that drive the absorption of drugs across the human pulmonary epithelium are not yet entirely understood. Numerous in vitro and in vivo reports have described the role of ATP‐binding cassette (ABC) drug transporters at the human pulmonary epithelium as potential drivers of drug exposure in the lung. However, available reports show conflicting results in terms of expression patterns and functional activity [1.].

To contribute to a better understanding of the role of ABC transporters in the human lung, we performed in vitro studies using human‐derived pulmonary epithelial cells from upper and lower airways (16HBE14o‐, Calu‐3, NCI‐H441, A549 and CI‐hAELVi) that were cultured on Transwells^®. Expression levels were measured by qPCR and Western Blot and bidirectional efflux studies were conducted to assess the functional activity of the ABC transporters.

The results indicate that ABC transporter expression and activity differs between the tested cell lines. This implies caution when using in vitro models to estimate pulmonary drug absorption and when comparing data across different models.

References

[1] Nickel, S., Clerkin, C. G., Selo, M. A. et al. (2016). Expert Opin Drug Deliv 13, no. 5: 667‐90. doi:10.1517/17425247.2016.1140144.

D. 13 Co‐Amorphous Drug Combinations for Inhalation Therapy

Johanna Dieplinger,¹Stefan Mitsche,^2,3Hartmuth Schröttner,^2,3and Sarah Neugebauer¹

¹Research Center Pharmaceutical Engineering GmbH, Graz, Austria.

²Institute of Electron Microscopy and Nanoanalysis (FELMI), Graz University of Technology, Graz, Austria.

³Graz Centre for Electron Microscopy (ZFE), Graz, Austria.

The treatment of tuberculosis (TB), asthma and COPD requires a multidrug therapy. While inhalation therapy is standard for the latter, TB treatment so far is oral or parenteral. However, inhalable anti‐TB drug formulations have gained research interest, since they offer direct delivery to the therapeutic target. In order to reduce drug dose variability while administering the combination products to patients, the generation of co‐amorphous systems (COAMS) is suggested. COAMS have drawn attention due to favorable properties like increased solubility, dissolution and stability. To facilitate the experimental screening phase and save time and cost a machine learning model was developed to predict the formation of COAMS based on literature data [1]. The model accuracy was 79% and after model building (training + validation), predictions were made for 35 drugs used in inhalation therapy as input factors. 18 API‐API combinations were experimentally tested and co‐milling experiments confirmed 10 positively predicted COAMS out of 13 and 3 negatively predicted non‐COAMS out of 5 correctly (experimental accuracy 72%). Therapeutic relevant combinations identified by our model that now will be further developed and tested for its application as inhalation powder are mometasone–glycopyrronium bromide (GB) or budesonide–GB. For TB treatment, respective first line drug combinations identified are ethambutol–rifampicin (RIF) and RIF–pyrazinamide.¹Fink et al. 2023, Pharmaceutics, 15(2), 347

D. 14 Evaluation of Photodynamic Efficiency of Nebulized Liposomesin vitroandin ovo

Lena Bender,¹Damiano Librizzi,²Jens Schäfer,¹and Udo Bakowsky¹

¹Department of Pharmaceutics and Biopharmaceutics, University of Marburg, Marburg, Germany.

²Department of Nuclear Medicine, Center for Tumor Biology and Immunology (ZTI), Marburg, Germany.

Lung cancer is one of the most common causes of cancer‐related deaths worldwide. Treatment strategies as chemotherapy, radiation therapy or a combination of both are used. One non‐invasive therapeutic option is photodynamic therapy (PDT). Next to the photosensitizer delivered by a carrier system and the light, PDT requires molecular oxygen. By irradiating the photosensitizer oxygen molecules react to cytotoxic reactive oxygen species [1].

Due to their low bioavailability, photosensitizers like curcumin should be encapsulated in a suitable nanoscale drug delivery system. For investigation of photodynamic efficacy curcumin‐loaded liposomes containing Dipalmitoylphosphatidylcholin (DPPC) and cholesterol were prepared by two different methods. Afterwards they were characterized physicochemically before and after nebulization including determination of output rate and emitted dose.

The cytotoxicity and efficacy were tested in vitro and in ovo with adenocarcinoma (A549) cells. Simulating physiological conditions, liposomes were nebulized onto xenografted A549 tumors on the extraambryonic chorioallantoic membrane of fertilized hen's eggs with subsequent PDT. Effects were investigated by Positron emission tomography‐computed tomography and histology. Finally, nebulized curcumin‐loaded liposomes offer an innovative treatment option.

References

[1] Duse, L., Agel, M., Pinnapireddy, S. et al. (2019). Pharmaceutics 11 (6). doi: 10.3390/pharmaceutics11060282

D. 15 NO ABSTRACT

D. 16In vitroActivity of Different siRNA‐Loaded Lipid Nanoparticle Formulations in an Air‐Liquid Interface Culture System Mimicking the Lungs

Stina Rademacker,¹Simone Carneiro,¹Federica Catapano,²Richard Wibel,³Christoph Heidecke,³Peter Hölig,³and Olivia Merkel¹

¹LMU, Munich, Germany.

²Sapienza University of Rome, Rome, Italy.

³Lipoid GmbH, Ludwigshafen, Germany.

To downregulate lung disease‐associated overexpression of specific genes, therapeutic delivery of siRNA against such pathologically (over‐)expressed genes is a promising approach. siRNA induces cleavage of specific mRNA resulting in inhibition of protein biosynthesis and thus in gene silencing. To apply siRNA successfully, a carrier is needed¹. In this study, we optimised the lipid composition of lipid nanoparticle (LNP) formulations aiming at their siRNA delivery efficiency and gene silencing performancein vitro. LNP formulations were prepared via microfluidics. The most promising candidates were selected by analysing the physicochemical properties such as zeta potential, size, and encapsulation efficiency. The optimised LNPs were successfully tested for cellular uptake and gene knockdownin vitro. To better addressin vivoconditions related to lung delivery, their performance was tested in an air‐liquid interface (ALI) culture system. Compared to conventional cell culture models, ALI models mimic the characteristics of the respiratory tractin vitromore realistically as a pseudostratified epithelium and a mucociliated differentiation can be achieved². Finally, we showed the impact of the lipid composition on the performance of siRNA‐loaded LNPs in an ALI culture system.

References

[1] Cullis PR, Hope MJ. (2017) Mol Ther. 25(7), 1467‐1475. doi:10.1016/j.ymthe.2017.03.013

[2] Baldassi D, Ambike S, Feuerherd M, et al. (2022) J Control Release 345, 661‐674. doi:10.1016/j.jconrel.2022.03.051

D. 17 The Novel Monoclonal Human Alveolar Epithelial Cell Line “Arlo” asin vitroModel of the Air‐Blood‐Barrier

Tobias Neu,^1,2Patrick Carius,^1,2Clémentine Richter,^1,2Lorenz Latta,¹Brigitta Loretz,¹Nicole Schneider‐Daum,¹and Claus‐Michael Lehr^1,2

¹Helmholtz‐Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Delivery across Biological Barriers, Saarbrücken, Germany.

²Department of Pharmacy, Saarland University, Saarbrücken, Germany.

In vitromodels of the respiratory tract are important tools for investigating and modeling drug transport in the deep lung. At present, hAEpC (primary human alveolar epithelial cells) are considered the gold standard in this field, but cell lines can also be a promising alternative. Unfortunately, most available cell lines are insufficient for such applications due to their inappropriate barrier properties. We addressed this need by immortalizing hAEpC and established the hAELVi (human alveolar epithelial lentivirus immortalized) cell line [1], which was further developed via single‐cell printing to a monoclonal cell line called “Arlo”. Arlo shows pronounced and reproducible barrier properties with monolayer formation and TEER (transepithelial electric resistance) of approximately 3000 Ω*cm²[2]. Therefore, Arlo appears well‐suited to mimic the human alveolar epithelium. Accordingly, we are characterizing Arlo in a wide range ofin vitroapplications as a model of the air‐blood barrier, e.g., for drug or drug candidate permeability, inhaled substance toxicity, or inflammatory response as part of deep lung co‐culture models.

References

[1] Kuehn, A., Kletting, S., de Souza Carvalho‐Wodarz, C. et al. (2016). ALTEX, vol. 33, no. 3, pp. 251–260. doi:10.14573/altex.1511131

[2] Carius, P., Jungmann, A., Bechtel, M. et al. (2023). Adv. Sci. 2023, 2207301. doi:10.1002/advs.202207301

D. 18 Surface Modification of PLGA Nanoparticles with Poly(vinyl alcohol) to Tackle Lung Barriers in Antimicrobial Peptide‐Based Inhalation Therapy

Pouria Savadi,¹Gemma Conte,^1,2Bruno Casciaro,³Maria Luisa Mangoni,³Francesca Ungaro,²and Ivana d'Angelo¹

¹Di.S.T.A.Bi.F., University of Campania "Luigi Vanvitelli", Caserta, Italy.

²Dept. of Pharmacy, University of Napoli “Federico II”, Napoli, Italy.

³Dept. of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.

Inhaled antimicrobial peptides (AMPs) are promising for treating lung infections, but their efficacy is limited by barriers imposed by infected lungs (mucus and bacterial biofilm). Surface‐engineered inhalable nanoparticles (NPs) represent the choice to tackle lung barriers, enabling AMP delivery to its target site. Polyvinyl alcohol (PVA) is commonly used as a stabilizing agent in the production of poly(lactide‐co‐glycolide) (PLGA) NPs, but it can also impart muco‐inertia to NPs. This study systematically investigates the effect of PVA coating on the interactions of PLGA NPs with model mucus and biofilm. NPs were prepared using PVAs with different hydrolysis degree (HD) and molecular weight (Mw) and optimized NPs were loaded with a model AMP (colistin). Preliminary results show that PVA with low HD and Mw provides an optimal shell on the NP surface, which shields interactions with the main components of mucus and biofilm. Additionally, the diffusion across lung barriers, evaluated by Transwell™‐assisted assay, is affected by PVA Mw, with low Mw PVA/PLGA NPs diffusing more rapidly through both mucus and bacterial alginates. The results obtained with colistin‐loaded NPs are consistent with those achieved for bare NPs. Partially hydrolyzed low Mw PVA (88% HD, Mw 31KDa) is revealed as a promising polymer to achieve mucus‐ and biofilm‐penetrating NPs. In vitro and in vivo studies are ongoing to characterize the antimicrobial potential of the optimized AMP‐loaded NPs.

D. 19 Dry Powder Inhaler (DPI) Resistance: Human Behaviour and Psychology of Patient Inspiratory Effort

Monbi Stefanova Chakma,¹Sally Meah,¹Martyn Biddiscombe,¹Liangfeng Han,²Bryan Newman,²Darragh Murnane,³and Omar Usmani¹

¹National Heart and Lung Institute, Imperial College London, London, United Kingdom.

²Division of Therapeutic Performance I, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA.

³School of Life and Medical Sciences, Department of Pharmacy, University of Hertfordshire, Hatfield, United Kingdom.

Background:DPI airflow resistance (Af‐Rs) may influence how respiratory patients perceive inhaler performance. Yet, limited data exist on how patients perceive the process of active inhalation and whether their perception of Af‐Rs affects their attitudes towards a certain DPI.

Aim:Explore the psychological and physiological aspects of patients' inspiratory effort through different Af‐Rs DPIs to develop a questionnaire to evaluate patients' perception of Af‐Rs.

Method:Patients (COPD, asthma) of different disease severities participated in focus groups and semi‐structured cognitive interviews that were transcribed to allow for coding of data and thematic analysis to cluster the emerging themes.

Results:COPD (n = 26; age = 67.2yr [SD = 7.1]; FEV1 = 1.6L [SD = 0.65]; and asthma patients (n = 26; age = 54.2yr [SD = 14.5]; FEV1 = 2.5L [SD = 0.85]; associated inspiratory effort with 3 key themes: Routine, Confidence, Control. The DPI administration routine appeared to be the greatest influence in 85%, regardless of the perceived difficulty of DPI Af‐Rs. Although all patients acknowledged the role of confidence, this was the second most important factor (80%). Surprisingly, only 58% felt they could control their inspiratory effort for a given DPI.

Conclusion:Our results suggest that perception of inspiratory effort is variable across asthma and COPD and may be influenced by interpretation of the observed 3 themes. Studies need to evaluate how these themes can gauge patient perception to Af‐Rs.

E. Inhalation in Disease and Age

E. 01 Aerosol Antibiotics and Immunizations

Bruce Rubin

Virginia Commonwealth University, Richmond, USA.

Antibiotic deivery by aerosol goes back more than 75 years with reports published in Science and the Diseases of Chest discussing aerosolization of penicillin to treat pneumonia. Aerosol antibiotics produce a high concentration in the airway with minimal systemic effect and toxicity and are user friendly compared to intravenous drugs. However they do not penetrate into the most distal airway, so antimicrobial resistance invariably develops. General considerations for administering antibiotics by aerosol are that the medication should be concentration dependent with a high AUC/MIC, well tolerated, and not inactivated in the airway. The aminoglycosides were the first antibiotics to be given by aerosol in the modern era to treat CF. Colomycin, azetreonam, and others are now also being used to treat CF and non‐CF bronchiectasis.

Immunizations can also be delivered by aerosol to the lung and, if deposited in the acinus, can have systemic absorption. In 1983, Dr. Albert Sabin and colleagues showed the feasibility of aerosolizing measles vaccine. Since then clinical trials have demonstrated that aerosol measles vaccine gives a superior boosting response compared to injectable vaccination in school‐aged children. However studies performed in infants under 10 months of age show that primary seroconversion rates are lower with aerosol than with subcutaneous vaccine. Immunizations are being developed for the treatment of other viral diseases including an aerosolized Ebola vaccine.

E. 02 Inhaled Targeting of NTM (NonTuberculous Mycobacteria), a Serious Lung Infection

Thomas Hofmann,¹Marshall Grant,¹Michael Castagna,¹Sebastian Canisius,²and Stefan Ufer³

¹MannKind Corporation, Danbury, USA.

²ACliRA, Tonder, Denmark.

³MannKind Corporation, Raleigh, USA.

Infection with nontuberculous mycobacteria (NTM) is a serious chronic condition, affecting >90,000 in the US alone. First‐line drugs for treating NTM are delivered to the lung through systemic circulation. Inhaled administration can achieve higher drug concentrations in the lung with a reduction in systemic exposure and associated adverse drug reactions. Inhaled liposomal amikacin, with limited approval for NTM, is the first such product. MNKD‐101, an inhaled suspension of clofazimine (CFZ), is currently in development at MannKind Corporation for the treatment of pulmonary NTM infections.

In a Phase 1 study in healthy volunteers, MNKD‐101 was well tolerated, no SAEs were reported, and most AEs were mild. Over the dose range 30 mg – 90 mg, CFZ Cmax and AUC0‐24, and AUC0‐t were dose proportional following administration of single and multiple doses. The mean terminal half‐life following a single dose was 24‐26 h but increased to 290‐300 h after 7 daily doses. Accumulation over 7 days was meaningful and increased with dose: Cmax ratios of 2.5 and 3.4 at 30 mg and 90 mg, respectively; corresponding AUC ratios were 2.8 and 3.9. Dose‐dependent accumulation enables flexible dosing regimens consisting of relatively short dosing periods followed by drug “holidays” during which the action of CFZ continues.

MNKD‐101 and inhaled liposomal amikacin differ in both their mechanisms of action and PK profiles, suggesting they could be complementary inhaled treatments for NTM.

E. 03In vitroActivity of OHet72 Alone or in Combination with First‐ and Second‐Line Drugs against Mycobacterium tuberculosis: Insight into its Mechanism of Action

Margaret Bourlon, andLucila Garcia‐Contreras

University of Oklahoma Health Sciences Center, Oklahoma City, USA.

Patients receiving treatments for tuberculosis (TB) experience severe side effects, which decrease their compliance. SHetA2, a novel anti‐cancer drug, also has anti‐TB activity and does not cause side effects. OHet72, a compound in the same class has also shown activity against drug‐susceptible and drug‐resistant TB. Aim: Identify the type of drug interaction (synergy, addition, or antagonism) between OHet72 and five first‐ and second‐line agents against Mycobacterium tuberculosis (MTB). Methods: The MIC of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), levofloxacin (LEV), moxifloxacin (MOX), and OHet72 against MTB was determined using microtiter alamar blue assay (MABA). The interaction between OHet72 and each drug was evaluated with the checkerboard and MABA assays using combinations of drug concentrations. The type of interaction was determined using the fractional inhibitory concentration index (FICI), where FICI <1 was synergism, 1‐4 was additive, and >4 was antagonism. Results: OHet72 had an MIC = 0.625 μg/mL against MTB and the MICs of all other drugs were within published values. The interactions of OHet72 with PZA, INH, LEV or MOX were all additive (FICI = 1.333, 1.125, 1.167, 1.375, respectively), but the interaction of OHet72 with RIF was synergistic (FICI = 0.5625). Conclusions: As INH and PZA act on the cell wall, while LEV and MOX act on DNA gyrase, it is possible that OHet72 acts at the cell wall level, but an intracellular mechanism may not be ruled out.

E. 04 Encapsulation of Clofazimine in Mesoporous Silica as a Potential Dry Powder Formulation for Treating Tuberculosis

Jesús Enrique Campos Pacheco,^1,2Azra Riaz,^1,2Peter Falkman,^1,2Adam Feiler,^3,4Mikael Ekström,⁵Georgia Pilkington,³and Sabrina Valetti^1,2

¹Biomedical Science, Faculty of Health and Society, Malmö University, Malmö, Sweden.

²Biofilms – Research Center for Biointerfaces (BRCB), Malmö University, Malmö, Sweden.

³Nanologica AB (publ), Södertälje, Sweden.

⁴Surface and Corrosion Science, KTH Royal Institute of Technology, Stockholm, Sweden.

⁵Iconovo AB, Lund, Sweden.

Mesoporous silica particles (MSPs) have generated significant interest for the delivery of poorly insoluble drugs for oral administration [1]. However, only more recently has their use for drug delivery via inhalation been considered, harnessing the particles' free flowing and aerodynamic properties towards delivery to the airways. The present study demonstrates the formulation of a dry powder composed of micron‐sized MSPs loaded with clofazimine (CLZ), a drug that has been shown to be effective for the treatment of multidrug‐resistant tuberculosis. Solid state analysis indicated that the drug was fully amorphous when confined in the nanometre pores (9‐10 nm) of MSPs, with a drug loading content around 6‐10% w/w. The Aerodynamic Particle Size Distribution (APSD) of the CLZ loaded particles actuated from an ICOone^®inhaler at 4 kPa showed a Fine Particle Fraction (<5 μm) of 48%. Under simulated lung fluid conditions, 50% of the encapsulated dose of CLZ was released in 2.5 h. The concentration of drug released from CLZ loaded MSPs was greater than the measured solubility of the crystalline drug. In vitro results revealed that the encapsulated CLZ permeate across Calu‐3 epithelium monolayer, with high retention in the monolayer without affecting monolayer integrity. The study indicates that CLZ‐MSPs could have value as a potential inhaled formulation for pulmonary infections.

References

[1] Valetti, S., et al. (2017). Nanomedicine (Lond). 12(8): 831‐844. Doi: 10.2217/nnm‐2016‐0364.

E. 05 Has the Time Come to End Use of the Blue Inhaler?

Robert Bals

Saarland University Hospital, Homburg, Germany.

Asthma treatment mainly depends on inhaled drugs. During the last decades concepts have been changed according to data from clinical trials. This presentation will summarize concepts that have been applied in treatment of asthma and COPD patients. Most recent developments in asthma do not recommend treatment with short‐acting beta‐agonists but favor the use of combined long‐acting substances and inhaled corticosteroids (ICS). For severe asthma several non‐inhaled biological are available. In COPD, the use od ICS has been discussed for many years. Most recent data highlight the use of tripple inhaled – single device therapy for selected COPD patients.

E. 06 Aerosol Transmission of Pathogens: Hospital Acquired Infections in Children and Adults

Christopher O'Callaghan

University College London (ULC) Great Ormond Street Children's Hospital Institute of Child Health, London, United Kingdom.

Greater understanding of how bacteria and viruses are transmitted and how to break the chain of infection in the hospital environment are urgently needed. It is likely that innovative solutions, from experts in fields such as aerosol medicine, that impact this problem will be rapidly tested and adopted.

The problem is huge. It is estimated that approximately 21% of hospital bed days are due to patients who acquire and infection whilst in hospital. In the UK alone in 2016/17, hospital acquired infections accounted for 7.1 million occupied hospital bed days at a cost of £2.7 billion. Large numbers of hospital staff also get infections due to their work.

The chain of infection may be broken, preventing infection, at various stages. These include tackling the pathogen itself, the reservoir of the pathogen, it's portal of exit, mode of transmission, portal of entry and by protecting the susceptible host. The aim of this presentation is to outline the chain of infection with a particular focus on aerosol transmission of pathogens.

Novel insights into the earliest interactions with the ciliated respiratory epithelium will be shown for aerosolised bacteria, in particular the attachment of Moraxella Catarrhalis and Haemophilus influenza to motile cilia. A hypothesis we have generated of how rhinovirus infection may facilitate bacterial aerosolization and lung dissemination will also be discussed.

Areas where research and product development are needed to help prevent aerosolization of bacterial pathogens will be highlighted.

E. 07 The Effect Of Discontinuing Aerosolized Hypertonic Saline Or Dornase Alfa On Mucociliary Clearance In Trikafta‐Treated CF

William Bennett,¹Timothy Corcoran,²Beth Laube,³Peter Mogayzel,³Steven Rowe,⁴Agathe Ceppe,⁵Jihong Wu,⁵Kirby Zeman,⁵and Scott Donaldson⁵

¹Department of Medicine and the Marsico Lung Institute, University of North Carolina at Chapel Hill,, Chapel Hill, NC, USA.

²Department of Medicine, University of Pittsburgh, Pittsburgh, USA.

³Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA.

⁴Department of Medicine and the Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA.

⁵Department of Medicine and the Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

A reduction in inhaled aerosol treatment burden after initiation of Trikafta is desired by many people with CF (pwCF). The SIMPLIFY trial previously demonstrated that discontinuation of aerosolized hypertonic saline (HS) or dornase alfa (DA) was non‐inferior to continuation of these treatments over a 6‐week treatment period. In this randomized, open‐label SIMPLIFY substudy, we used standardized MCC methods including gamma scintigraphy at multiple study sites to determine whether discontinuation of either HS or DA was associated with deterioration in the rate of in vivo mucociliary clearance (MCC). No significant differences in MCC endpoints were associated with HS discontinuation. For example, mean whole lung clearance (%) through 60 minutes (Ave60Clr) with discontinuation (N = 9) was 14.2 (5.1) (baseline) vs. 15.2 (4.4) (6‐weeks) (NS). With continuation of HS (N = 8) Ave60Clr was 15.5 (8.2) (baseline) vs.15.3 (11.9) (6 weeks). By contrast, while the continuation of DA showed no change in Ave60Clr, the discontinuation of DA (N = 6) showed an unexpected improvement of Ave60Clr, = 14.6 (3.9) (baseline) vs. 20.7 (4.9) (6 weeks) (p < 0.05). These results suggest that pwCF on Trikafta with mild disease do not experience a subclinical deterioration in MCC that could later impact health outcomes after discontinuing HS, and in fact may benefit from improved MCC after stopping DA treatment. Supported by the SIMPLIFY MCC Study teams and the CF Foundation.

E. 08 In Silico Modeling of Dosimetry in Sarcoidosis Patients with Airway Disease

Matthew Eden,¹Daniel VanDerhoef,²Maneesh Bhargava,³andJessica Oakes¹

¹Northeastern University, Boston, USA.

²Health Partners and Regions Hospital, St. Paul, USA.

³University of Minnesota, Minneapolis, USA.

Sarcoidosis is an inflammatory disease which occurs throughout the world, with lung involvement in most subjects. Airway involvement can occur in both large and small airways but is frequently under‐recognized as current diagnostic tools lack accuracy. Here, we created airway geometries from chest CT scans of three sarcoidosis patients. Computational fluid dynamic (inlet flow rate = 250mL/s) and particle transport simulations (3 μm diameter particles) were performed. Patient A and B both had abnormal spirometry tests. Patient A exhibited airway deformity, including abnormal left lower lobe bronchus. With several areas of airway remodeling, Patient B's airways featured airway constriction in all five lobes. Central airway resistance was largest in Patient B 2.33E‐5, compared to Patient A: 1.15E‐5 and Patient C: 1.52E‐5 g/mm⁴‐s. This elevated resistance was likely due to diffuse remodeling of the several of the airways. Airway deformity in Patient A resulted in regions with high‐speed flows, with adjacent areas receiving little to no flow. In Patient B, airway remodeling resulted in high‐speed jets in several airways. Airflow patterns appeared normal in Patient C. Particle deposition was elevated in patients A and B (11 and 12.8%) while remained near normal levels for Patient C (3.1%). In sarcoidosis subjects, in silico modeling has the potential to differentiate the severity and site of airway involvement with discreate narrowing, diffuse remodeling and normal airflow.

E. 09 Overview of the Upcoming CaspoNEB Clinical Trial: a Phase II Randomized Blind Placebo‐Controlled Study to Address the Treatment ofPneumocystisPneumonia

Stephan Ehrmann^1,2,3,4,Guillaume Desoubeaux,^2,5Adrien Lemaignen,⁶Alexandre Alanio,^7,8Cendrine Godet,⁹Elie Azoulay¹⁰, Antoine Roux¹¹, Pierre Tattevin¹², Stéphane Bretagne,^7,8Jeoffrey Pardessus,²Déborah Le Pennec,²Marine Andre,²Souleiman El Balkhi¹³, Nathalie Heuze‐Vourc'h,²Marie‐Sara Agier¹⁴, Elie Guichard,³Elodie Mousset¹⁵, and Agnès Caille³

¹Médecine intensive Réanimation, CHRU Bretonneau, Tours, France.

²Centre d'Etude des pathologies respiratoires, Inserm UMR1100, Université de Tours, Tours, France.

³Inserm CIC1415, CHRU Bretonneau, Tours, France.

⁴CRICS‐TriggerSEP F‐CRIN research network, Limoges, France.

⁵Parasitologie – Mycologie – Médecine tropicale, CHRU Bretonneau, Tours, France.

⁶Maladies infectieuses et tropicales, CHRU Tours, Tours, France.

⁷Parasitologie – Mycologie, Hôpital St‐Louis, AP‐HP, Paris, France.

⁸National centre of reference for fungal infection, Pasteur Institute of Paris, Paris, France.

⁹Pneumologie, Hôpital Bichat – Claude Bernard, AP‐HP, Paris, France.

¹⁰Réanimation médicale, Hôpital St‐Louis, AP‐HP, Paris, France.

¹¹Pneumologie, Hôpital Foch, Suresnes, France.

¹²Maladies infectieuses, CHU Rennes, Rennes, France.

¹³Pharmacologie – Toxicologie & Pharmacovigilance, CHU Dupuytren, Limoges, France.

¹⁴Centre de Pharmacovigilance, CHRU Bretonneau, Tours, France.

¹⁵Direction de la recherche et de l'Innovation, CHRU Bretonneau, Tours, France.

In spite of adequate antifungal therapy, the mortality rate ofPneumocystispneumonia remains ≥20%, the duration of hospital stay is ≈30 days, the requirement of invasive mechanical ventilation can reach 50% in some populations, and the fungal clearance is usually prolonged over 21 days. To improve the condition of infected patients, one could question about the adjunction of alternative drug(s). Based on previous in vitro studies, the echinocandin class,e.g.caspofungin, was evidenced to significantly reduce the fungal load, to decrease the lung inflammation, and also exhibited a good safety profile when administrated in rats infected withPneumocystis. Moreover, three trials provided proofs of clinical success, when caspofungin was given IV in inpatients. However, administrating the drug directly into the airways would sound more relevant to efficiently target the pathogen thriving in the lung alveoli.

The CaspoNEB project is a multicentre randomized controlled trial that will compare daily‐nebulized caspofungin to placebo in addition to conventional antifungal therapy duringPneumocystispneumonia in adult patients with respiratory assistance. As primary endpoint, a composite criterion will assess the survival and the relative reduction of the respiratory assistance (or the occurrence of increase in oxygenation) at day 7. Inclusions will take place in 28 centers in France, and 90 patients are expected to be randomized. The trial is supposed to be filled in the late 2026.

E. 10 Inhalable Nanostructured Microparticles Containing Antibiotic Adjuvants for Targeting Lung Infections in Cystic Fibrosis

Kristela Shehu,^1,2Annette Kraegeloh,²and Marc Schneider¹

¹Saarland University, Saarbruecken, Germany.

²Leibniz Institute for New Materials, Saarbruecken, Germany.

The presence of a dense mucus layer, that makes an ideal habitat for bacterial growth is a hallmark of Cystic Fibrosis (CF) [1]. Improving the penetration of antibiotics through CF mucus is pivotal for an efficient therapy and a lower bacterial resistance. One promising approach for this is the use of antibiotic adjuvants, which may potentiate the efficacy of antibiotics, reduce their therapeutic dose, thus tackle bacterial resistance [2].

Spray‐dried powders comprising Mannitol (Man), Azithromycin (AZI) and different Leucine concentrations (0‐15%), were prepared. Leucine impacts particle morphology (spherical to corrugated) and improves their aerodynamic behaviour (FPF 40‐70%, MMAD 1.5‐3.5 μm). Menadione (MEN) was successfully loaded into acetalated Maltodextrin [3]. Menadione potentiates the activity of Azithromycin against AZI‐resistantE. coliandP. aeruginosa. The adjuvant mechanism of MEN involves both generation of ROS and cell membrane disturbance.

References

[1] De Boeck, K et al. (2016). Progress in therapies for cystic fibrosis. Lancet Respir Med. 4, 662‐74.

[2] Douafer, H. et al. (2019). Antibiotic Adjuvants: Make Antibiotics Great Again! J. Med. Chem 62, 8665‐81.

[3] Abdel‐Hafez, S. et al. (2022). Formulation attributes, acid tunable degradability and cellular interaction of acetalated maltodextrin nanoparticles. Carbohydrate Polymers. 288

E. 11 Experimental Tools for Assessing and Optimizing Nasal Spray Delivery in Health and Allergic Rhinitis

Jihong Wu,¹Guilherme Garcia,²Julia Kimbell,¹and William Bennett¹

¹UNC‐Chapel Hill, Chapel Hill, USA.

²Marquette University and The Medical College of Wisconsin, Milwaukee, USA.

Introduction:The efficacy of nasal sprays is limited by their ability to deliver drug beyond the nasal valve (%Pen). We measured %Pen using two‐part 3D‐printed nasal replicas derived from CT scans of the nasal cavity. A patient‐specific nozzle positioning tool was designed to aim the spray at a 45o angle with respect to the nasal floor.

Methods:Experiments were performed by a single operator in triplicate on both the left and right sides of six models (3 healthy and 3 allergic rhinitics) at a constant unilateral inhalation rate of 7.5 L/min through the sprayed nostril. The radioisotope Tc‐99m was mixed into a bottle of Nasacort which was placed within a hand actuation monitor (HAM) to track actuation travel velocity and force. Following each spray administration, 2D gamma camera images were acquired with and without the anterior soft nose. %Pen was quantified as activity without the anterior soft nose as a percentage of total deposited activity.

Results:Over all tests (3 repeat measures in left and right nostrils) %Pen was 44.1 ± 14.4% for healthy and 53.3 ± 10.6% for rhinitics (p < 0.05). There was no correlation between %Pen and either HAM actuation travel velocity (29.8 ± 7.9 mm/s) or actuation force (4.48 ± 0.45 kg).

Conclusion:At a relatively constant actuation force the %Pen was greater in rhinitic than healthy models likely due to variability in spray position relative to each individual's nasal anatomy.

NO E. 12

E. 13 A New Passive Inhalation Chamber forin vivoPreclinical Testing of Dry Powder Inhalation Formulations

Jeffrey Mariner‐Gonzalez,¹Ilham Alshiraihi,²Ha Lam,²Sara Maloney,³Amarinder Singh,⁴Hyunseo Park,⁴Bernd Meibohm,⁴Anthony Hickey,⁵andMercedes Gonzalez‐Juarrero²

¹Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University,, Fort Collins, CO, USA.

²Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA.

³Technology Advancement and Commercialization, RTI International,, Research Triangle Park, NC, USA.

⁴Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA.

⁵Technology Advancement and Commercialization, RTI International, Research Triangle Park, NC, USA.

There is an unmet need for testing efficacy of dry powders in preclinical studies. Studies (e.g lung infections or other lung diseases) requiring daily and long‐term administration of drugs (months) administered via inhalation are limited by lack of adequate inhalation devices. A newly developed passive inhalation chamber was made and allows for creation of aerosol of dry powder within a mixed air‐isoflurane environment. The aerosol chamber is made of hollow cylinder containing a small quantity of dry‐powder formulation. The chamber has nose cones, an inner tube and exhaust filter and is connected to an electronic system that injects a mix of air/isoflurane anesthesia into the chamber with regular intervals. The system allows for creation of an internal aerosol cloud inside the chamber. Animals under anesthesia with their noses affixed to the nose‐cones breath into the aerosol of dry powder circulating inside the chamber. The system allows for administration of small quantities of drugs to be administered daily and for long periods of time to animals. When the lungs and plasma after 30 minutes of dry powder inhalation in the chamber contained sufficient drug to reach therapeutic levels.

E. 14 NO ABSTRACT

E. 15 Selection of a Nebulizer for the Efficient Treatment of Tuberculosis Infected Mice with Aerosolized OHet72 Nanocrystals

Alexa Beathard, andLucila Garcia‐Contreras

University of Oklahoma Health Sciences Center, Oklahoma City, USA.

Purpose:Select a nebulizer to generate aerosols for the efficient administration of OHet72 nanocrystals (NCs) to tuberculosis (TB) infected mice.

Methods:Before using the NC suspension, we evaluated the Pari‐LC Star and Hudson Updraft nebulizers with the Vios Pro compressor as well as the Aeroneb Lab nebulizer using a 3 mg/ml sodium fluorescein (NaF) solution. The aerosol performance was assessed in terms of their mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD) and fine particle fraction (FPF) using a New Generation Impactor (NGI) at a 15 L/min flow rate and 15 minutes run time. OHet72 NCs were prepared by the bottom‐up‐solvent‐antisolvent precipitation method and suspended in saline (3 mg/ml) to determine its aerosol performance.

Results:Aerosol droplets generated by the Hudson nebulizer had MMAD = 21 ± 10.6μm, GSD = 20.62 ± 19.09 and FPF = 0.09 ± 0.08%, whereas those from the Aeroneb had MMAD = 15.67 ± 5.51μm, GSD = 5.29 ± 0.51 and FPF = 3.22 ± 1.97%. In contrast, the aerosols generated by the Pari had a smaller droplet size (MMAD = 4.37 ± 0.76μm), tighter size distribution (GSD = 3.41 ± 0.80) and largest FPF = 8.74 ± 5.25%, thus it was selected for evaluation with the NCs. OHet72 NCs had a width = 255nm and length = 1.7μm.The OHet72 NC aerosols generated with the Pari were similar to those of NaF, with MMAD = 4.92 ± 2.61μm, GSD = 2.62 ± 0.29 and FPF = 6.35 ± 2.88%.

Conclusion:Based on aerosol performance, the Pari nebulizer was selected for efficacy studies with TB‐infected mice.

E. 16 Development of a Nintedanib Technosphere Powder for Targeted Lung Delivery in the Treatment of Idiopathic Pulmonary Fibrosis

Thomas Hofmann,¹Marshall Grant,¹Stefan Ufer,²Michael Castagna,¹and John Freeman¹

¹Mannkind, Danbury CT, USA.

²Swabian, Raleigh NC, USA.

Nintedanib is approved as an oral treatment for IPF (Ofev^®). MNKD‐201, a dry powder formulation of nintedanib for oral inhalation, consists of nintedanib coated onto Technosphere^®particles. The primary component of Technosphere particles is fumaryl diketopiperazine (FDKP), an FDA‐approved excipient for inhalation.

The current formulation of MNKD‐201 contains 20 wt% nintedanib as the free base. Laser diffraction on 51 lots of powder dispersed at 3 bar yielded x50 = 1.68 ± 0.14 μm and x90 = 3.16 ± 0.34 μm (mean ± SD) for the primary particles.

In a non‐GLP PD study, rats were treated for 21 days starting on day 7 after bleomycin was administered. Two doses of inhaled MNKD‐201 (5 and 10 mg/kg/day nintedanib at the nose) were compared with negative controls (bleomycin+placebo), positive controls (60 mg/kg/nintedanib by oral gavage), and healthy animals (saline+placebo). At sacrifice on day 28, lung weight and lung index, mechanical properties (inspiratory capacity, resistance, compliance and elastance), fibrotic foci and collagen content in the bleomycin‐treated groups were statistically comparable to each other but different from the healthy controls. When the extents of improvement from day 7 for all measures, including plethysmography, were rank‐ordered from best to worst, the consensus ranking was: high‐dose MNKD‐201, positive control, low‐dose, and negative control.

Inhaled nintedanib may be efficacious at doses significantly lower than the current oral therapy.

F. Lung Models ‐ Where are We Now?

F. 01 Real‐Time Monitoring of Cell Mechanics Improves Clinical Relevance of anin vitroFibrosis “Mini‐Lung”

Ali Doryab,^1,2andOtmar Schmid^1,2

¹Helmholtz Munich – German Center for Environmental Health, Institute of Lung Health and Immunity, Munich, Germany.

²Comprehensive Pneumology Center (CPC‐M), CPC‐M bioArchive, Helmholtz Munich, Member of German Center for Lung Research (DZL), Munich, Germany.

Background:Lung fibrosis is a progressive, non‐curable, and ultimately fatal disease. In vitro fibrosis models of the lung express pro‐fibrotic/‐inflammatory markers, but clinically relevant functional diagnostic parameters are lacking.

Methods:Here, we introduce an in vitro “mini‐lung” fibrosis model consisting of three key cell types of lung fibrosis cultured under an air‐liquid interface, cyclic stretch (“breathing”), and medium (“blood”) perfusion conditions. At the core of this technology is a novel BETA membrane mimicking the alveolar basement membrane, being ultra‐thin (<1μm), highly permeable, and extremely elastic (<10kPa), yet resilient to continued cyclic stretch (∼days). In addition, the CIVIC mini‐lung system permits aerosolized drug delivery and real‐time monitoring of tissue (“lung”) compliance.

Results:This fibrosis model captures key markers of lung fibrosis, such as enhanced soluble/deposited collagen, αSMA, and proinflammatory cytokines, as well as reduced compliance, which were alleviated by both aerosolized apical (topical) and liquid basal (oral) delivery of Nintedanib, an FDA‐approved fibrosis drug for oral delivery. Cyclic stretch was found to enhance drug efficacy as compared to static culture conditions.

Conclusion:Dynamic culture conditions and real‐time monitoring of cell mechanics improve clinical relevance of in vitro fibrosis models, paving the way for concomitant longitudinal in vitro studies of pharmacodynamics and pharmacokinetics.

F. 02 Complexin vitroModels of the Human Air‐Blood‐Barrier for Developing Anti‐Infective Aerosol Medicines

Claus‐Michael Lehr

HIPS ‐ Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany. Saarland University, Dept. of Pharmacy, Saarbrücken, Germany.

Complex in‐vitro models aim to reflect the (patho)physiology of specific organs or tissues either in healthy or diseased state in order to generate clinically meaningful readouts. Besides the ethical advantage to replace or reduce animal experiments, such models may save time and costs to translate new therapeutic modalities to the clinic.

To model the air‐blood barrier of the human lung, we started with monolayers of human alveolar epithelial cells (hAEpC) in primary culture, developing functional tight junctions and high transepithelial electrical resistance (TEER). Later we introduced a polyclonal human alveolar epithelial (hAELVi) and just recently a monoclonal cell line (Arlo) with similar properties. These epithelial cells may be implemented in various micro‐physiological systems, also to study the effect of breathing and co‐cultivated with other cells types, like e.g., macrophages or endothelial cells. A particular challenge is the mixed culture with bacterial biofilms to model chronic lung infections, which can meanwhile be realized most elegantly by 3D bioprinting.

Our models have been used for developing novel anti‐infectives, like e.g., quorum sensing inhibitors, aiming to eradicate pathogens without inducing antimicrobial resistance. Aerosolizable nano‐antibiotics are also being investigated to combat intracellular infections, such as e.g., tuberculosis or viral infections by Crispr/CAS‐like approaches.

Selected references:

Ho et al. (2020) Angew Chem 59:10292; Horstmann et al. (2022); ACS Infect Dis 8:137; Carius et al. (2023) Adv Sci 10:2207301; Huck et al. (2022) Adv Healthc Mater 11:2102117; Aliyazdi et al. (2023) Biofabrication 15:035019

F. 03 Mitigation of Quartz Aerosol Effects in Reconstituted Human Airway Epithelial Models Combined with Primary Macrophages

Sandeep Keshavan,¹Ruiwen He,¹Mauro Sousa de Almeida,¹Loretta Lina Müller‐Urech,²Alke Petri‐Fink,¹and Barbara Rothen¹

¹Adolphe Merkle Institute Université de Fribourg, Fribourg, Switzerland.

²Inselspital, Bern University Hospital, Department for BioMedical Research, Bern, Switzerland.

Hazard assessment using human lung tissue models is essential and relevant for identifying effects of aerosolized materials [1][2]. In the present study, we investigated the functional, morphological, and reactivity features of reconstituted human airway epithelium (MucilAir™) derived from healthy and asthmatic donors. Tissues were combined with primary human monocyte‐derived macrophages (MDMs) from healthy volunteers, and response to a single Dörentrup Quartz (DQ₁₂) dosage (10 μg/cm²) exposure was investigated up to 10 days. In addition, we found that in healthy tissues but not in asthmatic tissues, the overexpression of transforming growth factor (TGF)‐β and interleukin (IL)‐8 was more pronounced following exposure to DQ₁₂, but the addition of MDMs mitigated the inflammatory reaction of DQ₁₂. This emphasizes the potential role of macrophages in modulating the severe inflammatory responses induced by DQ₁₂exposure. Pre‐stained macrophages seeded on top of the healthy cell cultures moved towards the insert's edges, whereas a more constrained movement was observed for macrophages seeded on top of the asthmatic tissues. The reduced macrophage motility in asthmatic cell cultures could be attributed to stronger mucus production under pathophysiological conditions. In conclusion, coculture with MDMs in healthy and asthmatic tissues suggests that exposure to DQ₁₂does not impair ciliary movement, enhances mucociliary clearance, and facilitates phagocytosis.

F. 04 Human Primary Alveolar Epithelial Cells in Air‐Liquid Interface Transdifferentiate into Basal Like Intermediates

Christopher J. Herbst^1,2,3,Elena Lopez‐Rodriguez,⁴Gluhovic Vladimir,⁴Sabrina Schulz,¹Raphael Brandt,⁴Sara Timm,⁵Jubilant K. Abledu,¹Falivene Juliana,¹Peter Pennitz,⁶Holger Kirsten,⁷Geraldine Nouailles,⁶Martin Witzenrath,⁶Mathias Ochs^4,3,5, and Wolfgang M. Kuebler^1,2,3,8,9

¹Institute of Physiology, Charité ‐ Universitätsmedizin, Berlin, Germany.

²German Center for Cardiovascular Research (DZHK), Berlin, Germany.

³German Center for Lung Research (DZL), Berlin, Germany.

⁴Institute of Functional Anatomy, Charité ‐ Universitätsmedizin Berlin, Berlin, Germany.

⁵Core Facility Electron Microscopy, Charité ‐ Universitätsmedizin Berlin, Berlin, Germany.

⁶Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Department of Infectious Diseases, Respiratory Medicine and Critical Care, Berlin, Germany.

⁷University of Leipzig, Institute for Medical Informatics, Statistics, and Epidemiology, Leipzig, Germany.

⁸Keenan Research Centre, St. Michael´s Hospital, University of Toronto, Toronto, ON, Canada.

⁹Departments of Surgery and Physiology, University of Toronto, Toronto, ON, Canada.

Due to its large surface area, the alveolar blood‐gas barrier presents an attractive route for non‐invasive drug delivery. Pharmacokinetic studies across this barrier are traditionally hampered by the limited availability of primary alveolar epithelial cells and limited applicability of respective cell lines. Of late, human primary alveolar epithelial cells (hPAEpC) have become commercially available. Here, we characterized these cells at a morphological, functional, transcriptional and proteomic level.

Optimal growing conditions for hPAEpC were determined as 8 days in a gas‐liquid interface with an initial seeding number of 7,500 cells per cm²resulting in a tight cell monolayer as determined by TEER. Ussing chamber recordings detected an amiloride‐sensitive short circuit current. Western blot and immunocytochemistry revealed specific markers of both type I and type II alveolar epithelial cells, yet marker expression decreased rapidly as a function of passage number. Electron microscopy showed polarized cells with apical tight junctions, microvilli and lamellar bodies in all passages. In single cell transcriptomics, specific alveolar hallmark genes like NKX2.1 or surfactant proteins were absent, while respiratory epithelial character was confirmed.

We conclude that hPAEpCs share morphological and functional characteristics of alveolar epithelial cells without fully replicating their transcriptomic/proteomic expression profile.

Funded by the DFG, SFB 1449 ‐ 431232613; B01.

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F. 06 Multiscale Modeling of Aerosol Dosimetry: Validation with Subject‐Specific Deposition Data

Chantal Darquenne,¹Owen Price,²Bahman Asgharian,²Rajesh K. Singh,³Sean Colby,³Kevin Yugulis,⁴Richard A. Corley,⁵and Andrew P. Kuprat³

¹University of California, San Diego, La Jolla, CA, USA.

²Applied Research Associates, Raleigh, NC, USA.

³Pacific Northwest National Laboratory, Richland, WA, USA.

⁴Batelle Memorial Institute, Columbus, OH, USA.

⁵Greek Creek Toxicokinetics Consulting LLC, Boise, ID, USA.

In‐silico modeling of aerosol dosimetry is playing an increasingly important role both in assessing the potential health hazard of airborne particulate matter and in the design and development of new inhaled drugs. We developed a subject‐specific multiscale lung model of a healthy male subject to simulate aerosol transport over multiple breath cycles using computational fluid‐particle dynamics (CFPD) in a 3D model of the oral cavity and large airways bidirectionally coupled with a recently improved Multiple Path Particle Dosimetry (MPPD) model. Predictions were obtained for four conditions matching experimental aerosol exposures in the same subject from which the model was developed. These include two particle sizes (1 and 2.9 μm) and two subject‐specific breathing rates of ∼300 ml/s (slow breathing) and ∼750 ml/s (fast breathing) at a target tidal volume of 1 L. In‐silico predictions of retained fraction were 0.31 and 0.29 for 1 μm and 0.66 and 0.62 for 2.9 μm during slow and fast breathing, respectively, and compared well with experimental data (1 μm: 0.31 ± 0.01 (slow) and 0.27 ± 0.01 (fast), 2.9 μm: 0.63 ± 0.03 (slow) and 0.68 ± 0.02 (fast)). These results strongly suggest that hybrid models can accurately predict site‐specific deposition of aerosol within the lung. Such models could serve as an effective tool to explore the connection between disease and particulate matter exposure and/or inhaled therapy outcome.

This study was funded by NIH/NIEHS 1U01ES028669.

F. 07 A PBPK Model of Intranasal Administration with Detailed Nasal Compartment Architecture for Optimization of Nasal Drug Delivery

Théo Galland,¹Cristhyne León,¹Igor Faddenkov,¹Lorenz Lehr,²Caterina Sansone,²Anne Vaslin Chessex,²Christian Pasquali,²Alexander Kulesza,¹and Simon Arsène¹

¹Novadiscovery, Lyon, France.

²OM Pharma, Meyrin, Switzerland.

For drugs targeting the respiratory tract (RT), intranasal administration represents a very promising route but requires careful assessment of device‐dependent parameters. We developed a translational model of intranasal deposition, calibrated with deposition data in rodents [1], to simulate the impact of deposition patterns on drug exposure in humans, taking anatomical differences into account. The model was used in the context of intranasal administration of the bacterial lysate OM‐85, currently investigated for its use in asthma [2]. Inspired by the ICRP 66 model, the model separates the upper and lower RT [3]. It includes deposition, permeation and mucociliary clearance, and takes into account main device‐specific parameters. On top of the upper RT's subdivision into anterior and posterior nasal compartment, we divide the posterior nasal space in equally large compartments. This subcompartmentalization enabled us to simulate a variety of deposition patterns observed in nasal cast experiments. In particular, simulations highlight that uniform deposition limits the maximum local concentration yet leads to an overall lower exposure than localized deposition.

References

[1] Southam, D.S. et al. (2002). Am J Physiol Lung Cell Mol Physiol. 282(4), L833‐L839. doi: 10.1152/ajplung.00173.2001

[2] Pivniouk, L. et al. (2021). Allergy Clin Immunol. 149(3), 943‐956. doi: 10.1016/j.jaci.2021.09.013

[3] ICRP Publication 66. (1994). Ann ICRP. 24, 1‐482.

F. 08 Leveraging Low‐Radiatation Imaging to Inform Computations in the Context ofin silicoPopulation Studies

Stavros Kassinos,¹Fotos Stylianou,¹Pantelis Koullapis,¹Bo Olsson,²and Jousé Sznitman³

¹University of Cyprus, Nicosia, Cyprus.

²Emmace Consulting AB, Lund, Sweden.

³Technion, Haifa, Israel.

A promising new direction in respiratory care and inhaled drug delivery is the transition towardsin silicopopulation studies. Virtual population studies offer several important advantages. Not only can these be conducted much faster compared toin vivopopulation studies, conditions and parameters can be more precisely controlled such that they can be quickly repeated to explore “what‐if” scenarios. To reap the benefits of this emerging technology several key open questions must be addressed. One is the availability of shared community libraries hosting well‐documented datasets of chest imaging obtained with complementary modalities. Another issue is the establishment by the community of good‐practice recommendations to safeguard the fidelity and relevance ofin silicopopulation studies. These are challenging issues needing the coordination of various key players, a theme that will be addressed in a dedicated round table discussion during the 24th ISAM Congress. In this talk, we showcase the potential ofin silicopopulations studies through an example of anin silicopopulation study for a cohort of 200 healthy adults. We present results for regional aerosol deposition and airway resistance obtained via subject‐customized anatomies and a well‐established 1D generational model. We show that the degree of inter‐subject variability is significantly larger than what one would have obtained if simulations were customized using only subject lung volumes and inhalation profiles.

F. 09 NO ABSTRACT

F. 10 NO ABSTRACT

F. 11 Novel Fully Primary Human Airway Epithelium‐Alveolar Macrophagesin vitroCo‐Cultures Models to Study Host Pathogen Interactions

Bernadett Boda,Xiao‐Yann Huang,Ophélie Le Guen, Gowsinth Gunasingam, and Samuel Constant

Epithelix, Plan‐Les‐Ouates, Switzerland.

Being the first line of defense of the organism against airborne pathogens the airway respiratory epithelium is also a potent immune‐regulator which orchestrates both innate and adaptive immune responses upon bacterial or viral infections.

Here we established a new co‐culture model using well characterized, standardized human airway epithelium such as MucilAirTM, SmallAirTM and human lung primary macrophages (CD45+,HLA‐DR+, CD206+, CD11b+and CD14‐) for studying bacterial and viral infections. The alveolar macrophages were not only able to adhere to the epithelial cells, but also functional: The macrophages were capable of phagocytosis, evaluated using pHrodo™ Red (S cerevisiae Bio‐particles Conjugate). Moreover, the co‐culture models respond to pro‐inflammatory stimuli such as LPS, TNF‐a and Poly(I:C) with an increased IL‐8 secretion.

Upon bacterial infection with methicillin‐susceptible Staphylococcus aureus strain (MSSA), compared to MucilAir™ monocultures, MucilAir™‐macrophages showed stronger immune responses: (i) a reduction of bacterial growth (up to 1.5Log10 CFU) and (ii) decreased upregulation of IL‐8 and b‐defensin‐2 secretions. Interestingly, greater difference was observed for Streptococcus pneumonia (Sp19F): The presence of macrophages led to a decrease of 3.5Log10 CFU after 24 hours of culture versus MucilAir™ alone.

These novel in vitro models might find applications in understanding the role of immune‐epithelial cell interactions in infection disease.

F. 12 Bioprinting of Human Lung Models to Study Viral Infection

Johanna Berg,¹Beatrice Tolksdorf,¹Daniela Niemeyer,^2,3Julian Heinze,^2,3Christian Drosten,^2,3and Jens Kurreck¹

¹Department of Applied Biochemistry, Technische Universität Berlin, Chair of Applied Biochemistry, Berlin, Germany.

²German Centre for Infection Research (DZIF), Berlin, Germany.

³Institute of Virology, Charité‐Universitätsmedizin Berlin, Berlin, Germany.

Viral induced airway infections are a major threat of global health and destruction to infrastructure. Beside the outbreak of known viruses, the emergence of unknown viruses poses a constant menace. 3D model systems are needed to understand host/pathogen interactions in a human micro tissue environment and to develop suitable forms of therapy. The presented printed human lung model consists of a base layer containing endothelial cells. A second layer contains fibroblasts together with monocytic and endothelial cells, on top of which a seven‐layer cell‐free rim structure is printed. The cell‐containing hydrogel contains alginate, gelatin, collagen, hyaluronic acid, and laminin. Three weeks after submerged cultivation either A549 cells (alveolar epithel) or Calu3 cells (bronchial epithel) are seeded in the recess of the rim and the models are cultured for another seven days at air‐liquid‐interface. During cultivation cell viability and expression of specific cell markers were analysed. Finally, the models were infected with an influenca influenza A virus, which infected both types of epithelial cells and showed efficient replication in both model types. The Calu3 cells in the model could as well be infected by SARS‐CoV‐2.The printed lung model systems enable the investigation of pulmonary pathogenicity to support development of new therapeutics. Due to the possibility of quick adaptation to susceptible cell types the models may also serve as a tool to address further pathogens.

F. 13 Fibrous Aerosols as Potential Drug Carriers for Inhalation Therapy

Frantisek Lizal,Ondrej Misik, Miloslav Belka, Matous Cabalka, Ondrej Cejpek, Milan Maly, and Jakub Elcner

Brno University of Technology, Brno, Czech Republic.

The ability of inhaled fibres to penetrate deep into human lungs is well known due to the infamous health effects of asbestos. However, that ability can be advantageous if the fibres are made of biodegradable material and serve as carriers for therapeutic agents. Such an idea is not new, its applicability was demonstrated by Chan and Gonda [1]. Recent research brought magnetically guided fibres that can serve for targeted delivery of inhaled medicine [2]. The main problem remains with the precise prediction of the flow and deposition site of fibres and hence the precise dose. Our research hence focuses on improving the precision of computational models for predicting the transport of fibres in human airways. Experimental data on the orientation of fibres in the replica of the human first bifurcation was presented by Lizal et al. [3].

The work was funded by project GA22‐20357S.

References

[1] Chan, H. K. and I. Gonda. 1989. Aerodynamic properties of elongated particles of cromoglycic acid. Journal of Aerosol Science 20:157‐168. doi: 10.1016/0021‐8502(89)90041‐4

[2] Nikolaou, M., Avraam, K., Kolokithas‐Ntoukas, A et al. (2021). Materials Science & Engineering C‐Materials for Biological Applications 126. doi: 10.1016/j.msec.2021.112117

[3] Lizal, F., M. Cabalka, M. Maly, J. Elcner, M. Belka, E. L. Sujanska, A. Farkas, P. Starha, O. Pech, O. Misik, J. Jedelsky, M. Jicha. (2022). Aerosol Science and Technology. doi: 10.1080/02786826.2022.2027334

F. 14In silicoAirway Deposition of Inhaled Particles with Anti‐Cancer Therapeutic Ingredient

Ondrej Mišík,¹Jana Szabová,²Jakub Elcner,¹Filip Mravec,²and František Lízal¹

¹Faculty of Mechanical Engineering, Brno University of Technology, Brno, Czech Republic.

²Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic.

Lung cancer is the second most diagnosed cancer and the most frequent cause of cancer death [1]. Erlotinib is an epidermal growth factor receptor inhibitor used for non‐small cell lung cancer treatment.

Within this study, the erlotinib was incorporated into the 3 liposomal systems [2] and nebulized by 2 mesh nebulizers (Aerogen Solo, eFlow Rapid) and 2 air‐jet nebulizers (PARI LC Sprint, PARI LC Star). Aerosol aerodynamic particle size was measured by Aerodynamic Particle Sizer according to the methods described in [2] and the measured size distribution was set in the computational fluid dynamic simulations in realistic airway replica described in [2]. These calculations were performed by Reynolds‐averaged Navier Stokes equations with a stationary inhalation flow rate of 20 l/min (the average flow rate of the normal breathing inhalation).

For all of the measured nebulizers, a high fraction of the aerosol mass penetrated below the 7th generation of airway branching. This fraction was a bit higher for the case of the air‐jet nebulizers (86.79 ± 2.66 %) than for the case of vibrating mesh nebulizers (74.36 ± 4.61 %). According to the results, the nebulization of the liposomal form of erlotinib is a promising way of anticancer drug delivery.

References

[1] Sung H, Ferlay J, Siegel RL et al. (2021). CA Cancer J Clin 71, 209–49. doi: 10.3322/caac.21660.

[2] Szabová J, Mišík O, Fučík J et al. (2023). Int J Pharm 634. doi: 10.1016/j.ijpharm.2023.122695

F. 15 Human 3Din vitroAirway Models for Aerosol Testing of RNA Inhibitors against COVID‐19

Alexandra Hübl,Florentin Baur, and Maike Windbergs

Goethe University, Frankfurt/Main, Germany.

Due to SARS‐CoV‐2 pandemic,in vitroaerosol testing of appropriate drug candidates has become increasingly important. Current approaches for evaluation of inhalation toxicology of new actives often rely on cancer‐derived cell lines, submerged culture as well as submerged drug treatment leading to results that might not reflect reality.

In our approach, we are using primary human alveolar and bronchial cells cultured on porous membranes at air‐liquid interface to achieve 3Din vitroairway models consisting of ATI and ATII cells (lung) or ciliated and goblet cells (bronchia). Several novel RNA inhibitors against COVID‐19 are applied onto the cells as aerosols using the VITROCELL^®Cloud exposure system and toxicity is evaluated based on analysis of the cell viability (MTT assay), immunohistological staining with specific markers (e.g., SFTPC, Caveolin‐1, ZO‐1), and morphological changes (CLSM, SEM). Based on our toxicological study, we are developing an aerosol formulation for the most promising drug candidate.

Compared to cell lines and submerged culture of cells, our models show higher sensitivity to potentially toxic drug effects, offering a more predictive approach to investigate toxicology, absorption, and transport of inhalable drugs.

F. 16 Development of Fully Primary Human 3D Alveolar Model (AlveolAir™)

Cindia Ferreira, Bernadett Boda, Song Huang, Caroline Chojnacki, Mendy Bouveret, Samuel Constant, Ophélie Le Guen,Guy Barbin,and Mireille Caulfuty

Epithelix, Plan‐Les‐Ouates, Switzerland.

We herein describe the characterization and functionality of a full primary human epithelial‐endothelial 3D alveolar model, AlveolAir™. This novel in vitro model represents a relevant and reliable tool for inhalation toxicity assessment of drugs.

To characterize AlveolAir™, long‐term parameters were measured: Biomarkers for ATIs, ATIIs and tight junctions (CAV‐1, HTII‐280 and ZO‐1 respectively, Immunofluorescence); Morphology and lamellar bodies' presence (Histology & TEM); Existence of a maintained alveolar epithelial barrier (TEER) and SPC secretion (ELISA). Using these techniques, the evolution of the culture was monitored for several weeks.

Functionality of AlveolAir™ was evaluated by exposure to pro‐inflammatory compounds. TEER, cytotoxicity (LDH) and cytokines quantification were monitored daily. It revealed that AlveolAir™ is able to display an inflammatory response by secreting IL‐6, IL‐8 and RANTES significantly.

As proof‐of‐concept, effect of a flagellin‐based formulation was assessed on AlveolAir™. The tissues were exposed to several doses of flagellin apically, for 5 days. TEER, LDH, pro‐inflammatory cytokines (IL‐6 & IL‐8) and a panel of genes were evaluated. Altogether, flagellin was well tolerated by alveolar epithelia. Apical exposure induced biomarkers upregulation, demonstrating flagellin's immunomodulatory potential on alveoli.

Finally, co‐culture model between AlveolAir™ and primary alveolar macrophages has been developed.

F. 17 NO ABSTRACT

F. 18 Modelling Alveolar Inflammation on a Breathing Lung‐On‐Chip for Estimation of Anti‐Inflammatory Drug Responsein vitro

Clémentine Richter,^1,2Patrick Carius,^1,2Nuria Roldan,³Janick Stucki,³Nina Hobi,³Tobias Krebs,⁴Brigitta Loretz,¹Lorenz Latta,¹Alberto Hidalgo,¹Nicole Schneider‐Daum,¹and Claus‐Michael Lehr^1,2

¹Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany.

²Saarland University, Saarbrücken, Germany.

³AlveoliX AG, Bern, Switzerland.

⁴Vitrocell^®Systems GmbH, Waldkirch, Germany.

Animal testing remains the most widespread method for the pre‐clinical evaluation of inhaled drugs due to the absence of validatedin vitromodels. This study describes an inflamed alveolus model on a breathing lung‐on‐chip¹to predict human anti‐inflammatory drug response. This MPS allows for the investigation of the role of lateral stress in cellular responses upon diverse stimuli and their modulation by aerosolized drugs in a complex in vitro system.

The inflammation of the human alveolar epithelial cell line Arlo²was investigated in the presence of key physiological parameters such as macrophages, 3D breathing‐like stretch, and nebulization of anti‐inflammatory agents to improve the model's validity.

For both inflammatory stimuli (LPS and a combination of TNFα and IFNγ), the presence of macrophages was necessary to simulate the inflammatory response. Further, we demonstrated the efficacy of budesonide as an anti‐inflammatory model drug, protecting against the effects of both inflammatory stimuli. Remarkably, in the case of TNFα and IFNγ, barrier weakening and protection by budesonide were most notable in stretching conditions. Therefore, this model might be suitable for drug efficacy testing as an alternative to animal studies.

References:

[1] Sengupta A., Roldan N. et al. (2022). Front Toxicol. 4, 840606. doi: 10.3389/ftox.2022.840606.

[2] Carius, P.; Jungmann, A. et al. (2023). Advanced Science. e2207301 doi:10.1002/advs.202207301.

F. 19 Assessing Asthma Protection by the Standardized Bacterial Lysate OM‐85 in Mild and Severe Asthma Models

Claire Abadie,¹Helena Obernolte,²Katherina Sewald,²Armin Braun,²Anne Vaslin Chessex,¹and Christian Pasquali¹

¹OM Pharma, Geneva, Switzerland.

²Fraunhofer, Hannover, Germany.

Allergic asthma is a heterogeneous chronic disease, characterized by the activation of the Th2 immune response in the lung in response to an environmental stimulus, leading to airway hyper‐responsiveness and short breath. OM‐85 is a well‐tolerated oral bacterial lysate used in children and adults for the prevention of recurrent respiratory tract infections.

OM‐85 efficacy was investigated on two treatment regimens (therapeutic and prophylactic) in models of mild and severe allergy‐induced asthma in sensitized BALB/c mice challenged with ovalbumin.

In both models, prophylactic treatment presented significant reduction of lung resistance and improvement in eosinophilia in BAL while the therapeutic treatment was only effective in the mild model. The characterization of specific Cluster of Differentiation proteins highlighted a clear increase of MHC II in treated groups; CD80 was predominantly expressed in the therapeutic regimen, whereas CD40 and CD86 levels were increased in the prophylactic regimen. OM‐85 evidenced beneficial effects on cytokine release in both models and both regimens. The inflammatory score showed a clear improvement by prophylactic treatment only in the severe model.

OM‐85 showed protective effects against asthma regardless of severity, more marked in a prophylactic than therapeutic treatment. OM‐85 seems a good candidate for the prevention of mild asthma.

F. 20 Micro‐Alveolar Ducts – a Novel Kind of Pulmonary Airways Appearing Late during Alveolarization

Taha V. El Moudden, David Haberthür, andJohannes C. Schittny

Institut of Anatomy, University of Bern, Bern, Switzerland.

Pulmonary gas‐exchange takes place in the acini, which are defined as small trees of gas‐exchanging airways distal of the terminal bronchioles. Limited knowledge exists about their development because the required high‐resolution three‐dimensional (3D) imaging techniques were not available until recently. Using synchrotron radiation‐based X‐ray tomographic microscopy we studied the 3D‐structure of pulmonary acini in rats at postnatal days 4, 10, 21, and 60. We discovered novel alveolated micro‐airways which branch off from the alveolar ducts. We named them micro‐alveolar ducts and estimated their volume density. They appeared at day 21 and increased in number at day 60, but where not present at days 4 and 10. Furthermore, large acini facing the pleura possess a higher density of micro‐alveolar ducts than smaller, central acini. Because the micro‐alveolar ducts are forming long after branching morphogenesis ceased, we postulate that they are formed by an elongation and subsequent septation of existing alveoli. Furthermore, we postulate that the micro‐alveolar ducts are forming to ensure that all spaces between the growing tree of pre‐existing alveolar ducts are filled with alveoli as homogeneously as possible. Most likely, the mechanism of ventilation and particle deposition resembles the one in the sacculi (most distal part of the alveolar ducts) and not the one at the entrance of the acini.

F. 21 Assessing Inflammatory Effect of DQ12 Exposure in a Harmonized Lung Model

Isidora Loncarevic,¹Sandeep Keshavan,¹Barbara Rothen‐Rutishauser,¹Alke Fink,¹Fabian Blank^2,3,4, and Seyran Mutlu^2,3,4

¹Adolphe Merkle Institute, Fribourg, Switzerland.

²University of Bern, Bern, Switzerland.

³Department for BioMedical Research DBMR, Bern, Switzerland.

⁴Inselspital, Bern University Hospital, Bern, Switzerland.

In the field of inhalation toxicology, current in vitro lung models require the evaluation of model predictivity. Based on previously standardized and harmonized protocols within the H2020 EU project PATROLS, we applied a lung model consisting of the human bronchial cell line Calu‐3 combined with human monocyte‐derived macrophages cultured at the air‐liquid interface. We focused on lung inflammation as a result of crystalline quartz (DQ12) exposure as it occurs in both, humans and rats. First, the dose regimen was aligned with data obtained from the rat model. Reported doses that caused persistent inflammation and lung tissue remodeling in vivo showed no cytotoxic and proinflammatory effects in our model. However, experiments are ongoing to determine how repeated or higher DQ12 exposure in the lung model affects the gene expression profiles. Understanding the immunological capabilities of macrophages and their contribution to these changes can highlight their usefulness to predict in vivo response patterns when exposed to hazardous nanomaterials.

F. 22 Fit‐for‐Purpose Models to Assess the Alveolar Barrier: Health, Disease and Therapeutics

Léa Todeschini,¹Lea de Maddalena,¹Laurène Froment,¹Nicole Albrecher,¹Giulia Raggi,¹Nuria Roldan,¹Janick Stucki,¹and Nina Hobi²

¹AlveoliX AG, Bern, Switzerland.

²AlveoliX, Bern, Switzerland.

The distal lung represents one of the largest areas of exposure from the human body. Hence, the inhalation route can be exploited as a non‐invasive and efficient delivery system for API and biologics with lung‐targeted effects, but also for systemic administration.

To evaluate molecule effects locally with clinical relevance, we have developed different fit‐for‐purpose alveolar models accounting for breathing‐like dynamics and a cell culture substrate simulating the thin, soft and elastic lung parenchyma (^AXLung‐on‐chip System).

To extrapolate our results clinically, we assess established endpoints including barrier integrity, vascular leak, molecule transport, induced toxicity (apoptotic and necrotic events), and hit/target identification to match clinical parameters.

Additionally, we have established human models replicating important hallmarks of known lung alterations including pulmonary fibrosis (increased ECM protein secretion, barrier impairment, increased profibrotic gene expression), and molecule‐induced exacerbated inflammation (immune cell activation, vascular leak, increased toxicity). These models have not only been proven as reliable efficacy (Nintedanib) and safety predictors (FolR1‐TCB), but also can be used to estimate potential side effects of newly tested molecules.

With the shifting regulatory landscape towards animal free testing, the^AXLung‐on‐chip System appears as a promising predictive technology to safety speed up human‐focused applications.

F. 23 NO ABSTRACT

F. 24 NO ABSTRACT

F. 25 A Novel, Comprehensivein silicoModel for Respiratory Drug Delivery

Maximilian Grill,¹Karl‐Robert Wichmann,¹David Rudlstorfer,¹Maximilian Rixner,¹Marie Brei,¹Jakob Richter,¹Joshua Bügel,¹Nina Pischke,¹Wolfgang A. Wall,²Jonas Biehler,¹and Kei W. Müller¹

¹Ebenbuild GmbH, Munich, Germany.

²Technical University of Munich, Institute for Computational Mechanis, Garching b. München, Germany.

Generally, the huge potential of in silico models in drug development has been highlighted and demonstrated by many players, including the FDA. For respiratory drug delivery, however, existing models are severely limited by morphological truncation or spatial resolution and thus fail to realize the full potential.

We propose a novel whole‐lung model that predicts aerosol deposition throughout the entire lung, i.e. the full tree of conducting airways and, most notably, the alveolar tissue. Its high spatio‐temporal resolution allows detailed tracking of drug deposition during inhalation and exhalation. Built upon a previously published and validated, patient‐specific lung model for airflow and local tissue deformation¹, it thus enables the prediction of drug delivery results for various aerosols or any lung morphology with unprecedented level of detail. Further details of the model will be disclosed at the congress after IP has been secured.

Validation against 3D SPECT/CT images from 10 inhalation experiments of 6 healthy human subjects²shows excellent accuracy of the predicted regional deposition. Our scalable solution enables rapid in silico studies with large cohorts. Further extensions, e.g. towards diseases, are already in progress.

References

[1] Roth, C.J., Becher, T., Frerichs, I. et al. (2017). J. Appl. Physiol. 122, 855–867. doi:10.1152/japplphysiol.00236.2016

[2] Conway, J., Fleming, J., Majoral, C. et al. (2012). J. Aerosol Sci. 52, 1–17. doi:10.1016/j.jaerosci.2012.04.006

F. 26 ADAM10 and ADAM17 Are Key Elements of Barrier Development Determining 3D Model Systems

Ahmad Aljohmani,¹Clementine Richter,²Federico Guillermo Gharzia,¹Lorenz Latta,²Claus‐Michael Lehr,²and Daniela Yildiz¹

¹Institute of Experimental and Clinical Pharmacology and Toxicology, PZMS, ZHMB, Saarland University, Homburg, Germany.

²Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany.

The pulmonary epithelium is a complex system made up of various cell types differing between the proximal and distal part, which work together to maintain barrier integrity and to protect against harmful external factors. The regulation of junction and adhesion molecules, including E‐cadherin, occludins, claudins, and JAM‐A, is crucial for maintenance of epithelial integrity and homeostasis. These barrier molecules are proteolytically released into the extracellular space, with a disintegrin and metalloproteinase (ADAM) 10 and 17 being key elements of these cleavage events. Thus, adequate in vitro models have to be carefully evaluated for the reflection of this in vivo situation. Therefore, the barrier development of both Calu‐3 cells and the newly established human alveolar epithelial cell line Arlo was investigated in both air‐liquid interface (ALI) and liquid‐liquid interface (LLI) cultures. Our findings revealed differential regulation of ADAM10 and ADAM17 in these cell types under varying growth conditions, correlating with the differences in barrier strength. Gene silencing experiments could confirm the critical role of ADAM10 in barrier development only in Arlo but not in Calu‐3 cells. Furthermore, activation of ADAM17 was found to modulate barrier integrity through the cleavage of adhesion molecules while ADAM10 was less essential. Thus, both ADAM10 and ADAM17 are crucial for epithelial barrier homeostasis and well reflected in Arlo cell‐based 3D cell culture models.

F. 27 Personalized Cell Lines By Reproducible And Functional Immortalization

Tobias May,Kristina Nehlsen, and Anne Dittrich

InSCREENeX, Braunschweig, Germany.

A major limitation of current research is the shortage of functional personalized cells. To generate cells in sufficient numbers in vitro cell expansion is an attractive alternative. However, conventional immortalization regimens are unpredictable, cumbersome and often lead to drastic alterations of cellular physiology.

To overcome these limitations, we established a novel cell immortalization approach which relies on a lentiviral transduction of a small gene library. Thereby, we are able to reproducibly generate novel functional and immortalized cell lines within 3‐4 months. Until now we have successfully applied this process to more than 25 different cell types including e.g., endothelial cells, astrocytes, smooth muscle cells, thyrocytes, lung and intestinal epithelial cells.

Resulting cell lines can be cryopreserved, cultivated for more than 100 cumulative population doublings and are amenable to genetic engineering. Functional characterization demonstrated that the novel cell lines maintain a primary‐like phenotype and show cell type‐specific functions. The phenotype can be further improved by 3D culture conditions like e.g. spheroids, organoids, Air‐Liquid‐Interface demonstrating their suitability as a reproducible and functional cell source.

Once an immortalization regimen was established for a certain cell type, it could be easily adapted for the reproducible immortalization of the same cell type from a different donor paving the way for personalized cell lines.

F. 28 A Human Bronchus‐On‐Chip Model: a Promising Tool for Respiratory Research

T.N. Mai Hoang,Katja Graf, Martin Raasch, and Knut Rennert

Dynamic42 GmbH, Jena, Germany.

Respiratory conditions have become an increasingly pressing issue for public health in recent years. However, R&D of new drugs and therapies still lacks models that can recapitulate the human lung physiology on an organ or tissue level, and enable better data transferability from preclinical to clinical applications than currently available models.

The aim of our work was to establish a human bronchus‐on‐chip model recapitulating the human bronchial airway architecture and function under air‐liquid interface. The model consists of a bronchial compartment, and a vascular compartment separated by a porous membrane that allows for cell‐cell communication. The highly differentiated bronchial epithelium is composed of basal cells, ciliated cells, secretory club cells and mucus‐producing goblet cells. Expression of bronchial cell type‐specific markers were investigated. Furthermore, we established a pipeline for quantitative analysis of ciliary beating frequency using high‐speed video recordings and the opensource software CiliarMove.

The established bronchus‐on‐chip model could be used to investigate various aspects of airway biology, including the response of lung cells to drugs, toxins or microbial infections¹and help to accelerate the development of new treatments for lung diseases such as asthma and chronic obstructive pulmonary disease (COPD).

References

[1] T.N.M. Hoang, Z. Cseresnyés, S. Hartung et al. (2022). Biomaterials, 283: 121420. doi:10.1016/j.biomaterials.2022.121420

F. 29 Development of anin vitroFull‐volume Airway Approximation for Assessing Breath‐dependent Regional Aerosol Deposition

Ian Woodward,Yinkui Yu, and Catherine Fromen

University of Delaware, Newark, USA.

Existing preclinical models of aerosol deposition comprise a range ofin vivo,in vitro, andin silicoapproximations. Yet the need remains for an extensible, patient‐specific platform that incorporates the coupled effects of device, formulation, anatomy, and breath, to predict spatial aerosol deposition of an entire inhaled dose. Here, we describe a modularin vitroplatform, enabled by additive manufacturing and computational design, that combines patient‐specific anatomy from the tracheobronchial airways with two regions of filtration approximations within each lung lobe, thus enabling quantification of a central‐to‐peripheral (C/P) deposition ratio. Derived from features of an adult male, the lung model comprises ∼5 L total volume and generates physiologically relevant tidal inhalation and exhalation maneuvers at flow rates up to 50 SLPM through coordinated lobe‐level mechanical control, with tunable waveforms, lobar volume distributions, and breath holds. In experiments over 60 breath cycles corresponding to clinical benchmarks [1], the collected C/P ratio of nebulized aerosols ∼3‐5 μm in diameter ranged from 1.57‐3.93 for various approximation configurations, in close agreement with published findings. Through continued efforts, this platform may complement current approaches and fill an outstanding need in advanced preclinical development of aerosol medicine.

References

[1] Conway, J., Fleming, J., Majoral, C., et al. (2012) J Aerosol Sci 52, 1‐17. doi:10.1016/j.jaerosci.2012.04.006.

F. 30 3D‐Bioprinting of Bacterial Biofilm on Monolayer of Human Lung Cells as Advancedin vitroModel for Chronic Lung Infections

Aghiad Bali,^1,2Samy Aliyazdi,^1,2Lorenz Latta,¹Brigitta Loretz,¹Nicole Schneider‐Daum,¹and Claus‐Michael Lehr^1,2

¹Helmholtz Institute for Pharmaceutical Research (HIPS), Saarbrücken, Germany.

²Department of Pharmacy, Saarland University, Saarbrücken, Germany.

The development of new therapeutic approaches is very urgent to combat antibiotic‐tolerant biofilm‐related infections, which represent a global health threat [1]. The current standard to test novel anti‐infectives for safety and efficacy is by using animal models, which are, however, ethically problematic and limited by major differences to humans. Moreover, currentin vitromodels of bacterial biofilms do not allow addressing any host‐pathogen interactions.

Expanding on recently reported pipetting of biofilm microclusters [2], we here report a novel approach using 3D‐bioprinting to generate an improved human‐basedin vitroinfection model. Essentially, pre‐formed mature biofilms ofP. aeruginosaare printed using a self‐established agarose‐alginate bioink on confluent layers of human bronchial epithelial cells (Calu‐3) grown at Air‐Liquid‐Interface. Biofilm properties were maintained after printing. Furthermore, printing blank layers of the bioink is compatible with epithelial cell culture and does not impair drug permeation. After further validation, this model could be a potential alternative to animal experiments for preclinical anti‐infectives testing.

References

[1] Wagner and Iglewski (2008). Clin Rev Allergy Immunol, 35(3),124–134. doi:10.1007/s12016‐008‐8079‐9

[2] Horstmann et al. (2022).ACS Infect Dis,8(1),137–149. doi:10.1021/acsinfecdis.1c00444

F. 31 Integratedin vitro/ex vivoApproach for Early Efficacy and Respiratory Toxicity Assessment of New Inhalational Drug Candidate

Dorothee Winterberg,Helena Obernolte, Sabine Wronski, Detlef Ritter, Katherina Sewald, Armin Braun, and Katharina Schwarz

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany.

Background:Developing new anti‐infective strategies is lengthy and difficult. For a fast and predictive non‐clinical development, we established an integrative platform for evaluation of inhalational drug candidates. Here, we used Nafamostat, a repurposing drug with excellent anti‐viral efficacy against SARS Cov‐2.

Methods:For human proof of concept Precision Cut Lung Slice (PCLS) model as an ex vivo organotypic infection model was integrated with rat ex vivo isolated perfused lung (IPL) system and in vitro inhalation assays to address safety and efficacy.

Results:In‐vitro inhalation studies (A549 cells) indicate at a LOAEL for Nafamostat of ∼2 μg/ cm2. A dose dependent efficacy could be demonstrated in PCLS infection models (SARS‐CoV‐2). Toxicity set in only at an ∼100‐fold higher dose compared to efficacy, thereby revealing an excellent therapeutic window. In IPLs, a LOAEL of 0.2 mg/kg bw was observed with adverse effects only in the upper airways. In a 28‐day in vivo inhalation study, animals showed adverse effects for 0.3 mg/kg dose in the larynx/larger airways, while effects in the pulmonary region were smaller and no toxicity was observed in the nasal area.

Conclusion:Application and combination of different methods can provide data, that can be incorporated into the non‐clinical development of inhaled drug candidates. Therefore, these studies can contribute to a faster and more effective early pre‐clinical development and can help to refine regulatory studies.

F. 32 Determination of Local Efficacy and Safety for the Inhaled Antibiotics Tobramycin and Ciprofloxacin by an Integratedin silicoandex vivoApproach

Sven Cleeves, Sabine Wronski, Norman Nowak, Katharina Bluemlein, Monika Niehof, Katherina Sewald, Armin Braun, andKatharina Schwarz

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany.

Background:Pulmonary drug delivery is an attractive option for treatment of respiratory infections. For the development of inhaled antibiotics improved models for early assessment of efficacy, toxicity and respiratory pharmacokinetics are of particular relevance.

Methods:To assess the local therapeutic window for ciprofloxacin and tobramycin in the lungs, ex vivo organotypic Precision Cut Lung Slices (PCLS) were used in a Pseudomonas aeruginosa infection model. Isolated Perfused Rat Lungs (IPL) providing a fully intact organ system was established to investigate respiratory pharmacokinetics. Data integration was conducted using the PBKit inhalation‐focused PBK model developed by Fh ITEM.

Results:A dose dependent efficacy was observed for both antibiotics investigated, while local toxicity only set in at much higher doses, thereby defining the local therapeutic window. Comparison of effective doses for ciprofloxacin and tobramycin showed good agreement with corresponding MIC values. Absorption parameters determined in the organotypic IPL model for inhaled ciprofloxacin (permeability 4*10‐7 cm/s, blood tissue distribution 1:4.6) were used as input parameter in the inhalation‐focused PBK model. Predicted concentration‐time profile in humans corresponds well to published clinical data.

Conclusion:The combination of the different ex vivo models with PBK modeling provided predictive data, that can be incorporated into the non‐clinical development of inhaled drug candidates.

F. 33 NO ABSTRACT

F. 34 Understanding the Role of Asymmetric Branching in the Lung: Towards Targeted Drug Delivery

Debjit Kundu,and Mahesh Panchagnula

Indian Institute of Technology Madras, Chennai, India.

Understanding the intricate physics of fluid flow and aerosol transport in the lung is crucial for answering questions such as how airborne diseases like COVID‐19 infect our lungs or how atmospheric pollutants affect our health. Also, in a constructive sense, it can enable us to optimize and customize pulmonary drug delivery for specific patients. Our research explores airflow and aerosol deposition in the airways through Monte Carlo simulations of a Stochastic Asymmetric Multi‐Path Model. Our findings unveil the evolutionary role of asymmetric bifurcations in the human lung.

Moreover, we apply our models towards studying targeted drug delivery. On the basis of target regions, drugs administered in this manner can be categorized into two classes: those targeting the lung tissue itself, absorbed in the tracheobronchial region, and those intended to reach the deep lung and be absorbed into the bloodstream for systemic effects. We examined how asymmetry, particle size, and bronchoconstriction (a common symptom of several lung diseases) influence the distribution of regional deposition in the lung. We located regions which minimise variability. We showed that bronchoconstriction increases deposition in the upper lung and decreases it in the acinus. Furthermore, we identified two particle sizes which are optimal for drug delivery to the acinus. These insights would prove useful in precise dosage determination for medications as well as the choice and design of delivery devices.

F. 35 Development of a Clinically Validatedin vitro / in silicoMethod for the Determination of the Pharmacokinetics of Inhaled Agents

Ali Doryab,¹Thorsten Keller,²Bo Olsson,³Per Bäckman,³Anne Hilgendorff,⁴Jürgen Groll,²and Otmar Schmid¹

¹Helmholtz Munich, Munich, Germany.

²University of Würzburg, Würzburg, Germany.

³Emmace Consulting AB, Lund, Sweden.

⁴Ludwig‐Maximilians‐University of Munich (LMU), Munich, Germany.

We leverage our recently developed advancedin vitrobreathing miniaturized (alveolus) lung model initially focused on fibrosis (1) to develop anin silico‐enhancedin vitromethod for pharmacokinetics (PK) measurements of inhaled drugs as an alternative to animal studies. This alveolar barrier model consists of i) a bioinspired lung‐like BETA basement membrane lined with a co‐culture of epithelial and endothelial cells and ii) a millifluidic cell‐stretch CIVIC bioreactor system. The millifluidic system also allows metered aerosolized drug delivery and real‐time non‐invasive monitoring of lung function (compliance, stiffness). Using our BETA/CIVIC mini‐lung model, we can study i) the effectiveness and kinetics of aerosolized drug delivery, ii) the physicomechanical properties of cell substratum, such as stiffness and permeability, and iii) mechanobiology. Our model improves the functional (clinical) relevance ofin vitrofibrosis models and grants the combined longitudinal mechanistic or pharmacodynamics/‐kinetics studies. We anticipate that by utilizing computationalin silicoPBPK models, we can accurately predict in vitro drug transport measurements in a patient's lungs for the first time. This will be validated with clinically obtained PK profiles for selected drugs. This work was partly supported by the German Federal Ministry of Education and Research (BMBF; 16LW0133K, Project “KLIMA”).

References

[1] Doryab et al. (2022) Advanced Materials 34, 2205083. doi:10.1002/adma.202205083.

F. 36 Investigating Human Pneumonia Model Infection in an iPSCs Alveolus‐On‐Chip with Flexible Collagen‐l Membranes

Mona Amiratashani,¹Ina prade,²Frank Sonntag,³and Alexander Mosig¹

¹University Hospital, Jena, Germany.

²FILK Institute gGmbH, Freiberg, Germany.

³Fraunhofer, Dresden, Germany.

Organ‐On‐Chip (OOC) devices are emerging technologies that simulate the biologically perfused milieu of tissues. Although biomechanical cues, such as strain related to breathing, plays a significant role in priming cells and the course of infections, this aspect is often neglected in vitro studies. While most OOC systems contain membranes, their mechanical actuation is not often possible. Further, the faithful recreation of the flexible extracellular matrix, critical for cell attachment, differentiation, and morphogenesis in development, is still a challenge in current lung‐on‐chip systems [1]. We aim to recreate breathing mechanisms in an OOC system and to replace existing membranes with flexible Collagen‐I membranes. Tissue engineering techniques generate an immunocompetent, isogenic alveolus‐on‐chip model composed of alveolar type II, endothelial cells, and macrophages derived from human pluripotent stem cells, and the cell types will be co‐cultured on a Collagen‐I membrane. Immunofluorescence imaging is used to assess viability, confluency, and barrier formation. This isogenic model will be employed in infection studies for Influenza Virus A to investigate the molecular mechanisms of pneumonia and for testing antiviral drugs. The effects of simulated breathing biomechanics will be addressed using strain to assess functional tissue alterations throughout the infection.

References

[1] Zamprogno et al., (2021). ACS Biomater Sci Eng, 7(7), 2990‐2997. doi:10.1021/acsbiomaterials.0c00515

G. Recent Trends and Developments in Inhaler Technology

G. 01 The Ever Expanding Universe of Inhaler Technology

Andy Clark

Aerogen Pharma, San Mateo, USA.

The modern era of inhaler technology began in 1956 with the pMDI. In the 1960's DPIs were added to the technology list and in 1989 Dunne Miller and Weston patented the liquid atomizer technology that lead to Soft Mist Inhalers [1]. Since then, progress has been made in improving delivery efficiencies, dose ranges and patient interfaces [2].

It has not been smooth sailing however, in 1987's the propellants used in pMDIs were band due to global warming potential and there was an explosion of prototype inhalers aimed at ensuring patients would have alternatives should the pMDI get removed from the market. However, it soon became apparent that alternative propellants were viable and pMDIs would continue. As a result, most of these prototype inhaler technologies fell by the wayside.

The next wave of innovation, still happening today, is driven by patent expirations of the common respiratory drugs and generic inhalers being developed to take advantage of the “new” markets.

In addition to devices progress has also been made in formulation technologies used in pMDI and DPIs. Arguably, in terms of performance, these advances have had a larger impact than new devices (Clark, 2022).

It is interesting to speculate as to what improvements the next decade will bring.

References

[1] Stein S., and Thiel C. (2017) J. Aerosol Med. Pulmo. Drug Deli. 30 (1), 20‐41, doi:10.1089/jamp.2016.1297

[2] Clark A R (2022) Front. Drug Deliv. Respiratory Drug Delivery, Vol 2, doi.org/10.3389/fddev.2022.871147

G. 02 Recent Developments in Soft Mist Inhalation Devices

Wilbur de Kruijf

Thaerapy BV, Enschede, Netherlands. Resyca, Munich, Germany.

Soft mist inhalers (SMIs) are hand held multi dose liquid nebulisation devices. Currently only one device in this category is on the market, the Boehringer Ingelheim Respimat inhaler, for several drugs in COPD. In this lecture the author attempts to list the other SMI devices that are currently in development and to describe and categorize them by spray principle and performance characteristics.

In the development pipelines for inhaled drugs, a large portion is of biologic nature, nanobodies, antibodies, mRNA, peptides, etcetera. Many of the current developments require large volumes to be inhaled. That is why most of the early clinical trials are done with conventional nebulisers. And it is not easy to transfer these biologics into conventional dry powder inhalers. Some formulations are so fragile that certain types of nebulisers destroy the formulation upon spraying (1). Moreover, for many patient groups a nebuliser is not a convenient on‐the‐go treatment.

For these upcoming biologic drugs, soft mist inhalers have a good chance to create a user friendly and pocket size therapy.

References

[1] Klein, D., Poortinga, A., Verhoeven, F.,et al., Degradation of lipid based drug delivery formulations during nebulization, Chemical Physics vol 547, 111192, 2021.

G. 03 Accuracy and Repeatability of Inhalation Volume Data Recorded by Standard and High Flow Electronic Multidose Dry Powder Inhalers: anin vitroStudy

Henry Chrystyn,¹James Forrestal,²Dong Yang,²Guilherme Safioti,³andMark Milton‐Edwards⁴

¹Inhalation Consultancy Ltd., Leeds, United Kingdom.

²Teva Pharmaceuticals Ireland, Waterford, Ireland.

³Teva Pharmaceuticals Europe BV, Amsterdam, Netherlands.

⁴Teva Pharmaceuticals, Harlow, United Kingdom.

Introduction:The Digihaler^®System (Teva Pharmaceuticals, Israel) comprises an electronic Multidose Dry Powder Inhaler (eMDPI) with integrated electronic module that records usage and inhalation parameters, and transmits data to a patient mobile App and clinician Dashboard. Data on the accuracy of eMDPI peak inspiratory flow and inhalation volume (inhV) measurements have previously been reported.^1,2Here we describe accuracy and repeatability of inhV data recorded by standard‐ (SF) and high‐flow (HF) eMDPIs.

Methods:The accuracy and repeatability of eMDPI inhV measurements (L/min) were characterised using a critical flow controller and calibrated digital flowmeter for SF and HF eMDPIs, for volumes 0.5–4.00 L with durations 0.5–4.0 s. Accuracy of inhV data displayed on the clinician Dashboard was also verified.

Results:For SF (flow rates 30–90 L/min), mean difference was ‐0.01 L (95% CI: ‐0.020 to ‐0.003). For HF (flow rates 30–120 L/min), mean difference was 0.04 L (95% CI: 0.030 to 0.046). Relative standard deviation for volumes ≥0.5 L ranged from 0.4–4.7% and 0.5–4.2%, respectively. InhV data displayed on the Dashboard were consistent with those recorded by the eMDPI.

Conclusions:The SF and HF eMDPIs provide accurate and reliable inhaled volume data suitable for use in respiratory clinical management.

References

[1] Chrystyn, H et al (2018). Respir Drug Deliv 2018:527–32.

[2] Chrystyn, H et al (2022). JAMPDD 35:166–77. doi:10.1089/jamp.2021.0031.

G. 04 VHC Whistles – Not Fit for Purpose?

Mark Sanders,and Darragh Murnane

University of Hertfordshire, Hatfield, United Kingdom.

Aim:To assess alert whistle activation flow rate (AWAFR) for different pMDIs to determine suitability. Using AeroChamber Plus Flow‐Vu (AC), manufactured by Trudell Medical, and 12 current pMDIs.

Scope: Aerochamber should be used at <45 LMin⁻¹(1).

In‐vitro assessment conducted using University of Hertfordshire calibrated flow rig, Copley; HCP5, TPK, DUSA; TSI Certifier Plus. 3 new AC devices tested.

Results:Mean AWAFR ranged from lowest 35.8 LMin⁻¹(Atrovent, BI) to highest 131.6 LMin⁻¹(Flutiform, MundiPharma). Presence of counter can make a difference if it contributes to resistance, e.g. Ventolin Evohaler (GSK) without counter AWAFR = 96.5 LMin⁻¹, whereas same‐style Seretide Evohaler (GSK) with counter AWAFR 57.6 LMin⁻¹.

Conclusion:Arguably, the range for AWAFR should be 45‐60 Lmin⁻¹, thus discouraging patients from inhaling too fast. Only 2 (out of 14) pMDIs tested (Airomir (Teva), Seretide) were in this range. 5 of 12 had AWAFR in excess of 80 Lmin^−1,(Ventolin, Symbicort (AZ), Flutiform, Salamol (Teva), ProAir (Teva)). Such variation has potential to confuse multi‐medicated patients about purpose of whistle. Chamber located whistles depend on pMDI resistance for AWAFR, and each pMDI has a different resistance – consideration to relocating the whistle should be given.

References

[1] Mitchell JP, Nagel MW, Wiersema KJ, et al. 2000 Proceedings of drug delivery to the Lungs XI, London, UK, 11–12 December 2000, pp. 52–55.

G. 05 Drug Dispersion in an Optically Accessible Dry Powder Inhaler at Two Different Reynolds Numbers

Ajit Kumar,¹Agisilaos Kourmatzis,²Hak‐Kim Chan,²and Gajendra Singh^1,2

¹Indian Institute of Technology Mandi, Mandi, India.

²The University of Sydney, Sydney, Australia.

High‐speed microscopic imaging was used for the characterization of powder fluidization and deagglomeration in an optically accessible inhaler‐like device. The device consisted of a swirling chamber connected to two inlets, acting as a physical model of commercial devices such as the Aerolizer^®and Osmohaler^®. The dimensions of the inhaler were kept similar to the commercial devices to mimic realistic flow scenarios. The study focused on investigating the effects of the Reynolds number on two mechanisms of the drug dispersion process, (i) the powder evacuation from the powder pocket and (ii) shear‐based de‐agglomeration within the device. Powders tested included mannitol (M3 and M7 having aerodynamic diameters D50 = 2.92 and 6.77 μm) and lactose carriers from DFE Pharma‐SV010, having a D50 = 106.1 μm. Inhalation flow rates of 30 SLPM and 60 SLPM were used. The flow rates were selected to represent transitional (Re = 3297 for 30 SLPM) and turbulent (Re = 6593 for 60 SLPM) flow conditions, respectively. The study investigated the effect of the Reynolds number on some key statistics like particle and agglomerate mean velocity, size distribution, and root mean square of the velocity fluctuations. At turbulent flow conditions, high powder dispersion, high de‐agglomeration, and effective evacuation were observed. Interestingly, the effect of powder properties was more significant at transitional flow conditions compared to the turbulent flows for the case of mannitol powder.

G. 06 Role of Device Characteristics on the Aerodynamic Particle Size Distribution of Tigecycline Dry Powder Aerosols

Sara Maloney,¹Leanna Levin,¹Christine Strickland,¹Ilham Alshiraihi,²Jeffrey Mariner Gonzalez,²Amarinder Singh,³Hyunseo Park,³Mercedes Gonzalez‐Juarrero,²Bernd Meibohm,³and Anthony Hickey¹

¹RTI International, Research Triangle Park, USA.

²Colorado State University, Fort Collins, USA.

³University of Tennessee Health Science Center, Memphis, USA.

Tigecycline (TGC), a glycylcycline antibiotic, has demonstrated potential in treating patients with nontuberculous mycobacterial pulmonary infections. TGC's limited stability when reconstituted in aqueous solution and dose‐limiting adverse effects upon intravenous delivery at high doses have led to technical challenges and poor patient compliance. An attractive, alternative TGC delivery method uses dry powder inhalers (DPIs), where the drug is stored in its solid form, mitigating stability concerns, and delivered directly to the primary site of infection, reducing systemic reactions. Two optimized dry powder formulations of TGC were prepared containing either 70:0:20:10 or 50:20:20:10 weight ratios of TGC:lactose monohydrate:L‐leucine:phosphate buffer salts. Powders (varied loading weights) were delivered using RS‐01 DPIs (low, medium, high, or ultra‐high resistance with pressure drop of 1.2, 2.3, 3.6, or >6 kPa at 60 L/min, respectively), custom murine dosators, or a custom murine passive inhalation chamber, and the aerodynamic particle size distribution (APSD) was monitored. Results demonstrate mass median aerodynamic diameters of ∼2.6 μm, geometric standard deviations of ∼2.0, and fine particle fractions relative to the emitted dose of 65‐70% for the two formulations when delivered from a low resistance RS‐01 inhaler. Understanding the APSD as a result of device characteristics is crucial for proper animal dosing in preclinical studies and ultimately clinical translation.

G. 07 Assessment of Patients' Ability to Use Dry Powder Inhalers with a Perceived Activity and Weakness Score (PAWS) Questionnaire

Rajiv Dhand,Martin Valdes, Daniel Faradji, and Jennifer Ferris

University of Tennessee College of Medicine, KNOXVILLE, USA.

Rationale:PAWS is a 5‐item Likert scale‐based validated questionnaire comprising elements of activity, shortness of breath, strength, ability to carry, and tiredness/fatigue. Each component is rated on a scale of 1 to 5, and a patient's PAWS score is the sum of all components, thus scores range from 5 to 25 and a higher score indicates more perceived weakness. Previously, we found that PAWS correlates with respiratory muscle strength (maximal inspiratory pressure (MIP)), and peak inspiratory flow rate (PIFR). We assessed whether PAWS could predict patients' ability to use a dry powder inhaler (DPI) effectively.

Methods:After IRB approval, 70 adult patients completed the PAWS questionnaire followed by MIP and MEP measurements with a MicroRPM meter and PIFR with an In‐Check DIAL set at R2 resistance. The average of 3 consecutive measurements was recorded. Data were analyzed by Pearson correlation and p value <0.05 was significant with Bonferroni's correction for multiple comparisons.

Results:PAWS showed significant negative linear correlation with MIP (r = ‐0.5818, p < 0.05), and PIFR (r = ‐0.6054, p < 0.05). PAWS of <14 was associated with PIFR >40 L/min.

Conclusion:PAWS showed negative linear correlation with MIP and PIFR. A PAWS of <14 predicted an adequate PIFR with medium resistance DPIs, such as an Ellipta or a Diskus inhaler, suggesting that PAWS could be a rapid subjective screening tool to assess patients' ability to use DPIs.

G. 08 Excellent Colloidal Stability of Rugose Lipid Particles in Established and New Propellants

Zahra Minootan,¹Hui Wang,¹Nicholas Carrigy,²Kellisa Lachacz,²David Lechuga‐Ballesteros,²Andrew Martin,¹and Reinhard Vehring¹

¹Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada.

²Inhalation Product Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Durham, NC, USA.

Part of the effort to reduce the carbon footprint is associated with propellants used in medical devices such as pressurized metered dose inhalers (pMDIs). For example, HFO‐1234ze is a low global warming potential (GWP) propellant currently in research and development to investigate its suitability as a replacement for established pMDI propellants. As most drugs are not soluble in propellants and do not suspend well, engineered porous microparticles have been used to form uniform suspensions in pMDIs [1]. For this purpose, novel DSPC (distearoyl phosphatidylcholine) rugose lipid particles (RLPs) have recently been introduced and their simple manufacturing process eliminates the need for pore‐forming agents [2].

In this study, pMDI canisters containing spray‐dried RLPs were filled with either established HFA propellants (HFA‐227ea and HFA‐134a) or the new propellant HFO‐1234ze. Canisters were stored at 40°C for up to three months. Colloidal stability of RLPs, evaluated by shadowgraphic imaging method, was excellent in all three propellants. Also, particles were physically stable in the propellants, retaining their highly rugose morphology. This study suggests the co‐suspension formulation approach is a promising option for developing green pMDI products.

References

[1] Vehring, R., Lechuga‐Ballesteros, D., Joshi, V et al. (2012), Langmuir 28, 15015‐23. doi:10.1021/la302281n

[2] Wang, H., Ordoubadi, M., Connaughton, P et al. (2022), Pharm Res 39, 805–823. doi:10.1007/s11095‐022‐03242‐w

G. 09 Innovative Soft Mist Nebulizers Enable an Easy‐to‐Use Drug Delivery for Targeting the Upper and the Lower Respiratory Tract

Marie‐Christine Klein,Caroline Hoffmann, Horst Zimmer, and Andreas Bilstein

Ursatec GmbH, Tholey, Germany.

Targeted drug delivery is key to exploit state‐of‐the‐art drug developments to their full potential. In order to support the targeted drug delivery, application devices combine a number of characteristics to ensure performance and patient's safety: a precise dosage, the technical prerequisite to reach the target of interest and a good patient adherence to the treatment protocol, which goes hand in hand with a device that is easy‐to‐use and not too explanation‐extensive. Ursatec and its partners develop soft mist nebulizers, that are characterized by a very fine and innovative spray mist embedded in a standard inhalation device or a nasal spray and therefore ensure an easy and safe usability profile. The devices are designed to suit different treatment sites. The fine mist nasal spray allows some coverage of the olfactory region and supports nose‐to‐brain applications, which can be a powerful tool to bypass the blood‐brain barrier. Moreover, by using different spray nozzle units in the head of the device, it is possible to generate different droplet sizes, which enables laryngeal and pulmonary deposition. At the same time no propellants (CFCs, HFAs or VOCs) are being used. Furthermore, the devices allow innovative drug formulation without the use of preservatives, since its sophisticated construction prevents microbiological contamination.

G. 10 NO ABSTRACT

G. 11 Unsteady Aerosol Measurements: Assessing the eFlow Aerosol Bolus

Benjamin Heine,Carolin Hein, Amed Njoya, and Stefan Seemann

PARI Pharma GmbH, Gräfelfing, Germany.

Two experimental methods to quantify the aerosol bolus of nebulizers with an aerosol storage chamber were developed, applied to PARI's eFlow Technology nebulizer and compared to Philipps InnoSpire Go (no aerosol storage).

A delivered dose measurement setup was expanded such, that the cumulated aerosol output over a breathing cycle could be measured. Derivation of this function resulted in the unsteady aerosol output rate (uAOR) where the aerosol bolus could be quantified. To investigate the unsteady development of the droplet size over a cycle (uMMD) a breath simulator was attached to the nebulizer while a laser diffractor measured the droplet size. Using uAOR and unsteady respirable fraction the respirable dose over one cycle (uRD) was derived.

Using the new method, the unsteady behavior of AOR and MMD was observed. While for the eFlow the uAOR peak value reached more than 300% (t = 500ms) of the continuously operated aerosol head's total output rate, only 125% were observed for the InnoSpire Go, demonstrating the strong bolus effect of systems using an aerosol storage chamber. During storage, coalescence leads to a 5% increase in MMD, resulting in a slightly reduced uRD with a peak value of 1750 mg/min. Using the InnoSpire Go, the peak value in uRD was 440 mg/min, hence 4 times lower than for the eFlow.

The use of a storage chamber offers the advantage of high RDDRs while a strong aerosol bolus is delivered to the patient at the beginning of inhalation.

G. 12 Facing Unmet Needs in Nebulized Therapies – the Journey of Kolibri

Carolina Dantas,and Ulf Krueger

Pulmotree Medical GmbH, Munich, Germany.

Introduction:Despite constant evolution, nebulized therapies continue to face important unmet needs. First, uncontrolled particle deposition, related to aerosol characteristics and the quality of the breathing manoeuvre, remains a limitation for effective targeted drug delivery into the lungs. Additionally, difficulties with monitoring and high variability in results increase the risks associated with clinical development. Finally, the patient's needs: improving the nebulizer´s usability, providing feedback for correct use, and ultimately reducing the disease burden.

Results:Focused on these challenges, Pulmotree developed Kolibri, a connected mesh nebulizer, whose technology is hereby described. Kolibri was created to be customized to each drug product, clinical indication and strategy. Optimal particles are produced by a tailored aerosol generator, and an inhalation control technology guides the patient to achieve the ideal inspiration manoeuvre, according to the targeted deposition area. Remote monitoring is possible due to automatic data transfer about inhalation, adherence and device status, while using a centralized interface platform and a mobile app. To enhance the patient's experience and usability, Kolibri is a portable device of simple assembly and cleaning, with a modern design that alleviates the treatment's onus.

Conclusion:Kolibri´s technology can provide solutions to unanswered challenges and in drive progress in nebulized therapies.

G. 13 NO ABSTRACT

G. 14 Usability and Inspiratory Flow Profiles of Patients with Pulmonary Hypertension Inhaling with a Novel Soft Mist Inhaler (SMI)

Bernhard Müllinger,¹Nicolas Buchmann,¹Robin Huedepohl,²and Gerian Prins³

¹Resyca GmbH, Munich, Germany.

²Harvey Medical Consulting Ltd, Cambridge, United Kingdom.

³Resyca BV, Enschede, Netherlands.

Good inhaler technique is key for an effective inhalation treatment and reduction of critical inhaler errors. Recent publications show that treatment effects can be improved if inhalers are easy to use and support patients with pulmonary hypertension in their inhaler technique. The aims of this study were the usability assessment of the soft mist inhaler (PFSI, Resyca, Netherlands), evaluation of inhalation flow profiles, and observation of ability to safely use the inhaler.

A formative human factors study was conducted in 15 patients with pulmonary hypertension, functional class II, III, or IV, thereof 3 patients with handling difficulties like scleroderma. User device interaction, inhaler flow profiles and use tasks were assessed for all patients.

All patients were able to inhale with an inhalation duration of 3.6 seconds. Median inhalation duration was 6.0 seconds, median inhaled volume was 1.1 L and median flow rate was 13.5 L/min. Inhalation flow profiles for 10th and 90th percentile was 8.8 and 23.6 L/min respectively.

87% of patients rated the inhalation treatment with several breaths and performing of required use steps as comfortable. 73% assembled the device correctly with instructions for use.

Results from this human factors study show that patients comfortably inhaled slow and deep with the high resistance soft mist inhaler. The intuitive device design supported patients in applying steps to good inhalation technique.

References

[1] Gessler, T. et al. Pulmonary Circulation 2017; 7(2) 505‐513

G. 15 Assessment of Aerosol Delivery during Volume Control and Pressure Support Ventilation

Barry Murphy,Mary Joyce, and Ronan MacLoughlin

Aerogen, Galway, Ireland.

Introduction:Aerosol drug delivery is often prescribed concurrently with invasive mechanical ventilation in the treatment of respiratory disease. Ventilator mode selection is based on the condition of the patient and is an important factor in improving patient outcome. Here we assessed the aerosolised delivery of Salbutamol during different ventilation modes to a simulated adult patient.

Methods:5000μg of Salbutamol was aerosolised using the Aerogen Solo nebuliser (Aerogen, Ireland). The nebuliser was placed at the patient side of the wye within a dual limb breathing circuit with active humidification (RT111, MR850 F&P, NZ). Two modes, volume control (VCV) and pressure support ventilation (PSV), were selected using a ventilator (Bellavista, Vyaire Medical, US) with a collection filter placed between the ETT and test lung (IMT Analytics, Switzerland). Ventilation settings for VCV were RR15, Vt:500mL I:E 1:1 and PEEP 5 mbar Pressure support 8 mbar I:E 1:1 for PSV. The quantity of drug available at the lung was determined using UV spectrophotometry at 276nm.

Results:Results are expressed as a percentage of the nominal dose placed in the nebuliser medication cup. For VCV 26.42 ± 0.95% of Salbutamol was available at the end of the endotracheal tube while with PSV 18.06 ± 0.60% was available, p = 0.00.

Conclusions:This study found that ventilation mode had a significant impact on aerosol drug delivery with VCV facilitating delivery of a larger dose during normal adult ventilation.

H. Inhaled Biotherapeutics

H. 01 Lentiviral Vector Based Gene Therapy for Respiratory Diseases

Eric WFW Alton,^1,2A Christopher Boyd,^1,3Jane C Davies,^1,2Deborah Gill,^1,4Uta Griesenbach,^1,2Stephen Hyde,^1,4and Gerry McLachlan^1,5

¹UK Respiratory Gene Therapy Consortium, London, United Kingdom.

²National Heart and Lung Institute, Imperial College, London, United Kingdom.

³Edinburgh, Edinburgh, United Kingdom.

⁴Oxford, Oxford, United Kingdom.

⁵Roslin, Edinburgh, United Kingdom.

Inefficient pulmonary gene transfer is a major factor that has limited the development of a clinically suitable gene therapy for cystic fibrosis (CF). While Sendai virus‐mediated gene transfer to airway epithelial cells is highly efficient, the short duration of expression and strong immunogenicity have rendered Sendai virus‐based vectors ineffective for CF gene therapy.

We have developed a lentiviral vector pseudotyped with the Sendai virus F and HN envelope proteins (rSIV.F/HN), to deliver a normal copy of the CFTR cDNA into the genomic DNA of airway epithelial cells of patients with CF.

With this vector, we have demonstrated efficient and lifelong transduction in the airways of multiple species. In addition it shows the ability to redose, efficiently transduces all of the major airway epithelial cell types and demonstrates restoration of CFTR function in intestinal organoids.

A mutation‐agnostic gene therapy for CF may benefit patients who are genetically ineligible for CFTR modulator therapy, as well as eligible patients who experience adverse reactions or suboptimal responses to this therapy.

The above data support the progression of BI 3720931 towards the clinic, with a first‐in‐human trial in final preparation for regulatory submission.

H. 02 Topical Application of Nebulized Polyclonal IgG: from Proof of Concept into Clinical Development

Anna Schnell,^1,2Joseph Bain,³Ilka Schulze,^4,2and Alexander Schaub^5,2

¹CLS Behring, Bern, Switzerland.

²Swiss Institute for Translational Medicine, sitem‐insel, Bern, Switzerland.

³CSL Behring Innovation, Marburg, Germany.

⁴CSL Limited, Bern, Switzerland.

⁵CSL Behring, Bern, Switzerland.

The path from preclinical into the clinical development of a nebulized biological is neither straightforward nor well‐defined. CSL787 is a medicine made from human plasma‐derived immunoglobulins and intended as a potential treatment for Non‐cystic Fibrosis Bronchiectasis (NCFB). It is developed as a combination product with a mesh nebulizer. Different preclinical models and application routes were chosen to cover the broad features of the mode of action of immunoglobulins and the characteristics of airway disease. Due to the lack of standard model systems, the translatability into humans with NCFB is challenging and knowledge about disease biomarkers and the fate of IgG in diseased airways is scarce. With a summary of our published preclinical work^{1, 2, 3}insights into challenges, and the clinical development status, we would like to share our experience and contribute to a better understanding of inhaled immunoglobulin therapies.

References

[1] Koernig, S., Campbell, I.K., Schaub, A. et al. (2019). Mucosal Immunology 12, 1013‐1024. doi: 10.1038/s41385‐019‐0167‐z

[2] Vonarburg, C., Loetscher, M., Spycher, M.O. et al. (2019). Respiratory Research 20, 99. doi: 10.1186/s12931‐019‐1057‐3

[3] Schnell, A., Davrandi, M., Saxenhofer, M. et al. (2022). The Journal of allergy and clinical immunology 149, 2105–2115. doi: 10.1016/j.jaci.2021.12.778

H. 03 From Antifibrotic to Antiviral Therapy

Elisabeth Zeisberg

University of Goettingen, Göttingen, Germany.

Bacteria have been fighting viruses successfully for 3 billion years. For that purpose, they have developed the CRISPR/Cas 9 system against DNA viruses and the CRISPR/Cas13 system against RNA viruses. This talk will be about using the CRSIPR/Cas13 system to develop antiviral therapies for humans against RNA viruses like SARS‐CoV‐2. It will cover both the potential as well as the challenges of this system as an antiviral platform technology.

H. 04 Evaluation of the Efficacy of an Innovative Triparatopic Anti‐IL‐13 VhHs in a Murine Preclinical Model of Respiratory Inflammation

Nathalie Wauthoz,¹Philippe Gevenois,¹Elena Menchi,¹Coralie Lambot,¹Thami Sebti,²and Karim Amighi¹

¹Université libre de Bruxelles, Brussels, Belgium.²Laboratoires S.M.B., Brussels, Belgium.

This study evaluatedin vivoactivity of a developed triparatopic V_HH targeting human IL‐13 (“the trimer”)^[1]. The preclinical model of respiratory inflammation was adapted from^[2]by delivering 2*25 μg human IL‐13 in mouse lung. Then, the trimer was delivered at different doses (0.3, 1.0, 3.0, 10 or 30 mg/kg/group) by endotracheal route. The airway resistance (Rs, Rn and G) and elastance (Ers and H) parameters were determined by forced oscillation technique. For that, a dose‐response study to methacholine from 0.0 to 50.0 mg/ml was carried out to compare the treated groups to a positive disease control group (no treatment) and to a negative treatment control group (VHH unrelated to IL‐13). In parallel, several inflammatory mediators (eotaxin‐1, periostin, MMP9, IgE) and cells were quantified. The pulmonary administration of the trimer significantly and specifically improved the lung condition for parameters that were significantly altered by human IL‐13. From 0.3 mg/kg up to 1.0 mg/kg, a dose‐dependent efficacy was observed. However, from 3.0 mg/kg to 10 mg/kg, a lung remodelling and inflammation was observed, certainly due to a immune effect from the foreing (llama) nature of the trimer.

References

[1] Gevenois, P., De Pauw, P., Schoonooghe, S., et al. (2021). Journal of Immunology 207 (10) 2608‐2620. DOI: 10.4049/jimmunol.2100250

[2] Blanchard, C., Mishra, A., Saito‐Akei, H. et al. (2005). Clinical and Experimental Allergy 35, 1096–1103. DOI: 10.1111/j.1365‐2222.2005.02299.x

H. 05 NO ABSTRACT

H. 06 Spray‐Dried Protein Particles for Dry Powder Inhalation

Johanna Dieplinger,¹Andreina Isabel Afonso Urich,¹Joana T. Pinto,¹Michael Dekner,²and Amrit Paudel³

¹Research Center Pharmaceutical Engineering GmbH, Graz, Austria.

²Takeda Manufacturing Austria AG, Vienna, Austria.

³Institute of Process and Particle Engineering, Technical University of Graz, Graz, Austria.

When targeting respiratory diseases, direct delivery of APIs to the lungs presents various advantages, i.e., lower doses, etc. Dry Powder Inhalers (DPIs) are easy to use and deliver drug aerosols directly to the lungs. To reach them, it is generally agreed that drug particles must present an aerodynamic diameter da(1) between 1–5 μm. Spray‐drying(SD) allows production of micron‐sized particles for inhalation. L‐leucine (Leu) is often reported as acceptable excipient to yield flowable particles without carriers(2). This study discusses the engineering of inhalable particles of a protein drug in presence of Leu via SD. Therefore, miniaturized screening (MS) was performed. Based on that, a Design of Experiment (DoE) was carried out on a Büchi‐290 SD. Before the DoE(5 fac, 2 lev), MS was performed to select an appropriate Leu ratio. Inlet temperature(110‐150°C), pump setting(2‐4 mL/min), aspirator setting(80‐100%) and airflow(400‐800 L/min) and the particle size span, da, aggregates, yield, and moisture content were studied. Higher Leu content in MS increased aggregation, showing highest for 20%w/w. In SD, lowest aggregation was observed with 10% w/w Leu, suggesting more Leu is beneficial for protein stability and a yield of 87‐93%. A higher airflow in the DoE led to particles with adequate size.

References

[1] Alhajj, N, O'Reilly, N J, & Cathcart, H(2021).Drug Discov, 26. doi:10.1016/j.drudis.2021.04.009

[2] De Boer, A H et al(2002).Int J Pharm 249, 219–231.doi:10.1016/S0378‐5173(02)00526‐4

H. 07 siRNA‐Mediated Gene Silencing by Nebulised Lipid‐Based Delivery Vectors:In vitroPerformance in A549 Cells

Michael Neary,^1,2Abina Crean,^1,2Piotr Kowalski,¹and Katie Ryan^1,2

¹School of Pharmacy, University College Cork, Cork, Ireland.

²SSPC, the SFI Research Centre for Pharmaceuticals, Cork, Ireland.

Gene silencing by siRNA offers great potential in the treatment of many respiratory diseases. Efficient delivery of siRNA into the lungs, however, remains a challenge. We aimed to investigate the effectiveness of nebulisation for delivery of siRNA‐containing liposomes and lipid nanoparticles (LNPs) and its resultant impact on their physical properties and in vitro performance. Microfluidic mixing was used to produce several liposome and LNP‐based formulations which contained siRNA targeting luciferase as a reporter gene. Samples were nebulised using an Aerogen Pro vibrating mesh nebuliser and particle size, zeta potential and siRNA encapsulation % were measured prior to and post nebulisation. Cell viability and luciferase knockdown efficiency were also assessed in A549 lung cells. Particle size significantly increased post nebulisation particularly in PEGylated formulations. siRNA encapsulation % was unchanged by nebulisation in the liposomes as it remained >99% but was reduced by approximately half in the LNPs. Cell viability post treatment with all nebulized samples was high indicating their cytocompatibility. Generally, luciferase knockdown rates were altered after nebulising. In the nebulised LNPs however, luciferase knockdown remained high demonstrating excellent transfection efficiency. In summary, our data demonstrates the potential of nebulisation for pulmonary administration of siRNA, however the choice of lipid delivery vector is an important consideration.

H. 08 Aspherical, Nanostructured Microparticles for Delivery of CRISPR Cas to the Lungs

Jannis Fries,and Marc Schneider

Saarland University, Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarbrücken, Germany.

This study aims to describe a microparticulate, nanostructured carrier system designed for pulmonary application that possesses three key features:

The first feature is the actual active pharmaceutical ingredient (API), a CRISPR Cas, which is a novel and revolutionary tool for gene editing with an incredible potential for future therapies. At present, there is a lack of efficient and non‐toxic carrier systems for CRISPR Cas with a potential applicability. The second feature of the carrier system is the rod‐like shape of the microparticles. Regarding pulmonary application of drugs, aspherical, rod‐like microparticles are a promising approach that combines a greater deposition efficiency in deeper lungs and a bigger loading capacity of drugs in comparison to their spherical counterparts. Additionally, the elongated‐shaped particles enables kinetically controlled uptake by alveolar macrophages, which play a crucial role in cancerous and inflammatory diseases. Using a layer‐by‐layer method for the stabilization of the particles allows the delivery of a broad range of genetic material due to the negatively charged phosphate backbone of DNA, including plasmids coding for a CRISPR Cas. The third feature of the carrier system is the nanostructure achieved by calcium phosphate nanoparticles (CaP). These particles are a well‐known, non‐viral vector for transfection, which are considered to be highly biocompatible.

H. 09 Using Aspherical Nanostructured Microparticles to Target Alveolar Macrophages in Chronic Lung Diseases

Elena Haettig,and Marc Schneider

Saarland University, Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarbruecken, Germany.

Acute inflammation in the lung tissue is mostly a beneficial process to combat lung infections or injuries. Chronic lung inflammation on the other hand is associated with chronic respiratory diseases like COPD and asthma and induces remodeling of lung tissue and subsequent decrease of functionality in the affected tissues. One of the immune cells evoked in the aforementioned process are alveolar macrophages (AM) that can be found deep within the respiratory tract. Inhibition of AMs through local inhalation therapy could yield a more targeted approach causing fewer side effects and a lower necessary API amount.

To reach these alveolar macrophages, a nanostructured aspherical microparticle was developed with a template‐assisted approach. Mesoporous silica nanoparticles (mSNPs) are used to provide the framework and crosslinking with an LbL‐film stabilizes the nanoparticles in the rod‐like shape. By using an aspherically shaped microparticle, the intrinsic phagocytotic behavior of AM [1] is utilized to attain an uptake retention of the microparticles and subsequently of the API. SiRNA is used as an anti‐inflammatory drug to inhibit inflammatory pathways and incorporated in the LbL‐film. To achieve an improved endosomal escape of the siRNA, chloroquine or amantadine are loaded into the mSNPs.

References

[1] Mathaes R., Winter G., Besheer A. et al. (2014) Int J Pharm. 465, 159‐64. doi:10.1016/j.ijpharm.2014.02.037

H. 10 Inhalation of an Immunomodulatory Adjuvant to Treat Drug‐Resistant Bacterial Pneumonia

Jeoffrey Pardessus,^1,2Lilou Guillot,^1,2Alexie Mayor,^1,2Maria Cabrera,^1,2Déborah Le Pennec,^1,2Ronan MacLoughlin,³Mara Baldry,^4,5Linda B.S. Aulin,⁶Robin Michelet,⁶Charlotte Kloft,⁶Jean‐Claude Sirard,^4,5and Nathalie Heuzé‐Vourc'h^1,2

¹INSERM, Research Center for Respiratory Diseases, Tours, France.

²University of Tours, Tours, France.

³Research and Development, Science and Emerging Technologies, Aerogen, Galway, Ireland.

⁴INSERM, Center for Infection and Immunity of Lille, U1019, Lille, France.

⁵Institut Pasteur, Lille, France.

⁶Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany.

Bacterial pneumonia is a major cause of morbidity and mortality worldwide. Antibiotics constitute the standard of care but face the emergence of antimicrobial resistance and curative failure. Moreover, antimicrobial agents are often administered orally or intravenously (I.V) regardless the site of infection, as they are expected to distribute to this site. Inhalation is the obvious way of matching the delivery route to the target's location to treat pneumonia, is suitable for protein therapeutics1, and usually improves the therapeutic index of drugs.

Here, we investigated inhalation of FLAMOD, a Toll‐Like receptor 5 agonist, which enhances airway innate immune defenses and improves the therapeutic outcome relative to antibiotic alone during pneumonia. Evidences showing that the FLAMOD‐mediated immune protective effectors are regionally compartmentalized in the lungs further support the relevance of developing inhalation.

FLAMOD has been delivered either by nebulization into the lungs of macaques (Aerogen Solo^®) or I.V. Blood and bronchoalveolar lavages were collected to analyze FLAMOD pharmacokinetics and pharmacodynamics, in the systemic and local compartments.

Our results demonstrate that inhalation resulted in a local response, associated with a transient and 10‐fold lower systemic response, as compared to the I.V route. Overall, our findings indicate that inhalation is paramount to achieve and restrict the immunomodulatory activity of FLAMOD into the lung compartment.

H. 11 Mucin Remodeling for the Development of Novel Antiviral or Mucolytic Inhalable Biomaterials

Cosmin Butnarasu,¹Robyn Diehn,¹Tatyana Povolotsky,¹Fabian Nußhardt,¹Jolly Thomas,¹Shoresh Memkhezri,¹Nico Boback,¹Sonja Visentin,²and Daniel Lauster¹

¹Freie Universität, Institut für Chemie und Biochemie, AG Biophysikalische Chemie, Berlin, Germany.

²Department of Molecular Biotechnology and Health Science, University of Turin, Turin, Italy.

Mucins are large glycoproteins vital for protecting the airways by forming a mucus layer that traps and clears pathogens. Our growing understanding of their structure and functionalities has proposed mucins as a source for innovative functional biomaterials. Previously, we have been able to synthesize mucin nanoparticles that showed excellent drug loading capacity [1]. Most recently, it has been shown that mucin and mucin‐like materials are potential antiviral agents due to their ability to inhibit virus entry and replication[2].

In an attempt to expand the mucin toolkit, here we aim to develop mucin‐based antiviral or mucolytic nanomaterials to be administered by inhalation.

We obtain mucin‐derived peptides by either enzymatic digest, recombinant production or rational screening procedures. Functional peptide candidates will then be bioconjugated to polymeric scaffolds to achieve multivalent binding enhancement. After proving in vitro activity, the nanosystem will be formulated for inhalation administration to achieve a rapid onset of action and reduced systemic side effects.

If effective, the mucin‐derived nanosystem could lead to the development of novel therapies with broad applicability in respiratory viral infections or mucus‐related diseases.

References

[1] C. Butnarasu, P. Petrini, F. Bracotti, et al. (2022). Adv. Healthcare Mater. 11, 2200340, 10.1002/adhm.202200340

[2] C. Wardzala, A. M. Wood, D. M. Belnap, et al. (2022). ACS Cent. Sci. 8, 3, 351‐360, 10.1021/acscentsci.1c01369

H. 12 Pulmonary Nucleotide Transfection for Antiviral Therapy or Genome Editing

Achim Biesel,^1,2Brigitta Loretz,¹and Claus‐Michael Lehr^1,2

¹Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI), 66123 Saarbrücken, Germany.

²Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany.

The COVID‐19 pandemic brought mRNA drugs as novel therapeutics in the spotlight. While large investigations were made for parenteral applications of vehicles as lipid nanoparticles, their translation to pulmonary delivery is lagging behind – especially due to their insufficient stability upon aerosolization [1].

We demonstrated lipid‐polymeric nanoparticles (LPNs) consisting of a biocompatible polymer and cationic lipids [2]. These LPNs survived the shear stress of nebulization with a vibrating mesh nebulizer and largely remained their efficiency to transfect human lung epithelial cells (A549). A549 cells were further transfected with siRNA and mRNA, indicating the potential of the used carriers for dual delivery of small and large nucleotides, as needed for the delivery of CRISPR/Cas systems.

Overall, our hybrid carriers combine the high transfection efficiency of lipid formulations and the beneficial stability of polymeric vehicles, demonstrating their potency for pulmonary delivery of mRNA. Currently, we are further optimizing and characterizing these particles as vehicle for CRISPR/Cas, since this antiviral defense system is promising in the fight against respiratory viruses as SARS‐CoV‐2.

References

[1] Lokugamage, M.P., Vanover, D., Beyersdorf, J. et al. (2021). Nat Biomed Eng 5, 1059–1068.https://doi.org/10.1038/s41551-021-00786-x

[2] Kliesch, L., Delandre, S., Gabelmann, A. et al. (2022). Pharmaceutics 14(12), 2675;https://doi.org/10.3390/pharmaceutics14122675

H. 13 Characterisation of Extracellular Vesicle Delivery Using a Vibrating Mesh Nebulizer

Ciaran O Leime,¹Katie Gilligan,²Celine Larkin,²Gerry McCauley,²and Ronan MacLoughlin¹

¹Aerogen Limited, Galway, Ireland.

²OmniSpirant, Kilcolgan, Ireland.

Introduction:Inhaled Extracellular Vesicles (EVs) have been proposed as a method of targeted delivery for gene‐based therapies in the lung. Vibrating mesh nebulizers (VMN) have reportedly been the predominant nebuliser type used to deliver EV nanosuspensions given their ability to deliver large doses of therapy to the lung. We investigate the effect of basic formulation modifications on EVs aerosol characteristics when nebulised. These formulation modifications include PEGylation of EVs which improves their transport through the mucus layer of the lung.

Methods:Non loaded EVs suspended in saline were used. Combinations of PEGylated and non‐PEGylated EVs were assessed. Droplet size characterization was carried out using laser diffraction. Flow rate of the EV solution through the nebulizer was calculated in ml/min. An estimation of EV particles that successfully passed through the VMN (Aerogen Solo, Aerogen) was also calculated.

Results:PEGylated and non‐PEGylated EVs were successfully nebulized using VMN with acceptable aerosol output rates and droplet sizing to be inhaled efficiently into the lungs. Flow rate and VMD for both were comparable at approximately 0.5ml/min and 4μm respectively.

Conclusion:Both PEGylated and non‐PEGylated EVs can be successfully aerosolized using a VMN and can produce aerosol droplets suitable for lung‐based therapies. These results demonstrate that low dose PEGylation of EVs does not significantly impact the ability to aerosolize EVs.

H. 14 Assessment of the Aerosolised Delivery of Common Therapeutics to Neonatal Patients

Marc Mac Giolla Eain,Mary Joyce, and Ronan MacLoughlin

Aerogen Ltd, Galway, Ireland.

Introduction:Supplemental oxygen is commonly administered to neonatal patients. The addition of aerosol therapy concurrently allows the delivery of therapeutics for inhalation improving clinical outcomes. Here we assessed the aerosolised delivery of two commonly prescribed therapeutics to a simulated neonate patient receiving supplemental oxygen by means of an oxygen concentrator.

Methods:500μg Salbutamol and 250μg Budesonide were aerosolised using the Aerogen Solo nebuliser and Pro‐X controller (Aerogen, IRE) in combination with the Oxy6000 system (Bitoms GmbH, DE) and a nasal cannula (RCI Comfort flo, Flexicare Medical, UK) at 0.2 & 0.5LPM to a 3D‐printed anatomically correct head model of a 4‐month‐old. A neonatal breathing pattern (RR:40BPM, Vt:25mL, I:E:1:3) was generated using a breathing simulator (ASL5000 IngMar Medical, USA). The quantity of drug available at the level of the trachea was determined using UV spectrophotometry.

Results:Results are expressed as a percentage of the nominal dose placed in the nebulisers medication cup. At 0.2LPM, 0.74 ± 0.16% & 0.78 ± 0.45% of Salbutamol and Budesonide was available, while at 0.5LPM, 2.51 ± 0.37% & 2.29 ± 0.38% was available.

Conclusions:This study found that both therapeutics can be successfully nebulised and delivered at a variety of flow rates to a neonatal patient.

H. 15 Disordered Mesoporous Silica Particles as Emerging Platform to Deliver Biologic Molecules to the Lungs

Aura Rocío Hernández,^1,2Ekaterina Bogdanova,^1,2Jesús Enrique Campos Pacheco,^1,2Vitaly Kocherbitov,^1,2Adam Feiler,³Georgia Pilkington,³Mikael Ekström,⁴and Sabrina Valetti^1,2

¹Biofilms – Research Center for Biointerfaces (BRCB), Malmö, Sweden.

²Biomedical Science, Faculty of Health and Society, Malmö University, Malmö, Sweden.

³Nanologica AB, Södertälje, Sweden.

⁴Iconovo AB, Lund, Sweden.

Mesoporous silica particles (MSP)[1] have great potential as platforms for the pulmonary delivery of biologic molecules. This proof‐of‐concept work is focused on the demonstration use of disordered MSPs as an emerging carrier for dry powder inhalable formulation of proteins using lysozyme (LYS) as a model. During loading into the MSP, the LYS sorption capacity was shown to be directly proportional to the ionic strength of PBS buffer reaching a maximum loading capacity of 30% for 50 and 150 mM PBS. Desorption of LYS in simulated lung fluid showed a sustained release of LYS during the first ten hours reaching a plateau of 70% release of the initial encapsulated amount. The enzymatic activity after desorption of the LYS was demonstrated to be also high (71 ± 10%). In addition, the formulation of LYS in MSP was shown to generate a free‐flowing dry powder with a Fine Particle Fraction (FPF, d < 5 μm) of 73% of the delivered dose in an ICOone^®inhaler. This FPF is in the upper range of previously reported results for protein formulations (60‐80%)[2]. This proof‐of‐concept formulation thus demonstrates the possibility of encapsulation of a model protein, effective potential delivery to the lungs in high doses, and release with the high remaining enzymatic activity.

References

[1] Vallet‐Regí, M., Schüth, F., Lozano, D. (2022). Chem Soc Rev 51, 5365‐5451.doi:10.1039/D1CS00659B

[2] Adhikari, BR., Gordon KC., Das SC. (2022). Adv Drug Del Rev 189:114468.doi:10.1016/j.addr.2022.114468

H. 16 NO ABSTRACT

H. 17 OM‐85 Direct Airway Administration to Prevent Viral Infections and Its Potential Use as Inhalation Therapy

Anne Vaslin Chessex,Claire Abadie, and Christian Pasquali

OM Pharma, Meyrin, Switzerland.

OM‐85, a standardized soluble bacterial lysate is widely used via oral route to prevent recurrences of respiratory infections.

In an RSV model, OM‐85 airway administration in mice prior to viral instillation prevented infection, resulting in reduction of viral replication associated with less weight loss and lung inflammation. In contrast to oral intake, these protective effects were dose and time dependent (Antunes et al. 2022) and confirmed previous work in asthma models (Pivniouk et al 2021). In Rhinovirus models, similar protective effects were observed. Finally, assessing OM‐85 using upper vs lower lung airway administration resulted in a significant reduction of morbidity and mortality in animals infected with influenza and superinfected with bacterial infections. In this model, reduced viral titers and in lung tissue bacterial infection was robustly obtained. Again, these protective effects were dose dependent and showed higher efficacy as compared to oral administration.

To further support the potential development of a new route of administration via inhalation, feasibility tests were conducted using liquid formulation of OM‐85 to determine particle size and usability of vibrating mesh nebulizers.

The mean particle diameters measured by diffraction spectroscopy were in the inhalable size range for all nebulizers investigated. Results showed that OM‐85 liquid concentrate is compatible with liquid aerosolization paving the way for further development.

H. 18 Development of a Nebulization Approach of Fusion Inhibitory Lipopeptides to Protect Nonhuman Primates against Lethal Respiratory Nipah Virus Infection

Olivier Reynard,¹Mathieu Iampietro,¹Claire Dumont,¹Sandrine Le Guellec,²Marie Moroso,³Stéphanie Durand,¹Elise Brisebard,⁴Cyrille Mathieu,¹Christopher Alabi,⁵Laurent Vecellio,⁶Maria Cabrera,⁶Déborah Le Pennec,⁶Mateo Porotto,⁷Anne Moscona^7,8,9, and Branka Horvat¹

¹CIRI, Centre International de Recherche en Infectiologie, INSERM U1111, CNRS, UMR5308, Université Claude Bernard Lyon 1, École Normale Supérieure de Lyon, 21 Avenue Tony Garnier, Lyon, France.

²DTF‐Aerodrug, R&D aerosoltherapy department of DTF medical (Saint Etienne, France), Faculté de médecine, Université de Tours, Tours, France.

³INSERM‐Laboratoire P4 Jean Mérieux, Lyon, France.

⁴UMR703, PAnTher APEX, INRAE/Oniris, Nantes, France.

⁵Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

⁶CEPR, INSERM U1100, Université de Tours, Tours, France.

⁷Center for Host‐Pathogen Interaction, Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, USA.

⁸Department of Microbiology and Immunology, Columbia University Vagelos College of Physicians & Surgeons, New York, USA.

⁹Department of Physiology & Cellular Biophysics, Columbia University Vagelos College of Physicians & Surgeons, New York, USA.

Nipah virus (NiV) is a fatal zoonotic paramyxovirus that can be transmitted from person to person by the respiratory route, causing encephalitis and severe respiratory disease with an average fatality rate of 75%. There is currently no approved treatment or vaccine for this serious emerging infection. A lipopeptide‐based fusion inhibitor strategy has been developed and previously evaluated for efficacy against NiV in vitro and in vivo in hamster and non‐human primate (NHP) models using intratracheal administration. For human administration by inhalation, a mesh nebuliser and face mask were developed and evaluated in an African green monkey model that mimics human NiV infection. Three consecutive aerosolized doses of the lipopeptide (4mg/kg/24h) resulted in a uniform distribution of the lipopeptide in the respiratory tract. Nebulisation of the peptide was not associated with any apparent allergic reaction, toxicity or adverse haematological and biochemical effects. Peptide treatment significantly delayed lethality in monkeys following intratracheal NiV challenge. The results support the feasibility of nebulisation of antiviral lipopeptides for emergency response to NiV outbreaks. Aerosol delivery of fusion‐inhibiting peptides may be used to protect against NiV and other airborne viruses that use the class 1 fusion mechanism for entry.

The study was funded by ANR ASTRID‐Maturation (Nebunipastop project).

H. 19 Installing Covalent Depots of Biologicals on Lung Tissue

Max Kappus,Niklas Hauptstein, Marcus Gutmann, Tessa Lühmann, Theresa Brand, Kristina Lorenz, and Lorenz Meinel

University of Wuerzburg, 97074 Wuerzburg, Germany.

Lasting lung depots for biologicals are a technological gap with immediate application for the treatment of local lung diseases. We are enzymatically establishing covalent depots for biologics on lung surfaces following inhalation. The enzyme is a transglutaminase (TG), catalyzing an iso‐peptide bond between the γ‐carboxy group of glutamine (e.g. as present on the extracellular matrix or mucus component) and the ɛ‐amino group of lysine (e.g. as present on the biologic). To verify that the system is compatible with the generally available jet nebulizers, the aerodynamic diameter was tested using an Andreasen Cascade Impactor. The size distribution was between 0.7 and 3.3 μm, allowing drug delivery to the deep lung. The activity after nebulization of both the enzyme formulation and the designed biological were investigated using an Impinger followed by cell and fluorescence‐based assays. Although the transglutaminase activity decreased approximately 30 %, it still is sufficient for enzymatic immobilization. The biological showed no significant loss of activity.

H. 20 Mucosal Administration of Anti‐bacterial Antibody Provides Long‐term Cross‐Protection against Pseudomonas aeruginosa Respiratory Infection

Aubin Pitiot,^1,2Marion Ferreira^1,2,3, Christelle Parent,^1,2Chloé Boisseau,^1,2Mélanie Cortes,^1,2Laura Bouvart^1,2,3, Christophe Paget,^1,2Nathalie Heuzé‐Vourc'h,^1,2andThomas Sécher^1,2

¹INSERM, Tours, France.

²University of Tours, Tours, France.

³CHU of Tours, Tours, France.

Bacterial respiratory infections are one of the leading causes of mortality worldwide. Therapeutic antibodies (Ab) have proven to be a valuable tool in the clinical armamentarium. Anti‐infective Ab are usually administered parentally and rely on direct pathogen neutralization. Their potency is often suboptimal, as they poorly exposed airways where the bacteria is located. We previously showed that local delivery of antibacterial Ab directly into the respiratory tract offers a suitable approach to improve their therapeutic index. Using a mouse model of Pseudomonas aeruginosa infection, we found that an Ab delivered through the airways harnessed genuine innate and adaptive immune responses to provide long‐term immunity, protecting against a secondary bacterial infection.

In vitro antigen‐presenting cells stimulation assay, as well as in vivo bacterial challenges and serum transfer experiments indicate an essential contribution of immunes complexes, formed between the Ab and the pathogen, in the induction of a sustained and protective antibacterial humoral response. Interestingly, the long‐lasting response protected partially against secondary infections with heterologous P. aeruginosa strains. Overall, our findings suggest that mucosal delivery of Ab promotes rapid pathogen containment and provides long‐term protection against secondary infection. This novel modality associated with anti‐infective antibody opens new perspectives for the treatment of respiratory infections.

H. 21 Dose Determination of Aerosolized Fluorescently Tagged Oligonucleotides ex vivoin Sprague Dawley Rat Lung Tissue

Peter Jaques,Christopher Stutz, Tyler Price, Joseph Georges, Emily Torres, Allie Tornes, Michael Stonerook, and Mark Perry

AmplifyBio, West Jefferson, USA.

Fluorescently tagged test‐articles have been used to track biodistribution in animals and humans (1). Since delivery of aerosolized therapeutics by inhalation can more effectively treat some diseases, we developed an approach to deliver and visualize fluorescently tagged oligonucleotide particles in live rodents (2). Initially, the signal generated by a whole‐body spectrometer (IVIS Spectrum^®) was correlated to known masses of fluorescent particles delivered onto glass fiber filters. The signal will then be used to predict unknown masses of oligonucleotides tagged with fluorophore. The tagged‐oligonucleotide particles will be directly deposited onto and injected into rodent lung tissue and measured by the IVIS. A filter‐based calibration factor will be used to estimate the deposited mass, which will be validated by PCR. The results show that from 0.2 to 20 mg of fluorescent test article there was a linear relationship of fluorescence to mass. The fluorescent oligonucleotide mass results and that infused onto the rodent lung tissue will be presented. The results from this study will guide the research of a follow‐up study, designed to deliver, and measure the mass of tagged oligonucleotide aerosol in the lungs of live rodents using the whole body IVIS spectrometer.

References

[1] Aspinall, R., Prentice, A.M. and Ngom, P.T. (2011). PLOS ONE 6(6), 1‐7. doi:10.1371/journal.pone.0020812.

[2] Patton, J.S. Brain, J.D. Davies, L.A., et al. (2010). J Aerosol Med & Pulm Drug Delivery, 23, S71‐S87.

H. 22 NO ABSTRACT

H. 23 An Increase to Medium Scale Production of a Spray Dried Inhalable Tuberculosis Vaccine Using a Laboratory Scale Spray Dryer

Maximilian Aisenstat,¹Zahra Minootan,¹Joseph McCollum,²Mani Ordoubadi,¹Hui Wang,¹Kelvin Duong,¹Scott Tavernini,¹Alana Gerhardt,²Andrew Martin,¹Christopher Fox,²and Reinhard Vehring¹

¹University of Alberta, Edmonton, Canada.

²The Access to Advanced Health Institute (AAHI), Seattle, USA.

The production process for a spray dried inhalable tuberculosis GLA+SE and ID93 vaccine¹was optimized, leading to a tenfold increase in production rate. The process model²for a custom research spray dryer was used to maximize production rate while maintaining the moisture content, a predictor for stability. The increase from 3.6 g/hr to 36 g/hr was made by doubling the feed concentration and quintupling the feed rate. The drying gas flow rate and temperature were increased such that the predicted outlet conditions of 7% RH and temperature below 50°C were maintained for both processes. Using this technique, small scale production of this vaccine was increased to medium scale, allowing a benchtop method to be viable for animal or toxicology studies. A total of 97.6 g vaccine powder was produced in a 6 hour‐spray drying run with a yield of 49%. Post‐scale‐up powder was compared to the pre‐scale‐up powder. Morphology and solid phase were maintained on scale‐up. Squalene, GLA, and ID93 content were maintained on spray drying for both pre‐ and post‐scale‐up powders. Upon actuation from a DPI, emitted dose increased from 79 ± 19% to 100 ± 2% on scale up, and lung dose was maintained at 16 ± 2%. Moisture content increased from 2.35 ± 0.07% to 2.6 ± 0.2%.

References

[1] Gomez, M., McCollum, J., Wang, H et al. (2021). Vaccine 39, 5025‐5036. doi:10.1016/j.vaccine.2021.07.002

[2] Ivey, J., Vehring, R. (2010). Comput Chem Eng 34, 1036‐1040. doi:10.1016/j.compchemeng.2010.02.031

H. 24 Exploiting the Role of Ionizable Lipids in Lung Delivery of RNAs through Hybrid Lipid–Polymer Nanoparticles

Gabriella Costabile,¹Susy Brusco,¹Ersilia Villano,¹Agnese Miro,¹Fabiana Quaglia,¹Ivana d'Angelo,²andFrancesca Ungaro¹

¹Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.

²Di.S.T.A.Bi.F., University of Campania Luigi Vanvitelli, Caserta, Italy.

Inhalable hybrid lipid‐polymer nanoparticles (hNPs) comprising a poly(lactide‐co‐glycolide) (PLGA) core and a dipalmitoylphosphatidylcholine (DPPC) shell are very promising carries for RNA delivery to the lungs.1,2 Nonetheless, one of the main challenges to fully exploit the potential of inhaled RNA therapeutics is the need of a delivery platform able to escape degradation in the acidic endo/lysosomal compartment. The crucial role played by ionizable lipids in that sense has been demonstrated in marketed RNA‐loaded lipid nanoparticles, as Onpattro and mRNA‐based vaccines. In the attempt to optimize hNPs for lung delivery of RNAs, we developed hNPs modified on the surface with ionizable lipids (DODAP and DODMA). Ionizable lipids‐modified hNPs were fully characterized for size, surface properties, aerosolization performance and interactions with the mucin‐rich lung environment. Preliminary data on optimized formulation support the rationale and prompt further in vitro studies aimed to test uptake and endosomal escape of ionizable lipids‐modified hNPs in human lung fibrosis cell models.

This work is part of “National Center for Gene Therapy and Drugs based on RNA Technology”‐CN00000041 funded by MUR in the context of PNRR–M4C2 NextGenerationEU (DD n.1035 of 17.06.22).

References

[1] 1d'Angelo I., Costabile G. et al (2018), J Aerosol Med Pulm Drug Deliv. doi: 10.1089/jamp.2017.1364.

[2] 2 Conte G., Costabile G. et al. (2022) ACS Appl. Mater. Interfaces 14, 7565–7578, doi: 10.1021/acsami.1c14975.

H. 25 Tackling the Mucus Barrier in siRNA Delivery to the Lungs with Inhalable Hybrid Lipid‐Polymer Nanoparticles

Gabriella Costabile,¹Gemma Conte,¹Domizia Baldassi,²Agnese Miro,¹Fabiana Quaglia,¹Ivana d'Angelo,³Olivia M. Merkel,²and Francesca Ungaro⁴

¹Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.²Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig‐Maximilians‐Universität, Munich, Germany.³Di.S.T.A.Bi.F., University of Campania Luigi Vanvitelli, Caserta, Italy.⁴Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.

RNA interference is a potent and specific gene silencing approach at limelight for the local treatment of severe lung diseases. Nonetheless, a key challenge to earn the full potential of inhaled siRNA is the need of a delivery system able to assist siRNA transport through the lung barriers. In particular, a prominent production of mucus is distinctive of patients with progressive lung fibrosis. To tackle the mucus barrier, non‐PEGylated and PEGylated siRNA‐loaded hybrid lipid/poly(lactide‐co‐glycolide) nanoparticles (hNPs) were successfully developed.1 Important knowledge was gained on the effect of PEGylation for crossing mucus barrier, and guided the choice of the formulations to move towards further in vitro/in vivo investigations. Optimised mucus‐penetrating hNPs exhibited good aerodynamic behaviour, high encapsulation efficiency and prolonged release of siRNA in simulated lung fluids. Toxicity, uptake and silencing activity of siRNA‐loaded hNPs have been assessed in different human airway cell culture models, providing a tool to optimise hNP properties for in vivo inhalation. A proof of concept of the in vivo silencing activity of selected hNPs was provided in a murine model.

This work is part of “National Center for Gene Therapy and Drugs based on RNA Technology”‐CN00000041 funded by MUR in the context of PNRR–M4C2 NextGenerationEU (DD n.1035 of 17.06.22).

References

[1] Conte G., Costabile G. et al. (2022) ACS Appl. Mater. Interfaces 14, 7565–7578, doi: 10.1021/acsami.1c14975.

H. 26 Establishment of an Isolated Perfused Lung Model for Analysis of Local Induced Immune Response of Inhaled Immuno‐Modulating Agent OM‐85

Katharina Schwarz,¹Helena Obernolte,¹Sven Cleeves,¹Anne Vaslin Chessex,²Claire Abadie,²and Christian Pasquali²

¹Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, 30625 Hannover, Germany.

²OM Pharma, Preclinical Research Department, 1217 Meyrin, Switzerland.

OM‐85 is a soluble bacterial lysate effective via its anti‐microbial and immunomodulatory properties against infections. For development of an inhalable formulation, we established a novel approach to assess the regional deposition pattern of OM‐85 and the associated potential to induce immune response in airways and distal lung.

The approach integrates Isolated Perfused Rat Lung (IPL) model to provide an intact organ system with cellular, structural and functional integrity together with Precision‐Cut Lung Slices (PCLS) as immunocompetent model. From IPLs exposed to fluorescence‐labelled OM‐85 aerosols (20 min, 4 deep breaths), PCLS were prepared and analyzed for regional fluorescence pattern and cytokine release profile after 24h.

Regional deposition pattern analysis showed highest local deposition in the airways. Cytokine release of e.g. IFNs and IL‐1β was elevated in a dose‐dependent manner in tissue lysate and supernatant with max. 2000‐fold and 7‐fold increase in the airway and alveolar region. This demonstrates innate immune response activation from inhaled OM‐85 throughout the lung including alveolar region even for short exposure.

The combination of the twoex vivomethods, IPL and PCLS, allows in vivo relevant modeling of the regional deposition pattern and corresponding immune response profile in the lung. These data can be directly incorporated in proof‐of‐concept studies in early pre‐clinical development of new inhaled drugs, including immunomodulating agents.

I. Hot Topics

I. 01 Towards the Sustainable Discovery and Development of New Antibiotics

Rolf Müller

Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany.

The global rise of antimicrobial resistance, mainly due to the mis‐ and overuse of antibiotics, is one of the most pressing issues of our time. To counteract this development, novel resistance‐breaking antibiotics are urgently needed. Historically, the vast majority of antibiotics have been derived from microbial natural products. As compared to traditional bacterial producers, such as actinomycetes and bacilii, myxobacteria have been studied less extensively and thus harbor a large potential for the discovery of entirely new natural product scaffolds exhibiting promising bioactivities. Comparisons of myxobacterial metabolite profiles with the number of underlying biosynthetic gene clusters encoded in their large genomes show, that many compounds still remain unknown. Further, recent studies indicate that the order of myxobacteria likely comprises many more biodiverse representatives than previously assumed. According to metagenomics analyses, myxobacteria (including many underexplored representatives) are highly abundant in the soil microbiome, where they play a crucial role in soil nutrient and carbon cycling. Taken together with our recent genomic analyses, these findings suggest that the biosynthetic potential of myxobacteria is a long way from being exhausted.

We recently demonstrated that chemical diversity correlates with taxonomic distance in myxobacteria. Accordingly, we are more likely to isolate novel compound classes from strains which are phylogenetically distant from previously characterized strains as compared to closely related strains. This knowledge can be applied to prioritize strains for natural product discovery, thus increasing the chance of discovering compound classes with yet unknown chemical structures and biological activities.

I will discuss recent results from our laboratory regarding the identification of novel bioactive NPs from myxobacteria based on different approaches, and show our recent advances in their preclinical development.

I. 02 NO ABSTRACT

I. 03 Adoptive Transfer of HGF Overexpressing T Cells as a Potential Therapeutic Approach in the Bleomycin Injured Mouse Lung

Seyran Mutlu,¹Kleanthis Fytianos,¹Céline Ferrié,¹Melanie Scalise,¹Sofia Mykoniati,²Amiq Gazdhar,¹and Fabian Blank¹

¹Department for BioMedical Research DBMR, Department of Pulmonary Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.

²Unisanté, Lausanne, Switzerland.

Introduction:Idiopathic pulmonary fibrosis (IPF) is a lethal disease emerging from aberrant wound healing mechanisms disturbing the alveolar epithelial (AE) integrity. The immune system (IS) is known to be involved too. Hepatocyte growth factor (HGF) showed antifibrotic properties. We aim to study if adoptive transfer of HGF modified T cells (HGF‐T cell treatment) in bleomycin injured mice (BLM model) may promote re‐balancing of the pulmonary IS and repair of the AE.

Methods:Mice were instilled with 1.52U/kg of BLM and were sacrificed on days 7, 10 and 14 following administration. Mice received HGF‐transfected CD3+ T cells by intratracheal instillation 7 days following BLM administration and were sacrificed 7 days later. Analyses of lung tissue and bronchoalveolar lavage fluid were done by flow cytometry, histology, qRT‐PCR, ELISA and hydroxyproline assay. In vitro co‐cultures were screened for potential mechanisms induced by HGF‐T cell treatment in the BLM model.

Results:Immune cells showed a disturbed homeostasis of the IS after BLM treatment, which was, to some extent, re‐balanced by HGF‐T cell treatment in comparison to non‐treated BLM group. HGF–T cell treatment in the BLM model also reduced collagen content and fibrosis scoring compared to the non‐treated BLM control.

Conclusion:HGF‐T cell treatment induced elevated HGF level in the lung together with a decrease of fibrosis scoring and shows great potential for a promising therapeutic approach for IPF.

J. Aerosol Exposure and Health

J. 01 Assessing Exposures from Indoor Air Pollution in Low‐Resource Settings

Laura Nicolaou^1,2,3,and William Checkley¹

¹Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University, Baltimore, USA.

²Center for Global Non‐Communicable Disease Research and Training, School of Medicine, Johns Hopkins University, Baltimore, USA.

³Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA.

Three billion people worldwide, the majority from low‐ and middle‐income countries, rely on solid fuels for domestic cooking and heating. Incomplete combustion of these fuels in open fires and inefficient cookstoves results in high concentrations of harmful pollutants, including particulate matter (PM). Household air pollution (HAP) is a leading cause of illness and death worldwide and was responsible for 2.3 million deaths and 3.6% (91.5 million) of the disability‐adjusted life years lost in 2019. Portable, robust, and accurate methods are required to adequately characterize HAP exposures in low‐resource settings, determine exposure‐response relationships, and assess HAP mitigation strategies. I will provide an overview of exposure assessment methods that can be applied to better characterize personal exposures to HAP, focusing on novel techniques integratingin silicomodeling to estimate lung‐deposited doses as a more biologically relevant exposure metric.In silicomodels can also identify lung regions with enhanced PM deposition and increased likelihood of inflammation, which can contribute to understanding the pathophysiology of HAP‐related respiratory disease. I will present results from a study conducted in Puno, Peru, which utilized this method to examine the differences between cigarette smoking and biomass smoke exposure, the two main environmental risk factors for COPD.¹

References

[1] Nicolaou, L. and Checkley, W. (2021). Env Res 197, 11116. doi:10.1016/j.envres.2021.111116

J. 02 Toxic Potential and Health Effects of Mineral Dust

Khanneh Wadinga Fomba,¹Eduardo José dos Santos Souza,¹Gerrit Bredeck,²Roel Schins,²Martinique Frentrup,³Ulrich Nübel,³Sofia Gómez Maqueo Anaya,¹Dietrich Althausen,¹Kerstin Schepanski,⁴Sandra Freire,⁵João Gomes Cardoso,⁵Vanusa Rocha,⁵Isabel Ines Araujo,⁵Thomas Müller,¹Christian Nehls,⁶Honey Alas,¹José Luís Lima Spencer,⁷Ofélia Monteiro,⁸Thomas Gutzmann,⁶and Hartmut Herrmann¹

¹Leibniz Institute for Tropospheric Research in Leipzig (TROPOS), Leipzig, Germany.

²Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany.

³Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.

⁴Freie Universität Berlin, Berlin, Germany.

⁵University of Cabo Verde, Praia, Cape Verde.

⁶Research Center Borstel ‐ Leibniz Lung Center, Borstel, Germany.

⁷Dr. Baptist de Sousa Hospital ‐ Ministry of Health, Mindelo, Cape Verde.

⁸Agostinho Neto Hospital ‐ Ministry of Health, Praia, Cape Verde.

Mineral dust is a common natural constituent of air pollution in many regions of the world. Exposure to these particles has been associated with various respiratory, and cardiovascular diseases and mortality. However, the mechanisms through which these health effects are caused are not yet well understood. Here we investigate the toxic potential of Saharan dust particles to understand how dust particles can cause inflammation or generate oxidative stress in the body. As part of the DUSTRISK project, Saharan dust samples were collected in Cabo Verde in 2021 and 2022 and their chemical, microbial, physical, and toxic properties were investigated.

The results show that during dust events, inhabitants are exposed to high concentrations of fine and coarse particulate matter (PM2.5 > 150 μg/m³), which significantly exceeds the WHO guidelines. The dust contains transition metals such as Fe, Mn, Zn, and Cu that can generate excess reactive oxygen species in the body, causing oxidative stress, and activating the NLRP3 inflammasome pathway related to pulmonary disease. Its diverse chemical and microbial components suggest that multiple mechanisms can be triggered from their interactions leading to diverse health effects and deserves detailed investigations. The goal is to understand these mechanisms, develop effective strategies to mitigate their effects and inform public health officials to establish guidelines to reduce the health risk associated with dust exposure.

J. 03 Inhalable Aerosols from Carbon Fibres

Sonja Mülhopt,¹Manuela Hauser,¹Manuela Wexler,¹Jonathan Mahl,¹Werner Baumann,¹Silvia Diabaté,¹Susanne Fritsch‐Decker,¹Carsten Weiss,¹Alexandra Friesen,¹Matthias Hufnagel,¹Andrea Hartwig,¹Bastian Gutmann,²Christoph Schlager,²Tobias Krebs,²Ann‐Kathrin Goßmann,³Frederik Weis,³and Dieter Stapf¹

¹Karlsruhe Institute of Technology KIT, Karlsruhe, Germany.

²Vitrocell Systems GmbH, Waldkirch, Germany.

³Palas GmbH, Karlsruhe, Germany.

Carbon fibres (CF) and CF‐reinforced plastics (CFRPs) are innovative materials, which are increasingly produced, recycled, and disposed of, possibly releasing particles and fibres which could be fulfil the criteria of the World Health Organisation (WHO) to be respirable (critical aspect ratio >3:1, length >5 μm and diameter <3μm). This raises serious concerns about potentially harmful effects upon inhalation.

Based on a material flow analysis identifying relevant release scenarios of respirable dusts, investigations of CF/rCF/CFRP‐materials under thermal and mechanical stress are carried out. Inhalable aerosols are provided and characterized, which are delivered to the air‐liquid interface of human lung cells (ALI) in an exposure system. After exposure, toxicological investigations are carried out, i.e. directly on the apical surface of cell cultures, in order to simulate lung‐like conditions. Lung epithelial, macrophage, and fibroblast cell cultures in mono‐ and co‐culture are used for toxicological evaluation of respirable CF fragments focussing on determination of cytotoxicity, gene expression analyses and assessment of proinflammatory, profibrotic and genotoxic potential. Fibre characterisation and especially the generation of WHO fibres is discussed in the context of biological responses caused by inhalable CF (1).

References

[1] Friesen, A., Fritsch‐Decker, S., Mülhopt, S., et al. (2023). Int. J. Mol. Sci. 3, 1927. DOI: 10.3390/ijms24031927.

J. 04 Assessment of CYP1A1 Induction from Respiratory Cell Lines Exposed to Diesel and Urban Particulate Matter Using a Granisetron 7‐Hydroxylation Activity Assay

Hyunki Cho,¹Seungyun Baik,²Sang Kyum Kim,³andChang Seon Ryu¹

¹KIST Europe, Saarbrücken, Germany.

²KIST Europe, sbaik@kist‐europe.de, Germany.

³Chungnam National University, Daejeon, Republic of Korea.

Particulate matter (PM) is typically composed of agglomerated particles containing trace amounts of metals and chemical pollutants such as polycyclic aromatic hydrocarbons (PAHs). One extensively studied biomarker, cytochrome P450 (CYP) 1A1, is highly inducible when PAHs activate the aryl hydrocarbon receptor (AhR). Elevated CYP1A1 inducibility has been shown to be associated with increased pulmonary DNA adduction from PAH exposure and a higher risk of lung cancer. The aim of this study was to develop an LC‐MS/MS‐based assay to evaluate CYP1A1 induction potential following exposure to PM. The assay employed a CYP1A1 selective reaction of granisetron 7‐hydroxylation in response to an AhR inducer, 6‐formylindolo[3,2‐b]carbazole (FICZ), 3‐methylcholanthrene, benzopyrene in A549 cells. Exposure to FICZ (10 nM) significantly increased granisetron 7‐hydroxylation levels. The granisetron 7‐hydroxylation assay exhibited a better dose response from 0 to 10000 nM FICZ treatment than the ethoxyresorufin‐O‐deethylation (EROD) assay Furthermore, application of the granisetron assay to diesel and urban PM exposure showed a concentration‐dependent induction of CYP1A1 in A549 and human nasal epithelial cells. The granisetron assay exhibited better selectivity for CYP1A1 than the conventional EROD assay, which overlaps with the reactions of CYP1A2 and CYP1B1. The granisetron 7‐hydroxylation assay showed great potential for future respiratory toxicity evaluations.

J. 05 Combining Analytical Techniques to Assess Translocation of Diesel Particles across the Alveolar Tissue Barrierin vitro

Gowsinth Gunasingam,¹Ruiwen He,¹Sandor Balog,¹Alke Petri‐Fink,^1,2and Barbara Rothen‐Rutishauser¹

¹Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.

²Chemistry Department, University of Fribourg, Fribourg, Switzerland.

PM2.5 can cross the alveolar epithelium to the blood circulation, reaching secondary organs beyond the lungs. However, methods to quantify the rate of translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, we used a combination of different techniques to qualitatively with transmission electron microscopy (TEM) and quantitatively with ultraviolet‐visible spectroscopy (UV‐Vis) and lock‐in thermography (LIT) analyze the translocated fraction of SRM2975 diesel exhaust particles (DEP) across the alveolar epithelium in vitro. A549 cells were grown on permeable PET membranes, and then exposed to DEP at concentrations from 0 to 80 μg/mL. After 24‐hour, the basal fraction was collected for DEP analysis by TEM, UV‐VIS and LIT. All methods were able to detect DEP in the basal fraction when empty inserts were used, with limitations observed at 0.1 μg/mL. In presence of cells the detection of DEP in the basal fraction was challenging at all applied concentrations. With UV‐VIS and LIT technics DEP translocation rate across the tissue was determined around 1% at 20 and 40 μg/mL, and no particles were detected in the basal fraction at higher concentration, probably because of big agglomerates. Our results confirm a very low translocation rate of DEP across the alveolar epithelium. We will use the combination of analytical methods to further assess the rate across lung tissue barriers and upon repeated exposures to DEP.

J. 06 Proceedings from a Special Symposia and Panel Session on the Topic of the Aerosol Science of Infectious Diseases‐ What We Learned and What We Still Need to Know

Justin Taylor,and Shanna Ratnesar‐Shumate

Noblis‐ESI, Reston, USA.

The COVID‐19 pandemic has driven considerable research and public discussion about the role of aerosols in infectious respiratory disease transmission. The aerosol sciences research community has been at the forefront of this process, leading scientific studies of how SARS‐CoV‐2 and other respiratory pathogens, such as influenza and tuberculosis, are transmitted through the air and how to mitigate their transmission to protect public health. At the 2022 Annual Conference for the American Association for Aerosol Research (AAAR), a special symposia panel session was held entitled “Aerosol Science of Infectious Diseases: What we've learned and still need to know about transmission, prevention, and the one health concept.” The panel included leading aerosol scientists, who were heavily involved in the COVID‐19 response. The first session discussed “Lessons Learned from the COVID‐19 Pandemic” and the second session discussed “Gaps in Aerosol Science Identified During the COVID‐19 Pandemic.” The questions and answers were documented, and the panelists are currently preparing a Letter to the Editor of the journal of Aerosol Science and Technology to capture the outcomes of the special symposia which will inform future research area into emerging infectious diseases. This presentation will highlight the key findings from 2022 AAAR special symposia panel on the aerosol science of infectious diseases.

J. 07 Monitoring Diesel Exhaust Particles Deposition on Lung Cellsin vitroby Lock‐in Thermography

Ruiwen He,¹Olivier Schaub,²Christoph Geers,²Gowsinth Gunasingam,¹Maik Schultheiß,³Bastian Gutmann,³Tobias Krebs,³Alke Petri‐Fink,¹and Barbara Rothen‐Rutishauser¹

¹Adolphe Merkle Institute, Fribourg, Switzerland.

²NanoLockin GmbH, Fribourg, Switzerland.

³Vitrocell Systems GmbH, Waldkirch, Germany.

Diesel exhaust particles (DEPs) can deposit onto the respiratory epithelial surface upon exposure. Cellular burden of DEPs is important for dose‐response relationships in the cells, however, non‐invasive methods to continuously monitor DEPs on cells during exposure are still challenging and not well established. In our study, an alternative particle detection system, i.e. the miniaturized lock‐in thermography (Calorsito mini, NanoLockin GmbH, Switzerland) was explored. Lock‐in thermography is a thermosensitive detection method that applies light illumination to induce heat of carbon‐based particles. By testing a standard DEPs sample (SRM2975) under a light wavelength of 525 nm, a positive linear relationship (R2 = 0.97) was established between the thermal emission signals and DEPs levels ranging from 0 to 5 μg/cm2, with a limit of detection (LOD) at around 0.2 μg/cm2. It indicates that lock‐in thermography can be used to detect DEPs at a relatively low concentration. In addition, our preliminary results suggest that thermal emission signals from DEPs may change during internalization by lung cells, which can be used as a potential indicator of particle uptake by cells. Further studies on continuously monitoring DEPs are still ongoing to evaluate the interaction of DEPs with lung cells during exposure and to verify it with other methods.

J. 08 Stability of Aerosol Concentration on a Confined Volume forin vitroExposure Experiment

Chang Gyu Woo,¹Seoyeon Park,¹and Noo Li Jeon²

¹KOREATECH, Cheonan‐si, Republic of Korea.

²Seoul National University, Seoul, Republic of Korea.

Aerosol exposure can have huge impact on lung. Inhalation toxicity is one of the important factors for various chemical usage. In‐vitro test methods are developed for understanding mechanism better. Here we prepared in‐vitro test setup for aerosol exposure. We have tested aerosol stability in number concentration over time and position in the exposure chamber. Aerosol particles are generated using atomizer. To minimize diffusion losses, electrostatic dissipative tubes between experimental elements.

Other aerosol generators such as nebulizer, sparging liquid aerosol generator and etc. Air flowrate and particle characteristics over time were analyzed for aerosol concentration stability. This research can be applied to enhance in‐vitro aerosol exposure experiments for understanding in‐vivo mechanisms.

This work was supported by the National Research Foundation of Korea Grant funded by the Korean government (2019R1I1A3A01060938).

J. 09 Cell Viability and Inflammatory Responses of Amorphous Mesoporous Silica Particles on Different Macrophage Cells

Tetiana Yalovenko,^1,2Jesús Enrique Campos Pacheco,^1,2Emilie Schousboe,^1,2Anna Gustafsson,^1,2Georgia Pilkington,³and Sabrina Valetti^1,2

¹Biofilms – Research Center for Biointerfaces (BRCB), Malmö, Sweden.²Biomedical Science, Faculty of Health and Society, Malmö University, Malmö, Sweden.³Nanologica AB, Södertälje, Sweden.

The searching for safe and effective therapies for lung infections has been one of the major research focus[1]. Delivery anti‐infectives to the site of infection may increase the efficacy of treatment and reduce the systemic exposure of the drugs. MSPs are of interest as a drug carrier and promising carrier for pulmonary delivery[2]. To assess the potential toxicity and inflammatory responses, we investigated the impact of 3 type of placebo MSPs (0.008‐1mg/mL) on PMA‐differentiated THP‐1, M‐CSF‐stimulated and GM‐CSF‐stimulated macrophages using cell viability and cytokine release as assessment of potential toxicity to the doses delivered. When the cells were incubated with suspended MSPs in PBS for 4 h with MSPs concentrations up to 0.5mg/mL their viability was maintained between 80% and 100% compared to untreated cells. Cell viability decreased when incubation was extended to 8,12 h, however MSPs concentration of 0.125mg/mL and below maintained cell viability higher than 80% after 24 h incubation. The results of this study open a new perspective on the possibility of using MSPs as a nanocarrier of therapeutic agents and demonstrate the need for furthermore investigation. This work is a precursor of conducting studies with drug product where the herein obtained results could be related to the potential clinical use.

References

[1] 1.Hittinger M.,et al.(2015)Adv Drug Deliv Rev 85,44‐56.doi:10.1016/j.addr.2014.10.011

[2] 2.Valetti S.,et al.(2017)Nanomedicine 8,831‐844.doi:10.2217/nnm‐2016‐0364

J. 10 NO ABSTRACT

J. 11 Simulating Breathing Motions to Study Mechanical Stretch Induced Changes in Lung Epithelial Cell Behaviour

Sandeep Keshavan,¹Mira Witzig,¹Ludovica Cacopardo,²Nicole Guazzelli,²Arti Ahluwalia,²Alke Petri‐Fink,¹and Barbara Rothen‐Rutishauser¹

¹Aldolphe Merkle Institute, Université de Fribourg, Fribourg, Switzerland.

²Research Center “E. Piaggio”, University of Pisa, Italy, Pisa, Italy.

To improve the physiological relevance of lung cell models, a Dynamic Alveolar Interface (DALI) was developed to replicate the lung's air‐liquid interface (ALI) and breathing motions.1 The human alveolar epithelial cell line A549 was used to investigate the underlying mechanical effects under dynamic motions (5% strain). No significant differences in cell adhesion, viability, or gene expression of focal adhesion markers (E‐cadherin and Protein Tyrosine Kinase 2) were observed in static and dynamic conditions. However, there was a significant increase in Interleukin (IL)‐8 gene expression under stretching conditions in comparison to the static controls. To summarize, A549 cells formed a uniform monolayer in the DALI‐bioreactor even under breathing‐like stretching conditions, with an increase in IL‐8 gene expression as observed in several reports. In future experiments, A549 cells will be exposed to aerosolized crystalline quartz silica using a DALI‐bioreactor with and without stretching/breathing to assess proinflammatory and focal adhesion markers.

References

[1] Nossa, R., Costa, J., Cacopardo, L. & Ahluwalia, A. Engineering a dynamic model of the alveolar interface for the study of aerosol deposition. Biomed Sci Eng 3, (2020).

J. 12 Biomechanical Changes in Murine Lungs Following Chronic E‐Cigarette Exposure

Yasmeen Farra, Hector Millan Cotto, Jacqueline Matz, Chiara Bellini, andJessica Oakes

Northeastern University, Boston, USA.

Electronic cigarette (e‐cig) usage has been rising for the last decade. All experiments were performed with the tobacco flavored JUUL^TM, 3% nicotine. Particle sizes (EEPS, TSI) and concentration (MicroDust Pro, Casella) were measured. Female Apoe‐/‐ mice were nose‐only (inExpose, Scireq) exposed to e‐cig aerosols (2 puffs/min, particle concentration of 300 mg/m³) or room air for 95 mins daily for 16 weeks. Airway resistance (R_N), elastance (H), and tissue resistance (G) were determined (flexivent, Scireq). Maximum airway resistance (R_{N, max}) was found with methacholine (1.5 – 25 mg/mL). Airway thickness and parenchyma dimensions (linear mean intercept, L_M; thickness) were measured. E‐cig aerosols exhibited a bi‐modal distribution with count median diameters of 13.3 and 116 nm. Compared to controls, airway resistances were larger in the e‐cig exposed mice (R_N,e‐cig = 0.253 ± 0.025 vs. R_N,Air = 0.174 ± 0.006 cmH₂O‐s/mL) with increases in airway thickness (B_e‐cig = 8.64 ± 0.62 vs. B_Air = 7.39 ± 2.02 mm). Elastance (H_e‐cig = 28.65 ± 1.78 vs. H_Air = 22.94 ± 1.13 cmH₂O‐s/mL) and resistance (G_e‐cig = 5.11 ± 0.81 vs. GAir = 4.26 ± 0.39 cmH₂O‐s/mL) were larger in exposed mice compared to controls. L_Mwas smaller and parenchyma tissue thickness did not change. Exposure to e‐cig aerosols enhanced airway hyperresponsiveness. Prolonged use of e‐cig devices may cause remodeling that detrimentally impacts lung function.

Work was supported by a R03 HL142472 and a NSF GRFP Fellowship (Y. M. Farra).

J. 13 Variability in Exhaled Aerosol Emission in Healthy Humans for Different Respiratory Activities

Katharina Schwarz,¹Liudmila Banari,¹Wolfgang Koch,¹Horst Windt,¹Nadja Struß,¹and Jens M Hohlfeld^1,2,3

¹Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany.

²Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany.

³Member of the German Center for Lung Research (DZL‐BREATH), Hannover, Germany.

Background:Knowledge on exhaled aerosol concentration and size is essential for assessment of airborne infection risk and efficacy of mitigation measures. Though several studies are available, information on intra‐ and inter‐individual variability is limited.

Methods:A set‐up has been established allowing for collection and analysis of the amount of respiratory aerosols over a wide size range under realistic conditions. 30 healthy volunteers repeated the respiratory activities on two visits: tidal/deep breathing, quiet/normal/loud speaking, coughing, singing.

Results:Aerosol mass emission was lowest in tidal breathing. For quiet/normal speaking and fast deep breathing it was about 10‐fold higher and for loud speaking and singing about 100‐fold higher. Intra‐individually, there was a high reproducibility. Inter‐individual variability was two orders of magnitude for droplets <80 μm, and three orders of magnitude for droplets >80 μm. For each respiratory activity, a small fraction of volunteers was classified as high‐emitters with a 10‐100‐fold higher aerosol emission compared to the median value of the study group. A general classification, however, of individuals as high, medium, or low emitters throughout the different respiratory activities could not be made.

Conclusion:Investigation of the distribution and reproducibility of the aerosol emission for different respiratory activities in a larger group provides a valuable data set for airborne infection risk assessment.

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