Bio

Davide D’Angelo is currently a PhD student at the University of Parma. He completes his bachelor degree in 2017 in biotechnology at the University of Perugia, Italy, with a thesis on the methylation level of A.thaliana. In 2017 he moved to Parma, to complete his studies with a master's degree in Medical, Veterinary and Pharmaceutical Biotechnologies at the University of Parma, graduating with honours in 2019 with a thesis on the formulation of a pulmonary dry powder containing a vaccine against HPV. From 2019 he starts his PhD in the “Biopharmaceutics-Pharmacokinetics” program in the laboratory of pharmaceutical technology of Food and Drug Department at the University of Parma, working under the supervision of Professors Francesca Buttini and Fabio Sonvico on a project entitled “Particle engineering applied for the development of dry powders for inhalation containing biotechnological drugs”. His research is actually focused on the development of an inhalable powder containing cyclosporine for the prevention of pulmonary rejection after transplantation and potentially for the containment of the "cytokine storm" caused by Sars-CoV-2 infection.

Project summary

While for inhaled products a great deal of effort has been dedicated in the last few years to improve the methods of characterization to go beyond the “simple” aerosol characterization requested by pharmacopoeias and guidelines, this has not been the case for nasal products.
However, when nasal delivery is suggested for vaccination, for systemic delivery of peptides and proteins or for nose-to-brain delivery, it appears clear that the evaluation of spray pattern and geometry characteristics and of the eventual respirable fraction with aerodynamic diameter below 5 µm, do not provide sufficient insight into the biopharmaceutical characteristics of the formulation under investigation. Nasal cavity regional deposition and formulation dissolution/permeation appear much more relevant indicator if the nasal product under development will perform in following in vivo and clinical studies.

Around these topics, in addition to the Advanced Drug Delivery Research Lab (ADDRes Lab) of the University of Parma, a broad collaboration of academic partners has been created including:
• School of Cancer and Pharmaceutical Sciences, King’s College London, UK (Prof. Ben Forbes).
• Department of Pharmaceutics and Biopharmaceutics, Kiel University, Germany (Prof. Regina Scherließ)
• Faculté de Pharmacie, Université Libre de Bruxelles, Belgium (Prof. Jonathan Goole)

The first part of the project consists in a joint effort from the various collaborating group to characterize with advanced methods that go beyond the pharmacopoeial tests a reference commercial nasal product, i.e. mometasone furoate nasal spray suspension. Each group will receive few nasal products from the same production batch of the same supplier and will apply their own in-house approach developed over the last few years.
In the case of UniPR group, we will perform the spray deposition in an anatomically correct, transparent, silicone human nose educational model (Koken, Japan). The evaluation of the deposition pattern in different region of interest (lower, middle and upper nasal cavity). At the same time the product will be tested with the Next Generation Impactor using a 3D printed expansion chamber designed to allow the collection of the nasal product droplets on Snapwell insert in view of dissolution experiments.

The second phase of the project consists in the organization of a one-and-a-half-day symposium to be held in Parma (tentative date 17-18 May 2021), that will see the participation of the groups that took part in the study. The event will focus not only on the presentation of the data collected by the different groups but also on a roundtable of discussion about the different techniques and methods employed with a critical appraisal of merits and limitations. This project and the concluding Symposium will contribute to establish and strengthen a network of collaboration on the field.

Career Development Award Proposal

Project Title:
Advanced nasal product characterization: focus on deposition and dissolution

Description and Project Goals:
The main purpose of this work is to perform and compare some of the most recent nasal product characterization approaches in terms of deposition and dissolution. In detail the purpose of the study is to use all available cast models within the collaboration group to assess nasal deposition of the same commercial product utilising the in-house assessment strategy of each individual lab.

A second purpose is consisting in the organization of a one-and-a-half-day symposium to be held in Parma where data from the network will be presented along with details of the cast model and experimental details. This will strength the collaboration among the network members, disseminate best practices and the harmonization among these product characterization procedures.

Background

There is a wide interest in nasal administration of drugs due to peculiar characteristics of this site, for many years this route of administration has been limited to local delivery and later the potential of nasal administration for systemic delivery, mucosal vaccination and nose-to-brain delivery has been considered. Nevertheless, factors that influence drug deposition in the nasal cavity are less studied because this cavity is somewhat inaccessible, and delivery of aerosols into this site is a complex process that depends on many parameters including formulation characteristics, the device used, and the way the patient handles this device and the anatomical complexity of the nose (1).
During the optimisation of products for nasal delivery, the delivered dose, the spray pattern and geometry and droplet size distribution are indicated by pharmacopoeias and guidelines as the critical features to be assessed in a nasal delivery system. However, while this appears sufficient for local acting nasal products, it appears not adequate for nasal products designed for systemic, vaccine or nose-to-brain delivery. Indeed, there are still no recommendations or guidelines in terms of standard approaches to evaluate nasal deposition and/or dissolution profile of the formulation (2). No specific restrictions or recommendations are made in terms of nasal cast coating, flow profile or flow rate, insertion angle or insertion depth, manual vs. automated actuation and sample collection from cast. However, when results are reported, the experimental description should contain all these aspects.

In this project, each lab from the collaboration group will use its in-house cast and routine methods for the assessment of nasal deposition of a reference suspension spray (commercially available mometasone suspension spray 50 µg/shot). If applicable, the testing should be performed without and with nasal inspiration and application should be done in both nostrils at the same time with equal number of actuations.

Dissolution rates of nasal products are determined by the physicochemical properties of the formulation, the dosage form, and potentially the region in which the drug is deposited. A major problem with nasal administration is the rapid clearance of the formulation from the cavity due to mucociliary transport which correlates strongly with the duration of the drug action, its efficacy and can also jeopardize patient compliance by involving frequent dosing. More viscous liquid formulation or the administration of powder can increase the residence time of the drug on the nasal mucosa (3). However, the impact of these formulation choices on drug dissolution and availability are often not fully understood. Thus, also in this case an effort has to be produced towards the definition of an optimal dissolution methodology for nasal products that would involve the collection of the drug formulation as emitted from the device, deposition on a surface representative of nasal environment and dissolution at the liquid interface allowing a sufficient resolution to discriminate among different formulation compositions.

Expected results:
- The results shall show the variety of cast models deposition and dissolution methods with their capabilities (and shortcomings) which will allow cast critical evaluation for specific research questions during the roundtable at the concluding symposium.
- Comparison of single laboratory assessments will show how comparable/dissimilar are general conclusions being drawn from the different cast models (such as nasal vs. post-nasal fraction and regional deposition) and dissolution methods (different dissolution rates according to experimental setups).
- The comparison among the single laboratory assessment protocols will allow defining parameters which need or can be harmonized across labs in view of the proposal of a guideline for advanced nasal products testing.

Research Plan
Duration: 4 months.
Location: University of Parma, Italy.
Assessment of mometasone nasal spray deposition pattern
Deposition pattern of mometasone suspension spray will be assessed using a colour-based method consisting of uniformly coat the inner surface of a silicone human nose model (Koken Co, Japan) with Sar-Gel®, a water-indicating paste which changes from transparent to purple on contact with water.
Qualitative and quantitative estimation of the drug deposition will be obtained taking pictures of the nasal cast after aerosolization, with a digital camera. Koken nasal cast is constructed in two separable structures that could allow the quantification of nasal deposition selective washing of specific region of interest (e.g. lower, middle and higher nasal cavity section) with solvent mixture capable of dissolve the drug. The amount of mometasone in each part will be evaluated by HPLC.
The present of the mucus and mucociliary clearance, which are key parameters for nasal drug delivery, cannot be evaluated in such artificial models. However, this study will include a step of coating the nasal cast surface that could be performed by either nebulization or simple application of mucin dispersed in simulated nasal fluid to mimic nasal environment.
The emitted formulation per each actuation will be quantified by measuring the weight difference of the device before and after each actuation and administration-related variables will be specified when results will be reported.

Mometasone formulation collection and dissolution method
For nasal administration, cascade impactor coupled with expansion chambers are recommended by the Guidance for Industry “Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products - Chemistry, Manufacturing, and Controls Documentation” for the estimation of the deposition in the respiratory tract. The deposition in the expansion chamber represents the nasal fraction, while particles that reached the impactor represent the respirable fraction.
In this study, a 2 L modified expansion chamber and a next generation impactor, NGI will be employed (4). The modified expansion chamber designed by 3D-printing is comprised of a lower part that presents the connection to the impactor and an inlet hole to trigger the nasal device at 30° from the axis, and the upper half that allows the incorporation of three Snapwell cell culture inserts located opposite to the inlet hole. The impactor will be connected to a pump at a flow rate of 15 L/min.

This method will allow at the same time to: i) characterize the aerosol performance in terms of fraction above and below 5µm; ii) quantify the fraction collected in the whole expansion chamber and iii) collect the formulation deposited on the Snapwell insets for dissolution/permeation experiments.

In the case of the dissolution/permeation experiments will be performed in two experimental setups.
In the first case, dissolution experiments will be performed using Snapwell inserts will be non-coated or coated with a mucin solution in simulated nasal fluid (SNF). Inserts will be placed in a plate with wells filled with SNF. Samples will be collected at predetermined time intervals and replaced with the same volume of fresh buffer to evaluate the amount of drug dissolved.

In the case of permeation experiments, Snapwells will be coated with RPMI2650 human nasal cells. These cells when cultivated at air-liquid interface conditions form a pseudo-monolayer, express tight junctions and secrete mucus on their apical surface, thus providing an interesting model of the nasal epithelium. RPMI2650 will be cultivated on Snapwells for 14 days and then three cell inserts will be fitted into the modified expansion chamber before the experiment. After the actuation of the nasal device in the expansion chamber adapted on the NGI, the Snapwells will be removed from the modified chamber and placed in 6-well plates containing the fresh pre-warmed SNF as described before, for drug permeation assessment. Samples will be collected from the basal chamber at set time intervals. Subsequently, cells will be scraped from the insert membrane and lysed in order to quantify the amount of mometasone inside the cells by HPLC.
At the end of the experiments, the NGI will be disassembled and each impactor stage will be washed with a suitable solvent solution and samples will be analysed by HPLC.

Bibliography:

1. Kundoor V. and Dalby R. (2011) Pharm Res. 28(8):1895-1904.
2. Salade L. et al. (2019). Int J Pharm. 561:47-65.
3. Tiozzo Fasiolo L. et al. (2018). Eur J Pharm Sci, 113 2-1.7
4. Pozzoli M. et al. (2016) Eur J Pharm Biopharm, 107:223-233.