Victor Mercier
email: victor.mercier@gmail.com

Frédéric Bastides

Eric Bailly

Garcia-Hermoso Déa

Elodie Miquelestorena-Standley

Zaki El Baz

Emilie Marteau

Emmanuelle Vermes

Anne De Muret

Louis Bernard

Éric Bailly

Dea Garcia-Hermoso

Anne De Muret

Guillaume Desoubeaux

Successful Terbinafine Treatment for Cutaneous Phaeohyphomycosis Caused by Trematosphaeria grisea in a Heart Transplanted Man: Case Report and Literature Review

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Dematiaceous fungi are ubiquitous molds colored in pale brown-to-black due to the presence of melanin within their cell wall. They can cause chromoblastomycosis, mycetoma or phaeohyphomycosis. Phaeohyphomycosis refers to as a group of infections characterized by the presence of pigmented septate mycelia -but neither grains (that are pathognomonic to mycetoma) nor Medlar sclerotic cells (pathognomonic to chromoblastomycosis)within tissues [START_REF] Ajello | A Case of phaeohyphomycosis caused by a new species of Phialophora[END_REF]. It is potentially caused by a great variety of fungal genera [START_REF] Rinaldi | Phaeohyphomycosis[END_REF]. Phaeohyphomycosis has been mostly reported in tropical areas, both in immunocompromised and non-immunocompromised hosts [START_REF] Revankar | Primary central nervous system phaeohyphomycosis: A review of 101 cases[END_REF][START_REF] Mccarty | Phaeohyphomycosis in transplant recipients: Results from the Transplant Associated Infection Surveillance Network (TRANSNET)[END_REF].

Clinical signs vary largely. Therefore, phaeohyphomycosis is nowadays usually classified in three distinct subtypes: local superficial infection, local deep infection or disseminated infection. In case of local superficial infections, phaeohyphomycosis is generally due to the inoculation of the causative agent directly into the skin following trauma; then, the fungus and the surrounding inflammation grow over several years to result in a painless pseudo-tumorous mass. Laboratory tests allow the species identification by the means of in vitro culture and molecular sequencing, but there is no diagnostic assay available for blood. The therapeutic management is challenging. We present herein an original case of phaeohyphomycosis in a heart transplanted man caused by Trematosphaeria grisea (syn. Madurella grisea), that is a fungal species usually rather involved in mycetoma [START_REF] Ahmed | Revision of agents of black-grain eumycetoma in the order Pleosporales[END_REF]. Curative treatment with terbinafine was successful.

Observation

A 49-year-old man was referred for a painless tumorous lesion located at the left hand. It was significantly expanding over a one-month period. The patient had never left France, except for a short stay in Tunisia, ten years before. He also reported the inoculation of a foreign body into his left hand, following a road accident in metropolitan France few years ago. Three months before the present consultation, he underwent cardiac transplantation because of heart failure. He was currently given an immunosuppressive treatment based on mycophenolate mofetil, prednisone and tacrolimus monohydrate. At the physical examination, a bluish four centimeter-long abscessed and suppurated lesion of the soft parts was noticed on the dorsal side of the hand. The lesion was facing the third and fourth Erreur ! Source du renvoi introuvable.extensor muscle's tendon (Figure 1a, Figure 2). There was neither paresthesia nor sensory-motor deficiency. No satellite adenopathy was reported.

The abscess was drained (but the pus was not investigated), and a biopsy of the lesion was concomitantly performed. Histological examination showed pigmented mycelial structures without grains (Figure 3). The macroscopic features of colonies that were isolated in vitro first suggested Madurella genus (Figure 4).

Eventually, the species Trematosphaeria grisea (syn. Madurella grisea) was confirmed by DNA sequencing through genomic amplification of the rDNA 28S D1-D2 region (Score GenBank = 1133; cover= 99%; identit.=100%; GenBank accession number MN581332). The absence of fructification and sporulation did not allow measurement of the antifungal MICs (minimum inhibitory concentrations). For the initial curative treatment, isavuconazole, at the conventional dosage of 200 mg qd (after an initial loading dose over 48h), was empirically chosen due to its theoretical efficiency against Madurella species and its drug-to-drug interactions that are assumed quite limited with immunosuppressive drugs [START_REF] Kloezen | In vitro antifungal activity of isavuconazole against Madurella mycetomatis[END_REF]. Furthermore, isavuconazole does not require dosage adaptation in case of kidney insufficiency, the patient having herein a glomerular filtration rate at 47 mL/min. No clear clinical improvement was noticed over the next nine months; so, daily application of topic terbinafine ointment was added to oral isavuconazole. Two months later, substantial reduction of the lesion size was noticed (Figure 1b), so that the antifungal combination strategy was pursued for six additional months.

Afterwards, side effects occurred with diarrhea, and the renal function decreased due to possible interaction of the oral antifungal with tacrolimus monohydrate. The therapeutic drug monitoring showed high residual concentrations of isavuconazole (5.20 mg /L in blood). Isavuconazole was then withdrawn, and instead oral therapy with terbinafine alone was initiated with a dosage at 250mg per day. The total excision was denied by the patient. After six months of terbinafine alone, the lesion almost disappeared (Figure 1c), while the tolerance was satisfactory, so that the dosage was reduced at 250 mg every other day. After one year of treatment with oral terbinafine, the lesion is no more visible.

Discussion

Burgeoning and invasive masses of the soft parts can evoke a skin tumour, like cutaneous carcinoma [START_REF] Berg | Skin cancer in organ transplant recipients: Epidemiology, pathogenesis, and management[END_REF].

Phaeohyphomycosis -and more largely fungal infection of the skin and subcutaneous tissues -is often overlooked, because not well known by most of the practitioners. As there are no rapid and reliable serologic or immunologic tests that can be easily implemented for diagnosis of phaeohyphomycosis, biopsies with mycological and histological investigations must be systematically performed in case of any doubt.

Nowadays, the number of pathogenic fungi documented as causative agents of phaeohyphomycosis has reached 70 genera and 150 species [START_REF] Revankar | Melanized fungi in human disease[END_REF]. The recent application of molecular and phylogenetic tools has largely revised the taxonomic placement of melanised mycetes. Using a multigene phylogenetic approach, Ahmed et al. concluded that Trematosphaeria grisea species (syn. Madurella grisea) is phylogenetically-affiliated to the Trematosphaeriaceae family, suborder Massarineae in the Pleosporales order [START_REF] Ahmed | Revision of agents of black-grain eumycetoma in the order Pleosporales[END_REF]. Although not so common, T. grisea has long been acknowledged as an exclusive agent of mycetoma in South America, more precisely in Brazil [START_REF] Severo | Eumycetoma by Madurella grisea: Report of the first case observed in the southern brazillian region[END_REF][START_REF] Vilela | A case of eumycetoma due to Madurella grisea in northern Brazil[END_REF] and in Chile (but also in a submandibular abscess in India [START_REF] Ahmed | Revision of agents of black-grain eumycetoma in the order Pleosporales[END_REF]. However, demonstration of its involvement in phaeohyphomycosis is more recent, maybe because there was previously a lack of correct identification [START_REF] Revankar | A mycoses study group international prospective study of phaeohyphomycosis: An analysis of 99 proven/probable cases[END_REF]. Morphologically, T. grisea colonies are usually described as rugose-cottony-brown of slow growth rate at 30° C. A brownish pigment diffuses into the agar. Microscopic examination reveals long septate hyphal elements with chains of arthroconidia. However, further analysis with yeast nitrogen base shows that T. grisea assimilates glucose, maltose and saccharose, but not lactose [START_REF] Vilela | A case of eumycetoma due to Madurella grisea in northern Brazil[END_REF], whereas M. mycetomatis is able to assimilate glucose, maltose and lactose, but not saccharose [START_REF] Liu | Molecular Detection of Human Fungal Pathogens[END_REF]. Nowadays, modern tools available such as mass spectrometry or DNA sequencing can identify whole unusual kinds of microorganisms from the subculture, as long as the references for fragmentation mass patterns or DNA sequences exist in the data bank. So herein, we are able to distinguish between T. grisea and Madurella mycetomatis, i.e. the major species of the Madurella genus which is usually more frequently involved in mycetoma.

The present case report is original since the patient did not come from endemic tropical area and had been always living in France, except for a unique travel in Tunisia ten years before the heart transplantation. However, recent molecular studies showed that T. grisea can be found worldwide in the environmental, especially in water supplies and humid conditions in northern Europe [START_REF] Ahmed | Revision of agents of black-grain eumycetoma in the order Pleosporales[END_REF][START_REF] Ahmed | Nigrograna mackinnonii, not Trematosphaeria grisea (syn. Madurella grisea), is the main agent of black grain eumycetoma in latin America[END_REF]. Thus, one could suggest that the foreign body which was inoculated into the patient's left hand during the road accident, several years ago, likely represents the route of infection. Thereafter, the immunosuppressive treatment became an adequate underlying condition to enhance the development of the fungus, then subsequently generating local inflammation responsible for the macroscopic lesion without developing grains.

In practice, curative therapy for superficial phaeohyphomycosis is closed to the management of fungal mycetoma, based on oral antifungal drugs in first-line strategy, then on surgical excision (and, in some cases, on cryotherapy). Guidelines from Chowdary et al. recommend oral antifungals, mainly azoles, as co-adjunctive therapies particularly in immunocompromised patients and to prevent dissemination (recommendation BIII-grade) [START_REF] Chowdhary | ESCMID and ECMM joint clinical guidelines for the diagnosis and management of systemic phaeohyphomycosis: Diseases caused by black fungi[END_REF]. In our case, an oral antifungal azole was not enough to allow complete or partial cure, so our second-line strategy finally included extended-spectrum azole drugs added with terbinafine, because MICs of the latter were demonstrated low for most dematiaceous fungi [START_REF] Revankar | A mycoses study group international prospective study of phaeohyphomycosis: An analysis of 99 proven/probable cases[END_REF]. Terbinafine is actually a fungistatic agent that inhibits the ergosterol synthesis of the plasma membrane of fungi. While its therapeutic role has been longtime confined to treatment of dermatophyte infections, recent medical interest has been raised for subcutaneous or refractory tropical fungal infection [START_REF] Hay | Therapeutic potential of terbinafine in subcutaneous and systemic mycoses[END_REF][START_REF] Revankar | Use of terbinafine in rare and refractory mycoses[END_REF]. A review of the literature [Table 1] showed 22 cases of phaeohyphomycosis treated with terbinafine: 12 were successfully cured; in eight cases, terbinafine was prescribed in first-line, including seven times in monotherapy (two patients underwent concomitant surgery).

Moreover, there were six cases of phaeohyphomycosis in solid organ recipients, including five who were cured with terbinafine. Durations of treatment are poorly codified, but they often extend beyond several months or even years [Table 1]. The main clinical results that one can expect consist in clearly delineating the lesion through the prolonged antifungal therapy. This is supposed to make easier the surgical excision, and also to reduce the subsequent risk of recurrence. A regular follow-up is encouraged throughout three years to ensure complete clinical cure.

Figure 1 a

 1 Figure 1 a) Aspect of the lesions on the dorsal side of the left hand, facing the third and fourth Erreur ! Source du renvoi introuvable.extensor muscle's tendon; b) Aspect of the lesion after two months of treatment by isavuconazole and terbinafine ointment on local apply; c) Aspect of the lesion after six month of treatment by isavuconazole and terbinafine ointment on local apply, followed by six months of oral terbinafine.

Figure 2 a

 2 Figure 2 a) T1-weighted magnetic resonance imaging (MRI) of the left upper limb according to axial view exhibited a cutaneous injury measuring approximately 32 x 11 mm with bone contact (shown by the yellow arrow) but with neither bone edema nor osteitis; b) T1-weighted MRI of the left upper limb according to coronal view showed a lesion facing the third and fourth Erreur ! Source du renvoi introuvable.extensor muscle's tendon (shown by the yellow arrow)

Figure 3 a

 3 Figure 3 a) Histological observation according to HPS staining (Hematoxylin Phloxine Saffron) showed inflammatory granulomatous giganto-epithelioid infiltrates, with polymorphonuclear cells and lymphocytes developed near mycelial structures. No obvious criterion of malignancy was observed. b) Gomori-Grocott methenamine silver staining, and c) PAS staining (Periodic Acid Schiff) preparations highlighted melanized hyphae and beadlike swellings. No grains were seen, so the diagnosis of mycetoma was clearly rejected.

Figure 4

 4 Figure 4 Different colony morphologies (obverse and reverse) of Trematosphaeria grisea after ten days of in vitro growth at 30°C. Colonies were velvety, a) light grey and radially folded on Sabouraud-dextrose agar; b) light brown on oatmeal agar (OA); c) heaped with dark grey reverse on 2% malt extract agar (MEA) and d) light tan on potato-dextrose agar (PDA).

Acknowledgements

The authors are thankful to the patient. He didn't opposite to the publication of his medical case.

Funding

None

Disclosure

All authors have seen and approved the manuscript. They all contributed significantly to the work. They have no conflict of interest to declare. The manuscript has not been previously published, nor is not being considered for publication elsewhere.


S Le Guellec

S Ehrmann

Laurent Vecellio
email: vecellio@med.univ-tours.fr

In vitroin vivo correlation of intranasal drug deposition

Keywords: nasal spray, deposition, cast, nebulizer, in vitro, in vivo, particle size, anatomical model, devices

In vitroin vivo correlation (IVIVC) allows prediction of in vivo drug deposition from a nasally inhaled drug based on in vitro drug measurements. In vitro measurements include physical particle characterization and, more recently, deposition studies using anatomical models. Currently, there is a lack of IVIVC for deposition measurements in anatomical models, especially for deposition patterns in various nasal cavity regions. Therefore, improvement of in vitro and in vivo measurement methods and knowledge about nasal deposition mechanisms should help IVIVC in the future.

Graphical abstract:

In vitro parameters to be considered to predict in vivo nasal drug deposition: particle characteristics generated by drug delivery devices, patient ventilation and anatomy using anatomical model tools, for example.

Introduction

Aerosol inhalation has been studied for many years in the fields of pollution and pharmaceutics. The main focus has been on lung deposition, to prevent lung contamination by pollutants or to administer drugs by inhalation to treat patients. The nose has generally been considered as a whole in terms of anatomy and described as a "filter." However, the nasal cavity has a complex structure, with specific anatomical regions to be targeted by drug delivery (Figure 1). It is a large, air-filled space above and behind the facial visible nose, corresponding to a surface area of about 160 cm 2 and volume of 15 mL. The nasal septum divides the cavity into two fossae, with each fossa extending from the vestibule of the nose to the nasopharynx. Behind the vestibule and along each outer wall, there are three turbinates (superior, middle and inferior), which theoretically serve as anatomical targets for locally acting corticosteroid and antibiotics [START_REF] Laube | Devices for aerosol delivery to treat sinusitis[END_REF], as well as drugs intended for systemic effects [START_REF] Pires | Intranasal drug delivery: how, why and what for?[END_REF]. Beside and above the superior turbinate is the olfactory region of the nasal cavity, with the surface area corresponding to 5 cm 2 [START_REF] Gizurarson | Anatomical and histological factors affecting intranasal drug and vaccine delivery[END_REF], which may be targeted for nose to brain drug delivery [START_REF] Djupesland | The nasal approach to delivering treatment for brain diseases: an anatomic, physiologic, and delivery technology overview[END_REF]. The frontal, maxillary and anterior ethmoidal sinuses open into the middle meatus, which represents the regions of interest (ROIs) for sinus targeting [START_REF] Laube | Devices for aerosol delivery to treat sinusitis[END_REF]. The upper part of the rhinopharynx represents the main lymphatic drainage region of the nose, making it an attractive target for vaccine delivery [START_REF] Gizurarson | Anatomical and histological factors affecting intranasal drug and vaccine delivery[END_REF].

Among nasal drug delivery, there is an increasing interest to deliver drugs targeting these specific anatomical regions. However, to date, there is no clear relationship between the anatomical site of drug deposition and clinical efficacy. Beyond targeting the nose, itself, nasal drug delivery can serve for targeting the brain, the immune system (e.g., vaccines) or other systemic effects. From a physiological point of view, the nose is a non-vital organ, and thus, presents advantages in terms of drug toxicity risk by comparison to the lung.

Conversely, nasal drug deposition is not always desired and one aims to avoid it when targeting the lung. For example, aerosol administration via nasal cannula during nasal high flow therapy is a new way for drug administration, potentially well tolerated, but exposing to significant nasal deposition [START_REF] Dugernier | Nasal High-Flow Nebulization for Lung Drug Delivery: Theoretical, Experimental, and Clinical Application[END_REF]. Therefore, deposition studies in the nose may have a double interest: either with the aim of favoring nasal deposition or with the aim of avoiding it when targeting the lung.

Devices used for nasal drug delivery generate particles from a few micrometers (nebulizers) to hundreds of micrometers (spray pumps) with a large range of particle velocity. The range of particle characteristics is much larger than for traditional lung aerosol devices, generating particles between 1-5 µm. Drugs can be delivered through the nostrils or during the expiratory phase through exhaled aerosol deposition [START_REF] Wiesmiller | The impact of expiration on particle deposition within the nasal cavity[END_REF]. Taken together, there are many differences between lung and nasal drug delivery in terms of aerosol characteristics, anatomy, and mechanism of particle deposition, and thus, in vitro -in vivo correlation needs distinct specific considerations.

From a scientific point of view, in vitro studies are conducted before clinical trials in order to optimize drug delivery methods or to demonstrate the in vitro targeting. The in vitro step is based on the hypothesis of a positive correlation between in vitro measurement and in vivo deposition.

In this review, we will present the tools available to comprehensively characterize particles and their nasal deposition using in vitro and in vivo methods and the correlation between both. Moreover, we will examine the unmet needs to improve the in vitro -in vivo correlation (IVIVC) in nasal deposition.

In vitro methods

2.1 Aerosol physical characteristics measurements 2.1.1. Regulatory methods Nasal spray pump performance can be evaluated in accordance with different regulations, such as those set out by the EMA, US FDA [START_REF]Guideline on the pharmaceutical quality of inhalation and nasal products[END_REF][START_REF]Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products--Chemistry, Manufacturing, and Controls Documentation Guidance for Industry[END_REF] or one of the pharmacopoeias [START_REF]Nasal Preparations/Nasalia[END_REF]. Physical characteristics of the spray are then measured with precise methods. Shot weight measurements consist of weighing single actuations and provide information about the consistency of the dose delivered to the patient; however, it does not measure the delivered active compounds per dose. The delivery dose consists of spray actuation in a suitable container to collect the drug and to assay it in order to determine the mass of active compound delivered per dose.

Droplet size distribution (DSD) is measured by laser diffraction at two distances from the actuator orifice. The spray generation over time can be broken down in three phases: flow formation, developed flow and flow dissipation [START_REF]Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays for Local Action April[END_REF]. The DSD is measured in vitro during the fully developed phase; however, patients receive the drug during the three phases. Results are generally reported in terms of D10, D50, D90, span and percentage of particles smaller than 10 µm to predict lung penetration. In addition, laser diffraction does not measure the drug contained in the droplets, nor aerodynamic diameter. In the case of a liquid solution, the particle size distribution should be similar in terms of the volume and drug mass but it could be different for a liquid suspension or a powder including active compounds and excipients.

The cascade impactor is the measurement device allowing drug quantification within the particles and measurement of the aerodynamic diameter. It has been initially designed for measurements of aerosols and not for spray delivery. Regarding nasal systems, particle size is measured by a cascade impactor using a glass expansion chamber and a pre-separator.

Preferably, the glass expansion chamber would have a volume larger than 2L [START_REF] Doub | Measurement of drug in small particles from aqueous nasal sprays by Andersen Cascade Impactor[END_REF]; however, its relevance is questionable regarding its volume and its geometry compared to nasal cavities [START_REF] Williams | Evaluation of Nasal Inlet Ports Having Simplified Geometry for the Pharmacopeial Assessment of Mass Fraction of Dose Likely to Penetrate Beyond the Nasopharynx: A Preliminary Investigation[END_REF]. Moreover, it is not able to measure the complete distribution of the spray due to its size range limitation, which is smaller than the spray DSD. Rather, cascade impactor testing is intended to quantify the mass of the drug in small droplets to evaluate the dose that could reach the lungs.

Evaluation of spray pattern and plume geometry consists in shape/geometry measurements of the spray. It uses imaging methods to determine the angle and the width of the spray plume, the ovality ratio and the area of the section of the spray. Measurements are generally performed at two different distances from the actuator. Moreover, spray pattern evaluation is very challenging due to a high measurement variability, which can be explained by the differences in terms of the testing method itself, including time lag between consecutive actuations, day-to-day variability and laboratory-specific variability [START_REF] Grmaš | Analytical Challenges of Spray Pattern Method Development for Purposes of In vitro Bioequivalence Testing in the Case of a Nasal Spray Product[END_REF]. These measurements do not take into account the amount of drug contained in the different parts of the spray, which may be heterogeneous.

Spray pattern and spray plume geometry measurements are generally performed using automatic mechanical actuators in order to control the stroke length, actuation velocity and actuation force and thus, favor reproducibility of the spray characteristics [START_REF] Trows | Analytical challenges and regulatory requirements for nasal drug products in Europe and the U[END_REF][START_REF] Guo | The influence of actuation parameters on in vitro testing of nasal spray products[END_REF], even if it does not correspond to patient variability observed in clinical practice [START_REF] Kippax | Applications for Droplet Sizing. Manual versus Automated Actuation of Nasal Sprays[END_REF][START_REF] Doughty | Automated actuation of nasal spray products: effect of hand-related variability on the in vitro performance of Flonase nasal spray[END_REF][START_REF] Farjas | Influence of the Human Factor on Nasal Drug Delivery Device Evaluation[END_REF].

For a suspension in a nasal spray, drug particle size of the active compound has the potential to influence the drug bioavailability to the anatomical target zone [START_REF]Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays for Local Action April[END_REF]. In this context, Morphologically-Directed Raman Spectroscopy (MDRS) has emerged as a new method able to characterize the particle size and the shape of active pharmaceutical ingredient (API) in the drug product. Although MDRS has been recommended for the approval of generic mometasone furoate nasal suspension spray [START_REF] Liu | Scientific Considerations for the Review and Approval of First Generic Mometasone Furoate Nasal Suspension Spray in the United States from the Bioequivalence Perspective[END_REF], it measures the API particle size in the total collected spray and not in the different droplets sizes produced by the spray, which could impact the API deposition distribution in the nasal cavity and, consequently, the clinical effect.

Regarding the nasal nebulizer's performance, there is no medical device standard regarding its in vitro evaluation, even if EN ISO 27427 [START_REF]Anaesthetic and respiratory equipment -Nebulizing systems and components[END_REF] could be technically applied to quantify the aerosol product (inhaled mass) and measure the particle size by cascade impaction.

Although the FDA Guidance describes tests for nasal aerosol, it concerns multidose devices such as nasal pressurized metered dose inhalers, and not nebulizers.

Alternative methods

Alternatives to regulatory methods which involve using imaging techniques for spray pattern and plume geometry evaluation have been recently developed. They consist in a modified United States Pharmacopeia (USP) induction port to characterize nasal spray plume geometry [START_REF] Moraga-Espinoza | A modified USP induction port to characterize nasal spray plume geometry and predict turbinate deposition under flow[END_REF]. It uses the inertial method to collect the drug along the segmented induction port reflecting the distribution of the angle of the droplets emitted from the nozzle. MMPA (mass median plume angle), is then calculated and corresponds to the angle where 50% of the drug is deposited. This method enables measuring the distribution of the active compounds in the spray angle, which cannot be done by imaging techniques.

The particle velocity is not measured by regulatory methods and could have an impact on nasal deposition. Phase-Doppler anemometry is a method allowing the simultaneous measurement of droplet velocity and size. It has been observed that nasal spray pumps generate large-sized droplets, which tend to have higher velocities than small generated droplets [START_REF] Liu | Assessment of the influence factors on nasal spray droplet velocity using phase-Doppler anemometry (PDA)[END_REF]. Aerosol characteristics are generally measured at different distances from the nozzle because the spray does not have the same characteristics at different nozzle distances.

Particle image velocimetry and particle/droplet image analysis is used to measure the external spray characteristics and the DSD from a continuous spray. A study from Inthavong et al. [START_REF] Inthavong | Measurements of droplet size distribution and analysis of nasal spray atomization from different actuation pressure[END_REF], which involves using this technique, has observed a heterogeneous distribution of particle size and velocity in the different regions of the spray over the atomization duration.

These high precision measurements could be very interesting for particle deposition prediction in situations where the spray plume cannot develop to its full size, as it may happen inside the nasal vestibule in vivo. Combined with the intrinsic heterogeneity of the spray this may have important consequences on deposition prediction. Indeed, particle image velocimetry has the advantage of measuring the particle size in different regions of the spray over time; however, it is not able to measure the drug contained in the droplets.

These new methods provide additional information to regulatory methods, but no single method completely characterizes the spray and enables comprehensive deposition prediction in the different regions of nasal cavities.

Anatomical models

The modelling of nasal cavities is widely used to study the intranasal deposition of nasally inhaled drugs. This method serves as an in vitro tool that mimics upper airway geometry more or less closely to human geometry.

Different types of models

Thirty-eight different anatomical models have been identified in the literature and listed in Table 1 . The in vivo representativeness of these models was analyzed and is presented in Figure 2. The first models were created using glass and were only composed of a simple tube with two openings (nasal fossae) communicating with a sinus model [START_REF] Badre | Librairie Arnette) La physiologie des sinues et ses applications cliniques et thérapeutiques[END_REF][START_REF] Kim | Aerosol deposition in the airway with excessive mucus[END_REF].

Several more recent models share the same kind of simplified geometry. Nasal fossae have been modelled by a plastic tube [START_REF] Maniscalco | Assessment of nasal and sinus nitric oxide output using single-breath humming exhalations[END_REF][START_REF] Möller | Nasal high flow clears anatomical dead space in upper airway models[END_REF], bottles [START_REF] Xi | Understanding the mechanisms underlying pulsating aerosol delivery to the maxillary sinus: In vitro tests and computational simulations[END_REF] or by using a plastic cavity with simulated turbinates [START_REF] Durand | In vitro study of sonic aerosol to maxillary sinuses treatment, Drug Delivery to the Lungs[END_REF][START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF]. The nasal valve is also simulated by a narrowing in the tubes [START_REF] Möller | Nasal high flow clears anatomical dead space in upper airway models[END_REF]. These first structures could be equipped with narrow openings and parallel cavities to simulate paranasal sinuses using glass vials or syringes [START_REF] Maniscalco | Assessment of nasal and sinus nitric oxide output using single-breath humming exhalations[END_REF][START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF][START_REF] Thieme | Ventilation imaging of the paranasal sinuses using xenon-enhanced dynamic single-energy CT and dual-energy CT: A feasibility study in a nasal cast[END_REF].

Cadaver heads may intuitively be considered as an ideal representation of human geometry [START_REF] St Martin | Rimell Deposition of aerosolized particles in the maxillary sinuses before and after endoscopic sinus surgery[END_REF][START_REF] Hilton | Differential deposition of aerosols in the maxillary sinus of human cadavers by particle size[END_REF][START_REF] Moffa | Nasal delivery devices: A comparative study on cadaver model[END_REF][START_REF] Valentine | A prospective controlled trial of pulsed nasal nebulizer in maximally dissected cadavers[END_REF][START_REF] Manes | Prospective evaluation of aerosol delivery by a powered nasal nebulizer in the cadaver model[END_REF]; however, preservation issues and tissue retractions can rapidly limit the uses of such models. Plastination is an old preservative method consisting in replacing water and lipids by silicon, adapted by Durand et al. [START_REF] Durand | Preliminary study of the deposition of aerosol in the maxillary sinuses using a plastinated model[END_REF] and used on cadaver heads to overcome these limitations. Their plastinated models enable a great degree of preservation of anatomical structures and have been validated by rhinomanometry in terms of nasal resistance [START_REF] Croce | In vitro Experiments and Numerical Simulations of Airflow in Realistic Nasal Airway Geometry[END_REF][START_REF] Durand | Plastinated nasal model: a new concept of anatomically realistic cast[END_REF].

These models obtained from cadaver specimens present a high level of complexity in vivo representativeness.

Other models have been designed to mimic human geometry with a higher precision. Specifically, 3D-printed nasal models have been made by stereolithography using fused plastic deposition [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF] or resin polymerization [START_REF] Leclerc | Impact of airborne particle size, acoustic airflow and breathing pattern on delivery of nebulized antibiotic into the maxillary sinuses using a realistic human nasal replica[END_REF][START_REF] Francis | vitro study of aerosol deposition in nasal cavities during inhalation and exhalation[END_REF].

Since the first 3D-printed model [START_REF] Swift | Inspiratory inertial deposition of aerosols in human nasal airway replicate casts: Implication for the proposed NCRP lung model[END_REF], this technology has gained great interest. This modelling method allows increased sophistication and accuracy in in vitro geometry realism.

So far, all reported models are relatively complete in terms of anatomical features (Figure 2, Table 1) . This technique was particularly used to mimic pediatric upper airways [START_REF] Janssens | The Sophia anatomical infant nose-throat (saint) model: A valuable tool to study aerosol deposition in infants[END_REF][START_REF] Laube | Deposition of albuterol aerosol generated by pneumatic nebulizer in the Sophia Anatomical Infant Nose-Throat (SAINT) model[END_REF][START_REF] Storey-Bishoff | Deposition of micrometer-sized aerosol particles in infant nasal airway replicas[END_REF][START_REF] Minocchieri | Development of the premature infant nose throat-model (PrINT-Model): an upper airway replica of a premature neonate for the study of aerosol delivery[END_REF] to replace in vivo studies among children, which are required strong ethical considerations and procedures. A 3D-file of a pediatric nose and throat model is available to download for free [59], with other models being commercially available (Koken®, Japan). At the same time, bench studies services using personalized models have developed [74, 75].

Deposition quantification

Global nasal deposition has been investigated in in vitro anatomic models according to different aerosol characteristics and physical parameters [START_REF] Zhou | Nasal deposition in infants and children[END_REF][START_REF] Maniscalco | Sounding airflow enhances aerosol delivery into the paranasal sinuses[END_REF], or in comparison to simulated lung deposition [START_REF] Laube | Deposition of albuterol aerosol generated by pneumatic nebulizer in the Sophia Anatomical Infant Nose-Throat (SAINT) model[END_REF][START_REF] Réminiac | Nasal high flow nebulization in infants and toddlers: An in vitro and in vivo scintigraphic study[END_REF]. Most studies investigated the distribution of the deposited aerosol in different parts of the nasal model [START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF][START_REF] Kaye | Development and testing of particulate formulations for the nasal delivery of antibodies[END_REF][START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF]. Therefore, dismantlable models have been developed. Turbinates, maxillary sinuses and the ethmoid region are the principal regions of interest (ROIs) studied for nasal aerosol deposition. Spray deposition patterns are usually investigated in models with one or two nasal fossae geometries containing the three turbinates [START_REF] Foo | The influence of spray properties on intranasal deposition[END_REF][START_REF] Kundoor | Assessment of nasal spray deposition pattern in a silicone human nose model using a color-based method[END_REF][START_REF] Kundoor | Effect of formulation-and administration-related variables on deposition pattern of nasal spray pumps evaluated using a nasal cast[END_REF][START_REF] Lungare | Development and Evaluation of a Novel Intranasal Spray for the Delivery of Amantadine[END_REF][START_REF] Xi | Visualization and Quantification of Nasal and Olfactory Deposition in a Sectional Adult Nasal Airway Cast[END_REF][START_REF] Warnken | Personalized Medicine in Nasal Delivery: The Use of Patient-Specific Administration Parameters to Improve Nasal Drug Targeting Using 3D-Printed Nasal Replica Casts[END_REF] without paranasal sinuses. Moreover, some authors have studied the influence of different parameters, such as the addition of vibrations or the particle size, on the capacity of the aerosol to penetrate into the maxillary sinuses [START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF][START_REF] Guillerm | A new method of aerosol penetration into the sinuses[END_REF][START_REF] Durand | Impact of acoustic airflow nebulization on intrasinus drug deposition of a human plastinated nasal cast: New insights into the mechanisms involved[END_REF][START_REF] Leclerc | Impact of acoustic airflow on intrasinus drug deposition: New insights into the vibrating mode and the optimal acoustic frequency to enhance the delivery of nebulized antibiotic[END_REF][START_REF] Sato | Intra-nasal distribution of aerosols with or without vibration[END_REF]. In the same way, deposition of aerosolized drugs in the ethmoid region is investigated with the aim to target the olfactory region [START_REF] Reychler | Effect of three-drug delivery modalities on Olfactory function in chronic sinusitis[END_REF] and/or to reach the brain [START_REF] Yarragudi | Formulation of olfactory-targeted microparticles with tamarind seed polysaccharide to improve nose-to-brain transport of drugs[END_REF]. In parallel, nasal anatomic models were also used to investigate enhanced lung deposition of inhaled aerosols [START_REF] Janssens | The Sophia anatomical infant nose-throat (saint) model: A valuable tool to study aerosol deposition in infants[END_REF][START_REF] Réminiac | Nasal high flow nebulization in infants and toddlers: An in vitro and in vivo scintigraphic study[END_REF].

For aerosol deposition experiments with nebulizers, the anatomic model is usually ventilated by connecting a respiratory pump to mimic the patients' breathing [START_REF] Laube | Deposition of albuterol aerosol generated by pneumatic nebulizer in the Sophia Anatomical Infant Nose-Throat (SAINT) model[END_REF][START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF]. DSD of the aerosols inhaled through the model may also be studied by connecting the model to a cascade impactor [START_REF] Janssens | The Sophia anatomical infant nose-throat (saint) model: A valuable tool to study aerosol deposition in infants[END_REF]. In most nasal spray deposition studies, no ventilation is generated through the nasal model [START_REF] Kundoor | Effect of formulation-and administration-related variables on deposition pattern of nasal spray pumps evaluated using a nasal cast[END_REF][START_REF] Sartoretti | In vitro qualitative and quantitative CT assessment of iodinated aerosol nasal deposition using a 3D-printed nasal replica[END_REF][START_REF] Foo | A Simplified Geometric Model to Predict Nasal Spray Deposition in Children and Adults[END_REF]. However, in clinical practices, different breathing recommendations are made to patients during and after nasal spray administration, such as sniffing. However, few authors have included specific breathing patterns in their in vitro testing with nasal sprays [START_REF] Foo | The influence of spray properties on intranasal deposition[END_REF][START_REF] Guo | The effect of formulation variables and breathing patterns on the site of nasal deposition in an anatomically correct model[END_REF].

Anatomical models have been improved beyond geometric realism to mimic human cavities by adding artificial mucus [START_REF] Kim | Aerosol deposition in the airway with excessive mucus[END_REF], humidification or a flexible nose and face. For example, different mucus preparations were used by Sawant [START_REF] Sawant | In vitro Assessment of Spray Deposition Patterns in a Pediatric (12 Year-Old) Nasal Cavity Model[END_REF] to coat a pediatric (12-year-old) model. Coating simulates the presence of a mucosal surfactant, or mucus, plays a role in entrapping inhaled particles and allows humidification of the model [START_REF] Kaye | Development and testing of particulate formulations for the nasal delivery of antibodies[END_REF][START_REF] Castile | Development of in vitro models to demonstrate the ability of PecSys®, an in situ nasal gelling technology, to reduce nasal run-off and drip[END_REF]. Humidification can also be obtained by connecting a heater/humidifier to the model [START_REF] Salade | How to characterize a nasal product. The state of the art of in vitro and ex vivo specific methods[END_REF]. Recently the possibility to print the vestibule or the face in flexible materials enables simulating aerosol administration more closely to in vivo conditions with an improved accuracy in interface positioning [START_REF] Sartoretti | In vitro qualitative and quantitative CT assessment of iodinated aerosol nasal deposition using a 3D-printed nasal replica[END_REF][START_REF] Carrigy | Effect of facial material softness and applied force on face mask dead volume, face mask seal, and inhaled corticosteroid delivery through an idealized infant replica[END_REF].

Nasal deposition within the anatomic models can be measured by different methods, some of which are also used for in vivo clinical evaluation. Specifically, gamma scintigraphy is widely used on nasal casts. It allows radiotracer quantification for total or regional nasal deposition calculations [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF][START_REF] Laube | Deposition of albuterol aerosol generated by pneumatic nebulizer in the Sophia Anatomical Infant Nose-Throat (SAINT) model[END_REF][START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF], but has limitations for precise quantification in small ROIs [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF]. Moreover, 3D single photon emission computed tomography coupled with computed tomography (SPECT/CT) enhances the accuracy of measurements [START_REF] Leclerc | Assessing sinus aerosol deposition: Benefits of SPECT-CT imaging[END_REF]. The attenuation property of the model, which depends on the material and the model type, is not systematically included in studies. Tracers may also be used to visualize and/or quantify nasal deposition, by photography and/or UV-spectrophotometry dosages. In this case, the model used should be transparent or dismantlable to collect and analyze the tracer deposition [54 71, 94]. Nasal endoscopy can be used to visualize the tracer deposition in closed models. Durand et al. have inserted a humidity sensor into maxillary sinuses to detect the aerosol penetration in these cavities [START_REF] Durand | Preliminary study of the deposition of aerosol in the maxillary sinuses using a plastinated model[END_REF]. Recently, iodinated aerosols deposition quantification was directly performed by computed tomography (CT) [START_REF] Sartoretti | In vitro qualitative and quantitative CT assessment of iodinated aerosol nasal deposition using a 3D-printed nasal replica[END_REF].

In vivo methods

In vivo assessment is a core requirement for IVIVC analysis. The anatomical complexity of the nasal cavity and its high interspecies variability renders most animal in vivo models unsuitable to study human nasal spray deposition. Therefore, human clinical studies are required to fully validate in vivo the results observed on nasal casts and in other in vitro experiments [START_REF] Réminiac | Nasal high flow nebulization in infants and toddlers: An in vitro and in vivo scintigraphic study[END_REF]. Given the various targets pursued through nasal drug deposition (nasal mucosa, sinuses, brain, systemic effects, etc.) and the complexity of setting up clinical studies, some authors evaluated direct or surrogate markers of biological efficacy. In such studies, the efficacy measurements may preclude concomitant nasal deposition measurement and thus, correlation to in vitro experiments. In fact, given the complexity of the nasal anatomy, the correlation between nasal drug deposition and some biological effects may be less straightforward than for other targets of inhaled drug delivery, such as lung deposition.

Direct nasal deposition quantification methods

A great number of studies have evaluated nasal deposition of various tracers delivered as nasal sprays to humans. The first studies used direct endoscopic visualization of nasal deposition of solutions containing a dye (methylene blue, fluorescein, etc.) sprayed in the nose of healthy subjects or patients [START_REF] Bleier | Temporospatial quantification of fluorescein-labeled sinonasal irrigation delivery[END_REF][START_REF] Hardy | Intranasal drug delivery by spray and drops[END_REF]. This initially qualitative technique was improved by some groups by implementing imaging post-processing techniques to quantify dye deposition. To improve quantification of deposition, a great number of studies used radiolabeled solutions coupled with scintigraphy measurement of deposition [START_REF] Möller | Pulsating aerosols for drug delivery to the sinuses in healthy volunteers[END_REF][START_REF] Hardy | Intranasal drug delivery by spray and drops[END_REF][START_REF] Emanuel | Nasal deposition of ciclesonide nasal aerosol and mometasone aqueous nasal spray in allergic rhinitis patients[END_REF][START_REF] Djupesland | Nasal deposition and clearance in man: comparison of a bidirectional powder device and a traditional liquid spray pump[END_REF][START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF][START_REF] Djupesland | Bi-directional nasal delivery of aerosols can prevent lung deposition[END_REF][START_REF] Djupesland | Breath actuated device improves delivery to target sites beyond the nasal valve[END_REF][START_REF] Bacon | Pulmonary and nasal deposition of ketorolac tromethamine solution (SPRIX) following intranasal administration[END_REF][START_REF] Möller | Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols[END_REF].

An important limitation of such imaging techniques is related to precisely delineating the ROIs within the nose to quantify deposition. In addition, anatomical landmarks external to the nose may be radiolabeled, while the nose size can be estimated by acoustic rhinometry in order to delineate specific ROIs to quantify deposition [START_REF] Hardy | Intranasal drug delivery by spray and drops[END_REF][START_REF] Djupesland | Bi-directional nasal delivery of aerosols can prevent lung deposition[END_REF][START_REF] Möller | Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols[END_REF]. To more precisely estimate regional deposition within the nose, deposition scintigraphy may be combined with anatomic imaging, such as magnetic resonance imaging (MRI) [START_REF] Hardy | Intranasal drug delivery by spray and drops[END_REF][START_REF] Möller | Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols[END_REF], CT [START_REF] Möller | Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols[END_REF] or radioactive gas ventilation scintigraphy [START_REF] Möller | Pulsating aerosols for drug delivery to the sinuses in healthy volunteers[END_REF][START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF]. Currently, SPECT/CT using various sprayed tracers represents a significant improvement from 2D planar scintigraphy [START_REF] Leclerc | Assessing sinus aerosol deposition: Benefits of SPECT-CT imaging[END_REF]. Positron emission tomography (PET) may enable to envision specific drug labeling or nasal physiologic effects quantification [104]. However, the nose is a relatively small organ and regions with different functional implication are located in very close proximity; thus, deposition analysis may be impacted by variability in ROIs delineation and small patient-to-patient anatomic variation may have an important impact on the accuracy of regional deposition evaluation within the nose. Correction for local tissue attenuation is also a challenge that has a major impact on quantitative deposition analysis, which is required to move beyond qualitative deposition patterns [START_REF] Skretting | A new method for scintigraphic quantification of deposition and clearance in anatomical regions of the human nose[END_REF].

Indirect quantification methods

Depending on study design and objectives, nasal deposition of an inhaled drug may be quantified indirectly. Thus, studies evaluating pulmonary drug delivery through the nasal route can be used to indirectly assess nasal deposition as well. Whatever the tracer and quantification technique, the mass balance principle considers that the part of a given amount of a tracer delivered through the nose not recovered in the lung, upper airways, digestive tract or systemic circulation has been deposited in the nose. This may be considered positively if the nose was the target and negatively otherwise [START_REF] Djupesland | Bi-directional nasal delivery of aerosols can prevent lung deposition[END_REF]. Systemic pharmacokinetics may also serve as a surrogate measurement of nasal deposition and absorption when a concomitant lack of pulmonary delivery is observed [START_REF] Suman | Validity of in vitro tests on aqueous spray pumps as surrogates for nasal deposition, absorption, and biologic response[END_REF]. However, although indirect quantification methods enable comparisons between different nasal administration techniques, they do not enable IVIVC.

Biological effect measurement methods

Beyond nasal deposition, direct biological effects or measurements of surrogate markers of such effects represent the final clinical objective of in vivo evaluation. Such biological effects to be measured are as numerous as the clinical targets associated with nasal drug delivery, reaching from local to regional and systemic effects. First, nasal tissue is relatively simple to biopsy, which may enable direct histological evaluation of drug effects on epithelial remodeling and inflammation, for example [START_REF] Mastruzzo | Impact of intranasal budesonide on immune inflammatory responses and epithelial remodeling in chronic upper airway inflammation[END_REF] Local effects of drugs can also be evaluated macroscopically, through endoscopic examination (e.g., polyp grading [START_REF] Sindwani | NAVIGATE I: Randomized, Placebo-Controlled, Double-Blind Trial of the Exhalation Delivery System With Fluticasone for Chronic Rhinosinusitis With Nasal Polyps[END_REF]) or functionally, through standardized olfactory function testing [START_REF] Reychler | Effect of three-drug delivery modalities on Olfactory function in chronic sinusitis[END_REF]. Sinus drug deposition has also been evaluated, indirectly measuring exhaled nitric oxide after nebulizing a nitric oxide synthase inhibitor in the nose [START_REF] Maniscalco | Sounding airflow enhances aerosol delivery into the paranasal sinuses[END_REF]. Patients symptom relief after drug administration can be evaluated through patient-centered symptom scoring systems, such as the sino-nasal outcome test (SNOT) [START_REF] Mainz | Sinonasal inhalation of dornase alfa in CF: A double-blind placebo-controlled cross-over pilot trial[END_REF], the medical outcomes study sleep scale (MOS-SS) or the morning congestion score [START_REF] Sindwani | NAVIGATE I: Randomized, Placebo-Controlled, Double-Blind Trial of the Exhalation Delivery System With Fluticasone for Chronic Rhinosinusitis With Nasal Polyps[END_REF]. When assessing effects of nose-to-brain drug delivery, direct biological measurements are not feasible in humans. Therefore, indirect assessments are required, such as quantification of cerebral blood flow changes using magnetic imaging [START_REF] Wingrove | Characterisation of nasal devices for delivery of insulin to the brain and evaluation in humans using functional magnetic resonance imaging[END_REF][START_REF] Kullmann | Dose-Dependent Effects of Intranasal Insulin on Resting-State Brain Activity[END_REF] after insulin administration. In addition, the patients' cerebral symptoms scoring may also be performed, such as in nasal migraine therapy evaluation [START_REF] Lipton | DFN-02 (Sumatriptan 10 mg With a Permeation Enhancer) Nasal Spray vs Placebo in the Acute Treatment of Migraine: A Double-Blind, Placebo-Controlled Study[END_REF].

In vitro -In vivo correlation

In the field of intranasal aerosols, IVIVC can be analyzed separately in terms of the global aerosol deposited in the nose, as well as in terms of the regional deposition within the nasal cavities.

Total nasal deposition is generally assessed to compare the efficacy of different nasal aerosol delivery devices to predominantly target the nasal cavity comparatively to the lower respiratory tract. The nasal deposition is, therefore, described as a filtration efficiency function [START_REF] Becquemin | Particle deposition and resistance in the noses of adults and children[END_REF][START_REF] Golshahi | Intranasal Filtration of Inhaled Aerosol in Human Subjects as a Function of Nasal Pressure Drop[END_REF]. Nasal pumps are designed for strict nasal drug deposition, with droplets sizes significantly larger than 10 µm, as required by regulatory bodies. Many studies have already investigated the relationship between the in vitro measured aerosol DSD and in vivo nasal aerosol deposition. Indeed, it is well established that in vivo nasal deposition increases with particle size and velocity [START_REF] Storey-Bishoff | Deposition of micrometer-sized aerosol particles in infant nasal airway replicas[END_REF][START_REF] Becquemin | Particle deposition and resistance in the noses of adults and children[END_REF][START_REF] Cheng | Aerosol Deposition in the Extrathoracic Region[END_REF], which has also been observed in in vitro studies [START_REF] Shah | Design of experiments to optimize an in vitro cast to predict human nasal drug deposition[END_REF][START_REF] Newman | Drug delivery to the nasal cavity: in vitro and in vivo assessment[END_REF]. For example, Moller et al [START_REF] Möller | Pulsating aerosols for drug delivery to the sinuses in healthy volunteers[END_REF] have observed 100% of total nasal deposition with large droplets (60 µm) and only 70% with smaller droplets (3.5 µm). Most of these studies show a good IVIVC; however, some in vivo results are in contradiction with expected in vitro prediction based solely on particle size measurements [START_REF] Newman | Drug delivery to the nasal cavity: in vitro and in vivo assessment[END_REF]. Recently, Vecellio et al. [START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF] observed that a droplet size inferior to 10 µm can be associated with total nasal retention of a nasally inhaled aerosol without any aerosol penetrating the lung. Moreover, Djuspesland observed similar poor IVIVC among 16 healthy volunteers, with two 3.5 µm droplet-sized aerosols produced by two types of nasal devices [START_REF] Djupesland | Bi-directional nasal delivery of aerosols can prevent lung deposition[END_REF]. One aerosol was generated in one nostril by blowing into the device during an oral exhalation, while the other aerosol was generated during a nasal inhalation. These two conditions generate different deposition profiles, illustrating the importance of airflow impact on nasal deposition.

Given these in vivo studies, the DSD should not be considered alone to make an in vivo deposition prediction. Although outside the scope of regulatory requirements, it appears interesting to consider the in vitro airflow supply when evaluating nasally inhaled aerosols and its influence on nasal deposition. In particular, in cases where the anatomic geometry is strongly restricted, such as in children's nasal cavities, airflow and geometry may act in synergy to greatly amplify impaction phenomena in the nose of even micrometric particles.

Concerning regional nasal deposition, most in vitro studies were performed on anatomical models. However, this tool does not measure any aerosol characteristic per se, as it describes and quantifies an aerosol deposition pattern.

A great number of in vitro studies using different types of anatomical models [START_REF] Moffa | Nasal delivery devices: A comparative study on cadaver model[END_REF][START_REF] Francis | vitro study of aerosol deposition in nasal cavities during inhalation and exhalation[END_REF][START_REF] Reychler | Effect of three-drug delivery modalities on Olfactory function in chronic sinusitis[END_REF][START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF][START_REF] Saijo | Particle deposition of therapeutic aerosols in the nose and paranasal sinuses after transnasal sinus surgery: A cast model study[END_REF] as well as in vivo studies, have observed good IVIVC by measuring the deposition of radiolabeled aerosols [START_REF] Djupesland | Nasal deposition and clearance in man: comparison of a bidirectional powder device and a traditional liquid spray pump[END_REF][START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF][START_REF] Suman | Comparison of nasal deposition and clearance of aerosol generated by nebulizer and an aqueous spray pump[END_REF], or by indirectly measuring biological effects such nasal resistance [START_REF] Goh | Lidocaine/Phenylephrine Nasal Spray versus Nebulization Prior to Nasoendoscopy: A Randomized Controlled Trial[END_REF]. However, large and fast droplets as measured by in vitro studies can also have a deep penetration into the nasal cavity if one pays attention to specific implementation details, such as the angle and the insertion depth of the nasal device or the formulation viscosity [START_REF] Kundoor | Effect of formulation-and administration-related variables on deposition pattern of nasal spray pumps evaluated using a nasal cast[END_REF]. However, it has been demonstrated that the plume geometry pattern of two different nasal spray formulations shows a poor IVIVC in terms of the expected deposition in the turbinates of healthy volunteers [START_REF] Suman | Validity of in vitro tests on aqueous spray pumps as surrogates for nasal deposition, absorption, and biologic response[END_REF]. Spray formulations in development are usually tested for deposition in coated nasal models [START_REF] Wingrove | Characterisation of nasal devices for delivery of insulin to the brain and evaluation in humans using functional magnetic resonance imaging[END_REF][START_REF] Foo | The influence of spray properties on intranasal deposition[END_REF] without in vivo deposition validation. The formulation of an aerosol (powder, liquid, gel) may also influence its physical characteristics and, therefore, its deposition. A gel form of aerosol sprayed in a nasal anatomical model, coated with medical lubricant, exhibited a higher deposition in the upper turbinate, in comparison with a non-gel control solution with normal viscosity [START_REF] Castile | Development of in vitro models to demonstrate the ability of PecSys®, an in situ nasal gelling technology, to reduce nasal run-off and drip[END_REF]. Interestingly, a similar turbinate deposition was observed in vivo with a comparable gel formulation in healthy volunteers, quantified by endoscopy detection of fluorescein in comparison with a saline spray [START_REF] Bleier | Temporospatial quantification of fluorescein-labeled sinonasal irrigation delivery[END_REF].

Many studies have focused on targeting maxillary sinuses (MS) aerosol deposition. However, no previously described in vitro characteristics would predict the penetration and deposition of aerosol droplets into the maxillary sinuses without testing the deposition in an anatomical model. Sinus aerosol deposition can be achieved by modifying air flow [START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF][START_REF] Maniscalco | Sounding airflow enhances aerosol delivery into the paranasal sinuses[END_REF]. Most in vitro studies were carried out in anatomical models of various geometries [START_REF] Thieme | Ventilation imaging of the paranasal sinuses using xenon-enhanced dynamic single-energy CT and dual-energy CT: A feasibility study in a nasal cast[END_REF][START_REF] Durand | Preliminary study of the deposition of aerosol in the maxillary sinuses using a plastinated model[END_REF][START_REF] Leclerc | Impact of airborne particle size, acoustic airflow and breathing pattern on delivery of nebulized antibiotic into the maxillary sinuses using a realistic human nasal replica[END_REF][START_REF] Sato | Intra-nasal distribution of aerosols with or without vibration[END_REF][START_REF] Leclerc | Assessing sinus aerosol deposition: Benefits of SPECT-CT imaging[END_REF][START_REF] Merhie | A new strategy to improve drug delivery to the maxillary sinuses: The frequency sweep acoustic airflow[END_REF].

Moreover, a reasonably good correlation has been observed with in vivo deposition study evaluating laminar flow [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF][START_REF] Möller | Pulsating aerosols for drug delivery to the sinuses in healthy volunteers[END_REF][START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF]. Furthermore, a positive correlation may be assumed, given the favorable clinical studies observing positive results on clinical outcomes, such as for SNOT, nasal secretion analysis or nitric oxide measurements [START_REF] Maniscalco | Sounding airflow enhances aerosol delivery into the paranasal sinuses[END_REF][START_REF] Mainz | Sinonasal inhalation of dornase alfa in CF: A double-blind placebo-controlled cross-over pilot trial[END_REF][START_REF] Goektas | Treatment of chronic rhinosinusitis with pressure-pulsed corticosteroid inhalation[END_REF][START_REF] Videler | Nebulized bacitracin/colimycin: a treatment option in recalcitrant chronic rhinosinusitis with Staphylococcus aureus? A double-blind, randomized, placebocontrolled, cross-over pilot study[END_REF]. In addition, in vivo and in vitro data are in agreement, showing that large particles, such as spray droplets, do not reach the maxillary sinuses [START_REF] Möller | Pulsating aerosols for drug delivery to the sinuses in healthy volunteers[END_REF][START_REF] Leclerc | Impact of airborne particle size, acoustic airflow and breathing pattern on delivery of nebulized antibiotic into the maxillary sinuses using a realistic human nasal replica[END_REF][START_REF] Senocak | Sinonasal distribution of topically applied particles: computerized tomographic detection and the effects of topical decongestion[END_REF]. In terms of exact quantification of aerosol deposited in anatomical regions, IVIVC was observed for one anatomical model [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF]. Global and regional aerosol deposition in the nose is also influenced by the specific conditions of use by the patient, despite consistency in spray characteristics and administration parameters (depth, angle). Indeed, Homer et al. observed that vigorously inhaling whilst administering a spray made no difference in terms of its distribution in the turbinate region, in comparison with no inhalation [START_REF] Homer | An endoscopic photographic comparison of nasal drug delivery by aqueous spray[END_REF]. When regarding the patient position (supine vs. upright position), significant differences were observed in terms of deposition pattern of 50 µm sprays [START_REF] Bacon | Pulmonary and nasal deposition of ketorolac tromethamine solution (SPRIX) following intranasal administration[END_REF].

Despite some favorable results, IVIVC could be strongly questioned when considering anatomical characteristic features as influent parameters. First, with regards to the level of in vivo representativeness of anatomical nasal models and the modelling method, the results of IVIVC may not be the same, depending on the selected model used for in vitro deposition analysis [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF][START_REF] Kelly | Particle deposition in human nasal airway replicas manufactured by different methods. Part I: Inertial regime particles[END_REF]. Moreover, some limits in in vivo prediction should be considered with in vitro data deposition obtained from models with sinus volumes which were too small [START_REF] Möller | Ventilation and drug delivery to the paranasal sinuses: Studies in a nasal cast using pulsating airflow[END_REF] or with very smooth and reduced idealized ethmoid geometry [START_REF] Javaheri | An idealized geometry that mimics average infant nasal airway deposition[END_REF]. Creating a standard geometry tool has been an interesting approach to standardize in vitro nasal deposition analysis, but it could strongly decrease the in vivo inter-subject variation, also impacting IVIVC. Recent studies have taken into account these anatomical specifications by increasing modelling accuracy, for either ethmoid or for maxillary sinuses [START_REF] Xi | Visualization and Quantification of Nasal and Olfactory Deposition in a Sectional Adult Nasal Airway Cast[END_REF][START_REF] Yarragudi | Formulation of olfactory-targeted microparticles with tamarind seed polysaccharide to improve nose-to-brain transport of drugs[END_REF][START_REF] Xi | Nasal and Olfactory Deposition with Normal and Bidirectional Intranasal Delivery Techniques: In vitro Tests and Numerical Simulations[END_REF][START_REF] Hosseini | In vitro Measurement of Regional Nasal Drug Delivery with Flonase,® Flonase® Sensimist,TM and MAD Nasal TM in Anatomically Correct Nasal Airway Replicas of Pediatric and Adult Human Subjects[END_REF].

Finally, the internal nasal geometry can be summarized and considered as a tangle of different volumes and surfaces. In the field of nasal treatments, patients can present conditions such as ostium blockage, purulent secretions, nasal polyps, synechiae, turgescent turbinate and septal deviation, etc. In such cases, the surface area to volume ratio is strongly disturbed in comparison with healthy anatomy. Therefore, the in vitro aerosol characteristics that influence in vivo deposition in healthy subjects would not have the same behavior in patients. A small velocity of particles would increase along restricted passages. For instance, deep deposition of aerosols in vitro could be reduced in vivo, due to the presence of nasal polyps. Indeed, in vitro modelling of nasal cavities is limited by the lack of pathological models and consequently a lack of in vitro patients. In this case, a good IVIVC obtained between an "in vitro healthy subject" and an "in vivo healthy volunteer" should not support a good prediction for the "in vivo patient."

The modelling of nasal cavities by 3D-printing led to the creation of a new tool to measure intranasal deposition in vitro according to a large panel of characteristics. The measure of nasal aerosol deposition in these models is now quasi-systematically included in the panel of in vitro characterizations, on top of standard regulatory methods, as a preclinical requirement for in vivo studies. The aim of this complete pre-clinical evaluation is to personalize and design the optimal nasal treatment (formulation, administration, etc.) efficient for each individual patient [START_REF] Warnken | Personalized Medicine in Nasal Delivery: The Use of Patient-Specific Administration Parameters to Improve Nasal Drug Targeting Using 3D-Printed Nasal Replica Casts[END_REF], or to select the optimized setting to be used in the in vivo study [START_REF] Wingrove | Characterisation of nasal devices for delivery of insulin to the brain and evaluation in humans using functional magnetic resonance imaging[END_REF].

Most of the time, users directly consider the nasal model deposition as a quasi in vivo result, on the grounds that the 3D anatomical model is built from human CT-scan. Thus, only a few studies have elaborated an experimental set up to formally test IVIVC implementing the exact same delivery and measurement setup in vitro and in vivo [START_REF] Le Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF][START_REF] Maniscalco | Sounding airflow enhances aerosol delivery into the paranasal sinuses[END_REF].

In short, an improvement in accuracy of the in vivo prediction will be gained by overlapping the different sources of in vitro data.

Expert opinion 5.1 Parameters influencing nasal deposition

Three key parameters have been identified to influence total nasal deposition efficiency: particle characteristics, ventilation and nasal anatomy. Moreover, 10 µm is the limit of particle size reported by regulatory bodies, FDA and EMEA [START_REF]Guideline on the pharmaceutical quality of inhalation and nasal products[END_REF][START_REF]Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products--Chemistry, Manufacturing, and Controls Documentation Guidance for Industry[END_REF] and nasal studies [START_REF] Kelly | Particle deposition in human nasal airway replicas manufactured by different methods. Part I: Inertial regime particles[END_REF] to avoid lung deposition of nasal products. Deposition efficiency in the whole nasal cavities for aerosol particles larger than 1µm is governed by inertial impaction described by the Stokes number, which is a function of the aerodynamic diameter, air flow rate, and the characteristic nasal airway dimension [START_REF] Cheng | Aerosol Deposition in the Extrathoracic Region[END_REF]. Particles larger than 10µm generated by nasal sprays are also governed by the Stokes number, but high Stokes numbers (particles larger 20µm) could impact the sensitivity to other physical parameters, such as injection velocity, insertion angle, and spray cone angle on deposition distribution [START_REF] Inthavong | Measurements of droplet size distribution and analysis of nasal spray atomization from different actuation pressure[END_REF]. Indeed, the 10µm particle size limit has been commonly accepted; however, it remains questionable regarding new nasal devices using fine particles without lung deposition as described above [START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF][START_REF] Djupesland | Bi-directional nasal delivery of aerosols can prevent lung deposition[END_REF]. Moreover, this particle size limit has also raised the question regarding the difference between an aerosol and a nasal spray. Specifically, particles in a nasal spray have a higher particle velocity than aerosols and, consequently, a higher deposition efficacy for a similar particle size. Therefore, a unique limit of 10µm is questionable for a nasal spray.

The influence of air flow on nasal deposition has been clearly demonstrated by in vivo and anatomical model studies with nebulizers. However, the value of air flow is not the only parameter that needs to be considered: the direction of the air flow can also impact nasal and lung deposition.

The effects of nasal anatomy on nasal deposition have been demonstrated with an in vitro anatomical model and in vivo deposition studies [START_REF] Möller | Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols[END_REF]. One computational fluid dynamics study performed on 16 different anatomical nostrils demonstrated that olfactory deposition ranged from 4% to 100% when the injection location and spray characteristics were chosen to maximize olfactory deposition (particle diameter: 5-40 µm; particle velocity: 0-20m/sec; spray half cone angle: 17.5 to 30°, spray cone direction: upward to semi upward) [START_REF] Kiaee | Regional deposition of nasal sprays in adults: A wide ranging computational study[END_REF].

Moreover, an in vitro study using 10 realistic nasal models showed a decrease of 73% to 11% of cromolyn sodium deposition in the turbinate region with the increase of the administration angle from 30° to 75°. Moreover, correlation was also observed between the turbinate deposition, the minimum cross-sectional area of the models and the length of the models [START_REF] Warnken | Personalized Medicine in Nasal Delivery: The Use of Patient-Specific Administration Parameters to Improve Nasal Drug Targeting Using 3D-Printed Nasal Replica Casts[END_REF].

More recently, deposition in 5 realistic nasal casts for two different device orientations were examined by researchers at the University of Alberta in Canada [START_REF] Chen | In vitro assessment of an idealized nose for nasal spray testing: Comparison with regional deposition in realistic nasal replicas[END_REF]. Authors measured deposition in anterior turbinates from 25% to 65% and from 15% to 50% for 45° and 60° spray angles respectively. Moreover, the study's authors concluded that the idealized replica agreed well with average regional deposition in the realistic replicas. Deposition variability was higher with realistic replicas than with the idealized replica. Additional studies may be required with different nasal sprays and aerosols to validate the idealized model, which does not include sinuses [START_REF] Cheng | Aerosol Deposition in the Extrathoracic Region[END_REF].

A study examining in vivo deposition using a nasal spray on 10 volunteers has reported 91% variability in terms of deposition distribution between the upper and lower parts of the nasal cavities, and 92% variability between their inner and outer parts [START_REF] Suman | Comparison of nasal deposition and clearance of aerosol generated by nebulizer and an aqueous spray pump[END_REF]. Furthermore, a large variability (around 60%) for maxillary sinuses deposition was also observed when using a jet nebulizer on 6 healthy volunteers [START_REF] Vecellio | Deposition of aerosols delivered by nasal route with jet and mesh nebulizers[END_REF].

Unmet need for nasal drug IVIVC

IVIVC are generally based on few cases or represent mainly qualitative comparisons. The lack of strong IVIVC for nasal deposition can be explained by several different factors.

First, there is a lack of in vivo studies evaluating the influence of physical parameters on deposition patterns in nasal cavities, thus also limiting evaluation of IVIVC. During inhalation drug development, lung deposition is often measured before the clinical trial, which is generally not the case with nasal drugs, except for highly innovative devices. Based on this consideration, we recommend for the deposition measurements of new drugs to be performed before clinical trials to further elucidate their clinical drug efficacy. However, in vivo deposition studies require validated measurement methods in order to validate high quality results. For example, there is a need to standardize scintigraphy methods for nasal deposition measurement. Relationships between anatomical regions and pharmacokinetic or clinical effect are unclear to date. Scientific studies including deposition measurements and clinical effect evaluation should be conducted similarly to the inhalation studies performed for bronchodilators [START_REF] Usmani | Regional lung deposition and bronchodilator response as a function of beta2-agonist particle size[END_REF].

The lack of strong IVIVC can also be explained by the limitation of in vitro measurement methods. Standardized methods are not always adapted for new nasal drug delivery systems, which questions the relevance of these methods. Nasal sprays generate inhomogeneous particle sizes, as well as concentrations and velocities in the air; furthermore, their characteristics change over the time of the spray generation.

Furthermore, the use of active compounds in the form of a suspension adds complexity to this situation, as the drug concentrations inside droplets could be different. Regarding anatomical models to mimic in vivo deposition, technological advances could help to further improve IVIVC. But once again, key parameters will need to be defined to limit the complexity of the models.

Scientific key driver to improve IVIVC

The use of physics for identification of key parameters to characterize nasal drug delivery

Physics equations can be used to model the kinetics of particles, and thus, the deposition distribution. Based on this consideration, the use of physics formulas should help to select the most important parameters to be measured by in vitro methods. For example, for inhaled aerosol, the Stokes number is the key parameter to predict deposition by impaction [START_REF] Cheng | Aerosol Deposition in the Extrathoracic Region[END_REF]. It takes into account the DSD, the air velocity and the nasal airway dimension. Since sprays and aerosols are different in terms of generation and kinetics, this should be evaluated through the examination of physical impact of those differences and taken into account when performing measurements (when the spray is not fully developed for example).

The use of standardized ROIs

Nasal cavities are complex structures with different geometries. Therefore, ROIs used in studies should be precisely defined with regards to the therapeutic area. Moreover, further studies are necessary to evaluate the influence precise location of the drug deposition on the clinical efficacy. These measures will help to standardize ROIs in nasal cavities, as well as in central lungs for bronchodilator inhalation.

Consideration of the large in vivo variability vs. the in vitro standardized methods

When an IVIVC evaluation is conducted, the in vivo variability is generally higher than the in vitro variability (around 100% in terms of in vivo deposition variability in specific anatomical region vs. 10% for spray characteristic measurement [START_REF] Suman | Comparison of nasal deposition and clearance of aerosol generated by nebulizer and an aqueous spray pump[END_REF]). Therefore, in vitro measurements should be performed, while taking into consideration this in vivo variability as best as possible. Regarding particle characteristic measurement, it seems difficult to take the in vivo variability into consideration. Nasal cast models seem more suitable to reproduce these variabilities and could thus be more relevant for IVIVC evaluation.

Formulation consideration for nasal deposition

Particles can be solid or liquid, with different chemical and physical properties. Accordingly, standard deposition models consider the particle in terms of its aerodynamic diameter or geometric diameter to predict its deposition with no impact of formulation on its deposition. Indeed, this is theoretically true; however, formulation should be considered for IVIVC evaluation regarding the large delivered doses and the rapid change in drug compounds after deposition. The importance of this point can be explained by the total mass of deposited particles per area. If we compare nasal deposition and lung deposition in terms of deposited mass per area, we obtain the value of approximately 1mg/cm 2 for nasal deposition, whereas we obtain the value of less than 0.1µg/cm 2 for lung deposition.

Consequently, there is a 1000-fold higher mass of drug per cm 2 in nasal cavities compared to the lung, explaining, for example why dripping is observed after nasal drug deposition.

Based on this consideration, the deposited mass of particles can act like an anatomical geometry modification, reducing the passage for other particles, and thus, increasing the deposition in this deposited region. The bounce of the deposited powder should also be considered with nasal powder devices for the same reason. Moreover, liquid formulations can also quickly modify the surface recovery after deposition. Using a liquid with a high surface tension could increase the surface of recovery after deposition, and thus, modify rapidly the initial deposition of droplets.

Conclusion

Particles larger than 10 µm are deposited in nasal cavities, but particles smaller than this size may not necessarily penetrate the lungs. Deposition in nasal cavities depends on physical particle characteristics, ventilation and, patient anatomy. The lack of strong IVIVC for deposition patterns in the different regions of nasal cavities could be improved with the emergence of improved anatomical models. Therefore, it is necessary to develop relevant in vitro and in vivo measurement methods and to improve our knowledge regarding the nasal deposition mechanisms. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 1. 3D: three dimensions, CT: computed tomography, w/wo: with or without. Illustration n°1 was adapted from [START_REF] Badre | Librairie Arnette) La physiologie des sinues et ses applications cliniques et thérapeutiques[END_REF] and republished with the French society of Otorhinolaryngology permission. Illustration n°2 was adapted from [START_REF] Maniscalco | Assessment of nasal and sinus nitric oxide output using single-breath humming exhalations[END_REF] and republished with the European Respiratory society permission. Illustration n° 3 was adapted from [START_REF] Xi | Understanding the mechanisms underlying pulsating aerosol delivery to the maxillary sinus: In vitro tests and computational simulations[END_REF] and republished with permission by Copyright Clearance Center (CCC). Illustration n° 4 was republished with permission (CCC) from [START_REF] Kelly | Particle deposition in human nasal airway replicas manufactured by different methods. Part I: Inertial regime particles[END_REF]. Illustration n° 5 was republished with permission (CCC) from [START_REF] Javaheri | An idealized geometry that mimics average infant nasal airway deposition[END_REF]. Illustration n°6 was republished with permission (CCC) from [START_REF] Warnken | Personalized Medicine in Nasal Delivery: The Use of Patient-Specific Administration Parameters to Improve Nasal Drug Targeting Using 3D-Printed Nasal Replica Casts[END_REF]. Illustration n°7 was adapted from [START_REF] Xi | Understanding the mechanisms underlying pulsating aerosol delivery to the maxillary sinus: In vitro tests and computational simulations[END_REF] and republished with permission (CCC). Illustration n°8 was republished with Inhalation magazine permission from [START_REF] Azimi | Evaluating simulated patient use in realistic nasal airway models for the in vitro characterization of nasal spray products[END_REF]. Illustration n°9 was republished with permission (CCC) from [START_REF] Yarragudi | Formulation of olfactory-targeted microparticles with tamarind seed polysaccharide to improve nose-to-brain transport of drugs[END_REF]. Illustration n°10 is a laboratory photograph directly provided by authors, and published with APTARpharma, DTFmedical and University of Tours permissions. Illustration n°11 is a laboratory photograph directly provided by authors. Illustration n°12 was registered with permission from the CFD-Research Corporation website https://www.cfd-research.com/products/nasal-cavity-phantoms/, last accessed on 2020, July 30 th . Illustration n°13 was adapted from [START_REF] Hosseini | In vitro Measurement of Regional Nasal Drug Delivery with Flonase,® Flonase® Sensimist,TM and MAD Nasal TM in Anatomically Correct Nasal Airway Replicas of Pediatric and Adult Human Subjects[END_REF] and republished with permission (CCC). Illustration n°14 is a laboratory photograph directly provided by the authors and published with permission by model holder.

Figure 1 :

 1 Figure 1: Schematic representation of adult nasal cavities and theoretical therapeutic target regions for nasal drug deposition.

Figure 2 :

 2 Figure 2: Classification proposition of in vitro anatomical models of nasal cavities in regards to the complexity and in vivo representativeness of in vitro geometry. The models images are shown as an example to illustrate the proposed classification. The circle number refers to the first column the Table1. 3D: three dimensions, CT: computed tomography, w/wo: with or without. Illustration n°1 was adapted from[START_REF] Badre | Librairie Arnette) La physiologie des sinues et ses applications cliniques et thérapeutiques[END_REF] and republished with the French society of Otorhinolaryngology permission. Illustration n°2 was adapted from[START_REF] Maniscalco | Assessment of nasal and sinus nitric oxide output using single-breath humming exhalations[END_REF] and republished with the European Respiratory society permission. Illustration n° 3 was adapted from[START_REF] Xi | Understanding the mechanisms underlying pulsating aerosol delivery to the maxillary sinus: In vitro tests and computational simulations[END_REF] and republished with permission by Copyright Clearance Center (CCC). Illustration n° 4 was republished with permission (CCC) from[START_REF] Kelly | Particle deposition in human nasal airway replicas manufactured by different methods. Part I: Inertial regime particles[END_REF]. Illustration n° 5 was republished with permission (CCC) from[START_REF] Javaheri | An idealized geometry that mimics average infant nasal airway deposition[END_REF]. Illustration n°6 was republished with permission (CCC) from[START_REF] Warnken | Personalized Medicine in Nasal Delivery: The Use of Patient-Specific Administration Parameters to Improve Nasal Drug Targeting Using 3D-Printed Nasal Replica Casts[END_REF]. Illustration n°7 was adapted from[START_REF] Xi | Understanding the mechanisms underlying pulsating aerosol delivery to the maxillary sinus: In vitro tests and computational simulations[END_REF] and republished with permission (CCC). Illustration n°8 was republished with Inhalation magazine permission from[START_REF] Azimi | Evaluating simulated patient use in realistic nasal airway models for the in vitro characterization of nasal spray products[END_REF]. Illustration n°9 was republished with permission (CCC) from[START_REF] Yarragudi | Formulation of olfactory-targeted microparticles with tamarind seed polysaccharide to improve nose-to-brain transport of drugs[END_REF]. Illustration n°10 is a laboratory photograph directly provided by authors, and published with APTARpharma, DTFmedical and University of Tours permissions. Illustration n°11 is a laboratory photograph directly provided by authors. Illustration n°12 was registered with permission from the CFD-Research Corporation website https://www.cfd-research.com/products/nasal-cavity-phantoms/, last accessed on 2020, July 30

Figure 3 :

 3 Figure 3: Key considerations for In vitro -in vivo correlation of intranasal drug deposition

Table 1 :

 1 Anatomical models reported in the literature (not exhaustive list). The illustration number refers to figure 2. "One-part" indicates that the model cannot be dismantled, (?): the items is not precisely described by the authors, MS: maxillary sinuses, MRI: magnetic resonance imaging, CT: computed tomography, ABS: acrylonitrile butadiene styrene, w/v: weight/volume, FESS: functional endoscopic sinus surgery.

	Illustration number	Name of the model	Modelling process (material)	Geometry and particularities	Technical considerations
	Simplified geometry
	1	Glass Sinus [24, 25]	Traditional manufacture (glass)	-one-part -tube with one simulated sinus	-transparent -reusable, washable
		Airway model		-one-part (?)
		[26]	Traditional	-tube	-dismantlable model (?)
			manufacture	-coating with	-transparent (?)
			(glass)	simulated mucus	-different surface condition
	2	Sinus/Nasal model [27]	Traditional manufacture (plastic)	-two parts -tube and syringe sinus	-dismantlable model -transparent, -reusable, washable -variable geometry,
		ENT anatomical	Traditional	-two parts	-dismantlable model
		model	manufacture	-one square fossae	-transparent,
		[28]	(plastic)	and one simulate MS	-reusable, washable
		PARI nasal cast		-eight parts
		[29-31]	Method not described (polyoxymethylene)	-two plastic fossae with simulated turbinates (sagittal section) & glass vials	-dismantlable model -reusable, washable, -variable geometry
				for paranasal sinuses
		Tube Model			-dismantlable model
		[32]			-transparent,
			Traditional manufacture (sapphire & sodium chloride crystal)	-one-part -tube and nasal valve "nozzle"	-reusable, washable -not designed for nasal deposition but for gas clearance quantification
					(infrared spectroscopy and
					radioactive krypton imaging)
	3	Two-bottle model [33]	Traditional manufacture (plastic, soda bottle)	-two parts -one large bottle (500 ml) and one small (250 ml) + a plastic tube (neck)	-dismantlable model -transparent, -reusable, washable -variable geometry
	Sophisticated but incomplete geometry
		Nasal Airway models:		-mean of 70 parts
		Swift's replica,		(axial sections)
		Guilmette1 replica,		-built from MRI 53-
		Guilmette2 replica	Traditional	year-old male, no
		[34-37]	manufacture	paranasal sinuses,	-dismantlable model
			by hand carving or	-applied for 30 male &	-opaque model (?)
			by milling-machine	30 female geometries	-reusable, washable (?)
			(black acrylic)	from individuals of
				African, American,
				Caucasian, Asian
				ethnical backgrounds
	4	VIPER SLA (no illustration) [38]	Stereolithography on SLA 7000 or Viper Si2 machine printers (resin)	-one-part -built from MRI 53-year-old male -no paranasal sinuses	-transparent model -reusable, washable (?) -printer resolution VIPER > SLA
		Koken model	Method not	-two parts (sagittal	-dismantlable model
		(Koken Co. Ltd.,	described	section)	-transparent model

List of figure legends

List of figure legends

Abstract figure: List of physical parameters studied in previous in vitro studies that have an influence on in vitro nasal deposition.