WO2023017370A1

WO2023017370A1 - Composition in dry powder form for inhalation for use in a topical method of treatment of inflammation and/or oxidative stress of the respiratory system caused by air pollution

Info

Publication number: WO2023017370A1
Application number: PCT/IB2022/057241
Authority: WO
Inventors: Andrea BIFFI
Original assignee: Sofar S.P.A.
Priority date: 2021-08-09
Filing date: 2022-08-04
Publication date: 2023-02-16
Also published as: IT202100021602A1

Abstract

A composition in dry powder form for inhalation comprising: (i) a mixture M that comprises or, alternatively, consists of hyaluronic acid or an acceptable pharmaceutical grade salt thereof and, optionally, (ii) at least one acceptable pharmaceutical grade additive and/or excipient; wherein said composition is for use in a topical, preventive or curative, method of treatment of a disorder, or alteration, or symptom, or disease resulting from inflammation and/or oxidative stress of the respiratory system caused by air pollution.

Description

"Composition in dry powder form for inhalation for use in a topical method of treatment of inflammation and/or oxidative stress of the respiratory system caused by air pollution"

It is an object of the present invention a composition in dry powder form for inhalation comprising: (i) a mixture M comprising or, alternatively, consisting of hyaluronic acid or an acceptable pharmaceutical grade salt thereof, and optionally, (ii) at least one acceptable pharmaceutical grade additive and/or excipient; wherein said composition is for use in a topical, preventive or curative method of treatment of a disorder, or an alteration, or a symptom, or a disease resulting from inflammation and/or oxidative stress of the respiratory system caused by air pollution.

Additionally, it is an object of the present invention a dry powder inhaler operable by a subject by suction by mouth, wherein said inhaler comprises a disposable inhaler body and a disposable cartridge containing said composition in dry powder form; wherein said disposable cartridge is connected to said inhaler body so as to release an effective dose of said composition into the inhaler body.

Pollution is an alteration of the environment, whether anthropogenic or natural in origin, that produces permanent disruption or damage to life in an area and is out of balance with existing natural cycles.

Increasing world population, large urban development and the use of environmentally unfriendly technologies are among the main causes of pollution.

These causes lead to an increasing demand for consumer products and food, with a consequent increase in garbage and waste products, in a perverse spiraling circuit at the expense of the environment.

Pollution can affect air, water and soil, and can also be of noise and light type.

In large population centers or at manufacturing areas, the problem of air pollution is particularly present due to human activities, mainly related to the movement of people and things (by vehicles with internal combustion engines) and the heating or cooling of environments (obtained -directly or indirectly- by the combustion of fossil fuels).

Air pollution is of particular concern because it can cause severe cardiovascular and pulmonary diseases- including heart attack, stroke, atrial fibrillation, and pneumonia - even when exposure occurs over a long period of time at air pollution levels commonly considered to be within allowable regulatory limits.

The Applicant, after lengthy and intensive research and development activity, has developed a composition for use that is capable of protective or curative action on the respiratory system (and in particular on the epithelial cells of that system), in relation to possible damage caused by air pollution, where said composition is easy to administer and well tolerated by any subject, including children.

It is, therefore, an object of the present invention a composition in dry powder form for inhalation comprising: (i) a mixture M comprising or, alternatively, consisting of hyaluronic acid or an acceptable pharmaceutical grade salt thereof, and optionally (II) at least one acceptable pharmaceutical grade additive and/or excipient; wherein said composition is for use in a topical, preventive or curative method of treatment of a disorder, or an alteration, or a symptom, or a disease resulting from inflammation and/or oxidative stress of the respiratory system caused by air pollution, having the characteristics as defined in the accompanying claims.

Additionally, it is an object of the present invention a dry powder inhaler operable by a subject by suction by mouth, wherein said inhaler comprises: a disposable inhaler body and a disposable cartridge containing said composition in dry powder form; wherein said disposable cartridge is connected to said inhaler body so as to release an effective dose of said composition into the inhaler body, having the characteristics as defined in the accompanying claims.

In the present invention the term "composition(s)" means a pharmaceutical composition, or a medical device composition.

In the present invention, the term "dry powder" means a powder having a low moisture (water) content, e.g., a powder having a moisture content in the range from 0.01% to 10% by weight with respect to the total weight of the powder, preferably from 0.05% to 5%, more preferably from 0.1% to 3%.

In the present invention, the term "topical treatment" means that the composition of the invention exerts its effect on the mucosa where it is applied, its absorption being essentially nil.

Within this description, the expressions "PolmonYDEFENCE/DYFESA™", "PolmonYDEFENCE/DYFESA," "PolmonYDEFENCE," and "DYFESA™" will be used interchangeably as synonyms for the composition in dry powder form for inhalation that is the subject of the present invention.

Preferred embodiment of the present invention will be described below by way of example, and thus not as limitations, based on the attached drawings, which show:

- Figure 1 : Schematization of Franz's diffusion cell system employed in Example 1;

- Figure 2: Schematization of the in vitro absorption test of Example 1;

- Figure 3: Applied dose distribution in all compartments for inserts with both healthy and inflamed mucosa with an LPS solution, in the tests of Example 1;

- Figure 4: TEER values of PHASE 1 and PHASE 2 discussed in Example 1;

- Figure 5: TEER values of PHASE 3 discussed in Example 1 ;

- Figure 6: TEER values before and after the treatment of the inserts in PHASE 4;

- Figure 7: Calu-3 cell viability results by MTS assay. The percentage of viable cells compared with the negative cell control determined a range of concentrations of (A) sodium hyaluronate (Na-Hya), mannitol and mixture, (B) urban dust (UD). Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparisons test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001); - Figure 8: Measurement of the concentration-dependent induction of oxidative stress by DCF production (fold increment at 1 hr/O hr) in Calu-3 cells. (A) Sodium hyaluronate 0.02-2 mg/ml (B) PolmonYDEFENCE/DYFESA™ 0.04-5.56 mg/ml and (C) Urban dust 0.02-2 mg/ml in submerged culture. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparisons test (*p<0.033; **p<0.0021; ***p<0.0002; ****p<0.0001);

- Figure 9: Co-incubation study of oxidative stress-induced DCF production in Calu-3 cells over a range of concentrations and times. Statistical significance was calculated by comparing the following conditions: media only versus positive and negative controls and UD only; UD only versus each annotated compound concentration. One-way ANOVA with Sidak's multiple comparisons test was used (*p<0.033; **p<0.0021; ***p<0.0002; ****p<0.0001);

- Figure 10: Pre-incubation study of oxidative stress-induced DCF production in Calu-3 cells over a range of concentrations and times. Statistical significance was calculated by comparing the following conditions: media only versus positive and negative controls and urban dust only; urban dust only versus each annotated compound concentration. One-way ANOVA with Sidak's multiple comparisons test was used (*p<0.033; **p<0.0021; ***p<0.0002; ****p<0.0001);

- Figure 11 : Transepithelial electrical resistance (TEER) values (A), flow through epithelium over 4 hours (B) and apparent permeability (C) of Calu-3 cell epithelium after UD exposure. Statistical significance was calculated using one-way ANOVA test with Dunnett's post-test of multiple comparisons (****p<0.0001 when compared with control with HPFP and $$$$p<0.0001 when compared with media only);

- Figure 11d: Image of mucus staining with alcian blue of Calu-3 cells cultured in a model with ALI and exposed to different levels (from 5 to 250 pig) of urban dust using HPFP as vehicle. The black spots on the images represent the urban dust particles;

- Figure 12: ELISA assays detecting interleukin 6 (A) and 8 (B) in Calu-3 cells cultured with ALI using increased concentrations of urban dust. One-way ANOVA with Tukey's multiple comparisons test was used (*p<0.033; **p<0.0021; ***p<0.0002; ****p<0.0001);

- Figure 13: ELISA assays detecting interleukin 6 (A) and 8 (B) in Calu-3 cells cultured with ALI using prevention and triggering agents. One-way ANOVA with Tukey's multiple comparisons test was used. Significant changes between HPFP and all other conditions are indicated by ($p<0.033; $$$$ p, 0.0001) and significant changes between all conditions with urban dust are indicated by (*p<0.033; **p<0.0021; ***p<0.0002; ****p<0.0001);

- Figure 14: Deposition models in the ACI of PolmonYDEFENCE/DYFESA™ (A) normal stages (B) modified plate whose SO is replaced (C) percentage of deposition of the administered dose of PolmonYDEFENCE/DYFESA™ via ACI for Snapwell insert; - Figure 15: Transport study for PolmonYDEFENCE/DYFESA™ deposited in the ACI on the epithelium of Calu-3 cells in Snapwell inserts (A) percentage of PolmonYDEFENCE/DYFESA™ detected in the basolateral chamber of Snapwell inserts over 4 hours and on the epithelium (B) Flow through the epithelium and permeability coefficient after the 4 hours of transport;

- Figure 16: Wound healing study for PolmonYDEFENCE/DYFESA™ on Calu-3 cell epithelium in Transwell inserts. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparisons test. Where the symbol (#) denotes significant differences between Control and PolmonYDEFENCE/DYFESA™ (#p<0.033; ##p<0.0021; ###p<0.0002) and the symbol ($) denotes significant differences between sodium hyaluronate and PolmonYDEFENCE/DYFESA™ ($p<0.033);

- Figure 17: Schematization of the experimental design discussed in depth in Example 2.

- Figure 18: Permeability coefficient (A), TEER measurements (B) of Calu-3 epithelium after exposure to PolmonYDEFENCE/DYFESA. Statistical significance was calculated using one-way ANOVA with Dunnett multiple comparisons post-test (#p<0.033; ##p<0.0021 ; ####p<0.0001, **** p<0.0001) and Student's t-test (*p<0.05).

- Figure 19: (A) Mucus staining images of Calu-3 cells with Alcian blue cultured in the ALI ± PolmonYDEFENCE/DYFESA model. (B) Quantification of mucus, statistical significance was calculated using one-way ANOVA with Dunnett's post-test of multiple comparisons (*p<0.033; ***p<0.0002).

Therefore, it is on object of the presente invention a composition in dry powder form for inhalation comprising:

(I) a mixture M comprising or, alternatively, consisting of hyaluronic acid or an acceptable pharmaceutical grade salt thereof and, optionally,

(II) at least one acceptable pharmaceutical grade additive and/or excipient; wherein said composition is for use in a method of treatment, preferably a topical treatment, preventive or curative, of a disorder, or an alteration, or a symptom, or a disease resulting from inflammation and/or oxidative stress of the respiratory system caused by air pollution.

Said composition in dry powder form for inhalation preferably has an average particle diameter from 0.1 pm to 35 pm, preferably from 0.5 pm to 15 pm, more preferably from 1 pm to 10 pm. As an example, said average particle diameter could be determined by Eur.Ph. 2.9.34 and Eur.Ph. 2.9.31 (the latter for particles with average particle diameters > 20 pm).

In the present invention, the term "average particle diameter" refers to the size of a particle comprising all the components of the composition, for example, depending on the embodiment of the invention, of a particle comprising hyaluronic acid or a salt thereof, hyaluronic acid or a salt thereof and supporting agent (preferably mannitol), or hyaluronic acid or a salt thereof, supporting agent and the optional at least one acceptable pharmaceutical grade additive and/or excipient. The particles of a powder can be differentiated, primarily, into inhalable and respirable. The inhalable fraction is represented by a suspension of particles of various diameters (generally ranging from 10 microns to 100 microns) whose size is such that they result in interaction with the human respiratory system. The respirable fraction is represented by a suspension of particles with a particle size class (generally less than 4 microns) such that the non-ciliated part of the lungs (alveolar zone) is reached by respiratory motions.

Said composition is preferably in the form of dry powder for inhalation by mouth, more preferably by suction by mouth.

Hyaluronic acid (hyaluronan; Hya), an important component of the extracellular matrix, is a polymer consisting of glucuronic acid and N-acetyl glucosamine units. The central role of Hya in the respiratory system has been associated with tissue homeostasis through modulation of inflammation and oxidative stress, tissue remodeling, water balance, and biomechanical integrity.

Within said mixture M, hyaluronic acid or a salt thereof preferably has an average molecular weight from 20 kDa to 4000 kDa, preferably from 50 kDa to 1500 kDa, even more preferably from 150 kDa to 1000 kDa.

Preferably, said salt of hyaluronic acid is an alkaline or alkaline earth metal salt, most preferably sodium, potassium, magnesium, or calcium salt; even more preferably, the salt of hyaluronic acid is the sodium salt (sodium hyaluronate).

Preferably, hyaluronic acid or a salt thereof is of biofermentative origin, more preferably from fermentation of a bacterium of the genus Streptococcus, even more preferably of the species Streptococcus equi zooepidermicus.

Hyaluronic acid or a salt thereof preferably has an intrinsic viscosity ranging from 1.35 m³/kg to 1.60 m³/kg. Said inflammation or said oxidative stress is inflammation or oxidative stress preferably caused by urban dust (UD) pollution.

More preferably said inflammation or said oxidative stress is inflammation or oxidative stress of the upper respiratory tract (nasal cavity, pharynx and/or larynx) or lower respiratory tract (trachea, primary bronchi and/or lungs).

More preferably, said disorder, or alteration, or symptom, or said disease is selected from the group comprising or, alternatively, consisting of: bronchitis, asthma, acute airway inflammation, asthma attacks, cough and phlegm, decreased lung capacity, decreased respiratory function, chronic bronchitis, and respiratory tract tumor.

The effects on human health of urban dust (or fine dust) in the air, referred to as PM10 in relation to the size of the particles of which it is composed, depend mainly on its quantity (or concentration) as well as the nature of its components. They will, in fact, depending on their diameter, settle more or less deeply into the respiratory system. The type and severity of the effects determined on health are also influenced by the chemicals, organic and inorganic, present on the surface of the particles. Soluble substances, for example, can be absorbed by the organism where they are deposited, causing local disorders.

More severe effects, on the other hand, with disorders (symptoms) and changes in respiratory function (bronchitis, asthma that may even require hospitalization) have been observed after exposure (even if limited to one or two days) to high levels of PM10 and PM2.5 (particles less than 2.5 micrometers in diameter). The World Health Organization (WHO) has linked the spread of these effects to an increase (of 10 micrograms per cubic meter) in the average concentration of PM 10 and PM2.5 calculated over a 24- hour daily period.

When PM 10 contains high concentrations of metals, acute inflammation of the respiratory tract, asthma attacks , and alterations in the functioning of the cardiovascular system are common.

Prolonged exposure over time to even low levels of PM10 and PM2.5 is associated with increased respiratory disorders such as cough and phlegm, asthma, decreased lung capacity, reduced respiratory function and chronic bronchitis along with effects on the cardiovascular system. Exposure to smaller fine dust (PM2.5) has been associated with increased mortality from respiratory diseases and an increased risk of respiratory tract tumor. Tumors have also been linked to the presence of carcinogens attached to the surface of the particles (such as polycyclic aromatic hydrocarbons-IPAs in the case of soot), which, through PM2.5 can travel to the deepest part of the lungs, where they are absorbed by the organism.

In susceptible people such as asthmatics, individuals with lung disease, heart disease, and the elderly, it is reasonable to expect a worsening of their condition and disorders. Children up to 12 years old, having twice the breathing frequency, introduce larger volumes of air into the lungs than adults and may be at greater risk for some respiratory effects such as bronchial asthma attacks.

According to the World Health Organization, it is not possible to define a limit value for fine dust below which health effects do not occur in the population, so the concentration of PM10 and PM2.5 in the air should be kept as low as possible. However, the new WHO Air Quality Guidelines report that reducing PM 10 to 20 micrograms per cubic meter could result in a 15% reduction in mortality, by decreasing the incidence of diseases due to respiratory infections, heart disease, and lung tumor. For PM2.5, WHO proposes for health protection guideline values for population exposure of 10 micrograms per cubic meter on an annual basis.

Said mixture M preferably comprises, in addition to hyaluronic acid or a salt thereof, at least one supporting agent selected from the group comprising or, alternatively, consisting of lactose, a dextran, mannitol and mixtures thereof; preferably mannitol. More preferably said support agent comprises or, alternatively, consists of mannitol or D-mannitol.

The supporting agent facilitates the administration of said composition in dry powder form. Preferably, said D-mannitol comprises has a melting point range from 165°C to 170°C (determined according to Ph. Eur.) and, more preferably, a maximum conductivity of about 20 microS/cm.

Said mixture M preferably comprises or, alternatively, consists of:

- Hyaluronic acid or an acceptable pharmaceutical grade salt thereof, preferably sodium hyaluronate, in an amount from 15% to 60% by weight, preferably from 20% to 50% by weight, more preferably from 25% to 40% by weight, even more preferably from 28% to 38% by weight;

- said supporting agent, preferably mannitol, in an amount from 40% to 85% by weight, preferably from 50% to 80% by weight, more preferably from 60% to 75% by weight, even more preferably from 62% to 72% by weight; with respect to the total weight of said mixture M.

In said method of treatment, preferably a topical, preventive treatment, said composition preferably performs a barrier action or protective action of respiratory system epithelial cells from air pollution.

In said method of treatment, preferably a topical, curative treatment, said composition preferably has a healing and/or re-epithelializing action on respiratory system epithelial cells.

It is an object of the present invention also a mixture M for topical administration in dry powder form for inhalation consisting of:

- Hyaluronic acid or an acceptable pharmaceutical grade salt thereof, preferably sodium hyaluronate, in an amount from 15% to 60% by weight, preferably from 20% to 50% by weight, more preferably from 25% to 40% by weight, even more preferably from 28% to 38% by weight, with respect to the total weight of said mixture M; and

- mannitol, in an amount from 40% to 85% by weight, preferably from 50% to 80% by weight, more preferably from 60% to 75% by weight, even more preferably from 62% to 72% by weight, with respect to to the total weight of said mixture M.

The mixture M of the invention as such and for use according to the invention does not include N- acetylcysteine.

It is an object of the present invention also a dry powder inhaler operable by a subject by suction by mouth.

In the present invention, the term "suction by mouth" or "suction action by mouth" means that the subject inhales or aspirates by mouth in a single action (single blow suction action) with a force intensity such that the device of the invention delivers a predetermined dose of the composition of the invention in powder form present in the inhaler itself.

Said inhaler comprises a disposable inhaler body and a disposable cartridge containing said composition in dry powder form. Said disposable cartridge is connected to said inhaler body so as to release an effective dose of said composition into the inhaler body. Preferably, said inhaler has the characteristics given in document EP 3 386 575 B1 incorporated by reference in this description in the parts describing the inhaler (paragraphs from [0024] to [0041]).

The following are examples, given by way of example and not limitation, of the present invention. EXAMPLES

EXAMPLE 1: In vitro absorption test and evaluation of medical device properties on human mucociliary tissue (HmE).

The absorption profile of the key functional ingredient (hyaluronic acid) and the properties of "PolmonYDEFENCE" were tested on a 3D human mucociliary tissue (HmE) model used as a biological model that can best reproduce the conditions of the medical device application. Tests were conducted on both healthy and inflamed tissues with lipopolysaccharides (LPSs) following OECD 428 reference guidelines.

The barrier properties of the medical device in contact with the mucous membrane of the upper airway were also evaluated for a better understanding of its action after application.

The tested medical device is intended as an adjuvant in the protection of the mucous membranes of the respiratory tract, contributing to the maintenance of the physiological conditions of the upper and lower respiratory tract by restoring the homeostasis of the respiratory mucosa altered by acute or chronic inflammation.

The absorption assay was conducted to evaluate the penetration profile of the key functional ingredient (hyaluronic acid) using an in vitro 3D human mucociliary epithelium (HmE) by means of a Franz diffusion cell system.

At predefined times, selected to best simulate the application and contact time of the tested product, the receptor fluid was sampled.

To best reproduce physiological conditions of use, moreover, the absorption profile of "PolmonYDEFENCE" was evaluated on inflamed mucociliary epithelium. Inflamed mucociliary epithelium, in fact, may exhibit different characteristics than noninflamed epithelium, which impair the absorption of an applied substance.

A histological analysis was then performed to best highlight the characteristics of the medical device in contact with the mucosa and the effect on it. The morphological characteristics of a healthy untreated insert were compared with those of an insert treated with "PolmonYDEFENCE"; the same comparison was also made in the case of inflamed mucosa.

In order to evaluate the protective barrier effect of "PolmonYDEFENCE," histological analysis was performed by applying "PolmonYDEFENCE" on the mucosa before treatment with the inflammatory agent. Finally, the paracel lular permeation properties and overall integrity of the inserts were evaluated by testing mucosal inserts with Lucifer yellow. This test was performed on healthy mucosa, inflamed mucosa, and inflamed mucosa contacted with the medical device. In all these cases, the permeation profile of the marker molecule was studied.

Transepithelial electrical resistance (TEER) values were measured on all inserts. TEER values are used as index of the functionality of tight junctions, which constitute the epithelial diffusion barrier. TEER was measured on inserts both before and after exposure to the medical device of healthy and inflamed mucosa. These values were analyzed at the end of the study to show the impact of the inflammatory process and the action of the medical device on the tight junctions.

Description of the medical device

The characteristics of the PolmonYDEFENCE medical device are shown in Table 1 below.

Table 1

EXPERIMENTAL DESIGN

PHASE 1 : ABSORPTION STUDY ON HEALTHY MUCOSA

The absorption profile of hyaluronic acid within "PolmonYDEFENCE" was studied by means of a Franz diffusion cell system (Figures 1 and 2), wherein a mucociliary airway tissue was selected as an ideal 3D tissue model that could best represent the barrier function. An optimal exposure time of 24 hours to a tested substance was selecyed for a biological model, in line with what is suggested by the OECD 428 guidelines. Five time points were selected for sampling over the 24 hours to allow adequate characterization of the absorption profile.

The medical device was deposited for 24 hours on the apical side of the mucociliary insert: 6 mg of "PolmonYDEFENCE" (accurately weighed on an analytical balance at the time of application) was applied on 4 inserts and 250 l of PBS was applied to the reference control.

The receptor fluid was RCVR-FL, D-PBS solution containing calcium and glucose, selected to ensure both good cell viability during the test and better dispersion of the key ingredient. As mentioned earlier, predefined time points were selected to best characterize the absorption profile in order to simulate the application and contact time under the conditions of use: 0, 30 minutes, 2 hours, 6 hours and 24 hours.

At each time point, 1 ml was sampled from the receptor solution and stored in individual labeled Eppendorf tubes at 4°C.

After 24 hours, upon completion of the absorption test, the inserts were rinsed with PBS and stored in Petri dishes at -20°C for further analysis.

At the end of the test, the donor compartment, consisting of the insert, rings, ring covers and cap, was washed with PBS and stored at 4°C for further quantification of the deposited unabsorbed dose. Quantification of hyaluronic acid in PBS was conducted by validated ELISA assay. Hyaluronic acid contained in the biological samples (mucociliary airway tissue inserts) was processed and analyzed with an appropriate colorimetric kit (Purple Jelley) that was specifically validated for biological matrices by the supplier.

PHASE 2: ABSORPTION STUDY ON INFLAMED MUCOSA

"PolmonYDEFENCE" is intended for application to inflamed mucosa and potentially having different morphological and physicochemical characteristics from those found in non-inflamed mucosal tissues. Accordingly, it was evaluated that an absorption test on an in vitro model of inflamed mucosa would best represent the actual conditions that may occur during application of the tested device. The inflammatory process was induced on a 3D mucociliary epithelium using LPS as the inflammatory agent. The inserts were treated with LPS in accordance with what was suggested by the respective supplier: LPS was applied on the basolateral compartment for 16 hours before the absorption test procedures. Subsequently, the absorption test was performed to test the passage of hyaluronic acid, as conducted and described in PHASE 1.

PHASE 3: RECOVERY AND PROTECTION ASSESSMENT

A histological analysis was performed on both healthy and inflamed inserts, treated and untreated with the tested product, to evaluate the protective adjuvant behavior of "PolmonYDEFENCE" and to gain a deeper understanding of its action in contact with the mucosa.

Comparison between treated and untreated healthy mucosa was conducted to confirm tissue structure and to evaluate the effects of the medical device contacted with the biological model. Subsequently, the same evaluation was conducted on treated and untreated inflamed mucosa to verify the inflammatory status of the inserts and to assess the potential protective effect of the medical device on the mucosa: the inserts were inflamed by exposure to LPS (16 hours) overnight and than treated with the product and PBS, respectively.

In addition, to evaluate the potential barrier properties of the product, the insert was initially treated with the tested product and then LPS was added on the apical side. The application lasted 6 hours in order to best represent the intended conditions of use.

It was conducted to test the extent of LPS-induced inflammation after application of the medical device, and thus as an indicator of barrier properties.

PHASE 4: PERMEABILITY TEST (LUCIFER YELLOW)

To verify the integrity of tight junctions in the presence of the tested product, the permeability characteristics of human mucociliary tissue (HmE) were verified by measuring the passive passage of Lucifer yellow. Lucifer yellow is a fluorescent dye that is impermeable to the cell membrane. It is used to study the paracellular permeability of a substance. When the junctions are not broken, Lucifer yellow has very low permeability, whereas if the tight junctions are damaged, the passage of Lucifer yellow will be higher. The inserts therefore were treated with LPS overnight to induce the inflammatory process, after which one insert was treated with the medical device and the other with PBS. A third non-inflamed mucosal insert was treated with PBS only.

Comparison of Lucifer Yellow permeability in an inflamed insert with what is observed in a healthy insert should indicate the effect of LPS on tissue integrity.

PHASE 5: INTEGRITY TEST (TEER) Transepithelial electrical resistance (TEER) is the measurement of electrical resistance along a cell monolayer, and it is a very sensitive and reliable method for confirming the integrity and permeability of a cell monolayer. The TEER value was studied for all the inserts studied in the above steps, before the start and at the end of any procedure.

These values were compared to verify the possible impact on the integrity of tight junctions of all procedures performed. The effects obtained on inflamed mucosa and on mucosa treated with the medical device were compared with what was observed for untreated mucosa.

SAMPLES

Product specifications are given in Table 2 below.

Table 2

RESULTS

PHASE 1 and 2: ABSORPTION TEST

Figure 3 summarizes the applied dose distribution in all compartments for inserts with both healthy (samples A, B, C, D) and mucosa inflamed by an LPS solution at a concentration of 0.5 pig/ml (samples F, G, H, I). Samples E is a reference insert with healthy mucosa treated with PBS, while sample J is a reference insert with inflamed mucosa treated with PBS.

Results for total mass balance for all samples are shown in Table 3 below. All results meet the compliance criteria for 100% ±10% recovery according to OECD 428.

Table 3

PHASE 3: RECOVERY AND PROTECTION ASSESSMENT Histological evaluation demonstrated the presence of an epithelial layer with morphological features of the respiratory mucosa. The scaffolds were colonized by layers of epithelial cells with cilia on the free surface, mimicking the normal histological structure of the respiratory tract mucosa. Severe changes were observed only in the case of inflamed mucosa treated with PBS (control).

The summary of the results is shown in Table 4.

Table 4

+ = rare; ++ = few; +++ = numerous; ++++ = very numerous Healthy inserts treated with PBS or with the medical device confirmed the expected morphological structure of the airway epithelium as a valid in vitro model for the study.

The severe changes observed in the case of inflamed mucosa that has been in contact only with PBS show the inflammation achieved with LPS treatment.

Inflamed mucosa treated with the medical device showed less changes than untreated inflamed mucosa. This is an indicator of the recovery properties of the medical device.

Healthy mucosa contacted with the medical device and subsequently treated with LPS showed less alteration, and this is an indicator of the protective properties of the medical device when it is applied to the airway mucosa.

This conclusion is further supported by the results obtained in the absorption test described in phase 1 and 2 of the present study: the hyaluronic acid present in the medical device is not absorbed, and 100% of it was found in the apical compartment of the insert at 24 hours after application. This means that the hyaluronic acid remains dispersed in the mucus of the airway epithelium, thus inhibiting the passage of other molecules.

PHASE 4: PERMEABILITY TEST (LUCIFER YELLOW)

The results from phase four are shown in Table 5 below. In all cases, Lucifer yellow has a permeability > 3%, which means that in no case was the integrity of the inserts has been compromised.

Tab e 5

PHASE 5: INTEGRITY TEST (TEER)

Figure 4 shows the TEER values measured at the beginning and end of the studies for each insert. The starting TEER values are all above 400 ohms, indicating good tissue integrity and functional tight junctions.

The drop in TEER values is due to contacting the inserts with D-PBS for 24 hours instead of their culture medium. D-PBS, although enriched with calcium and glucose, can ensure cell survival for about 4 hours. Figure 5 shows TEER values measured from PHASE 3 inserts.

TEER values increase over time when the insert has been treated with the medical device. This can also be observed in the case of the insert treated with the medical device and later with LPS on the apical side. Figure 6 shows the TEER values before and after treatment of the inserts in PHASE 4. No significant differences in TEER values were noted for inserts treated only with PBS, while inserts contacted with the medical device showed higher TEER values after exposure.

This measurement can also be considered an indicator of the barrier action of the medical device. The barrier formed by "PolmonYDEFENCE" achieves higher TEER values by improving the integrity of tight junctions. The protective effect of "PolmonYDEFENCE" on an inflamed insert resulted in better recovery than an untreated inflamed mucosa, as higher TEER values were found.

CONCLUSIONS from EXAMPLE 1:

The results reported in PHASE 1 and PHASE 2 demonstrate that the absorption of HA contained in "PolmonYDEFENCE" after 24 hours in contact with both healthy and inflamed HmE is negligible.

The healthy mucosa contacted with "PlomonYDEFENCE" and subsequently treated with LPS showed less alteration than the untreated mucosa, indicating the barrier properties of "PolmonYDEFENCE" on HmE, as shown by the histological evaluation reported in PHASE 3 and confirmed by the TEER values reported in PHASE 5. This conclusion is further supported by the results obtained in the absorption test described in PHASE 1 and 2 of the study: the hyaluronic acid contained in the medical device is not absorbed, and remains 100% in the apical compartment of the insert at 24 hours after application. This means that the hyaluronic acid remains dispersed in the mucus of the airway epithelium, thus inhibiting the passage of other molecules.

Inflamed mucosa treated with "PolmonYDEFENCE" shows less changes than untreated inflamed mucosa, as shown by histological evaluation reported in PHASE 3, indicating a potential recovery effect of the medical device.

EXAMPLE 2: Biological evaluation regarding to the administration of sodium hyaluronate powder by dry powder inhaler (Pill Haler® ) targeting the upper respiratory tract up to the primary bronchi.

MATERIALS AND METHODS

Materials

Sample name, batch, expiration date and reference for the materials tested are shown in Table 6 below.

Table 6

Cell culture

Calu-3 cell line (human carcinoma-derived lung epithelial; HTB-55) was purchased from ATCC and cultured in 75 cm2 flasks containing F-12:DMEM (Sigma, Australia). Complete media supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Australia), 1% (v/v) nonessential amino acid solution (Gibco, Australia), and 2 mM L-glutamine (Gibco, Australia) were used in the assays, unless otherwise indicated, and maintained in a humidified atmosphere of 95% air, 5% CO2, at 37°C.

Cytotoxicity assay in liquid-covered culture (LCC)

Cells were seeded at a density of 5x10⁴ cells/well in a 96-well plate. After 48 hours, the media were removed and the cells were exposed to a series of 2-fold dilutions of UD, PolmonYDEFENCE/DYFESA™ mixture and the equivalent concentrations of crude Na-Hya and crude mannitol. After 24 h of incubation, 20 pl of MTS reagent was added to the wells and read after 2 h at 490 nm. Plates with urban dust were washed with phosphate buffer solution (PBS) to remove UD debris before adding the MTS solution (20% v/v), and readings were corrected by readings from a plate with UD alone (no cells). Media and 20% DMSO were used as controls. All dry samples were sterilized by UV irradiation for 30 min before the experiment.

Cellular model with air-liquid interface (ALI)

The ALI model of CALU-3 has been previously validated as an in vitro predictive model of upper airway epithelium physiology (1).

To make a model with ALI, cells were seeded at a density of 7.92 x 104 cells/insert on a Transwell polyester insert (growth area of 0.33 cm²) containing 100 pl in the apical chamber and 600 pl in the basolateral chamber. The apical chamber medium was removed 24 h after seeding and daily thereafter until an ALI was reached, while the basolateral chamber medium was replaced every other day until 14 days of culture. Snapwell polyester inserts (growth area of 1.12 cm2) were used for impact studies with a cell density of 1.58x105 cells/insert, and culture with ALI was achieved using 2 ml of medium in the basolateral chamber.

Integrity of the epithelial barrier The effect of the materials on epithelia cultured in a culture with ALI (14 days) was assessed by transepithelial electrical resistance (TEER) and by measuring the permeability of the cell layer to sodium fluorescein (Na-Flu), a marker of paracellular transporters, for 4 hours (1, 2).

TEER was measured after exposure to the test sample using an epithelial voltohmmeter (EVOM, World Precision Instruments, USA) connected to STX-2 rod electrodes. Before measurement, 100 l or 200 l of Hanks' balanced salt solution (HBSS) was added to the apical chamber of Transwell and Snapwell inserts, respectively.

For permeability studies, 100 l (Transwell) or 200 l (Snapwell) of Na-Flu (2.5 mg/ml) was added to the apical chamber, and 600 l (Transwell) or 2 ml (Snapwell) of HBSS was added to the basolateral chamber. At predetermined times, 100 l samples were taken from the basolateral chamber, then replaced with equal volumes of fresh HBSS. Fluorescence intensity was measured using a Spectramax plate reader at 485 (ex)/520 (em) nm. The apparent permeability coefficient (Papp) was calculated by equation (I) below:

Papp = (V/ACo )(dC/dt) (I) where V is the volume in the basolateral chamber, A is the membrane surface area of the Transwell inserts, CO is the initial concentration in the apical chamber, and dC/dt is the (cumulative) flux of Na-Flu through the membrane.

Mucus production

Mucus production after exposure to the test samples was assessed using alcian blue staining. After treatments, cells were washed with PBS and fixed in 4% (v/v) paraformaldehyde (PFA) solution for 15 min. After removal of the PFA residue with PBS, the cells were stained with alcian blue for 20 min. Excess dye was removed by subsequent washes with PBS, and the membranes were removed from the inserts and mounted on slides for microscopic analysis.

Antioxidant effect

The effects of the test samples alone and in combination with UD were evaluated using DCFDA (2',7'- dichlorofluorescein diacetate), a cell-permeable probe. After diffusion into the cell, DCFDA is deacetylated by cellular esterase, which are then oxidized by intracellular reactive oxygen species (ROS) into fluorescent DCF (2',7'-dichlorofluorescein). The intensity of DCF is an indicator of the overall levels of cellular ROS.

Cells were seeded in a black 96-well plate at 5x10⁴ cells/well and allowed to grow for 48 hours. After removal of media, cells were washed with phosphate buffered saline (PBS) and stained with DCFDA 10 piM (in serum-free media) for 20 min at 37 °C in the dark. Excess dye was removed by washing the cells with PBS. The samples were incubated with the test samples, and fluorescence intensity was measured immediately after incubation and after 1 h exposure at 480 (ex)/520 (em).

To evaluate the pro-/anti-oxidant effects of individual compounds, cells were individually exposed to Na- Hya, mannitol, UD, and PolmonYDEFENCE/DYFESA™ mixture.

To evaluate the preventive effect of Na-Hya or PolmonYDEFENCE/DYFESA™ against UD-induced ROS, two experimental models were tested:

(a) Co-incubation model-whereby cells were simultaneously exposed to the prevention agent (Na-Hya or PolmonYDEFENCE/DYFESA™ mixture) and the triggering agent (UD);

(b) Pre-incubation model-whereby the prevention agent was added to the cells 1 hour before the triggering agent (UD).

Cells without dye (without DCFDA) and a stimulating agent (DCFDA and media only) were included as controls for the assay. The anti-oxidant N-acetyl cysteine (NAC; 5 mM) and vitamin K3 (100 pM), a ROS generator, were used as controls for ROS. All measurements are calculated as fold change (change in times) at the O-hour time point.

Studies of inflammation in the model with ALI

The anti/pro-inflammatory effects of the materials were evaluated in the model with ALI. Test samples were deposited on the cell monolayer as a suspension in model propellant (HPFH-2H.3H- decafluoropentane, TCI America, USA), as previously reported (3). HPFH is a hydrophobic and highly volatile propellant that can be handled as a liquid at ambient pressure and evaporates rapidly after exposure to air, thus leaving the cell monolayer exposed only to dry powders (3).

After the ALI was achieved (14 days), 50 pl of UD suspensions (0.1 - 5 mg/ml) were added to the apical chamber, which determined the exposure of cells to 5 - 250 pg of UD/insert alone or in combination with specific amounts of Na-HYA and PolmonYDEFENCE/DYFESA™ mixture. Equal volumes of HPFP were used as control with vehicle. After incubation, the culture plate was left open for 10 to 15 min to allow the HPFP to evaporate. After 24 h of exposure, basolateral chamber media were collected for interleukin quantification. Interleukin (IL)-6 and IL-8 levels were determined by ELISA (BD OptEIA, BD Biosciences, USA), according to the manufacturer's instructions. Data are expressed as mean ± standard deviation of 3 biological replicates.

Andersen cascade impactor (ACI) deposition studies

The deposition profile of the PolmonYDEFENCE/DYFESA™ mixture by 30 mg PIIIHaler® (PolmonYDEFENCE /DYFESA™) through the stages of the ACI was studied using both conventional plates and the modified plate, containing Snapwell inserts, replacing stage 0 of the ACI (4). The ACI was connected to a rotary vane pump (Westech Scientific Instruments, UK) and the flow rate was adjusted to 60 l/min using a calibrated flow meter (TSI Model Instruments, Germany). To minimize particle bouncing, 50 pl of Brij 35:glycerol:ethanol solution (10:50:40 v/v/v) was used to coat the plates of the ACI (except the modified plate).

After deposition for 4 seconds on conventional plates or cell-free Snapwell inserts, the ACI was disassembled, and each stage was washed separately to chemically quantify the recovered Na-Hya. The modified plate containing the Snapwell inserts was removed and washed to calculate the total amount of drug deposited on this specific stage and each of the separate wells. Each Snapwell insert was also washed to determine the total dose deposited on each well. The samples were quantified by high- performance liquid chromatography (HPLC) according to the section "Quantification of sodium hyaluronate" below.

Transport studies in the model with ALI

Deposition of PolmonYDEFENCE/DYFESA™ on Calu-3 cell epithelia was achieved using ACI with a 3D- printed plate customized to receive Snapwell inserts (4). Prior to the experiment, 8 inserts were inserted into the modified plate and a total dose of 30 mg was delivered by Pill Haler® onto the cells (1 drive; 4 sec; 60 l/min). Snapwell inserts were transferred to the culture plates containing preheated HBSS in the basolateral chamber. At predetermined time points, 200 pl samples were collected from the basolateral chamber, replacing them with equal volumes of fresh HBSS. After 4 hours, the apical surface was washed with 400 pl of HBSS to collect the remaining Na-Hya from the PolmonYDEFENCE/DYFESA™ mixture on the monolayer. The cells were then scraped from the membrane and lysed for quantification of Na-Hya within the cells. Samples were quantified using HPLC according to the section "Quantification of sodium hyaluronate" below.

Quantification of sodium hyaluronate

Detection and quantification of sodium hyaluronate were conducted using an HPLC system equipped with a UV-Vis SPD-20A detector, LC-20AT liquid chromatography, a SIL-20A HT autosampler (Shimadzu) and a BioSep SEC-S2000 column (300 x 7.8 mm, 5 pm, 145A, Phenomenex, Torrance, USA), according to a validated method (provided by HollyCon). The mobile phase was KH2PO40.05M at pH 7.0. Samples were analyzed at 205 nm, a flow rate of 1 ml/minute and an injection volume of 10 pl, with a retention time of 5.0 min. Linearity was obtained between 2.5 and 500 pg/ml (R² = 0.99).

Study of wound healing with culture with ALI

The wound healing assay, also known as the scratch assay, was performed on the ALI model of Calu-3 cells. The experiments were performed after 14 days of culture with ALI. A scratch was made on the apical side of the cell layer with the tip of a pipette (P200 pl) along the membrane diameter of the Transwell insert. Prevention agents (Na-HyA and PulmonYDEFENCE/DYFESA™ mixture) using HPFP and control cells treated with an equal volume of HPFP alone were deposited over the wound. Transwell inserts were maintained in a humidified chamber at 37 °C in a 5% CO2 and 95% humidity atmosphere, and the wound was observed using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) with a Coolsnap ES2 camera. Photographs were taken every 20 minutes for the first 2 hours, followed by every 30 minutes up to 18 hours using NIS-Elements (version 3.22.01) after deposition of the prevention agent. Images were analyzed using Fiji Imaged, measuring the area of wound closure using an in-house macro. The wound closure percentage was calculated using equation (II) below:

% of wound size 00* (At/AO) (II) where At is the area of the wound at a given time and AO is the area of the initial wound.

Statistical analysis

Data are presented as mean ± standard deviation of three independent experiments. Statistical analysis was performed using Prism software version 8.0 (GraphPad, San Diego, USA). The averages were compared with annotated statistical analysis, followed by annotated tests of multiple comparisons.

RESULTS

Evaluation of cytotoxicity of Na-Hya, PulmonYDEFENCE/DYFESA™ mixture, mannitol and UD on Calu-3 cells. Cytotoxicity was tested with Calu-3 cells cultured in LCC culture and exposed to a concentration range of 0.01 - 2 mg/ml of Na-Hya, equivalent to a concentration range of 0.02 - 5.6 mg/ml of PolmonYDEFENCE/DYFESA™ mixture and equivalent to a concentration range (0.01 - 3.6 mg/ml) of Mannitol (Figure 7A). The results show that Na-Hya and Mannitol did not induce toxicity in Calu-3 cells in the concentration range tested, and both demonstrated a pro-proliferative effect. However, the mixture showed a statistically significant reduction in cell viability from concentrations of 0.35 mg/ml and above, probably caused by mechanical or osmotic stress. Therefore, exposure of Calu-3 cells to the mixture causes cytotoxicity from a concentration of 0.35 mg/ml when used in submerged culture.

Calu-3 cells were also exposed to 0.01 to 2 mg/ml of UD (environmental trigger) to clarify if the dust induced cytotoxic effects (Figure 7B). It was observed that UD did not induce toxic effects on Calu-3 cells up to the concentration of 1 mg/ml.

Evaluation of the anti-oxidant properties of the PulmonYDEFENCE/DYFESA™ mixture on Calu-3 cells Antioxidant properties were evaluated in order to assess the protective effect of the PolmonYDEFENCE/DYFESA™ mixture against UD-induced oxidative stress using the LCC model.

This part of the study was performed as described below: it was evaluated if Na-Hya, PolmonYDEFENCE/DYFESA™ mixture and UD alone induced ROS production on Calu-3 cells using a range of concentrations; it was evaluated if Na-Hya or the PolmonYDEFENCE/DYFESA™ mixture was able to reduce UD- induced oxidative stress in Calu-3 cells, with the following two experimental setups:

1) Co-incubation of Na-Hya or PolmonYDEFENCE/DYFESA™ mixture with UD;

2) Pre-incubation of Na-Hya or PolmonYDEFENCE/DYFESA™ mixture and later addition of UD.

Figure 8 shows the fold changes of ROS production at 1 hour after exposure to individual compounds (Na- Hya, PolmonYDEFENCE/DYFESA™ mixture and UD) in the different concentration ranges. for Na-Hya from 0.02 to 2 mg/ml, equivalent to a range for the PulmonYDEFENCE/DYFESA™ mixture of 0.04 to 5.56 mg/ml. for UD: from 0.02 to 2 mg/ml.

The results obtained showed that UD significantly increased oxidative stress in Calu-3 cells from concentrations of 0.5 mg/ml and above, while only Na-Hya (2 mg/ml) reduced baseline levels of ROS. The PolmonYDEFENCE/DYFESA™ mixture showed no changes in relation to ROS levels.

To evaluate if Na-Hya or the LungYDEFENCE/ DYFESA™ mixture was able to reduce UD-induced oxidative stress in a Calu-3 LCC model, two experimental setups (co-incubation and pre-incubation) were tested. The concentration of 1 mg/ml of UD was selected because it induced a significant increase in ROS without reduction in cell viability (Figure 7B).

Figure 9 shows data from the study of co-incubation of UD (1 mg/ml) with Na-Hya or PolmonYDEFENCE/ DYFESA™ mixture over a 2-hour time course. At all time points (0.5 hr, 1 hr, 1.5 hr and 2 hr), the positive control was significantly higher than the cells in the media alone, indicating the reliability of the assay. Exposure to UD significantly increased oxidative stress for most of the time points tested. A significant decrease in oxidative stress was observed after 1.5 hours in cells co-incubated with UD and PolmonYDEFENCE/DYFESA™ for a concentration of 1.4 mg/ml and higher, compared with UD alone. This observation was sustained at 2 hours.

In the pre-incubation study (Figure 10), no significant increase was detected in the positive control, indicating that the experimental setup was not appropriate for testing the anti-oxidant effect of the PolmonYDEFENCE/DYFESA™ mixture.

Evaluation of the integrity, permeability and mucus production of Calu-3 cell epithelium after UD exposure A cell culture model was made with ALI for Calu-3 cells to mimic airway epithelium. A series of experiments were conducted to test the integrity, permeability, and response of the epithelium to different concentrations of the environmental pollutant (UD) (from 5 pig 250 pig). Specifically, epithelial barrier integrity was assessed by TEER and Flu-Na measurements. TEER experiments show that the use of HPFP on the epithelium of Calu-3 cells significantly increased the electrical resistance compared with the control (Calu-3 cells only). In addition, 250 pg of UD also significantly increased electrical resistance compared with all other samples (Figure 11 A). No significant changes were observed in flux through the epithelium and apparent permeability between cells in the media compared with cells with HPFP or UD (Figures 11 B and 11C), demonstrating that HPFP and UD do not cause any significant detrimental effect to the barrier integrity of the cells.

The effects of UD on the mucus layer formed on the surface of Calu-3 epithelium in a culture with ALI were also evaluated using alcian blue staining (Figure 11D). Urban dust particles (5 to 250 pig) were deposited on Calu-3 cells in an culture with ALI model using an HPFP propellant. The mucus layer that can be identified by the blue color did not change due to the presence of UD. However, a quantitative analysis of the color intensity is not possible because of the presence of the black spots derived from the UD particles.

Evaluation of the anti-inflammatory properties of PolmonYDEFENCE/DYFESA™ on Calu-3 cells in culture with ALI

The advantage of using the model with ALI is that the media in which the interleukins are secreted are not exposed to UD, as it is added to the apical compartment, which is separate from the compartment containing the basal media, avoiding the problem encountered when using the submerged model (ABI- HCCN-PRE-210309).

The secretion of interleukins by Calu-3 cells exposed to different concentrations of UD (5, 50, and 250 pig) were evaluated before testing the protective effect of the PolmonYDEFENCE/DYFESA™ mixture.

Figure 12 showed that UD induced a significant increase in IL-6 and IL-8 secretion from 50 pg and above. UD (250 pg) was selected for the next series of experiments because it presented a significantly large increase compared with vehicle control (HPFP) in both IL6 and IL8, and also demonstrated that this concentration did not affect the integrity and permeability of Calu-3 epithelium after exposure to it (Figure 11).

The protective effects of the PolmonYDEFENCE/DYFESA™ mixture against UD-triggered inflammation are shown in Figure 13. The PolmonYDEFENCE/DYFESA™ mixture (69 pg) significantly reduced basal levels (without UD) of IL6 in Calu-3 cells. Na-Hya alone (12.5 and 25 pg) was able to significantly reduce IL6 production triggered by UD, whereas all concentrations of PolmonYDEFENCE/DYFESA™ (equivalent to the same amounts of Na-Hya tested alone) significantly reduced IL6 secretion induced by UD exposure. No significant changes in IL8 secretion were observed when exposed to UD for any concentration of Na- Hya or PulmonYDEFENCE/DYFESA™ (Figure 14B).

Evaluation of the effect of PolmonYDEFENCE/DYFESA™ on wound healing

The wound healing study was performed using the same amounts of Na-Hya (25 pig) and PolmonYDEFENCE/DYFESA™ mixture (69 pig) used in the inflammation assay. The wound healing study showed that the PolmonYDEFENCE/DYFESA™ mixture improved wound healing (Figure 16). In addition, when comparing the control with samples containing PolmonYDEFENCE/DYFESA™ deposited on the cell surface, a significantly higher rate of wound healing was observed and at the same time significantly greater reductions in wound size were observed, which could be observed as early as 3 hours after deposition. No significant differences were observed between control and sodium hyaluronate Na-Hya alone.

Significant differences in wound size were also observed between sodium hyaluronate Na-Hya and PolmonYDEFENCE/DYFESA™ mixture at 5 hours after wounding.

Therefore, the PolmonYDEFENCE/DYFESA™ mixture has a positive effect on wound healing on the Calu-3 cell epithelium.

Deposition in the ACI stages of PulmonYDEFENCE/ DYFESA™

The deposition percentage of Na-Hya delivered by the product PolmonYDEFENCE/DYFESA™ in the ACI stages, with and without the modified plate, is presented in Figure 14 with the data shown in Table 7.

Both conventional ACI and ACI with modified plate (Table 7) showed that more than 70% of PolmonYDEFENCE/DYFESA™ was deposited in the throat of ACI. No PolmonYDEFENCE/DYFESA™ was detected beyond stage 3 (cut-off 2.77 pm). The deposition percentage in individual Snapwell inserts showed some variation, but nothing statistically significant (Figure 14C). The Na-Hya deposited in each Snapwell insert corresponded to 9.5± 1.1 pig (equivalent to 26.5± 3.1 pig of PolmonYDEFENCE/DYFESA™ mixture).

Table 7: Deposition percentages of PolmonYDEFENCE/DYFESA™ through the stages of ACI using the conventional impactor and ACI with the plate modified with Snapwell inserts.

Table 7

1 N/A - not applicable

1 ND - not determined

Evaluation regarding to the transport of deposition administered in the ACI of

PolmonYDEFENCE/DYFESA™ through the Calu-3 cell epithelium

The purpose of this study was to evaluate the transport of Na-Hya, present in PolmonYDEFENCE/DYFESA™, through airway epithelial cells after deposition on Snapwell inserts placed on the modified ACI plate (60 l/min x 4 s).

Data obtained from the transport studies showed that 0.1 -0.2% of PolmonYDEFENCE/DYFESA™ was detected in the basolateral chamber within 30 minutes. However, the level did not increase over time, indicating that the traces detected were from the experimental setup. C was further validated by the fact that Na-Hya was not detected within the cells after lysis, and most was detected on the apical surface of the epithelium (SU). Therefore, PolmonYDEFENCE/DYFESA™ was not found to be transported through the Calu-3 epithelium, and furthermore, exposure to UD had no impact on the transport (Figure 15A).

Epithelial permeability was evaluated after the transport study to observe if any adverse effects on epithelial layer integrity were present due to exposure (4 hours) to PolmonYDEFENCE/DYFESA™. PolmonYDEFENCE/DYFESA™ alone had no negative impacts on the integrity of the Calu-3 cell epithelium, and the addition of UD alone or in combination with PolmonYDEFENCE/DYFESA™ had no negative impacts on the integrity of the Calu-3 cell epithelium after 4 hours of exposure (Figure 15B).

CONCLUSIONS from Example 2:

The work program aimed to evaluate the biological responses of dry sodium hyaluronate powder (PolmonYDEFENCE/DYFESA™), delivered by PillHaler device® , as a potential protective agent for the upper respiratory tract against environmental pollutants. The experiments, which employed the Calu-3 cell model in submerged culture, revealed that the PolmonYDEFENCE/DYFESA™ mixture was not cytotoxic after long-term (24-hour) exposure at concentrations below 0.35 mg/ml. At the concentration of 1.4 mg/ml and above, PolmonYDEFENCE/DYFESA™ was shown to reduce the oxidative stress triggered by urban dust (UD) exposure, which was observed as early as 1.5 hours after co-incubation.

PolmonYDEFENCE/DYFESA™ was found to reduce inflammation in both Calu-3 cell basal epithelium and epithelium with exacerbated inflammation triggered by UD, using the air-liquid interface (ALI) model. The effect of PolmonYDEFENCE/DYFESA™ was significantly superior to the use of the active ingredient alone (Na-Hya).

PolmonYDEFENCE/DYFESA™ mixture was also shown to have a positive effect in wound healing on Calu-3 epithelium, compared with control and Na-Hya alone.

Studies using the Andersen cascade impactor (ACI) demonstrated that PolmonYDEFENCE/DYFESA™ was mainly deposited in the throat stage (70%) and up to impactor stage 3 (cut-off 1.9 mm). PolmonYDEFENCE/DYFESA™ (alone or in combination with UD) that had deposited on the Calu-3 cell epithelium was not transported through the epithelium. The wound healing assay showed a positive effect induced by PolmonYDEFENCE/DYFESA™ compared with control and sodium hyaluronate alone.

This comprehensive body of work reports several beneficial properties of PolmonYDEFENCE/DYFESA™ as an antioxidant, anti-inflammatory, and protective barrier in the upper respiratory tract, using Calu-3 cells as a model.

These conclusions regarding to the formation of a protective barrier and the ability to heal or close wounds of this composition are in line with the conclusions of Example 1 .

EXAMPLE 3: Evaluation of mechanisms involved in the long-term protective effect of POLMONYDEFENCE/DYFESA on Calu-3 epithelium over 24-hour period.

Materials and methods

Table 8: Sample name, batch, expiration date and reference sample for materials tested.

Table 8

Cell culture

Calu-3 cell line (human carcinoma-derived lung epithelial; HTB-55) was purchased from ATCC and cultured in 75-cm² bottles containing F-12: DMEM (Sigma, Australia). A complete culture medium supplemented with 10% (v/v) Fetal Bovine Serum- (FBS, Gibco, Australia), 1% (v/v) nonessential amino acid solution (Gibco, Australia) and 2 mM L-glutamine (Gibco, Australia) was used in the tests unless otherwise indicated and maintained in a humidified atmosphere of 95% air, 5% CO2, at 37°C.

Cellular model of air-liquid interface (ALI).

The Calu-3 ALI model has been previously validated as an in vitro predictive model of upper airway epithelium physiology. To prepare an ALI model, cells were seeded at a density of 7.92 x 10⁴ cells/insert on a Transwell polyester insert (growth area of 0.33 cm² ), containing 100 pL in the apical chamber and 600 pL in the basolateral chamber. The medium from the apical chamber was removed 24 h after seeding and then daily until an ALI was reached, while the medium from the basolateral chamber was replaced every other day until 14 days of culture.

Integrity of the epithelial barrier

The effect of the materials on epithelia grown in an ALI culture (14 days), was assessed by transepithelial electrical resistance (TEER) and by measuring cell layer permeability with sodium fluorescein (Na-Flu), a marker of the paracel lular transporter, over 1 hour. TEER was measured after exposure to the test sample using an epithelial voltohmmeter (EVOM, World Precision Instruments, USA) attached to STX-2 rod electrodes. Before measurement, 100 pL of Hanks' balanced salt solution (HBSS) was added to the apical chamber of the Transwell. For permeability studies, 100 pL (Transwell) of Na-Flu (2.5 mg/mL) was added to the apical chamber, and 600 pL (Transwell) of complete medium was added to the basolateral chamber. At 15-minute intervals over 1 hour, 100 pL of samples were taken from the basolateral chamber and replaced with equal volumes of fresh complete medium. Fluorescence intensity was measured using a Spectramax plate reader at 485 (ex)/520 (em) nm. The apparent permeability coefficient (Papp) was calculated from Equation 1

Papp - (V/AC_a) dC/dtA

Equation 1 where V is the volume in the basolateral chamber, A is the surface area of the Transwell membrane, Co is the initial concentration in the apical chamber, and dC/dt is the (cumulative) flux of Na-Flu across the membrane.

Mucus production

Mucus production after exposure to the test samples was assessed using Alcian blue staining. After treatment, cells were washed with phosphate buffered saline (PBS) and fixed with 4% (v/v) paraformaldehyde (PFA) solution for 15 min. After removing residual PFA with PBS, the cells were stained with Alcian blue for 20 min. Excess dye was removed by successive washes with PBS, and the membranes were excised from the inserts and mounted on slides for microscopic analysis. The slides were visualized with the Nanozoomer Imager and the stained mucus quantified using Image J software. Statistical analysis

Samples were tested in triplicate and data are presented as mean ± standard deviation (StDev). Statistical significance was determined by conducting a one-way ANOVA the Dunnett's post-test of multiple comparisons and Student's t-test.

Results

Evaluation of the integrity and permeability of Calu-3 epithelium up to 24 hours of exposure to PolmonYDEFENCE/DYFESA

The integrity and permeability of Calu-3 epithelium were evaluated after deposition of PolmonYDEFENCE/DYFESA on its surface using HPFP propellant. No significant change was observed in epithelial permeability up to 6 hours compared with 0 hours. After 8 hours, a significant increase (oneway ANOVA) in permeability was observed (Figure 18). However, at 24 hours, a control sample (without PolmonYDEFENCE/DYFESA) showed significantly higher (t-test) epithelial permeability than epithelium with PolmonYDEFENCE/DYFESA (69 mg). The significant reduction in membrane permeability with PolmonYDEFENCE/DYFESA compared with the control at the 24-hour time point is most likely due to the barrier-forming abilities of the sodium hyaluronate in the formulation.

TEER experiments (Figure 18) were used to test epithelial integrity. No significant change in epithelial integrity was observed up to 8 hours. A significant increase in TEER measurements was observed at 24 hours with no significant differences between control and treated epithelium. The increase observed at 24 hours is most likely due to ongoing cell proliferation during the additional period of epithelial cell growth. Time-dependent fluctuations in barrier integrity (TEER) have been previously reported in Calu-3 ALI cultures in the literature.

Evaluation of mucus secretion within the Calu-3 epithelium up to 24 hours of exposure to PolmonYDEFENCE/DYFESA

The effects of PolmonYDEFENCE/DYFESA on the mucus layer formed on the surface of the Calu-3 epithelium grown on ALI was also evaluated using Alcian blue staining (Figure 19A). PolmonYDEFENCE/DYFESA (69 mg) were deposited on Calu-3 cells grown in the ALI model using HPFP propellant. Quantitative color intensity analysis (Figure 19B) showed a significant decrease in epithelial mucus at 8 and 24 hours compared with 0 hours. However, no significant difference was observed at any time point between control and treated epithelium.

Conclusions

PolmonYDEFENCE/DYFESA (69 mg) significantly reduced the permeability of Calu-3 epithelium compared with the control after 24 hours of incubation. When comparing membrane integrity, PolmonYDEFENCE/DYFESA had no adverse effects on Calu-3 epithelium up to 24 hours of exposure. PolmonYDEFENCE/DYFESA showed no significant changes in mucus production compared with the control.

Claims

1 . A composition in a dry powder form for inhalation including:

(I) a mixture M comprising or, alternatively, consisting of. hyaluronic acid or an acceptable pharmaceutical grade salt thereof in an amount from 15% to 60% by weight, with respect to the total weight of the mixture M; and at least one supporting agent selected from the group comprising or, alternatively, consisting of lactose, a dextran, mannitol and mixtures thereof in an amount from 40% to 85% by weight, with respect to the total weight of the mixture M; and optionally,

2. The composition for use according to claim 1, wherein said hyaluronic acid or an acceptable pharmaceutical grade salt thereof is present in an amount from 20% to 50% by weight, preferably from 25% to 40% by weight, more preferably from 28% to 38% by weight, with respect to the total weight of the mixture M.

3. The composition for use according to claim 1 or 2, wherein said supporting agent is present in an amount from 50% to 80% by weight, preferably from 60% to 75% by weight, more preferably from 62% to 72% by weight, with respect to the total weight of the mixture M.

4. The composition for use according to any one of the preceding claims, wherein said inflammation or said oxidative stress is inflammation or oxidative stress of the respiratory system caused by urban dust (UD) pollution.

5. The composition for use according to any one of the preceding claims, wherein said inflammation or said oxidative stress is inflammation or oxidative stress of the upper respiratory tract (nasal cavity, pharynx and/or larynx) or the lower respiratory tract (trachea, primary bronchi and/or lungs).

29

6. The composition for use according to any one of the preceding claims, wherein said (i) mixture M further comprises at least one supporting agent selected from the group comprising of or, alternatively, consisting of lactose, a dextran, mannitol and mixtures thereof; preferably mannitol.

7. The composition for use according to any one of the preceding claims, wherein said mixture M comprises or, alternatively, consists of:

8. The composition for use according to any one of the preceding claims, wherein, in said method of treatment, preferably a topical, preventive treatment, said composition has a barrier action or protective action of epithelial cells of respiratory system from air pollution.

9. The composition for use according to any one of the preceding claims, wherein, in said method of treatment, preferably a topical, curative treatment, said composition has a healing and/or re-epithelializing action on epithelial cells of respiratory system.

10. The composition for use according to any of the preceding claims, wherein said disorder, or alteration, or symptom, or said disease is selected from the group comprising or, alternatively, consisting of: bronchitis, asthma, acute inflammation of the respiratory tract, asthma attacks, cough and phlegm, decreased lung capacity, reduced respiratory function, chronic bronchitis, and tumor of the respiratory tract.

11. The composition for use according to any one of the preceding claims, wherein said composition is in the form of a dry powder for inhalation by mouth, preferably by suction by mouth.

12. A dry powder inhaler operable by a subject by mouth suction, wherein said inhaler comprises: - a disposable inhaler body;

30 - a disposable cartridge containing the composition in dry powder form according to any of the preceding claims; wherein said disposable cartridge is connected to said inhaler body so as to release an effective dose of said composition into the inhaler body.

13. A mixture M for topical administration in dry powder form for inhalation consisting of:

- mannitol, in an amount of from 40% to 85% by weight, preferably from 50% to 80% by weight, more preferably from 60% to 75% by weight, even more preferably from 62% to 72% by weight, with respect to the total weight of said mixture M.