Concentrated treignac mineral water for Anti-inflammatory use in pneumology, and aerosol device containing same

Abstract

The invention relates to a Treignac natural mineral water for use in aerosol therapy in the context of human and animal inflammatory diseases, the Treignac natural mineral water having the following characteristics: - a silica content, expressed as SiO2, of less than 30 mg / l, in particular from 6 to 9 mg / l; - a dry residue at 180°C of less than 30 mg / l; and - a pH of from 5.5 to 6, the mineral water being concentrated up to eight times by reverse osmosis, vacuum concentration or lyophilisation. The invention also relates to an aqueous composition for the prevention and / or treatment of inflammatory diseases, characterised in that it consists of a concentrated Treignac mineral water, optionally in combination with at least one co-agent chosen from compatible formulation agents. The invention further relates to a medical device comprising an aerosol-generating device and a Treignac mineral water or the composition of the invention, packaged in its sterile, concentrated form at the optimal volume for an aerosol therapy session.

Description

CONCENTRATED TREIGNAC MINERAL WATER FOR ANTI-INFLAMMATORY USE IN PNEUMOLOGY AND AEROSOL DEVICE CONTAINING IT

[0001] The present invention relates to Treignac natural mineral water and its use in aerosol therapy in the context of human and animal inflammatory pathologies.

[0002] Among these inflammatory pathologies, bronchial inflammatory pathologies affect a high proportion of the population and are currently treated systemically and locally with powerful anti-inflammatories not without side effects. A light basic treatment without side effects would therefore be desirable and expected.

[0003] By scanning electron microscopy, it has previously been demonstrated by the inventors' work that concentrated Treignac water was capable of inducing the formation of condensed silica fractal figures, silica being the majority compound of this mineral water, the latter being dissolved in the form of orthosilicic acid (33% of the dry residue) (Agnès Smith et al., Fractal structures and silica films formed by the Treignac water on inert and biological surfaces, Nanoscale Advances, 2020, 2, 3821-3828).

[0004] The advantage of concentrated water lies in its high concentration of natural hydroxysilicic acid and its respect for the ionic balance of natural mineral water. In vitro studies on the film-forming effect have shown that the properties of Treignac natural mineral water are preserved and even amplified in concentrated water.

[0005] Furthermore, patent FR1908259 describes a composition comprising a film-forming constituent and at least one extract of macroscopic edible mushroom cell walls, for use by application to an external biological barrier so as to form a film of condensed orthosilicic acid trapping the mushroom cell wall extract. The film-forming constituent may consist of natural Treignac mineral water, this mineral water in a concentrated state, in particular concentrated by reverse osmosis, or a dehydration product of this concentrated mineral water.

[0006] Following on from these previous studies on the moisturizing, soothing, film-forming and protective effect of this natural mineral water, and in vitro, by the inventors on the formation of fractal forms of condensed silica, the inventors wished to test the effect of this water on cultured cells from a human mucosa subject to numerous inflammatory pathologies, namely the respiratory mucosa.

[0007] They therefore previously tested the biological effect of this product on the release of cytokines involved in bronchial inflammatory processes on normal bronchial cells in culture compared to another mineral water of comparable composition, as well as to NaCl controls.

[0008] The inventors then surprisingly observed that natural Treignac mineral water sprayed onto bronchial cells in culture induced a significant inhibition of the secretion of cytokines IL-6, IL-8 and TNF-α directly involved in inflammatory processes.

[0009] Thus, the invention aims to provide a natural product with a moderate anti-inflammatory effect that is easy to use by patients in the form of an aerosol generated by commonly used aerosol-generating devices. This natural product is envisaged to make it possible to reduce the local or general drug doses that are given to patients in the context of pulmonology treatments. Pathologies that could directly benefit from the use of the present invention are, for example, asthma, chronic obstructive pulmonary disease (COPD) or bronchiectasis and cystic fibrosis.

[0010] The invention also provides a medical device for administering aerosol therapy to a subject, said medical device using concentrated Treignac mineral water as a natural product for anti-inflammatory purposes. More particularly, the medical device uses a breathing mask to administer the aerosol to the subject. In certain embodiments, the natural product may be administered prior to or following conventional anti-inflammatory therapy by taking corticosteroid or bronchodilator medications. Detailed description of the invention

[0011] The present invention therefore firstly relates to a natural mineral water from Treignac intended to be used in aerosol therapy in the context of human and animal inflammatory pathologies, the natural mineral water from Treignac having the following characteristics: a Silica content, expressed as SiO2, of less than 30 mg / L, being in particular 6 to 9 mg / L; a dry residue at 180°C of less than 30 mg / L; and a pH of 5.5 to 6.

[0012] In particular, Treignac natural mineral water can have the following composition:

[0013] Calcium 0.9 mg / L

[0014] Magnesium 0.42 mg / L

[0015] Sodium 2.80 mg / L

[0016] Potassium 0.26 mg / L

[0017] Silica 6.93 mg / L

[0018] Bicarbonates 3.3 mg / L

[0019] Chlorides 3.23 mg / L

[0020] Sulfates 0.65 mg / L

[0021] Nitrates 3.04 mg / L

[0022] Nitrites <0.01 mg / L

[0023] Fluoride <0.10 mg / L;

[0024] and the following properties:

[0025] pH 5.7

[0026] Dry residue at 180°C 22mg / L

[0027] Conductivity 27 µS / cm.

[0028] Treignac natural mineral water can be concentrated up to eight times before use by: reverse osmosis; vacuum concentration; or freeze-drying.

[0029] Reverse osmosis is a membrane separation technology based on diffusion selectivity. A semi-permeable membrane is placed between two compartments containing solutions of different salinities. Osmosis is a natural phenomenon that consists of the migration of water from the less concentrated solution to the more concentrated solution (osmotic flow). When a pressure greater than the osmotic pressure is applied to the more concentrated solution, the water flow is directed in the opposite direction to the osmotic flow. This is the principle that is applied.

[0030] The concentration of Treignac mineral water can also be carried out by vacuum concentration in a vacuum evaporator, or by freeze-drying, namely freezing then evaporation under vacuum.

[0031] For example, whether by reverse osmosis or vacuum evaporation the final product obtained after concentration is liquid and, targeting a final concentration of 8X, the values ​​of the corresponding dry residues vary from 160 to 220 mg / L with an average of 180 mg / L. The corresponding concentrations of silica expressed as SiO2 vary from 50 to 60 mg / L with an average of 54 mg / L.

[0032] In a particular embodiment, the concentrated water or the lyophilisate, after resolubilization in sterile distilled or exchanged water, is subsequently subjected to a sterilization step by filtration or preferably by gamma irradiation.

[0033] Filtration can be carried out on 0.22 µm PVDF membrane filters. For example, a sterile syringe filter ø 33 mm porosity 0.2 µm ClearLine® - membrane: PVDF can be used.

[0034] In the embodiment by which the natural mineral water of Treignac is concentrated by freeze-drying, the freeze-dried product is resolubilized by sterile distilled or permuted water so as to obtain a final concentration ranging from 50 to 60 mg / L of silica expressed as SiO2.

[0035] For example, starting from one liter of concentrated water with a dry residue of 180 mg / L and a silica concentration of 54 mg / L, we will theoretically obtain 180 mg of powder which must be dissolved in 125 mL of distilled or deionized water.

[0036] The inflammatory pathologies targeted by the present invention may be respiratory inflammatory pathologies, in particular the respiratory inflammatory pathologies are chosen from asthma, chronic obstructive pulmonary disease, cystic fibrosis, allergies, bronchial dilation, pulmonary interstitial pathologies.

[0037] Targeted inflammatory pathologies can also be inflammatory pathology of the nasal mucosa, in particular rhinitis or sinusitis.

[0038] The present invention also relates to an aqueous composition for the prevention and / or treatment of inflammatory pathologies, the composition possibly consisting of a mineral water as defined above, where appropriate in combination with at least one co-agent chosen from compatible formulation agents.

[0039] Exemplary co-agents may include, but are not limited to, a pro-hydrating agent, including hyaluronic acid.

[0040] Treignac mineral water or the composition of the invention is intended to be applied by aerosol.

[0041] The term "aerosol" means a set of fine particles, solid or liquid, suspended in a gaseous medium. To meet the needs of the invention, the aerosol must contain particles whose size is less than or equal to 3µm.

[0042] For this purpose, the aerosol is produced by an aerosol generation device of the Ultraneb ultrasonic nebulizer type and the size of the aerosol particles is less than or equal to 3µm.

[0043] An ultrasonic nebulizer has a piezoelectric crystal that converts the electrical impulse into vibrations. The aqueous contents then vibrate, and the solution particles dissolve and mix with the air. The higher the ultrasonic frequency, the finer the droplets obtained.

[0044] It is also possible to use a pneumatic nebulizer which is equipped with a motor and composed of two elements, the pneumatic compressor which produces the compressed air and the nebulizer chamber in which the solution is transformed into fine particles.

[0045] It is understood that any type of nebulizer or aerosol generating device suitable for aerosol production meeting the criteria of the present invention may be used in the medical device of the invention.

[0046] The aerosol generating device comprises a tank intended to receive the aqueous solution to be aerosolized.

[0047] Typically, the products used in aerosols in pulmonology are either physiological water (NaCl 9 g / L) to hydrate and cleanse the bronchial tree, or medications (bronchodilators or corticosteroids). Thermalism also uses aerosols (mineral water, sea water) with the use of a mask or by breathing an external mist, but none of the products used so far have shown anti-inflammatory activity on local cytokines.

[0048] The invention also relates to the use of this mineral water or of the composition for the basic treatment associated where appropriate with the medicinal treatment of inflammatory pathologies in humans and / or animals using an aerosol generator.

[0049] For this purpose, aerosol therapy with sterile concentrated Treignac mineral water can be combined with anti-inflammatory treatment based on corticosteroids or bronchodilators. Aerosol therapy sessions can be observed 2 to 3 times per day.

[0050] The present invention further relates to a medical device comprising an aerosol generating device and Treignac mineral water or an associated composition, wherein the Treignac mineral water or the composition is packaged in its concentrated, sterile form, at the optimal volume for an aerosol therapy session.

[0051] For this purpose, a volume of 5 to 10 mL and an aerosol therapy session lasting 10 to 15 minutes can be planned.

[0052] The medical device may be adapted for use in aerosol therapy by using an aerosol generating device coupled with a mask and / or mouthpiece or applied without a mask or mouthpiece in a dedicated enclosure or room.

[0053] In a particular embodiment, the mask is a nasobuccal mask.

[0054] In another particular embodiment, the aerosol therapy is carried out in a dedicated closed enclosure.

[0055] Furthermore, the medical device can be used in outpatient care in hospitals, doctor's offices or other care centers.

[0056] It can also be used at the subject's home.

[0057] Particular embodiments of the present invention will now be described, with reference to the appended figures.

[0058] In these figures:

[0059] is a graph representing the net release of IL-6 (average of triplicates) by normal bronchial cells in culture stimulated by Poly(I:C) at 0.3 µg / mL and after aerosolization under the different conditions of Example 6 (physiological water, concentrated Treignac water and concentrated Volvic water);

[0060] is a graph representing the net release of IL-8 (average of triplicates) by normal bronchial cells in culture stimulated by Poly(I:C) at 0.3 µg / mL and after aerosolization under the different conditions of Example 6 (physiological water, concentrated Treignac water and concentrated Volvic water);

[0061] is a graph representing the net release of TNFα (average of triplicates) by normal bronchial cells in culture stimulated by Poly(I:C) at 0.3 µg / mL and after aerosolization under the different conditions of Example 6 (physiological water, concentrated Treignac water and concentrated Volvic water);

[0062] is a graph representing the net release of IL-6 in pg / mL (mean of 9 measurements) by normal bronchial cells in culture under the different conditions of Example 7 (physiological water, NaCl 55 mg / L, concentrated Treignac water), with stimulation by Poly(I:C) at 0.3 µg / mL;

[0063] is a graph representing the spontaneous release of IL-6 (mean of 9 measurements) after aerosolization and without Poly(I:C) activator.

[0064] Example 1: Preparation of concentrated Treignac water

[0065] A concentrate of natural mineral water from Treignac is prepared by reverse osmosis.

[0066] The starting Treignac water has the following composition:

[0067] Calcium0.9 mg / LMagnesium0.42 mg / LSodium2.80 mg / LPotassium0.26 mg / LSilica6.93 mg / LBicarbonates3.3 mg / LClorides3.23 mg / LSulfates0.65 mg / LNitrates3.04 mg / LNitrites<0.01 mg / LFluoride<0.10 mg / L

[0068] Under the following technical conditions: pressure of 10 6 Pa (10 bar) and refrigeration with tap water at 12°C.

[0069] The reverse osmosis unit used was manufactured by Techniques Industrielles Appliquées (TIA). The model is the BL 200 membrane process. CSM (Customer Satisfaction Membrane) membranes are polyamide membranes made of thin-film composite (TFC).

[0070] The device is made up of: stainless steel tank with a volume of 45 L (diameter of 31.5 cm and height of 57 cm); CAT Pumps 311 pump; and support for installing the membrane.

[0071] The operating conditions for conducting this test are as follows:

[0072] Pressure: 10 6 Pa (10 bar)

[0073] Refrigeration: tap water (temperature 12°C). Carrying out the test

[0074] Before using the device, it was washed with reverse osmosis water with a conductivity of less than 3 µS / com. The stainless steel tank is then filled with natural mineral water from Treignac. Once this tank is filled, the reverse osmosis system is put into operation. The water in the stainless steel tank passes through this pipe, also made of stainless steel. The operating pressure is measured at this level. The water penetrates the membrane.

[0075] The quantity of liquid constituting the permeate is renewed by continuously adding Treignac natural mineral water directly into the tank. The operation of the device is maintained with recirculation of the concentrate until a volume of concentrate equal to one eighth of the initial volume of Treignac natural mineral water is obtained. This is a concentration by a factor of eight.

[0076] The concentrate present in the tank is then collected in a container. Silica dosage

[0077] Silica determination is performed using an ammonium molybdate test in a HACH Lange LCW 028 cuvette.

[0078] The very low minerality of Treignac natural mineral water (22 mg of dry residue at 180°C on average) makes it possible to obtain, according to the process described, a weakly mineralized osmosis water, having the same portion of minerals as the original water but containing 55 to 65 mg of silica / L (expressed as SiO2).

[0079] Example 2: Preparation of a concentrate by vacuum evaporation of concentrated Treignac water

[0080] Natural mineral water is subjected to a vacuum of -950 mbar at a temperature of 40°C. Regular boiling is maintained for 15 hours at a flow rate of around 6 mL / min in order to obtain an 8-fold concentrate.

[0081] Example 2bis: Preparation of a lyophilisate of concentrated Treignac water

[0082] In a polypropylene jar, 300 mL of concentrated Treignac water is frozen at -20°C. The frozen water is then introduced into the Christ alpha 1-4 LSC plus freeze-dryer and left until completely freeze-dried. This allows 56.61 mg of dry residue to be recovered. The resulting residue is stored in an airtight container, itself stored in a desiccator to prevent rehydration.

[0083] Example 3: Preparation of a spray (an aerosol) based on a composition of the invention

[0084] The composition of the invention is obtained in the form of an 8-fold concentrated solution and sterilized by gamma irradiation.

[0085] This solution is then introduced into the nebulizer reservoir. The aerosol generating device is connected to the mask.

[0086] Example 4: Development of aerosolization conditions for the in vitro test

[0087] Aerosol generating device: UltraNeb nebulizer (ref MC201206d1).

[0088] The tests resulted in the development of a production of a regular aerosol with a droplet size of <3µm and its deposition on a culture plate, in order to deposit around 1.5 to 2µL of aerosol per 2 cm well. 2The aerosolization conditions were standardized (saturation of a 5L enclosure for one hour and one minute of contact with the plates) and allowed regular aerosolization of the plates and the deposition of 1µL / cm 2 of the aerosolized product.

[0089] Example 5: Development of in vitro experimental conditions

[0090] The trial is taking place in the context of the fight against chronic inflammatory respiratory diseases, in particular chronic obstructive pulmonary disease (COPD), which represents the 3 ème leading cause of death worldwide.

[0091] The working hypothesis is therefore that the aerosolized concentrated Treignac water could form a protective film at the level of the bronchioles and exert its protective activity and / or reduce the intensity of the inflammatory symptoms.

[0092] The cells used in the study are primary bronchial epithelial cells HBEpC (ref: Bioalternatives HBEpC1, used in 5 ème passage, 50,000 cells / well).

[0093] Cell culture conditions: 37°C, 5% CO2.

[0094] Culture medium: Airway epithelial cell basal medium optimized for the test.

[0095] The stimulant that causes cell inflammation is Poly(I:C) (polyinosinic:polycytidylic acid), a synthetic analogue of double-stranded RNA, as double-stranded RNAs (TLR3 agonists) have been described to induce an inflammatory response similar to that observed in COPD.

[0096] The inflammation markers analyzed are IL-6, TNF-α

[0097] and IL-8. Tested products

[0098] Mineral water from Treignac concentrated by reverse osmosis: dry residue 55 mg / L, conductivity 72 µS / cm, SiO2 18.3 mg / L, Na+ 6.2 mg / L, Cl- 9 mg / L, pH 6.9 Mineral water from Volvic concentrated by reverse osmosis: dry residue 74 mg / L, conductivity 94 µS / cm, SiO2 18 mg / L, Na+ 7 mg / L, Cl- 8 mg / L NaCl 9 g / L (physiological water) NaCl 55 mg / L Established protocol

[0099] Normal bronchial cells (Bioalternatives HBEpC1) are distributed in 24-well plates so that they are adherent and not joined after 48 hours of incubation. The plates are emptied of their medium and the plates are placed in an enclosure saturated with a mist of the product to be tested from an aerosol generator used in human therapy (Ultraneb – ref MC201206d1), the droplets being calibrated (<3µm in diameter).

[0100] The fog deposition was previously standardized to 1 µg / cm2 in 1 minute.

[0101] The culture medium is then added by adding 0.3 or 0.1 µg / mL of the poly(I:C) activator mimicking a viral infection.

[0102] After 48 hours, cytokines are measured by ELISA in the culture supernatants and the results are expressed in pg / mL.

[0103] Example 6: Effect of concentrated Treignac water aerosolization on bronchial epithelial cells in vitro

[0104] In a first test, physiological water NaCl 0.9% and the waters Eau de Treignac Concentrat and Eau Volvic Concentrat were evaluated on the release of three inflammation markers, IL-6, TNF-α and IL-8, by HBEpC cells under aerosolization conditions and stimulated or not by the activator Poly(I:C).

[0105] Poly(I:C) (purity 10%, Sigma, ref. P9582) is in stock solution at 0.5 mg / mL in ultrapure water and used at a concentration of 0.3 µg / mL.

[0106] Quantification of the three inflammation markers was performed by multiplex assay using the BD method TM Cytometric Bead Array (CBA).

[0107] Tested compoundAppearance / storageApplicationTest conditionTested concentrationPhysiological water t NaCl 0.9%LiquidRoom temperatureAerosolizationNebulizer saturation 60 min, flow rate 4 power 4, 1 min misting, 20 min in incubator at 37°CPureEau de Treignac Concentrate*LiquidStorage: +4°CAerosolizationNebulizer saturation 60 min, flow rate 4 power 4, 6 min misting, 20 min in incubator at 37°CDilution 1 / 3 in ultrapure waterEau Volvic Concentrate*LiquidStorage: +4°CAerosolizationNebulizer saturation 60 min, flow rate 4 power 4, 1 min misting, 20 min in incubator at 37°CDilution 1 / 3 in ultrapure water

[0108] * sterilization by 0.22 µm filtration before use Cultures and treatments

[0109] Non-aerosolized conditions

[0110] HBEpC cells were seeded in 24-well plates and cultured in culture medium for 24 hours. The medium was then replaced with culture medium containing or not (controls and Eau de Treignac Concentrat condition) the reference bafilomycin (inhibitor of the activating effect of Poly(I:C)), tested at 3 nM, and the cells were preincubated for 24 hours. After preincubation, the medium was replaced with culture medium containing or not the reference bafilomycin or the Eau de Treignac Concentrat compound (tested systemically) and containing or not (non-stimulated conditions) the activator Poly(I:C), tested at 0.3 µg / mL. The cells were then incubated for 48 hours.

[0111] Aerosolized conditions

[0112] HBEpC cells were seeded in 24-well plates and cultured in culture medium for 24 hours. The medium was then replaced with culture medium containing or not the bafilomycin reference, tested at 3 nM, and the cells were incubated for 24 hours. The medium was removed from each well and the plates were placed in the chamber (previously saturated with mist for 45 / 60 minutes with the compounds to be tested). The cells were incubated in this chamber so that the droplets could settle on the surface of the cell lawn, then in an incubator at 37°C. At the end of the incubation, the culture medium containing or not the bafilomycin reference and containing or not the Poly(I:C) activator, tested at 0.3 µg / mL, was added. The cells were then incubated for 48 hours.

[0113] All experimental conditions were carried out in triplicate.

[0114] After incubation, culture supernatants were collected to assay the amount of IL-6, TNF-α, and IL-8 secreted. Multiplex assay (CBA system)

[0115] The amounts of cytokines present in the culture supernatants were measured using BD kits TM Specific CBA: Human IL-6 Flex Set (BD TM Biosciences, A7 Marbles, Ref. 558276), Human TNF Flex Set (BD TM Biosciences, D9 Beads, Ref. 558273) and Human IL-8 Flex Set (BD TM Biosciences, Balls A9, Ref. 558277). Results

[0116] The results observed on the non-misted cells (inhibition by bafilomycin, spontaneous release of the three cytokines tested and appearance of the cultures) are consistent with the expected results and validate the protocol.

[0117] Under the experimental conditions of the test, in basal conditions (absence of stimulation by Poly(I:C), aerosolization of HBEpCs with the different waters induced an increase in the basal release of IL-6 (between 305% and 448% of the non-aerosolized control) and IL-8 (between 272% and 397% of the non-aerosolized control), without modulating the release of TNF-α.

[0118] Stimulation of aerosolized cells by Poly(I:C) at 0.3 µg / l showed the following effects, the results being obtained in triplicates and expressed as net cytokine release, representing the difference between cytokine release after cell activation and spontaneous release in the absence of stimulation by the Poly(I:C) activator: IL-6 release: in all aerosolized conditions, net IL-6 release by bronchial cells was increased, but this stimulation was significantly lower in response to Eau de Treignac Concentrat (33.1% of the control "physiological water" (NaCl 9g / L)) (). IL-8 release: Comparison of the effect of Eau de Treignac Concentrat with its control physiological water NaCl 9g / L also showed an inhibitory effect of Eau de Treignac Concentrat (19.7%) ().TNF-α release: Comparison of the effect of Eau de Treignac Concentrat with its control physiological water Nacl 9g / L also showed a 78% inhibition by Eau de Treignac Concentrat (). Volvic water tested in parallel showed no statistically significant inhibitory effect on the release of the three cytokines tested. In all aerosolized conditions, bafilomycin showed the same effects against Poly(I:C) as in the absence of aerosolization.

[0119] The results obtained in this experiment suggest that Eau de Treignac Concentrat would have an inhibitory effect against the activation of bronchial cells induced by Poly(I:C) by reducing the release of IL-6, IL-8 and TNF-α.

[0120] Example 7: Effect of concentrated Treignac water aerosolization on IL-6 release by bronchial epithelial cells in vitro

[0121] In a second series of experiments, Treignac Concentrate water as well as NaCl 55 mg / L (salinity equivalent to Treignac water concentrated at 1 / 3) and physiological water NaCl 9 g / L were evaluated in hexaplicates on the release of a single inflammation marker IL-6 (by ELISA assay) by HBEpC cells under aerosolization conditions and stimulated or not by Poly(I:C).

[0122] Tested compoundAppearance / storageApplicationTest conditionTested concentrationSaline NaCl 0.9%LiquidRoom temperatureAerosolizationNebulizer saturation 45 min, flow rate 4 power 4, 2.5 min misting, 20 min in incubator at 37°CNaCl 9 g / LNebulizer saturation 45 min, flow rate 4 power 4, 2.25 min misting, 20 min in incubator at 37°CNaCl 55 mg / L (dilution of Nacl 0.9% in ultrapure water)Treignac Water Concentrate*LiquidStorage: +4°CAerosolizationNebulizer saturation 45 min, flow rate 4 power 4, 6 min misting, 20 min in incubator at 37°CDilution 1 / 3 in ultrapure water

[0123] * sterilization by 0.22 µm filtration before use

[0124] ** sterilization by filtration 0.22 µm after dilution

[0125] The Poly(I:C) activator (purity 10%, Sigma, ref. P9582) is in stock solution at 0.5 mg / mL in ultrapure water and used at a concentration of 0.3 µg / mL.

[0126] Aerosolized conditions

[0127] HBEpC cells were seeded in 24-well plates and cultured in culture medium for 48 hours with renewal of the medium after 24 hours of incubation. The medium was removed from each well and the plates were placed in the chamber (previously saturated with aerosol for 45 minutes with the compounds to be tested). The cells were incubated in this chamber so that the droplets could deposit on the surface of the cell lawn, then in an incubator at 37°C. At the end of the incubation, the culture medium containing or not (unstimulated controls) the activator Poly(I:C), tested at 0.1 and 0.3 µg / mL, was added. The cells were then incubated for 48 hours.

[0128] After incubation, culture supernatants were collected to assay the amount of secreted IL-6. ELISA assay

[0129] The amount of IL-6 present in the culture supernatants was measured using an ELISA assay kit according to the suppliers' instructions: Human IL-6 Duo Set (R&D Systems, Ref. DY206). Results

[0130] Due to the good stimulation of bronchial cells by Poly(I:C) at 0.3 µg / mL on the release of IL-6 and the marked inhibitory effect of Eau de Treignac Concentrate observed, this experiment was carried out to confirm the effect of this water on this mediator under the same technical conditions. The effect of Poly(I:C) in the absence of aerosolization having already been observed previously, these non-aerosolized conditions were not repeated.

[0131] Under experimental conditions, in the absence of stimulation by Poly(I:C), a basal release of IL-6 was observed in the different aerosolized conditions, with a statistically lower release after aerosolization by Eau de Treignac Concentrat in comparison with the two controls NaCl 55 mg / L and NaCL 9 g / L (83% and 80% of these respective controls).

[0132] Stimulation of aerosolized cells by Poly(I:C) globally induced an increase in IL-6 release by bronchial cells with an effect dependent on the concentration of Poly(I:C): NaCl 55 mg / L: 226% and 287% of the aerosolized control Eau de Treignac Concentrate: 215% and 245% of the aerosolized control NaCl 9 g / L: 196% and 296% of the aerosolized control.

[0133] Comparing the effect of aerosolization on the response of cells to stimulation by Poly(I:C), Eau de Treignac Concentrat showed an inhibitory effect on the release of IL-6 (41%)().

[0134] Statistical calculations performed on the 9 values ​​(from the two series of experiments) concerning the release of IL-6 by Poly(I:C) compared to physiological water show that Treignac water induces a statistically significant inhibition (34.7%, p<0.004).

[0135] Under the previous experimental conditions, the spontaneous release of IL-6 by bronchial cells aerosolized by concentrated Treignac water is not significantly different from the spontaneous release of IL-6 from these same cells aerosolized by physiological water (NaCl 9g / L) (). These results support the absence of toxicity of concentrated Treignac water on bronchial cells in culture.

[0136] To summarize, Treignac water sprayed onto cultured bronchial cells induced a significant inhibition of the secretion of the three cytokines tested. Repetition of the experiments for IL-6 (9 measurements) showed an inhibition of 37.4% for a significance of p<0.004.

[0137] A first experiment carried out in triplicate showed a significant inhibition of the release of cytokines IL-6 (33.1%), TNF (78%), IL-8 (19.7%) compared to the control physiological water NaCl 9 g / L. The activity of Volvic mineral water is not significant on the release of these cytokines.

[0138] A second series of experiments was carried out in hexaplicate to confirm the effect of Treignac mineral water on IL-6 release compared to two NaCl controls, namely physiological water NaCl 9g / L as previously, as well as NaCl 55 mg / L (corresponding to the salinity of concentrated Treignac mineral water). The inhibitory effect of Treignac water was again confirmed compared to the two controls. Analysis of the 9 values ​​obtained during experiments 1 and 2 shows a significant inhibition of 41% and an absence of toxicity on the spontaneous release of IL-6.

[0139] Concentrated Treignac mineral water also has an original biological effect on this model: this effect on the release of pro-inflammatory cytokines can be described as anti-inflammatory.

[0140] The inventors therefore have a concentrated mineral water, already used in cosmetics, with moderate anti-inflammatory properties on the main cytokines involved in bronchial inflammatory pathologies, all without the addition of chemical active ingredients.

[0141] One of the advantages of the present invention is that, unlike first-line treatments such as those using corticosteroids or bronchodilators, it remains devoid of toxicity. The results of in vitro and in vivo toxicity tests on bronchial cells support its use in pulmonology. In addition, regarding the implementation of the invention, the necessary volumes of Treignac mineral water per patient are relatively low and the raw material is accessible at low cost. Furthermore, the treatment of this water on an industrial scale is easy and inexpensive.

Claims

- Concentrated mineral water for use in aerosol therapy in the prevention and / or treatment of inflammatory respiratory or nasal mucosal pathologies, characterized by the fact that the natural mineral water of Treignac is, prior to its application, concentrated up to eight times by reverse osmosis, vacuum concentration or freeze-drying, the concentrated mineral water having the following characteristics: a Silica content, expressed as SiO 2 , ranging from 6 to 9 mg / L up to 50 to 65 mg / L; a dry residue at 180°C ranging up to 160-220 mg / L. - Concentrated mineral water according to claim 1, characterized in that the concentrated water or the lyophilisate, after total resolubilization in sterile distilled or exchanged water, is subsequently subjected to a sterilization step by filtration or preferably by gamma irradiation. - Concentrated mineral water according to any one of claims 1 and 2, characterized in that the respiratory inflammatory pathologies are chosen from asthma, chronic obstructive pulmonary disease, cystic fibrosis, allergies, bronchial dilation, interstitial pulmonary pathologies. - Concentrated mineral water according to one of claims 1 and 2, characterized in that the inflammatory pathology is an inflammatory pathology of the nasal mucosa, in particular rhinitis or sinusitis. - Aqueous composition for use in aerosol therapy in the prevention and / or treatment of inflammatory respiratory or nasal mucosal pathologies, characterized in that it comprises a concentrated mineral water as defined in any one of claims 1 to 4, where appropriate in combination with at least one co-agent chosen from compatible formulation agents. - Concentrated mineral water according to one of claims 1 to 4 or composition according to claim 5, characterized in that it is formulated in the form of an aerosol. - Concentrated mineral water according to one of claims 1 to 4, 6 or composition according to one of claims 5 or 6, characterized in that the aerosol is produced by an aerosol generation device of the ultrasonic nebulizer type and that the size of the aerosol particles is less than or equal to 3µm. - Concentrated mineral water according to any one of claims 1 to 4, 6 and 7 or composition according to any one of claims 5 to 7 intended to be used in aerosol therapy in combination, where appropriate, with the medicinal treatment of inflammatory pathologies in humans and / or animals. - Medical device, characterized in that it comprises an aerosol generating device and a concentrated Treignac mineral water according to any one of claims 1 to 4, 6 and 7 or a composition according to any one of claims 5 to 7, in which the Treignac mineral water or the composition is packaged in its concentrated, sterile form, at the optimal volume for an aerosol therapy session. - Medical device according to claim 9, characterized in that the medical device is suitable for use in aerosol therapy by using an aerosol generating device coupled to a mask and / or mouthpiece or applied without a mask or mouthpiece in a dedicated enclosure or room. - Medical device according to any one of claims 9 or 10, characterized in that the medical device is used in outpatient care in a hospital, medical office or other care center. - Medical device according to any one of claims 9 to 11, characterized in that the medical device is used at the subject's home.

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