CONCENTRATED TREIGNAC WATER AEROSOL DEVICE FOR ANTI-INFLAMMATORY USE IN PNEUMOLOGY

Abstract

The invention 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 from 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, the mineral water being concentrated up to eight times by reverse osmosis, vacuum concentration or freeze-drying. It also relates to an aqueous composition for the prevention and / or treatment of inflammatory pathologies, characterized in that it consists of a concentrated mineral water from Treignac, where appropriate 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 Treignac mineral water or the composition of the invention, packaged in its concentrated, sterile form, at the optimal volume for an aerosol therapy session. Figure to be published with the abstract: Figure 1A.

Description

Title of the invention: CONCENTRATED TREIGNAC WATER AEROSOL DEVICE FOR USE ANTI-INFLAMMATORY IN PNEUMOLOGY

[0001] The present invention relates to natural mineral water from Treignac 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 generally 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 the high concentration of natural hydroxysilicic acid and the respect of the ionic balance of natural mineral water. In vitro studies concerning 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 cell walls of macroscopic edible mushrooms, for use by application to an external biological barrier so as to form a film of condensed orthosilicic acid trapping the extract of cell walls of mushrooms. The film-forming constituent may consist of a 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] To follow up on these previous studies on the hydrating, 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-a 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 respiratory 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 less than 30 mg / L; and - a pH of 5.5 to 6.

[0012] In particular, Treignac natural mineral water may 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 22 mg / L

[0027] Conductivity 27 pS / cm.

[0028] Treignac natural mineral water can be, prior to its application, concentrated up to eight times 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 salinity. 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 by 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 pm PVDF membrane filters. For example, a sterile 0.33 mm syringe filter with 0.2 pm porosity 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] As an 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 will have to 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] The targeted inflammatory pathologies can also be an 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 3 pm.

[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 3 pm.

[0043] An ultrasonic nebulizer has a piezoelectric crystal that transforms the electrical impulse into vibrations. The aqueous contents therefore vibrate and the particles of the solution dissolve and are mixed 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 air tablet and the nebulizer chamber in which the solution transforms into fine particles.

[0045] It is understood that any type of nebulizer or aerosol generating device suitable for the production of aerosol 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] Generally, the products used in aerosol in pneumology are either physiological water (NaCl 9 g / L) to hydrate and clean 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 has 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 ranging from 5 to 10 mL and an aerosol therapy session lasting from 10 to 15 minutes can be provided.

[0052] The medical device may be adapted 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.

[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 a hospital, doctor's office or other care center.

[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] [Fig. 1 A] 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 pg / mL and after aerosolization under the different conditions of Example 6 (physiological water, concentrated Treignac water and concentrated Volvic water);

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

[0061] [Fig. IC] is a graph representing the net release of TNFa (average of triplicates) by normal bronchial cells in culture stimulated by Poly(EC) at 0.3 pg / mL and after aerosolization under the different conditions of Example 6 (physiological water, concentrated Treignac water and concentrated Volvic water);

[0062] [Fig.2] is a graph representing the net release of IL-6 in pg / mL (average 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 pg / mL;

[0063] [Fig.3] is a graph representing the spontaneous release of IL-6 (average 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] [Tables 1] Calcium 0.9 mg / L Magnesium 0.42 mg / L Sodium 2.80 mg / L Potassium 0.26 mg / L Silica 6.93 mg / L Bicarbonates 3.3 mg / L Chlorides 3.23 mg / L Sulfates 0.65 mg / L Nitrates 3.04 mg / L Nitrites < 0.01 mg / L Fluoride <0.10 mg / L

[0068] Under the following technical conditions: pressure of 106Pa (10 bar) and refrigeration by tap water at 12°C.

[0069] The reverse osmosis device used was manufactured by the company 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 constitution of the device is as follows: - 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 carrying out this test are as follows:

[0072] Pressure: 106 Pa (10 bar)

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

[0074] Before using the device, it was washed using osmosis water with a conductivity of less than 3 pS / 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 adding natural mineral water from Treignac continuously directly into the tank. The operation of the apparatus is maintained with recirculation of the concentrate until a volume of concentrate equal to one eighth of the initial volume of natural mineral water from Treignac is obtained. This is a concentration by a factor of eight.

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

[0077] The silica dosage is carried out by an ammonium molybdate test in a HACH Lange LCW 028 tank.

[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 pot, 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 a hermetic container, itself stored in a desiccator to prevent any 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 generation device: UltraNeb nebulizer (ref MC201206dl).

[0088] The tests resulted in the development of a production of a regular aerosol with a droplet size of <3pm and its deposition on a culture plate, in order to deposit around 1.5 to 2 pL of aerosol per 2 cm2 well. The aerosolization conditions were standardized (saturation of a 5L enclosure for one hour and one minute of contact with the plates) and allowed the regular aerosolization of the plates and the deposition of IpL / cm2 of the aerosolized product.

[0089] Example 5: Development of in vitro experimentation 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 third leading cause of death in the world.

[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 HBEpCl, used at the 5th 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 allows cell inflammation is Poly(I:C) (acid polyinosinic acid:polycytidylic acid), synthetic analogue of double-stranded RNA because it

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[0107] It has been described that double-stranded RNAs (TLR3 agonists) induce an inflammatory response similar to that observed in COPD. The inflammation markers analyzed are IL-6, TNF-a and IL-8. Tested products - Treignac mineral water concentrated by reverse osmosis: dry residue 55 mg / L, conductivity 72 pS / cm, SiO2 18.3 mg / L, Na+ 6.2 mg / L, Cl- 9 mg / L, pH 6.9 - Volvic mineral water concentrated by reverse osmosis: dry residue 74 mg / L, conductivity 94 pS / 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 Normal bronchial cells (Bioalternatives HBEpCl) 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 MC201206dl), the droplets being calibrated (<3pm in diameter). The fog deposition was previously standardized to 1 pg / cm2 in 1 minute. The culture medium is then added by adding 0.3 or 0.1 pg / mL of the poly(I:C) activator mimicking a viral infection. After 48 hours, cytokines are measured by ELISA in the culture supernatants and the results are expressed in pg / mL. Example 6: Effect of concentrated Treignac water aerosolization on bronchial epithelial cells in vitro In a first test, physiological water NaCl 0.9% and the waters Eau de Treignac Concentrât and Eau Volvic Concentrât were evaluated on the release of three inflammation markers, 1TL-6, TNF-a and 1TL-8, by HBEpC cells under aerosolization conditions and stimulated or not by the activator Poly(I:C). 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 pg / mL. Quantification of the three inflammation markers was performed by multiplex assay using the BD™ Cytometry Bead Array (CBA) method. [Tables 2] Compound tested Appearance / storage Application Test condition Concentration tested Physiological water NaCl 0.9% Liquid Room temperature Aerosolization Nebulizer saturation 60 min, flow rate 4 power 4, 1 min misting, 20 min in incubator at 37 °C Pure Treignac Water Concentrate* Liquid Storage: +4°C Aerosolization Nebulizer saturation 60 min, flow rate 4 power 4, 6 min misting, 20 min in incubator at 37 °C Dilution to 1 / 3 in ultrapure water Water Vol life Concentrate* Liquid Storage: +4°C Aerosolization Nebulizer saturation 60 min, flow rate 4 power 4, 1 min misting, 20 min in incubator at 37 °C Dilution to 1 / 3 in ultrapure water

[0108] * sterilization by filtration 0.22 pm 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 Concentrât 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 Concentrât compound (tested systemically) and containing or not (non-stimulated conditions) the activator Poly(I:C), tested at 0.3 pg / mL. The cells were then incubated for 48 hours. [YES] Aerosolized conditions

[0112] HBEpC cells were seeded into 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 can be deposited on the surface of the cell mat, 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 pg / mL, was added. The cells are then incubated for 48 hours.

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

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

[0115] The quantities of cytokines present in the culture supernatants were measured using specific BD™ CBA kits: Human IL-6 Flex Set (BD™Biosciences, Beads A7, Ref. 558276), Human TNF Flex Set (BD™ Biosciences, Beads D9, Ref. 558273) and Human IL-8 Flex Set (BD™Biosciences, Beads 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(LC), the aerosolization of HBEpC 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-a.

[0118] Stimulation of aerosolized cells by Poly(LC) at 0.3 pg / l exhibited 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, the net release of IL-6 by bronchial cells was increased, but this stimulation was significantly lower in response to Eau de Treignac Concentrât (33.1% of the “physiological water” control (NaCl 9g / L)) ([Fig.lA]). - IL-8 release: Comparison of the effect of Eau de Treignac Concentrât with its control physiological water Nacl 9g / L also showed an inhibitory effect of Eau de Treignac Concentrât (19.7%) ([Fig.lB]). - release of TNF-a: Comparison of the effect of Eau de Treignac Concentrât with its control physiological water Nacl 9g / L also showed an inhibition of 78% by Eau de Treignac Concentrât ([Fig.lC]). - Volvic water tested in parallel showed no statistically significant inhibitory effect on the release of the three cytokines tested. - Under 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 Concentrât 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-a.

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

[0121] In a second series of experiments, Treignac Concentrated 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 ELIS A assay) by HBEpC cells under aerosolization conditions and stimulated or not by Poly(I:C).

[0122] [Tables3] Tested compound Appearance / storage Application Test condition Tested concentration Physiological water NaCl 0.9% Liquid Room temperature Aerosolization Nebulizer saturation 45 min, flow rate 4 power 4, 2.5 min misting, 20 min in incubator at 37°C NaCl 9 g / L Nebulizer saturation 45 min, flow rate 4 power 4, 2.25 min misting, 20 min in incubator at 37°C NaCl 55 mg / L (dilution of NaCl 0.9% in ultrapure water) Treignac water Concentrate* Liquid Storage: +4°C Aerosolization Nebulizer saturation 45 min, flow rate 4 power 4, 6 min misting, 20 min in incubator at 37°C Dilution 1 / 3 in ultrapure water

[0123] * sterilization by filtration 0.22 pm before use

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

[0125] The Poly(LC) 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 pg / 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 pg / mL, was added. The cells were then incubated for 48 hours.

[0128] After incubation, the culture supernatants were collected in order to measure 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 pg / 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 the 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 Concentrât 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 - Treignac Water 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 Concentrât showed an inhibitory effect on the release of IL-6 (41%) ([Fig.2]).

[0134] Statistical calculations carried out 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) ([Fig.3]). 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 induces a significant inhibition of the secretion of the three cytokines tested. Repetition of the experiments for 1TL-6 (9 measurements) shows 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 in order to confirm the effect of Treignac mineral water on the release of IL6 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, having 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, 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 volumes of Treignac mineral water required 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

Claims

1. 1 - Natural mineral water from Treignac intended for use 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.

2. - Concentrated mineral water intended for use in aerosol therapy in the context of human and animal inflammatory pathologies, characterized in that the natural mineral water according to claim 1 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 SiO2, in a range from 6 to 9 mg / L up to 50 to 65 mg / L; - a dry residue at 180°C in a range up to 160-220 mg / L.

3. - Concentrated mineral water according to claim 2, 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.

4. - Mineral water according to any one of claims 1 to 3, characterized in that the targeted inflammatory pathologies are 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.

5. - Mineral water according to one of claims 1 to 3, characterized in that the inflammatory pathology is a pathology inflammatory disease of the nasal mucosa, particularly rhinitis or sinusitis.

6. - Aqueous composition for the prevention and / or treatment of inflammatory pathologies, characterized in that it comprises a mineral water or concentrated mineral water as defined in any one of claims 1 to 5, where appropriate in combination with at least one co-agent chosen from compatible formulation agents.

7. - Mineral water according to one of claims 1 to 5 or composition according to claim 6, characterized in that it is formulated in the form of an aerosol.

8. - Mineral water according to one of claims 1 to 5, 7 or composition according to one of claims 6 or 7, 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 3pm.

9. - Mineral water according to any one of claims 1 to 5, 7 and 8 or composition according to any one of claims 6 to 8 intended to be used in aerosol therapy in combination, where appropriate, with the medicinal treatment of inflammatory pathologies in humans and / or animals.

10. - Medical device, characterized in that it comprises an aerosol generating device and a Treignac mineral water according to any one of claims 1 to 5, 7 and 8 or a composition according to any one of claims 6 to 8, 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.

11. - Medical device according to claim 10, 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.

12. - Medical device according to any one of claims 10 or 11, characterized in that the medical device is used in outpatient care in a hospital, doctor's office or other care center.

13. - Medical device according to any one of claims 10 to 12, characterized in that the medical device is used at the subject's home.

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