Viscous composition comprising reactive oxygen species (ROS)

Abstract

The invention relates to a composition for medical and cosmetic use comprising a viscous or gelled matrix that encapsulates inside it an aqueous solution containing reactive oxygen species, in particular the superoxide ion O2 -, for the treatment and improvement of alterations and diseases that benefit from the administration of effective quantities of Reactive Oxygen Species (ROS).

Description

[0001] VISCOUS COMPOSITION COMPRISING REACTIVE OXYGEN SPECIES (ROS)

[0002] Technical field

[0003] The invention refers to a formulation in viscous or gel form capable of encapsulating reactive oxygen species (ROS) within it, in particular the superoxide ion. The formulation is used in the medical and cosmetic fields.

[0004] The invention is also related to cosmetic and pharmaceutical compositions and forms that comprise this gel, in particular cosmetic and pharmaceutical forms that can be administered orally or topically for the treatment of alterations and diseases that benefit from the administration of effective quantities of Reactive Oxygen Species (ROS). More specifically, the gel can be used for the topical treatment of dermis and mucous membranes infections in general, in particular bacterial and fungal infections, more particularly caused by fungal species belonging to the genus Candida.

[0005] Known art

[0006] ROS are provided for purposes useful to the body, such as: promotion of the expression of genes encoding molecules with antioxidant action and function as signal molecules and antimicrobial defence.

[0007] ROS can be useful in the treatment of various pathologies such as, but not limited to: inflammation, mycosis, viral diseases, tumors and are also often used in cosmetics.

[0008] Therefore, reactive oxygen species (ROS), although they are often considered harmful due to their role in oxidative stress, have several useful applications in both pharmaceuticals and cosmetics. This is how they are used in the pharmaceutical sector.

[0009] Anticancer therapy

[0010] Some drugs exploit the increase in ROS levels to induce selectiveoxidative stress in cancer cel I s, leading them to apoptosis (programmed cell death). Cancer cells are in fact more susceptible to high levels of ROS than healthy ones. In addition, ROS-based therapies are often used in combination with chemotherapy and radiotherapy, increasing the sensitivity of cancer cells to traditional treatments and improving their efficacy.

[0011] Antimicrobial therapies

[0012] ROS are used to damage membranes, proteins and DNA of bacteria, viruses and fungi, enhancing the effectiveness of antimicrobial treatments. In clinical settings, they are integrated into disinfectant solutions, advanced dressings and treatments for infected wounds, providing greater protection against resistant pathogens.

[0013] Therapies against neurodegenerative diseases

[0014] Although ROS are involved in the pathogenesis of diseases such as Alzheimer's and Parkinson's, some experimental treatments try to modulate ROS to restore redox homeostasis. In particular, these therapies aim to balance the ratio of antioxidants to oxidant species, reducing oxidative damage without compromising essential cell signaling processes.

[0015] Immunotherapy

[0016] ROS are used to stimulate or modulate the immune response, enhancing the activity of immune cells such as T lymphocytes. In addition, they can limit the tumor microenvironment, making it more favourable for immune cell activity and enhancing the effectiveness of immunotherapy in cancer patients.

[0017] Here's how they are used in the cosmetic field

[0018] Anti-aging

[0019] ROS are used in treatments that stimulate regenerative processes. Some cosmetics use the controlled effect of ROS to activate cell repair mechanisms, such as collagen synthesis, and to neutralize excess free radicals, reducing surface oxidative stress.Photodynamic Therapy (PDT)

[0020] In treatments for skin aging or blemish reduction, photodynamic therapy uses light-induced ROS production to eliminate damaged cells or improve cell renewal and treat conditions such as resistant acne, actinic keratosis, and rosacea.

[0021] Lightening treatments

[0022] ROS can be used to reduce skin pigmentation, promoting cell turnover and lightening the skin. This effect is particularly useful for treating dark spots and discoloration.

[0023] Skin repair

[0024] Some treatments use ROS to stimulate the skin's natural antioxidant response, improving the repair of skin damage.

[0025] In practice, although ROS have these positive applications, their use must be carefully balanced to avoid side effects such as chronic oxidative stress, which can damage healthy cells and accelerate aging or the pathogenesis of several diseases.

[0026] The patent document W02023057989 describes a method and equipment for making aqueous solutions containing reactive oxygen species (ROS) and for administering the solutions to a biological system. ROS are obtained by mechano-induced splitting of ozone in aqueous solution under pressure through submicron passages. Mechanoinduced heterolytic cleavages are obtained by passing at 7-15 bar through pores of 0.001-0.2 pm in the absence of electrodes and plasma. The solutions can be formulated; however, it does not go into detail which gels can be adequately used to have a negligible or acceptably negligible decay of the reactive oxygen species incorporated within it. In fact, it has been verified that compositions containing this solution, when not adequately formulated, generally exhibit too fast, unacceptable and unsatisfactory decay of reactive oxygen species.

[0027] In particular, there are no indications to allow stable gel compositions containing ROS to be obtained.However, in none of all the products and compositions / formulations described in the literature and possibly commercially available has been found the ability to maintain ROS stably within a gelled matrix both of which can be used in the cosmetic and pharmaceutical fields.

[0028] Gel formulations containing ROS and rheological modifiers for topical / cosmetic applications wherein the production of the reactive solution containing ROS is obtained by salination (NaCI) and electrolysis are also known in the literature. The reactive solutions thus obtained have chlorinated oxidizing species (e.g. HOCI / OCI-) and the formulations have decidedly low viscosities. It is also known in the dermatological literature that, although, some topical formulations based on HOCI can be well tolerated; however, the same literature shows that the physicochemical properties and the skin impact of HOCI after application strictly depend on formulation parameters (pH and stability) and conditions of use. In the NaOCI / "bleach" field, phenomena such as dryness, itching or burning sensation are also reported in some subjects or conditions of use, and in general it is recognized that hypochlorite solutions may present irritative potential if used improperly or at high concentrations. This technical framework reinforces the motivation of the expert in the field to look for alternative ways for repeatable cosmetic / dermal formulations suitable for topical use, without necessarily basing the solution on the "active chlorine" chemistry typical of salt electrolysis.

[0029] Therefore, the technical problem that the invention solves is to provide a gel / viscose composition that incorporates ROS in aqueous solution with stability over time and suitability for oral or topical use at room temperature, offering an alternative route to systems based on salt electrolysis and oxidizing chlorinated species, while maintaining high rheological characteristics suitable for encapsulation and controlled release.

[0030] Unless expressly excluded, this chapter is to be understood as part of the detailed description according to this invention.

[0031] Summary of the inventionThe invention aims to solve the problems of known art and to provide a formulation containing ROS and characterized by increased stability over time.

[0032] Therefore, the subject of the present invention is a formulation that is able to incorporate / embed / encapsulate / retain aliquots of an aqueous solution containing negative ions within a gelled or viscous matrix designed to protect negative ions from rapid decay. The characteristics of the gel are described below.

[0033] The composition of the invention comprises:

[0034] • a carrier; and

[0035] • an aqueous solution based on reactive oxygen species (ROS) deriving from the splitting of ozone such as superoxide anions (Oz‘ ), singlet oxygen Oz), hydroxyl radicals (OH*), oxidizing oxygen radical (O’);

[0036] and wherein said carrier is or comprises:

[0037] • water; and

[0038] • at least one rheological modifier selected from substances that are capable of keeping the composition viscous, at a temperature of <30°C.

[0039] Other objects of the invention are the cosmetic and pharmaceutical compositions comprising this formulation.

[0040] Another object of the invention is the process to obtain this formulation.

[0041] A further object of the invention is a kit comprising aliquots of the composition of the invention, packaged in single-dose form or in individual dosing units, possibly applicator devices and explanations, possibly readable on multimedia reading devices.

[0042] The solution according to the invention concerns a composition comprising:• a carrier; and

[0043] • an aqueous solution based on reactive oxygen species (ROS) deriving from the splitting of ozone such as superoxide anions {O2 ), singlet oxygen (1O2), hydroxyl radicals (OH*), oxidizing oxygen radical (O’);

[0044] wherein, said carrier is or comprises:

[0045] • water; and

[0046] • at least one rheological modifier selected from the substances that are able to maintain viscous a composition, at a temperature of <30°C.

[0047] This ROS-based aqueous solution is free from oxidizing chlorinated species (e.g. HOCI / OCI-) and is obtained by a process of mechanoinduced heterolytic cleavage of ozone in aqueous solution under pressure through submicron steps, preferably by means of the equipment and / or method described in W02023057989. Mechanoinduced heterolytic cleavages are obtained by passing at 7-15 bar through pores of 0.001-0.2 pm in the absence of electrodes and plasma. In addition, the compositions of the invention, not being generated through electrolysis processes and chloride-based solutions, are free from Cl2, HOCI, CIO-, secondary ROS species. On the contrary, they involve the primary production of O2-, O’, OH*,1O2, the mechanical splitting of ozone, they do not involve electrolysis or electrodes, nor anodic oxidation of chlorides.

[0048] Preferably, the composition, at a temperature <30°C, has a viscosity between 300,000-600,000 mPa-s, preferably between 400,000-480,000 mPa-s.

[0049] Preferably, the carrier, before the addition of the ROS-based aqueous solution, has a viscosity between 300,000-600,000 mPa-s, preferably between 400,000-480,000 mPa-s.

[0050] Preferably, viscosity is measured by rotational viscometer with R6 rotor at 20-25°C.Preferably, the above composition has a rheological modifier that is preemptively mixed with pharmaceutically and cosmetically acceptable additives to obtain a cosmetic or pharmaceutical formulation, Preferably, the rheological modifier of the composition is chosen from xanthan (xanthan gum), carbomer, and combinations thereof.

[0051] Preferably, the amount of solution based on oxygen reactive species of the composition according to the invention is from 2% to 98%, preferably in the range 5-50%, preferably between 10-40%, preferably in the range 15-35%, preferably between 20-30%, preferably in the range 23-27%, more preferably about 25%.

[0052] Preferably, the carrier in the composition is present in an amount between 2% and 95%, preferably in the range 50-95%, preferably in the range 60-90%, preferably between 65-85%, preferably between 70-80%, preferably in the range 73-77%, more preferably about 75%.

[0053] Preferably, the weight ratio between the amount of ROS-based aqueous solution and the carrier is between 0.5 and 2.

[0054] Preferably, the composition further comprises at least one additive chosen from:

[0055] • pH regulators chosen from: citric acid, malic acid, acetic acid, lactic acid, phosphoric acid, triethanolamine;

[0056] • preservatives chosen from: potassium sorbate, monosodium glutamate, ozonated glycerine, calcium propionate, imidazolidinyl urea;

[0057] • humectants chosen from: propylene glycol, sorbitol, glycerine; • stabilizers; such as tetrasodium glutamate diacetate (GLDA);

[0058] • antioxidants chosen from: tocopherol, ascorbic acid, disodium calcium ethylenediamine-tetraacetate;

[0059] wherein, said additive is added in an amount between 0.001-10%, preferably between 0.01-5%.Preferably, the composition comprises:

[0060] o the ROS-based aqueous solution in an amount ranging from 5 to 75%;

[0061] o carbomer, preferably in an amount between 2% and 20%; o potassium sorbate, preferably in an amount between 0.01% and 5%

[0062] o ozonated glycerine, preferably in an amount between 0.01% and 5%;

[0063] o imidazolidinyl urea, preferably in an amount between 0.01% and 5%;

[0064] o propylene glycol, preferably in an amount between 0.01% and 5%;

[0065] o disodium calcium ethylenediaminetetracetate, preferably in an amount between 0.01% and 5%;

[0066] o water up to 100%.

[0067] The method of preparing the composition according to the invention comprises the steps of:

[0068] • preparing the carrier by mixing water and the rheological agent, and optionally additives and / or stabilizers, to form the carrier; • at a temperature less than or equal to 30°C, adding the aqueous solution based on ROS deriving from the splitting of ozone to the carrier;

[0069] • mixing until homogenized, keeping the temperature below or equal to 30°C;

[0070] • using the composition thus obtained or transferring it to dispensers for packaging.

[0071] More specifically, the method of preparing the composition according to the invention comprises the steps of:

[0072] • mixing for a time between 5-45 minutes and at a temperaturebetween 0°C and 90°C, the rheological agent in the form of powder or viscous solution, water and, optionally, the additives and stabilizers necessary for the final formulation, to form the carrier;

[0073] • bringing the mixture of said thus obtained carrier to room temperature;

[0074] • at a temperature less than or equal to 30°C, adding the aqueous solution based on reactive oxygen species to the carrier, preferably according to a weight ratio between the ROS-based solution and the carrier between 0.5-2;

[0075] • mixing until homogenized, keeping the temperature below or equal to 30°C;

[0076] • using the resulting composition or transferring it to the dispensers for packaging.

[0077] Preferably, the mixing is practiced at a temperature of 0-59°C or at a temperature of 60-90°C.

[0078] Preferably, the composition of the invention comprises one or more cosmetically and pharmaceutically acceptable components.

[0079] Preferably, the composition of the invention can be formulated in the form of creams, lotions, ointments, capsules, ovules with the gel inside, sprays, aerosols, liposomal formulations.

[0080] Preferably, the composition of the invention can be administered orally or topically.

[0081] Preferably, the composition of the invention is a cosmetic composition. Preferably, the cosmetic composition of the invention can be used for applications such as: anti-acne; after-shave or post-hair removal; peeling; laser treatments; anti-aging, moisturizing, anti-redness, antipollution, protective, repairing, depigmenting face creams; repairing, protective, depigmenting hand creams; toning, regenerating body creams; trichological compositions, for the treatment of hair loss andthickening, to contrast alopecia; cleansers for face, body, hands, intimates; complete skin care, periocular microcirculation; cellulite; intimate lubricant; gel for foot mycosis; nail strengthening gel; slimming body cream; intimate cleanser; gel for nail regrowth; skin depigmentation.

[0082] Preferably, the cosmetic composition according to the invention can be used for applications in skin aging treatments; in combination with photodynamic therapy to eliminate damaged cells or improve cell renewal; to treat conditions such as resistant acne, actinic keratosis and rosacea; to stimulate the skin's natural antioxidant response, improving the repair of skin damage; to counteract the hair loss; to counteract or reduce the appearance of wrinkles and fine lines; to increase the skin's resistance to environmental insults; to reduce dark spots and hyperpigmentation caused by photoaging; to improve the ability to neutralize harmful free radicals generated by exposure to UV rays or pollution; to improve the texture of the skin and reduce the visible signs of aging; as an adjuvant to electromedical therapies, for instance treatments such as Shock Waves - Laser, Radio Frequencies, Pulsed Light.

[0083] Preferably, the composition of the invention is for use in the pharmaceutical field.

[0084] Preferably, the pharmaceutical composition of the invention can be used in all diseases and pathologies that benefit from the administration of effective quantities of ROS, such as: healing activity; anti-fungal activity; anti-inflammatory activity; anti-bacterial activity against gram-positive, gram-negative and anaerobic bacteria; antiseptic activity; anti-viral activity, in particular DNA or RNA viruses and lipid-enveloped viruses such as HIV, SARS-CoV-2, flu viruses, respiratory syncytial virus, Herpes simplex (HSV-1, HSV-2); anti-tumor activity; immune system modulation activities; immune stimulation: neuroprotection, tissue regeneration, wound healing.

[0085] Preferably, the pharmaceutical composition of the invention can be used in the treatment of inflammations, in particular skin inflammations such as sunburns, light, medium or severe burns, inflammation of themucous membranes and deep skin layers such as gastritis, haemorrhoids, rhinitis, conjunctivitis, muscle and musculo-skeletal inflammations such as muscle disorders, tendinitis and bursitis, arthritis, comprising rheumatoid arthritis, chronic and degenerative inflammation, such as fibromyalgia; neurodegenerative diseases such as multiple sclerosis; healing of skin wounds such as cuts and lacerations, surgical wounds to accelerate wound closure and reduce the formation of hypertrophic scars or keloids, post-surgical dental wounds, pressure sores; Inflammation and lesions of the mucous membranes, such as oral lesions, such as gingivitis, stomatitis or periodontitis, mouth ulcers, canker sores, vaginal or anal wounds: Gastric ulcers: Intestinal lesions: such as Crohn's disease or ulcerative colitis; fungal diseases such as candidiasis, in particular mucosal, cutaneous, systemic candidiasis; acne, bacterial acne, psoriasis, hair loss; atopic and contact dermatitis; Onychomycosis; Fungal infections affecting the nails, infections caused by dermatophytes such as Microsporum canis and Trichophyton mentagrophytes, Candida or non-dermatophytic molds; Dermatophytosis and superficial skin infections caused by dermatophytes such as Trichophyton, Microsporum and Epidermophyton; infections caused by Malassezia spp., deep and systemic mycosis such as pulmonary or systemic infections caused by Aspergillus spp.; urinary tract infections, systemic infections caused by Histoplasma capsulatum; infections caused by Cryptococcus neoformans; vaginal infections caused by Candida albicans, Candida glabrata, Candida parapsilosis, Candida kefir, Gardnerella vaginalis; Mycosis of the skin and scalp such as Tinea capitis and Tinea corporis in adult humans and children, fungal infections affecting skin folds, interdigital spaces or the nail and periungual areas; Treatment of Chronic Ulcerations: such as diabetic or venous ulcers, Skin abscesses, Bacterial rhinitis, Bacterial bronchitis, Sinusitis: Gastrointestinal infections, peptic ulcer disease caused by Helicobacter pylori, Bacterial cystitis: Bacterial prostatitis: antibiotic resistance, Methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa diseases, Viral infections such as HIV, SARS-CoV-2, Flu virus infections, Respiratory syncytial virus, Herpes simplex (HSV-1, HSV-2); HPV skin or mucosal viral infections such as human papillomavirus, Viral hepatitissuch as Hepatitis B (HBV) and Hepatitis C (HCV), Viral infections of the central nervous system such as neurotropic viruses (e.g. Herpes zoster, rabies virus), Systemic viral infections such as HIV, Infectious mononucleosis (Epstein-Barr virus, EBV), Gastro-intestinal infections by Rotavirus and Norovirus; Solid tumors such as breast cancer: Lung cancer: Liver carcinoma: Glioblastoma, Hematological cancers such as Leukemia: Acute myeloid leukemia (AML). Lymphoma: Gastrointestinal cancers such as gastric and colorectal cancer, Melanoma and skin cancers, Gynaecological cancers such as ovarian and cervical cancer; vascular diseases such as ischemia and hypertension: Alzheimer's or Parkinson's, anti-aging therapies such as oxidative hormones.

[0086] Preferably, the pharmaceutical composition of the invention can be used as an adjuvant in therapies for bacterial and fungal diseases such as candidiasis and irritation and inflammation of the mucous membranes and dermis.

[0087] Preferably, the pharmaceutical composition of the invention can be used in combination with antibiotic drugs, antivirals pro-oxidant drugs: drugs that block glutathione (GSH) or thioredoxin, radiotherapy, photodynamic therapies.

[0088] The invention is also related to a kit comprising the composition described here in a single pack or predefined dosing units, optionally a reusable applicator or a disposable applicator containing effective doses of the composition, instructions for use optionally accessible remotely through an electronic means.

[0089] Still further objects of the invention will be evident from the detailed description that follows.

[0090] Brief description of the Figures

[0091] The invention will be described below in at least one preferred embodiment for the sole purpose of explanatory and not limiting the scope of the present invention with the help of the figures in the annex, wherein:

[0092] Figure 1: Evaluation of cell viability, measured by the Crystal Violetmethod, complete culture medium for 24 hours;

[0093] Figure 2: Assessment of cell viability, measured by the MTT method, complete culture medium for 24 hours;

[0094] Figure 3: Assessment of cell viability, measured by the MTT method, complete culture medium for 48 hours;

[0095] Figure 4: The toxic effect of the tested compound on NCI-H292 cells;

[0096] Figure 5: Cell viability of the compound on human aerial epithelial cells NCI-H292;

[0097] Figure 6: Cell viability of the compound on human aerial epithelial cells NCI-H292;

[0098] Figure 7: Cell viability of the compound on human aerial epithelial cells NCI-H292;

[0099] Figure 8: Cell viability of the compound on human aerial epithelial cells NCI-H292.

[0100] Definitions

[0101] • In this document, the term "carrier" as used herein refers to a substance used as a base for embedding / incorporating / encapsulating / retaining an aqueous solution containing negative ions within a gelled or viscous matrix. The characteristics of the carrier are described below.

[0102] • In this document, the terms "approximately" or "about" as used herein when referring to a measurable value such as a quantity, a time duration, and the like, are intended to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, wherein such variations are appropriate to perform the methods described.

[0103] • In this document, the terms, "comprises," "includes," "has," "having," "contains," "containing," "characterized by," or any other variation of these terms, are intended to cover a non-exclusive inclusion, subject to any limitations expressly stated. For instance, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to these elements but may comprise other elements not expressly listed or inherent in that composition, mixture, process, or method.

[0104] • In this document, the percentages (%) referring to the quantities of the individual components constituting the composition of the invention are to be understood as % by weight, unless otherwise specified.

[0105] • In this document, the term "carbomer" indicates a family of synthetic homopolymers more or less cross-linked with high molecular weight and consisting of units of acrylic acid. When dispersed in water, carbomer is in the form of a viscous solution that can be further structured (gelled) by adjusting the pH, usually with bases such as sodium hydroxide or triethanolamine. At room temperature, it maintains a stable viscosity and does not spontaneously gel.

[0106] • Xanthan gum, or xanthan, is a natural polysaccharide produced by bacteria of the genus Xanthomonas. It is soluble in cold or hot water, creating a viscous solution, but it does not form a true rigid gel.

[0107] Detailed Description of the Invention

[0108] The invention refers to a composition in viscous or gel form, capable of encapsulating within it reactive oxygen species (ROS), in particular the superoxide ion, to be used in the cosmetic and medical fields.

[0109] The invention is also related to compositions and pharmaceutical forms that comprise this viscous or gel form, in particular pharmaceutical forms that can be administered orally or topically.

[0110] According to an embodiment, the composition according to the invention comprises a mixture of natural elements / substances having a high concentration of negative ions, said mixture being free of oxidizing species, which can be used in the cosmetic and pharmaceutical fields.More specifically, the gel can be used for the systemic or topical treatment on dermis and mucous membranes of imperfections, pathologies and diseases that benefit from the administration of pharmaceutically and cosmetically acceptable aliquots of compositions comprising ROS.

[0111] The ROS(s) formulated according to this invention make it possible to treat, prevent, control, alleviate symptoms and discomforts of the following diseases / pathologies both at a pharmaceutical and cosmetic level when administered in effective quantities to living animal beings, in particular mammals, more particularly humans, the elderly, newborns and infants comprised.

[0112] Some classes of conditions that may benefit from targeted ROS administration comprise:

[0113] Vascular diseases

[0114] • Ischemia-reperfusion: Small amounts of ROS can act as signals to activate protective mechanisms (ischemic preconditioning).

[0115] • Hypertension: Modulation of ROS can improve vascular tone. Oncology

[0116] • Tumor therapy: ROS can increase the efficacy of some cancer treatments (e.g., radiation therapy and chemotherapy), by inducing selective oxidative stress in cancer cells.

[0117] Immunology

[0118] • Immune stimulation: ROS are involved in immune defence, facilitating the response of macrophages and other immune system cells against infections.

[0119] Neurology

[0120] • Neuroprotection: In small doses, ROS can act as neurotrophic signals, stimulating neuronal growth and repair in settings such as Alzheimer's or Parkinson's disease (if properly balanced).Tissue regeneration

[0121] • Wound healing: ROS regulate the inflammatory response and promote angiogenesis, promoting healing.

[0122] Anti-aging therapies

[0123] • Oxidative hormesis: Small amounts of ROS can activate protective and adaptive cellular pathways, improving resilience to oxidative stress.

[0124] Compositions according to the invention can be administered successfully as they show the following activities.

[0125] Anti-inflammatory activity: with particular reference to skin inflammation such as, but not limited to:

[0126] 1. Skin inflammation

[0127] • Sunburn and burns and light, medium, or severe burns: ROS can modulate the skin's inflammatory response, reducing cell damage and promoting tissue regeneration.

[0128] • Psoriasis: ROS can be used to reduce the abnormal activation of the immune system that perpetuates psoriasis.

[0129] • Atopic and contact dermatitis: Regulation of oxidative stress can help reducing inflammatory responses.

[0130] 2. Inflammation of the mucous membranes and deep skin layers

[0131] • Gastritis: ROS, if properly modulated, can help reduce gastric inflammation and promote the restoration of the mucosal barrier.

[0132] • Haemorrhoids: The anti-inflammatory effect of ROS reduces swelling, pain, and promotes faster healing.

[0133] • Rhinitis and conjunctivitis: Controlled oxidative stress can reduce inflammatory overactivity of the mucous membranes, relieving symptoms of congestion and irritation.3. Muscle and musculoskeletal inflammation

[0134] • Muscle pain: ROS can be used to improve post-workout or posttrauma recovery by reducing inflammation and promoting the release of regenerative signals.

[0135] • Tendonitis and bursitis: The ability to decrease inflammation can shorten recovery time and relieve pain.

[0136] • Arthritis (comprising rheumatoid arthritis): ROS, in combination with antioxidant agents, can be used to reduce inflammatory processes.

[0137] 4. Chronic and degenerative inflammation

[0138] • Fibromyalgia: Although not directly an inflammation, ROS can act by improving mitochondrial metabolism and reducing associated symptoms.

[0139] • Neurodegenerative diseases (e.g. multiple sclerosis): In these diseases, ROS can help reduce local inflammation and associated oxidative damage.

[0140] Anti-fungal activity: with particular reference to diseases due to Candida and those affecting the skin, such as acne, psoriasis, hair loss.

[0141] Antifungal disorders

[0142] 1. Candidiasis

[0143] • Mucosal candidiasis: Candida albicans infections that affect the oral, vaginal, or oesophageal mucous membranes.

[0144] • Skin candidiasis: Superficial infections of the skin, often in moist areas such as skin folds.

[0145] • Systemic candidiasis: A serious infection involving the blood or internal organs, typical of immunocompromised patients.

[0146] 2. Onychomycosis• Fungal infections affecting the nails (caused by dermatophytes, Candida, or non-dermatophytic molds). ROS can destroy fungal cell walls by promoting the regeneration of the nail plate.

[0147] ermatophytosis (ringworm)

[0148] • Superficial skin infections caused by dermatophytes such as Trichophyton, Microsporum, and Epidermophyton. Modulation of ROS can reduce associated inflammation and damage microorganisms.

[0149] ityriasis versicolor

[0150] • An infection caused by Malassezia spp., characterized by hypopigmented or hyperpigmented skin spots. The oxidative action of ROS can counteract the growth of the fungus and restore skin balance.

[0151] eep and systemic mycosis

[0152] • Aspergillosis: Pulmonary or systemic infection caused by Aspergillus spp. ROS can damage the cell walls of the fungus in combination with standard treatments.

[0153] • Histoplasmosis: Systemic infection caused by Histoplasma capsulatum. The use of ROS in combination with conventional therapies.

[0154] • Cryptococcosis: Caused by Cryptococcus neoformans, typical of immunocompromised patients; ROS may play a role in reducing fungal burden.

[0155] aginal mycosis

[0156] • Vaginal infections with Candida albicans or other species {Candida glabrata, Candida parapsilosis) can be treated with ROS to reduce fungal load, inflammation and restore the balance of the vaginal flora.

[0157] ycosis of the skin and scalp• Tinea capitis'. Infection of the scalp, common in children, caused by dermatophytes.

[0158] • Tinea corporis: Infections in the torso or other areas not covered by hair.

[0159] 8. Paronychia and intertrigo

[0160] • Fungal infections affecting skin folds, interdigital spaces, or the periungual area. The antioxidant and antimicrobial action of ROS can reduce symptoms and promote healing.

[0161] Advantages of using ROS in fungal infections

[0162] 1. Selective damage to fungi: ROS attack the cell walls and lipid membranes of fungi, interfering with life processes without severely damaging healthy tissues.

[0163] 2. Reduction of inflammation: In addition to the antifungal action, ROS modulate the local inflammatory response, accelerating recovery.

[0164] 3. Improved efficacy of conventional treatments: ROS can enhance the action of systemic ortopical antifungals, reducingthe need for high dosages.

[0165] Healing activity: with particular reference to burns, sunburns.

[0166] Role of ROS in healing

[0167] 1. Activation of cell signaling pathways:

[0168] • ROS act as second messengers, activating molecular pathways involved in cell proliferation, angiogenesis, and collagen production.

[0169] • They stimulate fibroblasts and keratinocytes, promoting the formation of granulation tissue.

[0170] 2. Modulation of inflammation:• ROS, in controlled amounts, help recruiting immune cells such as neutrophils and macrophages to the wound area, which remove debris and microorganisms.

[0171] 3. Stimulation of angiogenesis:

[0172] • ROS promote the formation of new blood vessels, improving the supply of oxygen and nutrients needed for tissue regeneration.

[0173] 4. Promotion of tissue remodelling:

[0174] • They promote the deposition and maturation of collagen fibers, improving the strength and elasticity of the regenerated tissue.

[0175] Pathologies and scar healing applications of ROS

[0176] 1. Skin wounds

[0177] • Burns (mild, medium, and severe): Modulated ROS help reducing inflammation and accelerating the formation of new tissue.

[0178] • Chronic ulcerations: Such as diabetic or venous ulcers, where ROS can stimulate healing in difficult-to-treat wounds.

[0179] • Cuts and lacerations: ROS improve the healing process and reduce the risk of secondary infections.

[0180] 2. Surgical wounds

[0181] • After surgery, ROS can be used to accelerate wound closure and reduce the formation of hypertrophic scars or keloids.

[0182] 3. Pressure sores

[0183] • Pressure injuries, common in bedridden patients, can benefit from ROS to stimulate tissue regeneration and prevent infections.

[0184] 4. Inflammation and lesions of the mucous membranes

[0185] • Oral injuries: Mouth ulcers, canker sores, or post-surgical wounds in dentistry can benefit from ROS to reduce pain and speedhealing.

[0186] • Vaginal or anal wounds: ROS can be useful for regenerating mucosal tissues, such as after trauma, childbirth, or surgery.

[0187] 5. Internal scarring

[0188] • Gastric ulcers: Controlled ROS can promote the regeneration of damaged gastric mucosa.

[0189] • Intestinal lesions: In conditions such as Crohn's disease or ulcerative colitis, ROS can help improve tissue repair.

[0190] Benefits of using ROS for healing

[0191] 1. Shortening healing time: ROS stimulate the proliferative and reparative step, reducing the time required for wound closure. 2. Reduced risk of infection: Thanks to their intrinsic antimicrobial action, ROS help prevent infectious complications during the healing process.

[0192] 3. Improved scar quality: By promoting the formation of type I collagen, ROS contribute to the creation of a more uniform and resilient scar.

[0193] Anti-bacterial activity: with particular reference to diseases affecting the skin, such as acne, psoriasis, hair loss.

[0194] Antibacterial mechanism of action of ROS

[0195] 1. Damage to cell membranes:

[0196] • ROS oxidize the lipids of the bacterial membrane, causing the loss of cell integrity and leading to the death of the bacterium.

[0197] 2. Alteration of proteins:

[0198] • ROS denatures essential bacterial proteins, interfering with their enzymatic and structural functions.

[0199] 3. DNA damage:• The oxidation of DNA bases impairs the bacterium's ability to replicate and survive.

[0200] 4. Oxidative stress:

[0201] • Bacteria, especially pathogenic ones, are often unable to counteract a high production of ROS, making them particularly vulnerable.

[0202] Pathologies and antibacterial applications of ROS

[0203] 1. Skin infections

[0204] • Infected wounds: ROS can be used to treat wounds contaminated with bacteria, improving healing and preventing complications.

[0205] • Skin abscesses: Controlled oxidative action can help reduce bacterial load and inflammation.

[0206] • Bacterial acne: ROS can fight Propionibacterium acnes, reducing inflammation and improving the appearance of the skin.

[0207] 2. Infections of the mucous membranes

[0208] • Oral infections: Such as gingivitis, stomatitis or periodontitis, often caused by anaerobic bacteria, can benefit from the antibacterial action of ROS.

[0209] • Vaginal infections: ROS can reduce the growth of pathogenic bacteria (such as Gardnerella vaginalis), helping to restore balance to the vaginal flora.

[0210] • Bacterial rhinitis: In infections of the nasal mucous membranes, ROS can reduce the proliferation of bacteria responsible for the symptoms.

[0211] 3. Respiratory infections

[0212] • Bacterial bronchitis: ROS can help reduce the bacterial load in aerial infections.• Sinusitis: Local use of ROS can help fighting bacteria in the nasal cavities and sinuses.

[0213] 4. Gastrointestinal infections

[0214] • Gastritis and peptic ulcer disease: Modulated ROS can be used to reduce the proliferation of bacteria such as Helicobacter pylori, which is often responsible for these conditions.

[0215] • Intestinal infections: ROS could reduce the growth of pathogenic bacteria without compromising the intestinal flora too much.

[0216] 5. Urogenital infections

[0217] • Bacterial cystitis: The antibacterial action of ROS can help reduce the load of bacteria such as Escherichia coli, which often cause urinary tract infections.

[0218] • Bacterial prostatitis: Here, too, the use of ROS can support traditional treatments, reducing pathogenic bacteria.

[0219] 6. Systemic infections

[0220] • Sepsis: Although requiring extreme modulation, ROS could be explored to combat systemic bacterial infections in combination with antibiotics.

[0221] 7. Antibiotic resistance

[0222] • ROS can be used to target antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa. The oxidative action is not affected by conventional resistance mechanisms.

[0223] Advantages of the antibacterial action of ROS

[0224] 1. Wide spectrum of action:

[0225] • ROS are effective against gram-positive, gram-negative, and anaerobic bacteria.2. Reduction of risk of resistance:

[0226] • As ROS causes multiple, non-specific damage to bacterial cells, it is difficult for bacteria to develop resistance.

[0227] 3. Synergistic effect with antibiotics:

[0228] • ROS can enhance the effect of traditional antibiotics, reducing the need for high dosages.

[0229] 4. Targeted local action:

[0230] • Topical or local applications of ROS can directly affect the infected area without adversely affecting healthy tissues.

[0231] Anti-viral activity

[0232] Antiviral mechanisms of action of ROS

[0233] 1. Direct Virus Damage:

[0234] • ROS can damage viral genetic material (DNA or RNA) and proteins in the lipid capsid or envelope, inhibiting viral replication.

[0235] • In lipid-enveloped viruses (e.g., HIV, SARS-CoV-2), ROS can oxidize membrane lipids, destroying the integrity of the virus.

[0236] 2. Modulation of the immune system:

[0237] • ROS activate cells of the innate immune system, such as macrophages and neutrophils, enhancing these cells1ability to engulf and destroy viruses.

[0238] • They stimulate the production of antiviral cytokines (e.g.

[0239] interferons), enhancing the immune response against infection.

[0240] 3. Synergistic effect:

[0241] • ROS can be used in combination with conventional antiviral drugs, improving their efficacy.

[0242] Pathologies and antiviral applications of ROS1. Viral respiratory infections

[0243] • COVID-19 (SARS-CoV-2):

[0244] • ROS can contribute to damaging the lipid envelope of the virus, reducing its infectivity.

[0245] • In addition, modulating oxidative stress can reduce inflammation and associated lung damage.

[0246] • Flu:

[0247] • ROS can act against the flu virus, either directly or by improving viral clearance through the immune system.

[0248] • Respiratory syncytial virus (RSV):

[0249] • The antiviral action of ROS can help limit infection in the lower respiratory tract.

[0250] 2. Skin viral infections

[0251] • Herpes simplex (HSV-1, HSV-2):

[0252] • ROS can reduce virus replication in oral or genital lesions, speeding up healing and reducing viral load.

[0253] • Human papillomavirus (HPV):

[0254] • In HPV skin or mucosal infections, ROS can reduce the proliferation of infected cells and improve the local immune response.

[0255] 3. Viral hepatitis

[0256] • Hepatitis B (HBV) and Hepatitis C (HCV):

[0257] • ROS can help limit viral replication and improve hepatic immune function, when used in combination with standard antiviral therapies.

[0258] 4. Viral infections of the central nervous system• Neurotropic viruses (e.g. Herpes zoster, rabies virus):

[0259] • ROS can act as adjuvants, improving viral clearance in nerve tissues.

[0260] 5. Systemic viral infections

[0261] • HIV:

[0262] • Although the use of ROS requires caution to avoid immune damage, they may play a role in destroying viral particles and improving the effectiveness of antiretroviral therapies.

[0263] • Infectious mononucleosis (Epstein-Barr virus, EBV):

[0264] • ROS can help reduce viral replication and alleviate systemic symptoms.

[0265] 6. Gastro-intestinal infections

[0266] • Rotavirus and Norovirus:

[0267] • ROS can inhibit the replication of gastro-intestinal viruses and reduce the duration of symptoms.

[0268] Benefits of using ROS as antivirals

[0269] 1. Wide spectrum of action:

[0270] • ROS are effective against DNA and RNA viruses, regardless of their structure.

[0271] 2. Reduced risk of resistance:

[0272] • As ROS act via non-specific oxidative damage, viruses are unlikely to develop resistance mechanisms.

[0273] 3. Local effect:

[0274] • Local application of ROS (e.g., sprays, gels, solutions) can limit viral infection without significant systemic effects.

[0275] 4. Modulation of the immune response:• ROS not only destroys viruses but also help the immune system respond better to infections.

[0276] Anti-tumor activity

[0277] Anti-tumor mechanisms of action of ROS

[0278] 1. Induction of selective oxidative stress:

[0279] • Cancer cells have a higher metabolism than normal cells, which makes them already exposed to high levels of endogenous ROS.

[0280] • Increasing ROS beyond a critical threshold (via therapeutic interventions) can exceed the antioxidant capacities of cancer cells, leading to programmed cell death (apoptosis) or necrosis.

[0281] 2. Direct damage to DNA and proteins:

[0282] • ROS causes double-stranded DNA breaks and oxidation of nucleotide bases, blocking the replication and proliferation of cancer cells.

[0283] • Protein oxidation interferes with essential cellular processes. 3. Inhibition of shelter systems:

[0284] • ROS can impair the repair mechanisms of cancer cells' DNA and cell membranes, making them more vulnerable.

[0285] 4. Synergistic effect with traditional treatments:

[0286] • ROS enhance the efficacy of radiotherapy and chemotherapy, amplifying induced cell damage.

[0287] 5. Modulation of anti-tumor immunity:

[0288] • ROS can activate anti-tumor immune responses, improving the recognition of cancer cells by the immune system.

[0289] Types of tumors and applications of ROS

[0290] 1. Solid tumors• Breast cancer: Increased ROS can induce selective apoptosis in cancer cells without affecting healthy tissues.

[0291] • Lung cancer: ROS-based therapy is promising, particularly for nonsmall cell lung cancer (NSCLC).

[0292] • Liver cancer: ROS can damage liver cancer cells, reducing tumor progression.

[0293] • Glioblastoma: Brain tumor cells, which are particularly resistant, are vulnerable to therapies based on increased oxidative stress.

[0294] 2. Haematological cancers

[0295] • Leukemia: ROS can reduce the proliferation of leukemia cells, particularly in acute myeloid leukemia (AML).

[0296] • Lymphoma: The cancer cells in lymphomas are particularly susceptible to ROS-induced oxidative stress.

[0297] 3. Gastrointestinal cancers

[0298] • Gastric and colorectal cancer: The use of ROS can impair the ability of cancer cells to proliferate and survive.

[0299] 4. Melanoma and skin cancers

[0300] • ROS-based therapies can be applied topically to selectively destroy cancer cells without harming healthy skin.

[0301] 5. Gynaecological cancers

[0302] • Ovarian and cervical cancer: Controlled increase in ROS can improve the effectiveness of traditional treatments and reduce tumor proliferation.

[0303] Advantages of the use of ROS in anti-cancer treatments

[0304] 1. Selectivity for cancer cells:

[0305] • Cancer cells, already oxidatively stressed, are more vulnerable to increased ROS than normal cells.2. Reduction of resistance to treatments:

[0306] • ROS can reduce the resistance of tumors to chemotherapy drugs and radiotherapy.

[0307] 3. Synergistic effect:

[0308] • ROS enhance the effectiveness of traditional treatments without the need to increase doses.

[0309] 4. Activation of the immune system:

[0310] • They promote the recognition of cancer cells by the immune system.

[0311] ROS-based therapeutic approaches

[0312] 1. Pro-oxidant drugs:

[0313] • Molecules that selectively increase ROS levels in cancer cells (e.g., high-dose ascorbate, arsenic trioxide).

[0314] 2. Inhibition of tumor antioxidant systems:

[0315] • Drugs that block glutathione (GSH) or thioredoxin may increase the sensitivity of cancer cells to ROS.

[0316] 3. Radiotherapy and ROS:

[0317] • Radiation therapy increases ROS production, and combination therapies can amplify the cytotoxic effect.

[0318] 4. Photodynamics and ROS:

[0319] • Photodynamic therapies that exploit ROS to locally destroy solid tumors.

[0320] Cosmetic Activity: with particular reference to discomfort due to lack of lubrication in the intimate areas, derived from pathologies such as infections and inflammation or aging of the mucous membranes.

[0321] Volunteers suffering from alopecia and / or baldness of undefined originwho have used the composition of the invention report both an increase in hair density and a reduction in hair loss.

[0322] On a cosmetic level, the use of Reactive Oxygen Species (ROS) has gained interest for the potential benefits in improving the skin. In small amounts, ROS can stimulate cellular mechanisms useful for regeneration and improvement of skin quality.

[0323] Here are some cosmetic applications:

[0324] 1. Skin rejuvenation

[0325] • Collagen stimulation: ROS, when properly balanced, can activate fibroblasts and improve collagen synthesis, reducing the appearance of wrinkles and fine lines.

[0326] • Skin hormesis: Small oxidative stresses can activate cell repair processes and increase the skin's resistance to environmental insults.

[0327] 2. Acne treatments

[0328] • ROS can have an antimicrobial effect, helping to reduce the proliferation of bacteria such as Propionibacterium acnes, which is often responsible for acne.

[0329] 3. Uniformity of skin tone

[0330] • Treatments that take advantage of ROS (such as photodynamic therapy) can help reduce dark spots and hyperpigmentation caused by photoaging.

[0331] 4. Repair of skin damages

[0332] • Wound healing: ROS can promote angiogenesis (formation of new blood vessels) and improve skin regeneration processes after damage or invasive treatments.

[0333] 5. Paradoxical antioxidant protection• Controlled induction of ROS can activate the skin's endogenous antioxidant defence systems, enhancing the ability to neutralize harmful free radicals generated by exposure to UV rays or pollution.

[0334] 6. Cosmetic Photodynamic Therapies

[0335] • The combined application of photoactivatable substances and light can generate ROS that contribute to the improvement of skin textures and the reduction of visible signs of aging.

[0336] 7. Improvement of skin glow

[0337] • Controlled treatments with ROS can increase blood circulation and stimulate faster cell turnover, giving the skin a fresher and brighter appearance.

[0338] . Improvements in electromedical therapies

[0339] • All treatments such as Shock waves - Laser, Radio frequencies, Pulsed light

[0340] The present invention is intended to control, improve, prevent or treat diseases and therefore concerns a composition for the treatment of diseases that benefit from the administration of effective quantities of ROS.

[0341] The present invention also aims to mitigate or relieve imperfections and, in any case, can be applied to the skin for cosmetic purposes.

[0342] According to an embodiment, the composition according to the invention is stable and is suitable for topical or oral administration. This composition comprises an aqueous solution containing high charges of negative ions dispersed / solubilized within a viscous matrix (or viscous carrier). More precisely, the aqueous solution that is incorporated / encapsulated / retained within the viscous carrier is a solution based on reactive oxygen species (ROS) free of oxidizing species.

[0343] This solution is obtained by mechanical splitting of ozone, not byelectrolysis of chlorides, it is therefore free of chlorinated species generated electrolytically as ROS are obtained in the absence of anodic oxidation of chlorides.

[0344] According to an embodiment, the composition according to the invention comprises a mixture of natural elements / substances having a high concentration of negative ions that is able to express cosmetic and pharmaceutical activities as indicated above, such as antibacterial, antiinflammatory and antifungal activities.

[0345] More specifically, the composition according to the invention is in the form of a stable viscous gel and is suitable for oral ortopical applications on mucous membranes and skin layers affected by diseases that can be treated or whose symptoms can be controlled, improved, prevented, cured or alleviated by the administration or application of effective doses of ROS.

[0346] The composition according to the invention can be used as such or it can be formulated in the form of creams, lotions, ointments, capsules, ovules with the gel inside, sprays, aerosols, liposomal formulations. This composition comprises a solution containing high charges of negative ions that is incorporated / encapsulated / retained within a viscous matrix.

[0347] The same inventors of the present invention have already extensively described in the patent application WO2023057989 a device and a method for obtaining an aqueous solution containing reactive oxygen species (ROS) such as the superoxide anion O2. The reactive oxygen species (ROS) described in the patent application W02023057989 are by their nature free from oxidizing species resulting from electrolytic treatments.

[0348] The present document, therefore, aims to exploit, for instance, the technology and process reported in the aforementioned patent application to create a composition comprising a high concentration of negative ions encapsulated within it to be used in the cosmetic and pharmaceutical fields.The composition according to the invention comprises:

[0349] • a carrier; and

[0350] • an aqueous solution based on reactive oxygen species (ROS).

[0351] The solution based on reactive oxygen species (ROS), preferably obtained by the equipment and method described in WO2023057989, consists of an aqueous solution containing reactive oxygen species resulting from the cleavage of ozone such as superoxide anions (O2 ), singlet oxygen C ), hydroxyl radicals (OH*), oxidizing oxygen radical (0‘), preferably superoxide anions (O2 ) and oxygen oxidizing radical (0‘) and free from electrolytica lly generated oxidizing and / or chlorinated species.

[0352] The concentration of negative ions, preferably superoxide O ions, in the composition according to the invention is between 100-1200 ppm, preferably 200-1000 ppm, preferably 300-8700 ppm, more preferably 400-600 ppm.

[0353] Said carrier is or comprises:

[0354] • water; and

[0355] • at least one rheological modifier.

[0356] Optionally, the carrier according to the invention can further contain additives that are pharmaceutically and cosmetically known in themselves, necessary for the desired cosmetic or pharmaceutical formulation.

[0357] This rheological modifier is selected from substances that are able to keep the solution viscous at a temperature at least less than or equal to 30°C.

[0358] Preferably, this rheological modifier is or comprises xanthan (xanthan gum), carbomer, and combinations thereof.

[0359] In an embodiment, the rheological agent is in the form of a powder, said powder being mixable and able to be suitably mixed with water andpossibly other pharmaceutically and cosmetically acceptable ingredients under the conditions necessary to obtain the viscosity suitable for the uses according to the invention. The expert in the field will be able to identify the solubilization conditions suitable for the realization of the invention.

[0360] In an embodiment, the rheological agent is presented in the viscous form suitable for obtaining the composition of the invention as described below.

[0361] Preferably, the carrier according to the invention has a density between 0.1-1.5 g / cm3.

[0362] Preferably, the carrier according to the invention has a viscosity between 300,000-600,000 mPa-s, preferably between 400,000-480,000 mPa-s.

[0363] Viscosities are measured with a rotational viscometer, Fungilab brand, model Viscolead-ADV-R - R6 impeller, temperatures 20-25°C in accordance with ASTM 115, 789, 1076, 1084, 1286, 1417, 1439, 1638, 1824, 2196, 2336, 2364, 2393, 2556, 2669, 2849, 2983, 2994, 3232, 3236, 3716, - SO: 2555, 1652, - BS: 6075, 5350. In a preferred embodiment, the carrier according to the invention comprises Carbomer and has a viscosity of about 403000 mPa-s.

[0364] In a preferred embodiment, the carrier according to the invention comprises Xanthan gum and has a viscosity of about 462000 mPa-s. The rheological modifiers selected in accordance with the invention are substances that allow the "cold" solution to encapsulate to stabilize negative ions to make them available to cells.

[0365] In fact, in case of exposure to excessive heat during the encapsulation of the ROS-based solution in the carrier, the reactive oxygen species would denature and undergo degradation to O2. The precocious decomposition of ROS would lead to a drastic lowering of the therapeutic efficacy of the composition of the invention. For this reason, according to the method of the present invention, it is necessary to prepare this composition by working in the encapsulationstep at a temperature <30°C, preferably in the range 20-25°C, more preferably about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C. The expert in the field will choose the best temperature to practice the invention. It is therefore necessary to select a rheological agent that keeps the solution viscous at at least one of the indicated temperatures and that is able to encapsulate the ROS-based aqueous solution without being subjected to thermal cycling.

[0366] The rheological agents that can be used in the composition of the present invention are selected from those capable of keeping the carrier viscous at a temperature at least less than or equal to 30°C. A carrier that, for instance, gels below 25°C (thus losing its viscous nature) but that above this temperature maintains a fluid-viscous consistency is absolutely capable of being used for the realization of the composition of the invention.

[0367] These rheological agents are previously mixed with water and with additives typically used in cosmetics and pharmaceuticals in order to form a carrier. This carrier will then be mixed with the negative ionbased solution in order to incorporate / encapsulate / retain it inside. Appropriately, at a temperature at least less than or equal to 30°C, the carrier maintains a fluid consistency that allows sliding between adjacent layers. The ability of the rheological agent according to the invention to maintain the fluid-viscous solution at the working temperature (i.e., the temperature at which the ROS-based aqueous solution is encapsulated in the carrier) favours the subsequent encapsulation of the ROS-based aqueous solution.

[0368] The physical state of the carrier (e.g., viscous matrix, gel) according to the invention does not depend exclusively on the nature of the rheological agent chosen but can also be a function of its concentration in water, the temperature involved, the presence of additives or a combination thereof. The variation of one or more parameters can significantly alter the chemical-physical properties of the mixture.However, it is not always possible to establish exactly what is the physical state wherein a certain composition occurs. In other words, there may be intermediate / hybrid situations wherein classifying the composition as a mere viscous or gel solution can be quite complicated. In addition to Carbomer and xanthan gum that perfectly meet the formulation needs of the composition of the invention, there are several other polymers that form viscous solutions without becoming real gels, and which can therefore be implemented, among these are exemplified in a non-limited way:

[0369] • Hydroxyethyl cellulose (HEC) - Derivative of cellulose, dissolves in water forming viscous solutions. Viscosity depends on concentration and temperature but does not form rigid gels.

[0370] • Hydroxypropylmethylcellulose (HPMC) - Similar to HEC, it is used in cosmetic and pharmaceutical applications. It can form a stable viscous matrix.

[0371] • Modified Guar Gum (Guar hydroxypropyltrimonium chloride) - A chemically modified natural polysaccharide. It dissolves in water creating viscous solutions.

[0372] • Polyacrylamide - Synthetic polymer that can create very viscous solutions, but not rigid gels.

[0373] • Tara gum or locust gum (carob bean gum) - They form viscous solutions at room temperature without achieving complete gelation.

[0374] • PVP (Polyvinylpyrrolidone) - Synthetic polymer that, in aqueous solution, produces a viscous consistency without forming real gels.

[0375] • Carob gum and alginate gum -These substances also form viscous solutions in water.

[0376] • Carboxymethylcellulose (CMC): Excellent for clear gels and suspensions.

[0377] • Acrylates and polyethylene glycols, silicones, hyaluronic acid andhyaluronates, poloxamers, chitosans.

[0378] and mixtures thereof.

[0379] In any case, all rheological agents used in cosmetics and pharmaceuticals are suitable for use according to the present invention provided that they keep the carrier viscous at the processing temperature, identified as the temperature at which the mixing between the solution containing ROS and the carrier takes place.

[0380] This temperature is identified to be < 30°C, above which the solution containing ROS degrades quickly and loses its effectiveness.

[0381] A simple and effective test to measure whether a rheological agent is adequate to make the carrier at an appropriate temperature according to the present invention is the following.

[0382] Tube Reversal Test (Gel Strength Test) - Procedure:

[0383] at a temperature between 15° and 30°C, pour the test carrier comprising the solvent and the dissolved rheological agent into a tube with a diameter of 5-20 mm, usually 10 mm, and leak-proof (such as a test tube or glass cylinder),

[0384] Turn the tube upside down and observe the behaviour of what is poured into it:

[0385] • a "gel" will hold its shape and will not flow immediately,

[0386] • a viscous solution or, in some cases, a "viscous gel" (i.e., a state of matter intermediate between a gel and a viscous solution that nevertheless maintains a fluidity / mobility between adjacent layers capable of sliding over each other and generating a flow regime) will flow slowly inside the tube.

[0387] The additives to be added to make stable cosmetic and pharmaceutical formulations must also meet specific characteristics. In fact, adjuvants, preservatives and other additives known in themselves in the cosmetic and pharmaceutical fields can be used, but they must be chosen from those components that do not oxidize in the presence of an oxidizer atroom temperature and in general at storage temperatures, i.e. they must have an oxidation-reduction potential of 0.3 volts to 3.5 volts. These requirements and characteristics are known to the expert in the field who, also on the basis of what is indicated in this description, will be able to choose the right components for the final compositions. In some embodiments, the composition according to the present invention comprises:

[0388] • the carrier in an amount between 2% and 95%, preferably in the range 50-95%, preferably in the range 60-90%, preferably between 65-85%, preferably between 70-80%, preferably in the range 73-77%, more preferably about 75%;

[0389] • the solution based on reactive oxygen species (ROS) in an amount between 2% and 98%, preferably in the range 5-50%, preferably between 10-40%, preferably in the range 15-35%, preferably between 20-30%, preferably in the range 23-27%, more preferably about 25%.

[0390] In some embodiments, the ratio (by weight) between the amount of ROS-based aqueous solution and the carrier is between 0.5-2.

[0391] Preferably, when the rheological modifier is xanthan, the ratio (by weight) between the amount of ROS-based aqueous solution and the carrier is between approx. 1-2 (2,000 g of aqueous solution + 1,000 g of carrier).

[0392] More preferably, when the rheological modifier is xanthan, the ratio (by weight) between the amount of ROS-based aqueous solution and the carrier is about 2.

[0393] Preferably, when the rheological modifier is carbomer, the ratio (by weight) between the amount of ROS-based aqueous solution and the carrier is between 0.5-1 (1,000 gr of aqueous solution + 1,000 gr of carrier).

[0394] More preferably, when the rheological modifier is carbomer, the ratio (by weight) between the amount of ROS-based aqueous solution andthe carrier is about 1 (i.e. 50:50), obtained by combining 1 liter of ROS-based aqueous solution for each kilogram of carrier.

[0395] The rheological modifier of the composition according to the invention is present in an amount in the range 10-20% (with respect to the composition).

[0396] Optionally, the composition according to the invention may further comprise at least one additive / adjuvant chosen from:

[0397] • pH regulators, such as but not limited to citric acid, malic acid, acetic acid, lactic acid, phosphoric acid, triethanolamine;

[0398] • preservatives, such as but not limited to potassium sorbate, monosodium glutamate, ozonated glycerine, calcium propionate, imidazolidinyl urea;

[0399] • humectants, such as but not limited to\ propylene glycol, sorbitol, glycerine;

[0400] • stabilizers; as a non-limiting example, tetrasodium glutamate diacetate (GLDA);

[0401] • antioxidants, such as but not limited to tocopherol, ascorbic acid, disodium calcium ethylenediaminetetracetate;

[0402] wherein at least one additive optionally comprised in the composition of the invention, during the preparation of said composition, is preliminarily added to the carrier and therefore prior to the addition of the ROS-based aqueous solution.

[0403] In a preferred embodiment, the pH regulating agent is lactic acid.

[0404] Preferably, the at least one additive / adjuvant optionally comprised in the composition according to the present invention is added in an amount in the range 0.001-10%, preferably between 0.01-5%.

[0405] Optionally, the composition may additionally contain one or more of the following components: antibiotic agents, antimicrobial agents, antibacterial agents, antifungal agents, anti-inflammatory agentsknown in themselves.

[0406] In an embodiment, the composition according to the invention comprises:

[0407] • the aqueous solution based on ROS;

[0408] • at least one rheological modifier;

[0409] • at least one preservative;

[0410] • at least one pH regulator;

[0411] • at least one antioxidant;

[0412] • at least one humectant;

[0413] • water in a quantity sufficient to reach 100% of the composition. In a particularly preferred embodiment of the present invention, the composition comprises:

[0414] • the aqueous solution based on ROS in an amount between 5- 75%;

[0415] • carbomer, preferably in an amount between 2-20 %;

[0416] • potassium sorbate, preferably in an amount between 0.01-5%;

[0417] • ozonated glycerine, preferably in an amount between 0.01-5%;

[0418] • imidazolidinyl urea, preferably in an amount between 0.01-5%;

[0419] • propylene glycol, preferably in an amount between 0.01-5%;

[0420] • disodium calcium ethylenediaminetetracetate, preferably in an amount between 0.01-5%;

[0421] • water in a quantity sufficient to reach 100% of the composition. The composition according to the invention can have a pH between 4.5 and 7, preferably between 5 and 7.The composition according to the invention can have a viscosity between 300,000-600,000 mPa-s, preferably between 400,000-480,000 mPa-s. The composition according to the invention comprising the carrier and the ROS-based aqueous solution incorporated within it can be found in the form of gel, viscous gel, viscous solution.

[0422] The composition according to the invention may be formulated and used as such or may be formulated in the form of creams, lotions, ointments, capsules, liposomal formulations, spray formulations or aerosols; these alternative formulations comprise the composition according to the invention and at least one of the excipients, adjuvants, preservatives, emulsifiers, hydrophilic components, active agents, buffers, salts, viscosity modifiers or other agents such as fragrances and other additives chosen from those pharmaceutically and cosmetically acceptable and on the basis of the desired formulation.

[0423] The composition according to the invention can be used for topical applications, preferably on mucous membranes and skin layers affected by candidiasis, for instance, or by oral administration to treat and / or alleviate the symptoms of diseases that benefit from the administration of effective amounts of ROS. Formulations containing the compositions of the invention can be prepared according to standard procedures and suitable for administration and application to the skin and mucous membranes of a mammal, comprising humans, comprising newborn, infants and the elderly.

[0424] Method of realization

[0425] The procedure for preparing compositions according to the invention comprises the following basic operational steps:

[0426] • preparing the carrier by hot mixing, e.g. in a turbo-mixer operating for a time between 5-45 minutes and at a temperature between 60°C-90°C, the rheological agent in the form of powder or viscous solution, water and, optionally, the additives and stabilizers necessary for the final formulation, e.g. triethanolamine, propylene glycol, imidazolidinyl urea, potassium sorbate, EDTA, to form the carrier; alternatively:• mixing cold, e.g. in a turbo-mixer operating for a time between 5- 45 minutes and at a temperature between 0°C and 59°C, the rheological agent in the form of powder or viscous solution, water and, optionally, the additives and stabilizers necessary forthe final formulation, e.g. triethanolamine, propylene glycol, imidazolidinyl urea, potassium sorbate, EDTA, to form the carrier;

[0427] bringing to room temperature said carrier;

[0428] adding the aqueous solution based on oxygen reactive species to the carrier at a temperature of less than or equal to 30°C, preferably according to a weight ratio between the ROS-based solution and the carrier between 0.5-2;

[0429] mixing until homogenized, keeping the temperature below or equal to 30 °C;

[0430] using the resulting composition or transferring it to the dispensers for packaging.

[0431] The composition may also contain one or more of the following components: antibiotic agents, antimicrobial agents, antibacterial agents, antifungal agents, anti-inflammatory agents known in themselves. pH regulating agents can be added in the quantities necessary to obtain the desired pH, generally a physiological pH between 4.5 and 7, preferably between 5 and 7, wherein lactic acid is preferred.

[0432] Advantageously, the composition according to the invention is surprisingly able to stabilize the reactive oxygen species (ROS) contained within it. The gelled or viscous structure, in fact, allows a homogeneous distribution of the ROS-based aqueous solution, favouring the stability of the dispersion and slowing down the spontaneous degradation of the reactive species.

[0433] The composition according to the invention allows negative ions to be delivered through the skin and mucous membranes when applied in topical formulations such as creams, gels or ointments. The composition transports negative ions to the innermost layers of the skin and mucosawhere they must act, facilitating absorption and improving / preserving their stability. The composition therefore conveys the negative ions, assisting their uniform distribution on the mucous membranes and skin and transporting them deep into the tissues; improving their absorption and increasing the permeability of the skin and mucous membranes, thus making it easier for active negative ions to pass through the surface layers; all this thanks to the function of stabilizing the composition against the ions that are kept in a stable form that protects them from degradation.

[0434] The efficacy and activity of the reactive species-based composition can be advantageously modulated by varying the amount of ROS-based aqueous solution to be comprised / dispersed / encapsulated in the carrier.

[0435] Another unexpectedly advantageous aspect of the invention relates to the preferred use of carbomer or xanthan as rheological modifiers. In fact, said rheological modifiers, once dissolved in water to give the viscous solution, are able to encapsulate a host aqueous solution at about ambient temperature, without the need for a thermal heatingcooling cycle. In this way, exposure to high temperatures of thermolabile and reactive species such as ROS can be avoided.

[0436] Application and Uses

[0437] The composition according to the invention can be applied with the aid of an infuser, a dispenser, a nebulizer, a generic device for a controlled release of the product, or directly by hand.

[0438] The composition of the invention does not require any special precautions for conservation.

[0439] A further object of the invention is a kit, said kit comprising the composition of the invention, in a single package or in predefined dosage units, a reusable applicatoror a disposable applicator containing effective doses of the composition, instructions for use optionally accessible remotely through electronic means. According to another aspect of the invention, the kit can also or alternatively contain pre-dosed packages, single or multi-use, containing effective doses of the composition,

[0440] As reported in more detail in the following examples, the composition according to the invention has proved to be advantageously able to express antifungal activity, in particular towards the fungal species of the genus Candida.

[0441] Advantageously, the composition is further capable of expressing antibacterial activity. This composition is able, in fact, to inhibit the formation and promote the breakdown of biofilms involved in the chronicization of wounds on bacteria generally involved in infections.

[0442] Advantageously, the composition according to the invention also possesses anti-inflammatory properties.

[0443] Furthermore, as shown in the examples below, the composition according to the invention does not possess cytotoxic activity.

[0444] As highlighted below in the EXAMPLES section, the effects of the product of the invention on cell viability have been studied by testing a cellular model of human keratinocytes, HaCaT cells (Examples 7 and 8), also following preliminary, unpublished studies carried out on mucoepidermoid cells of NCI-H292 lung cancer that have shown that the product of the invention exerts an effect of cell viability recovery compared to the control carried out in presence of only the colloidal medium wherein it is dispersed (Example 8). Since the above results suggested that the origin of this effect could be due to the activation of cellular energy metabolism at the mitochondrial level, it was decided to evaluate cell proliferation using two different methods that would allow to discriminate cell viability as a function of mitochondrial activity (MTT test) from that measured as a function of the amount of DNA, proportional to the number of cells, present in the different cell samples treated with different concentrations of the invention compound (Crystal Violet test).

[0445] As is evident from the results discussed in Example 7, obtained on human HaCaT cells, chosen as a study model as it is the first permanentepithelial cell line of adult human skin that shows a normal capacity for epidermal differentiation and is now widely characterized, the compound of the invention - at the tested concentrations - is not only non-toxic to keratinocytes, but it is functional to the recovery of their vitality with respect to control; moreover, the compound was tested for 24 and 48 hours, a time well beyond the proper and reasonable conditions of use even of leave-on cosmetic products. In addition, the invention compound is a compound that has high affinities from a chemical point of view compared to products used for therapeutic purposes, so it can be considered safe for human health. From the data obtained and given the considerations expressed so far, the product consisting of the compound of the invention could, therefore, be proposed as a eutrophic skin cosmetic. Having adopted two methods of studying cell proliferation, moreover, the hypothesis that the invention compound acts as a "metabolic activator" or "mitochondrial metabolic activator" has gained even more strength. To verify this aspect in greater depth, other evaluations could be added, such as the citratesynthase enzymatic assay, rapid and cheap, or the evaluation of the energy charge by HPLC, also a rather fast and not very expensive assay, as well as the adoption of more sophisticated mitotracking techniques by flow cytometry and fluorescence microscopy.

[0446] Example 9 shows the results of the in vitro tests that demonstrated the antimicrobial activity of the composition of the invention carried on different bacterial strains and not only (Escherichia coli, Klebsiella pneumoniae, Candida glabrata, Candida albicans, Candida kefir, Candida parapsilosis, Streptococcus dysgalactiae, Enterococcus faecalis, Enterococcus faecium, S. aureus, Pseudomonas aeruginosa), at the tested concentrations. In addition, the anti-inflammatory activity of the composition of the invention delivered on ex vivo human leukocytes of healthy donors has been demonstrated by analysis of cytokine production in response to an inflammatory stimulus produced by lipopolysaccharides (LPS) (as known in art), at the concentrations studied. Other studies are currently underway to confirm the possibility of using the compound of the invention conveyed for the treatment of wounds. Examples 11-15 show studies carried out by the University of Genoa andprivate laboratories on products containing the composition of the invention conveyed by different matrices.

[0447] In particular, in Examples 10 and 11 the antioxidant power of the soothing invention compound on a human endotheliocyte cell line (HECV) is demonstrated and certified: the pretreatment with the soothing compound of the aforementioned cell model, in fact, is able to completely protect against oxidative damage caused by hydrogen peroxide at the tested concentrations.

[0448] In Example 11, the cosmetic Deep Oxygenizing Lightening was tested with the following composition:

[0449] Base Gel 73.70% (Water 90.76%, Carbomer 5.00%, Triethanolamine 1.64%, Glycerine 1.00%, Propylene Glycol 1.00%, Imidazolidinyl urea 0.30%, Potassium Sorbate 0.20%, Disodium EDTA 0.10%) Water + Oxygen 25.00%, (Water 50.00%, Oxygen 50.00%), Jojoba Oil dac 1.00%, Liposomes Hyaluronic Acid pf 0.10%, Kojic Acid 0.10%, Vitamin C (Ascorbic Acid) 0.10%

[0450] which contains the compound of the invention, for an in vitro evaluation of the depigmenting activity on melanocyte cell cultures. In particular, the tested product - at the tested concentrations - demonstrated a significant ability to reduce the production of melanin by B16 cells (mouse melanoma cells).

[0451] In Example 12 it was shown that the cosmetic Deep Oxygenizing Antiaging, having the following composition:

[0452] Base Gel 73.70% (Water 90.76%, Carbomer 5.00%, Triethanolamine 1.64%, Glycerine 1.00%, Propylene Glycol 1.00%, Imidazolidinyl urea 0.30%, Potassium Sorbate 0.20%, Disodium EDTA 0.10%) Water + Oxygen 25.00%, (Water 50.00%, Oxygen 50.00%), Argan Oil 1.00%, Liposomes Hyaluronic Acid pf 0.10%, Vitamin A 0.10%, Vitamin C (Ascorbic Acid) 0.10%, Vitamin E 0.10%

[0453] which contains the compound of the invention, has a protective andrestorative effect with respect to an "outdoor and indoor environmental stress" carried out in vitro on human keratinocytes (Huker cell; insults tested: urban dust, UV-rays anti-pollution test).

[0454] In Example 13 it was shown that the cosmetic Deep Oxygenizing Ultralight gel, having the following composition:

[0455] Base Gel 73.90% (Water 90.76%, Carbomer 5.00%, Triethanolamine 1.64%, Glycerine 1.00%, Propylene Glycol 1.00%, Imidazolidinyl urea 0.30%, Potassium Sorbate 0.20%, Disodium EDTA 0.10%) Water + Oxygen 25.00%, (Water 50.00%, Oxygen 50.00%), Macadamia Oil 1.00%, Hyaluronic Acid Liposomes pf 0.10%;

[0456] and containing the invention compound, is able to reduce the production of pro-inflammatory interleukins in in vitro tests on human keratinocytes treated with LPS (Huker cell), confirming what has been demonstrated in Example 9.

[0457] The potentials of the invention compound in the cosmetic field, after what has been reported, are numerous: having antiseptic, antiinflammatory and probably healing activity, it can be comprised in antiacne products, as well as in products to be used after-shave or after any face and body treatment that can cause, albeit slightly, an inflammatory state (hair removal, peeling, laser treatments, etc.).

[0458] Given its versatility, also linked to its activity at the level of cell proliferation, it is desirable to use it in all those products aimed at skin trophism, from face creams (anti-aging, moisturizing, anti-redness, antipollution, protective, repairing, depigmenting, etc.), hands (repairing creams, protective creams, depigmenting creams, etc.) and body creams (e.g. toning, regenerating treatments, etc.). In this context, a trichological line for the treatment of hair loss can also be inserted. In addition, always linked to the many functions described, in particular the antioxidant and antiseptic one, the compound of the invention is suitable to be comprised in cosmetics related to skin and intimate hygiene (cleansers for face, body, hands, intimates, etc.) in order toproduce a complete cosmetic line of skin care.

[0459] Empirical observations show that the invention compound could improve the periocular microcirculation. Ad hoc studies on volunteers are needed to have statistical significance.

[0460] If this were confirmed, the product of the invention could also be evaluated in treatments not only at the periocular level, but also for cellulite, always after ad hoc tests.

[0461] The following examples are provided to illustrate the invention and are not to be considered as limiting its scope.

[0462] EXAMPLES

[0463] The following composition was used in the following embodiments of the invention and tests related to the evaluation of antifungal, antibacterial and anti-inflammatory properties:

[0464] Example 1

[0465] Method of making the composition of the invention.

[0466] A method for preparing the composition according to the invention is described below. This method is to be understood as an example and absolutely not limiting towards the possible variations of the method or towards possible expedients within the reach of the expert in the field, and which may take the form, for instance, of changes in the rotation speed of the instruments, temperature, timing, speed of addition and other conditions known to the experts in the field.

[0467] The composition according to the present invention can therefore be obtained by the method described below or by slight modifications of this method which are in any case within the reach of the expert in the field.

[0468] Said method comprises the steps of:

[0469] • preparing the carrier as follows: mix hot, e.g. in a turbo-mixer operating at a temperature between C-80 / 90°C and mixing for atime necessary to bring the temperature back to C-20°C, the rheological agent in the form of powder or viscous solution, water and, optionally, the additives and stabilizers necessary forthe final formulation, e.g. triethanolamine, propylene glycol, imidazolidinyl urea, potassium sorbate, EDTA, to form the carrier; placing the carrier in a turbo-mixer;

[0470] • subjecting said carrier to a temperature between 8-45°C and a mixing speed between 3-100 rpm;

[0471] • adding the ROS-based aqueous solution at a rate of 1.6 - 12.7 ml / s at a temperature of 30°C or less;

[0472] • shaking for a time between 20-500 s at a temperature less than or equal to 30°C;

[0473] • transferring the composition thus obtained into the dispensers and packaging.

[0474] Carbomer Composition:

[0475] Water 94.03%, Carbomer 2.00%, Triethanolamine 1.47% Ozonated glycerine 0.80%, Propylene glycol 0.70%, Sodium benzoate 0.50%, Potassium sorbate 0.40%, Disodium EDTA 0.10%. The compositions were tested and the viscosity was measured, as shown in the table.

[0476]

[0477] Viscosity test

[0478] Tested Carbomer Carbomer + Xantan Xantan product Water (1+1) gum gum + Equipment

[0479] Trademark Fungilab

[0480] Model Vise Olead-ADV R

[0481] Equipment Rotational viscosimeter

[0482] type

[0483] Spindle Measurement with R6 Conformity Compliant with ASTM / ISO standards Brand and Vise Olead-ADV R

[0484] Model

[0485] Test executed

[0486] Ambient 21°C 21°C 21°C 21°C temperature

[0487] Rotational 2 rpm 2 rpm 2 rpm 2 rpm speed

[0488] Measurement 30 s 30 s 30 s 30 s time

[0489] Sample 100 ml 100 ml 100 ml 100 ml volume

[0490] Viscosity 403000 69200 462000 95000 mPa / s mPa / s mPa / s mPa / s

[0491] Example 2

[0492] Antifungal activity test.

[0493] The composition prepared in Example 1 was used to run the test.The composition according to the invention was initially tested for its antifungal activity and for the possible activity of inhibiting the formation and disintegration of biofilms involved in the chronicization of wounds on yeasts such as Candida albicans (CAF2-1).

[0494] In addition, some clinical cases of different Candida species (C. glabrata, C. albicans, C. kefir, C. parapsilosis) were also used in the experimentation.

[0495] The composition was tested at serial 1:2 dilutions and the MIC (minimum inhibitory concentration) was considered the highest dilution wherein no fungal growth was detected. The antibiotic Fluconazole was used as a positive control.

[0496] Antibiofilm activity was evaluated at concentrations 1:4, 1:8, 1:16.

[0497] The growth of the yeast Candida albicans CAF2-1 was inhibited up to a dilution of the composition of 1:16. At the same dilution, the formulation under investigation was able to significantly reduce biofilm formation; the results obtained are comparable to those obtained with Fluconazole treatment. On the other hand, as far as the breakdown of the biofilm is concerned, the composition according to the invention manages to reduce it only up to 1:4 dilution while fluconazole was ineffective.

[0498] Experiments practiced on the clinical isolate of C. albicans confirm the results obtained for the CAF2-1 strain.

[0499] The experimental study done on clinical isolates of Candida glabrata and Candida kefir reports growth inhibition with the composition of the invention up to 1:16 dilution. Not only that, this composition, at this dilution, allows the inhibition of biofilm formation in most clinical isolates, while no biofilm breakdown has been observed.

[0500] The growth of C. parapsilosis is inhibited after treatment with the composition of the invention diluted to 1:8. Both the reduction of biofilm formation and the breakdown of the biofilm are confirmed by treatment with the composition of the invention diluted to 1:8.Example 3

[0501] Antibacterial activity test.

[0502] The composition prepared in Example 1 was used to run the test The composition of the invention, in orderto use it in wound treatment, has been further tested for its antimicrobial activity and for the possible inhibition of the formation and breakdown of biofilms involved in wound chronicity on bacteria generally involved in infections, such as the Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 35984), the Gram-negative bacteria Pseudomonas aeruginosa (ATCC PAO-1) and Escherichia coli (ATCC 86963).

[0503] In addition, clinical isolates of E. coli (resistant to ESBL beta-lactams), Klebsiella pneumoniae (resistant to KPC carbapenems), Streptococcus dysgalactiae, Enterococcus faecalis, Enterococcus f aecium, S. aureus (resistant to MRSA oxacillin) and Pseudomonas aeruginosa were used in this trial.

[0504] The composition was tested at serial 1:2 dilutions and the MIC (minimum inhibitory concentration) was considered the highest dilution wherein no microbial growth was detected. The antibiotic Gentamicin was used as a positive control. Antibiofilm activity was evaluated at concentrations 1:4, 1:8, 1:16.

[0505] The growth of 5. aureus ATCC 25923 was inhibited up to 1:8 dilution of the composition. In addition, the composition of the invention was able to reduce the formation of biofilm up to 1:16 dilution while only 1:4 dilution was able to break down the formed biofilm after 24 hours of incubation.

[0506] Inhibition of growth of 5. epidermidis ATCC 35984 was observed up to 1:4 dilution of the composition. The composition was found to be able to reduce biofilm formation up to 1:16 dilution while a dispersive effect of the preformed biofilm with a 1:4 dilution was noted after 24 hours of incubation.The data obtained for E. coli ATCC 86963 show an inhibition of bacterial growth up to 1:4 dilution. The composition showed antibiofilm activity up to 1:16 dilution, while the dispersion of the preformed biofilm was observed only at 1:4 dilution.

[0507] The growth of P. aeruginosa PAO-1 was inhibited up to 1:4 dilution. In addition, the composition can inhibit biofilm formation by up to 1:16 dilution. On the contrary, no significant biofilm breakdown activity was observed. Also in this case, the results obtained for all ATCC strains are comparable to those obtained with Gentamicin treatment.

[0508] Experiments practiced on clinical isolates of S. aureus MRSA, P. aeruginosa and E. coli ESBLs confirm the results obtained for ATCC strains. Inhibition of growth of clinical isolates o Klebsiella pneumoniae KPC was observed after treatment with the 1:8 diluted composition of the invention. The same dilution led to a high reduction in biofilm formation and dispersion of the preformed biofilm in all eight clinical isolates.

[0509] Experimentation on three isolates of Streptococcus dysgalactiae demonstrated growth inhibition with the 1:8 diluted composition. Biofilm formation is reduced up to 81% after treatment with the 1:4 diluted composition, while biofilm breakdown has been detected up to 90% with treatment with the 1:8 diluted composition.

[0510] Three clinical isolates for E. faecalis were tested and growth was seen to be inhibited using the 1:8 diluted composition. The same dilution leads to the reduction of the biofilm, while the dispersive effect of the gel on the preformed biofilm was highlighted only when the isolates were treated with the 1:4 diluted composition.

[0511] For clinical isolates of E. faecium , growth was inhibited after treatment with the 1:8 diluted composition. The reduction in biofilm formation is present if the 1:4 diluted composition is used for all isolates, but the values reported for biofilm breakdown are not significant.

[0512] Example 4

[0513] Anti-inflammatory activity test.The composition prepared in Example 1 was used to run the test.

[0514] The anti-inflammatory activity on ex vivo human leukocytes from healthy donors was evaluated by analysis of cytokines production in response to an inflammatory stimulus (lipopolysaccharide, LPS, known in art). Prior to the test, the cytotoxic effect of ozone-gel at 4 and 24 hours was determined. The CC50 assessed after 4 and 24 hours of incubation was 1:4 and 1:32 dilution, respectively. At 4 hours, the percentage of living cells remains about 100% until 1:32 dilution of the gel, while after 24 hours of incubation with the diluted gel, 100% of living cells are observed only up to 1:128 dilution. On the basis of these results, the concentration of ozone-gel to be tested in the antiinflammatory response was chosen and, considering that the antiinflammatory activity is performed at 24 hours, it was decided to continue the experiments with a 1:100 gel dilution.

[0515] Leukocytes stimulated with LPS produce TNF-a at 4 and 24 hours of treatment. Co-treatment of leukocytes with LPS and ozone gel causes a significant reduction (P=0.02) in cytokine production, compared to leukocytes stimulated with LPS.

[0516] On the other hand, post-treatment with the gel after a 4-hour stimulation of cells with LPS does not reduce TN F-a production (P=0.56). Given the antimicrobial and anti-inflammatory characteristics highlighted in this study, the gel is suitable for use in wound treatment.

[0517] Example 5

[0518] Cytotoxicity test.

[0519] The composition prepared in Example 1 was used to run the test.

[0520] The cytotoxic effect of the composition of the invention was determined at 4 and 24 hours. The CC50 assessed after 4 and 24 hours of incubation was 1:4 and 1:32 dilution, respectively. At 4 hours the percentage of living cells remains about 100% up to 1:32 dilution of the composition, while after 24 hours of incubation with the diluted composition, 100% of living cells are observed only up to 1:128 dilution.Example 6

[0521] Anti-inflammatory activity test.

[0522] The composition prepared in Example 1 was used to run the test The anti-inflammatory activity of the composition of the invention was evaluated on ex vivo human leukocytes from healthy donors, by analysis of cytokine production in response to an inflammatory stimulus (lipopolysaccharide, LPS).

[0523] Based on the results of cytotoxicity studies and the fact that antiinflammatory activity is performed at 24 hours, 1:100 dilution was chosen as the composition concentration to be tested in the antiinflammatory response.

[0524] Leukocytes stimulated with LPS produce TNF-ot at 4 and 24 hours of treatment. The co-treatment of leukocytes with LPS and this composition causes a significant reduction (P=0.02) in cytokine production, compared to leukocytes stimulated with LPS. On the other hand, post-treatment with composition after a 4-hour stimulation of cells with LPS does not reduce TNF-a production (P=0.56).

[0525] Given the antibacterial, antifungal and anti-inflammatory characteristics highlighted in this study, the composition in gel form according to the invention can be used in the treatment of wounds and in general bacterial and / or fungal infections.

[0526] Example 7

[0527] Evaluation of the biological effect of the product of the invention (mixture of water-soluble oxygen and ozone) on human HaCaT keratinocytes, a cellular model of the skin.

[0528] HaCaT cells, spontaneously immortalized human keratinocytes isolated from healthy epidermis, were cultured in 75 cm2surface flasks and 10 cm diameter Petri dishes, in a volume of DMEM (Dulbecco's modified Eagle's Medium Low Glucose, 5.5 mM) medium of 15 ml and 10 ml respectively, enriched with 10% (v / v) fetal bovine serum (FBS), 1% Glutamine (Gin) (v / v) and 1% penicillin / streptomycin (v / v) (completemedium) and incubated at 37°C in a humidified environment in the presence of 5% CO2. Once a confluence of 60-70% was reached, the cells were removed by trypsinization and subsequently divided to continue with their proliferation. For the trypsinization procedure, the removal of the culture medium was followed by two washes with 3 ml of PBS (0.08% NaCI w / v; 0.002% KCI w / v; 0.002% KH2PO4w / v; 0.006% Na2H O4w / v) and a treatment with 1 ml of trypsin / EDTA (0.05% trypsin and 0.02% EDTA), which was left to act for 10 minutes at 37°C. This cell propagation and trypsinization procedure was repeated for a total of three steps in order to obtain an adequate number of cells to start the experimental procedures.

[0529] During the preparation for the experimental step, the cells, whose health status was assessed by morphological analysis by light microscope observation, were trypsinized as described above, counted under the light microscope using the Burker chamber and seeded in 24-well plates with a density of 30000 cells / cm2in a complete medium volume of 1 ml per well. After 72 hours of culture, the treatments were carried out for different times (24 and 48 hours) and with different concentrations of the composition of the invention conveyed by sterile double-distilled water (double-distilled water, composition of the invention) or by a preserved colloidal matrix (double-distilled water, glycerine, composition of the invention, carbomer, triethanolamine, propylene glycol, imidazolidinyl urea, potassium sorbate, disodium ethylenediaminetetraacetate). Two different cell viability assays were performed:

[0530] 1. CRYSTAL VIOLET

[0531] It is a cell viability assessment assay carried out on cells grown in 24-well plates: crystal violet is an intercalating agent that allows DNA to be quantified in direct proportion to the number of cells adhered to in the well. The method involves the removal of the culture medium from the wells of the plates, followed by a wash with 1 ml of PBS / well and then, the addition to each well of 200 pl of Crystal Violet 0.1% w / v dissolved in methanol. After incubating the plates at 37°C in agitation for 30 minutes, washes are carried out in water to remove excess dye,followed by a drying step at 37°C for 60 minutes. The next phase involves the addition of 600 pl of acetic acid 10% v / v to each well in water and a 15-minute stirring phase to solubilize the dye. Finally, 100 pL are taken from each well and transferred to a well of a 96-well plate to take the absorbance reading at 596 nm.

[0532] 2. MTT

[0533] The MTT assay, devised by Mosman in 1983, is a test that allows to assess the metabolic status of cells. The test, in fact, is based on the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mitochondrial dehydrogenases, with the production of formazan crystals. This assay is also carried out in 24-well plates: the method involves incubating the cells in the presence of MTT 0.5 mg / ml at 37°C for a period of time from 30 minutes to 1 hour, in order to have 800 pl / well. Subsequently, 800 pl of a solution of 2-propanol and 0.04 N HCI are added to each well, followed by an incubation with agitation of the plates at 37°C for 10 minutes; this step allows the formazan crystals to be solubilized. Finally, 200 pL taken from each well are transferred to a 96-well plate to take absorbance readings at 563 nm.

[0534] The viability of HaCaT cells treated for 24 hours with the composition of the invention diluted in the culture medium so as to result with 10% and 20% v / v concentration is significantly reduced; with a 48-hour treatment at the same concentrations, the viability of HaCaT cells evaluated with both the Crystal Violet assay and the MTT assay is almost zero.

[0535] Three different concentrations of the composition of the invention conveyed by the gelatinous matrix diluted in the culture medium were tested so that they were 1.25%, 2.5% and 5% v / v, with which the HaCaT cells were treated for 24 hours. Using the first evaluation method, there was no statistically significant reduction in the viability of HaCaT cells treated with 1.25%, 2.5% and 5% of the composition of the invention interdispersed in the colloidal vehicle provided by Bioxid-Ad sas when compared with cells grown in the culture medium and gelatinous matrix. The latter, however, have a significantly reduced viabilitycompared to cells grown in the complete culture medium. (FIGURE 1) If, on the other hand, we evaluate cell proliferation with the MTT assay, a significant decrease in the viability of the cells grown in the culture medium and gelatinous matrix is confirmed compared to those grown in the culture medium alone. (FIGURE 2) However, in this case, HaCaT cells treated with the lowest concentrations of the composition of the invention, respectively 1.25% and 2.5% v / v of the composition of the invention diluted in the culture medium, show a significant increase in their proliferation compared to cells grown in the culture medium to which an equal amount of gel had been added. (FIGURE 2)

[0536] The viability of HaCaT cells treated for 24 hours with the composition of the invention diluted in the culture medium so as to result with a v / v concentration of 10% and 20% is significantly reduced; with a 48-hour treatment at the same concentrations, the viability of HaCaT cells evaluated with both the Crystal Violet assay and the MTT assay is almost zero.

[0537] Three different concentrations of the composition of the invention conveyed by the gelatinous matrix diluted in the culture medium were tested so that they were 1.25%, 2.5% and 5% v / v, with which the HaCaT cells were treated for 24 hours. Using the first evaluation method, there was no statistically significant reduction in the viability of HaCaT cells treated with 1.25%, 2.5% and 5% of the composition of the invention interdispersed in the colloidal vehicle provided by Bioxid-Ad sas when compared with cells grown in the culture medium and gelatinous matrix. The latter, however, have a significantly reduced viability compared to cells grown in the complete culture medium. (FIGURE 1) If, on the other hand, we evaluate cell proliferation with the MTT assay, a significant decrease in the viability of the cells grown in the culture medium and gelatinous matrix is confirmed compared to those grown in the culture medium alone. (FIGURE 2) However, in this case, HaCaT cells treated with the lowest concentrations of the composition of the invention, respectively 1.25% and 2.5% v / v of the composition of the invention diluted in the culture medium, show a significant increase in their proliferation compared to cells grown in the culture medium towhich an equal amount of gel had been added. (FIGURE 2) In order to confirm the previous results, a further experiment was practiced, evaluating with the MTT assay alone the effect of the concentrations of 2.5% and 5% v / v of the composition of the invention diluted in the culture medium on HaCaT cells compared to incubation in the presence of the gel alone, after 48 hours of treatment. In this case, the concentration of 1.25% v / v of the composition of the invention diluted in the culture medium was not chosen, since there is no statistically significant difference compared to the concentration of 2.5% v / v when the cells were treated for 24 hours. The proliferation of HaCaT cells treated with the concentration of 2.5% v / v of the composition of the invention diluted in the culture medium for 48 hours is significantly increased compared to those grown in culture medium and gelatinous vehicle, while there is no statistically significant difference between these and those treated with 5% v / v of the composition of the invention, reconfirming previous results. After 48 hours of incubation, in particular, a greater recovery by the STL of any mitochondrial damage induced by the gel is observed compared to treatment carried out for 24 hours. (FIGURE 3)

[0538] The composition of the invention - at the tested concentrations - is not only non-toxic to keratinocytes but is functional to the recovery of their viability compared to control; moreover, the composition was tested for 24 and 48 hours, a time well beyond the proper and reasonable conditions of use even of leave-on cosmetic products.

[0539] Example 8

[0540] Ex-vivo evaluation of ionized oxygen on mucin-producing humans -effect of aerial epithelial cells NCI-H292

[0541] The RPMI medium, penicillin and streptomycin, fetal bovine serum (FBS) and trypsin were purchased from Euroclone (Pero, Milan, Italy). Ionized oxygen / oxygen-ozone in bi-distilled water and ionized oxygen / oxygen-oxygenated ionized ozone were supplied by Bioxid-AD (Forte dei Marmi, Lucca, Italy). The crystal violet dye was purchased from Sigma (Milan, Italy). All other chemicals were reagent grade. NCI-H292 mucin-producing human aerial epithelial cells were obtained from the University of Lyon.

[0542] NCI-H292 mucin-producing human aerial epithelial cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2) in 24-well multi-well plates (50000 cells / well). After 24 hours, the cells were treated with different concentrations of ionized oxygen / oxygen-ozone in double-distilled water (part A) or ionized oxygen / oxygen-oxygenated ionized ozone (part B), for different times, as indicated in the Results. As controls, NCI-H292 human aerial epithelial cells were incubated with the medium alone (part A and B) and with the medium containing the ionized oxygenated gel (part B) for the same time. Good cell culture practices were followed.

[0543] The cell proliferation assay was performed by crystal violet staining method. The cells were washed with PBS. After removing the PBS, a solution of 0.1% crystal violet in methanol was added, incubating for 30 minutes at 37°C under gentle stirring. Finally, the cells were washed three times with water and left to dry at room temperature. Acetic acid (0.6 mL, 10%) was added to the wells and kept for 15 minutes at room temperature under gentle stirring. One hundred microliters from each well were transferred to a 96 multi-well plate for quantitative analysis by absorbance measurement at 596 nm with an EL 808 automatic microplate reader from Bio-Tek Instruments Inc (Winooski, Vermont, USA). The percentages were calculated considering the absorbance of the negative control (untreated cells) as 100%.

[0544] PART A NCI-H292 mucin-producing human aerial epithelial cells were seeded 24 hours prior to treatment. Cells were incubated with ionized oxygen / oxygen-ozone in sterile double-distilled water at 5% (light grey), 10% (medium grey), 20% (dark grey) ionized oxygen / oxygen-ozone in the medium for 15, 120, 210 minutes (FIGURE 4).

[0545] As controls, NCI-H292 human aerial epithelial cells were incubated withmedium alone. The toxic effect of the tested compound is already evident after 15 minutes of treatment: a reduction in cell viability of about 32% is observed with 20% ionized oxygen / oxygen-ozone in the medium. After 210 minutes, only 21% of the cells treated with 5% ionized oxygen / oxygen-ozone in the medium are still alive. It was further investigated how mucin-producing NCI-H292 human aerial epithelial cells react to ionised oxygen / oxygen-ozone in sterile doubledistilled water at higher dilutions for different timeframes. Three samples were tested: 1:1000 (light grey), 1:500 (medium grey), 1:250 (dark grey) of the compound diluted in the medium for 120 minutes and for 24-48 hours, after 24 hours of incubation in RPMI. (FIGURE 5) As controls, NCI-H292 human aerial epithelial cells were incubated with medium alone. No changes in cell viability were observed for all dilutions and times tested. In conclusion, no toxic effects are observed even after a 48-hour treatment with a 1:250 dilution (which is 4 times higher than that used in therapeutic treatment).

[0546] PART B NCI-H292 mucin-producing human aerial epithelial cells were seeded 24 hours prior to treatment. Subsequently, the cells were incubated with ionized oxygen / oxygen-oxygenated ionized \ozone (Bioxid-AD) at different concentrations: 0.5 mg / ml (corresponding to 1:1.000.000 compared to the first experiment, (light grey); 1 mg / ml (medium grey) and 2 mg / ml (dark grey), for 24 to 48 hours (Figure 6). As controls, NCI-H292 human aerial epithelial cells were incubated with medium alone. Even when tested at the same ionized oxygen / oxygen-ozone concentrations as in the second experiment, the results are completely different: after 24- and 48-hour treatments, cell viability is drastically reduced with all concentrations tested.

[0547] In the second part of the experiment, mucin-producing NCI-H292 human aerial epithelial cells were seeded 24 hours before treatment. Subsequently, the cells were incubated with ionized oxygen / oxygen-oxygenated ionized ozone (Bioxid-AD) at different concentrations: 0.5 mg / ml (corresponding to 1:1.000.000 compared to the first experiment), 1 mg / ml and 2 mg / ml, for 60 and 120 minutes and for 24hours. (FIGURE 7) As controls, NCI-H292 human aerial epithelial cells were incubated with either the medium alone or the gel-containing medium. Cell viability increased after a 60-minute treatment with 2 mg / ml ionized oxygen / oxygen-oxygenated ionized ozone, while after a 120-minute treatment, testing 2 mg / ml ionized oxygen / oxygen-oxygenated ionized ozone, cell viability decreased. After 24 hours, cell viability is dramatically reduced compared to incubated cells with only the medium for all dilutions tested. Furthermore, if we consider as controls the cells grown in the medium containing only the gel, the cells treated with ionized oxygen / oxygen-oxygenated ionized ozone show a superior viability (FIGURE 8 - Gel without Oxygen (White bordered), gel with ROS (medium grey). In conclusion, the viability of cells incubated with the medium and gel alone is drastically reduced compared to cells incubated with the medium alone. In addition, considering how you control cells grown in the gel-only medium, cells treated with ionized oxygen / oxygen-oxygenated ionized ozone show superior viability.

[0548] The composition of the invention exerts a recovery effect of cell viability (mucoepidermoid cells of lung cancer NCI-H292) compared to the control carried out in the presence of only the colloidal medium wherein it is dispersed.

[0549] Evaluate cell proliferation using two different methods, one based on mitochondrial activity (MTT test) and one on DNA quantity (Crystal Violet test)

[0550] Example 9

[0551] The formulation of ozone-gel, in order to use it in wound treatment, was initially tested for its antimicrobial activity and for the possible activity of inhibiting the formation and breakdown of biofilms involved in wound chronicity on bacteria generally involved in infections, such as the Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 35984), the Gram-negative bacteria Pseudomonas aeruginosa (ATCC PAO-1) and Escherichia coli (ATCC 86963), and on yeasts such as Candida albicans (CAF2-1).

[0552] In addition, 58 clinical isolates were used in this trial: E. coli (resistant toESBL beta-lactams), Klebsiella pneumoniae (resistant to KPC carbapenems), several species of Candida (C. glabrata, C. albicans, C. kefir, C. parapsilosis), Streptococcus dysgalactiae, Enterococcus faecalis, Enterococcus faecium, S. aureus (resistant to MRSA oxacillin) and Pseudomonas aeruginosa.

[0553] Ozone gel was tested at serial 1:2 dilutions and the MIC (minimum inhibitory concentration) was considered the highest dilution wherein no microbial growth was detected. The antibiotic Gentamicin or Fluconazole was used as a positive control for bacteria and yeast respectively. Antibiofilm activity was evaluated at concentrations 1:4, 1:8, 1:16.

[0554] The growth of S. aureus ATCC 25923 was inhibited up to an ozone gel 1:8 dilution. In addition, the ozone-gel was able to reduce the formation of the biofilm up to 1:16 dilution while only the 1:4 dilution was able to break down the biofilm formed after 24 hours of incubation.

[0555] Inhibition of the growth of 5. epidermidis ATCC 35984 was observed up to the 1:4 dilution of the ozone gel. The ozone-gel was able to reduce the formation of biofilm up to 1:16 dilution while a dispersive effect of the preformed biofilm was noted with a 1:4 dilution after 24 hours of incubation.

[0556] The data obtained for E. coli ATCC 86963 show an inhibition of bacterial growth up to 1:4 dilution. The ozone-gel showed antibiofilm activity up to 1:16 dilution, while the dispersion of the preformed biofilm was observed only at 1:4 dilution.

[0557] The growth of P. aeruginosa PAO-1 was inhibited up to 1:4 dilution. In addition, ozone gel can inhibit biofilm formation up to 1:16 dilution. On the contrary, no significant biofilm breakdown activity was observed. Also in this case, the results obtained for all ATCC strains are comparable to those obtained with Gentamicin treatment.

[0558] The growth of the yeast Candida albicans CAF2-1 was inhibited up to 1:16 dilution of ozone-gel. At the same dilution, the study formulationwas able to significantly reduce biofilm formation; the results obtained are comparable to those obtained with Fluconazole treatment.

[0559] On the other hand, as far as biofilm breakdown is concerned, ozone-gel can only reduce it up to 1:4 dilution, while fluconazole was ineffective. Experiments practiced on clinical isolates of 5. aureus MRSA, P. aeruginosa and E. coli ESBL and C. albicans confirm the results obtained for ATCC strains.

[0560] Inhibition of growth of clinical isolates of Klebsiella pneumoniae KPC was observed after treatment with 1:8 diluted ozone gel. The same dilution led to a high reduction in biofilm formation and dispersion of the preformed biofilm in all eight clinical isolates.

[0561] The experimental study done on clinical isolates of Candida glabrata and Candida kefir reports an inhibition of growth with ozone-gel up to 1:16 dilution, the gel at this dilution, also allows the inhibition of biofilm formation in most isolates, while no biofilm breakdown was observed. The growth of C. parapsilosis is inhibited after treatment with 1:8 diluted ozone gel. Both the reduction of biofilm formation and the breakdown of the biofilm are confirmed by treatment with 1:8 diluted ozone-gel.

[0562] Experimentation on three isolates of Streptococcus dysgalactiae demonstrated growth inhibition with 1:8 diluted ozone gel. Biofilm formation is reduced up to 81% after treatment with 1:4 diluted ozone gel, while biofilm breakdown has been detected up to 90% with treatment with 1:8 diluted ozone gel.

[0563] Three clinical isolates for E. faecalis were tested and growth was seen to be inhibited using 1:8 diluted ozone gel. The same dilution leads to the reduction of the biofilm, while the dispersive effect of the gel on the preformed biofilm was highlighted only when the isolates were treated with 1:4 diluted ozone-gel.

[0564] For clinical isolates of E. faecium , growth was inhibited after treatment with 1:8 diluted ozone-gel. The reduction of biofilm formation is presentif 1:4 diluted ozone-gel is used for all isolates, but the values reported for biofilm breakdown are not significant.

[0565] Secondly, the anti-inflammatory activity of ozone-gel on ex vivo human leukocytes from healthy donors was evaluated by analysis of cytokine production in response to an inflammatory stimulus (lipopolysaccharide, LPS). Prior to the test, the cytotoxic effect of ozone gel was determined at 4 and 24 hours. The CC50 assessed after 4 and 24 hours of incubation was 1:4 and 1:32 dilution, respectively. At 4 hours, the percentage of living cells remains about 100% up to 1:32 dilution of the ozone gel, while after 24 hours of incubation with diluted ozone gel, 100% of living cells are observed only up to 1:128 dilution. On the basis of these results, the concentration of ozone-gel to be tested in the antiinflammatory response was chosen and, considering that the antiinflammatory activity is performed at 4 hours, it was decided to continue the experiments with an ozone-gel 1:100 dilution.

[0566] Leukocytes stimulated with LPS produce TNF-ot at 4 and 24 hours of treatment. Co-treatment of leukocytes with LPS and ozone gel results in a significant reduction (P=0.02) in cytokine production, compared to leukocytes stimulated with LPS.

[0567] On the other hand, post-treatment with ozone-gel after a 4-hour stimulation of cells with LPS does not reduce TN F-a production (P=0.56). Given the antimicrobial and anti-inflammatory characteristics highlighted in this study, ozone gel could be used in this formulation in the treatment of wounds.

[0568] In addition, since studies in the literature show a regenerative activity of ozone, further experiments are underway on keratinocytes and human fibroblasts to verify the effect of ozone-gel on cell proliferation.

[0569] Example 10

[0570] Evaluation of the anti-oxidant power of Soothing Ge!

[0571] Evaluation of protection from ROS in human endotheliocytes: the tested product was diluted 1:10 in cellular medium (1 g product / 9 ml ofmedium) and from that dilution the following were made: 1.25; 2.5; 5.0; 10.0; 20.0; 40.0 (weight / volume [mg / ml]; compared to the cellular medium). Untreated cells are used as negative control and cells treated with hydrogen peroxide (H2O2) 0.75 mM as positive control.

[0572] Day 1

[0573] After counting, the suspension (in culture medium) of HECV cells was transferred to 96-well plates: 200 pl per well. The cells contained in the plates were cultured in an incubator at 37°C and a humid atmosphere enriched with 5% CO2 for 24 hours.

[0574] Day 2 Treatment

[0575] After morphological analysis of the cells (using the phase contrast microscope) to verify the state of health of the cells and the absence of contamination, the culture medium was removed, and I proceeded to the treatment with the sample at different dilutions 1.25; 2.5; 5.0; 10.0; 20.0; 40.0 mg / ml. Negative control was maintained simultaneously. Subsequently, the plates were placed in an incubator at 37°C and the atmosphere was enriched with 5% CO2 for 24 hours.

[0576] Day 3

[0577] After morphological verification of the cell monolayer, the treatment was removed, the cells were further incubated for 2 hours with 0.75 mM hydrogen peroxide, and MTT cell viability test was performed thereafter.

[0578] MTT is a tetrazolium salt that in its oxidized and soluble form is yellow in color. It is converted into its reduced form into formazan salt by dehydrogenase (succinate dehydrogenase), and in this form the salt is insoluble and precipitates in the form of purple crystals. The addition of isopropanol solubilizes the crystals to form a purple solution whose intensity, evaluated by spectrophotometric reading at 570 nm, is proportional to the amount of living cells present at the time of the test. The viability of HECV cells in presence 1.25; 2.5; 5.0; 10.0; 20.0; 40.0 mg / ml. of tested product to which 0.75 mM hydrogen peroxide wassubsequently added is shown in the graph below. Values are normalized with negative checking ([- / -]; 100%) wherein the cells are kept in the same growing conditions in the absence of the tested product and hydrogen peroxide. The bars in the graph shown here show the cell viability values [%] calculated on the basis of the negative control. The concentration of 1.25 mg / ml of tested product was able to completely protect the cells from the damage caused by hydrogen peroxide (83% cell viability in the presence of hydrogen peroxide alone and 105% in the presence of hydrogen peroxide and the tested product).

[0579] In conclusion, in vitro tests have shown that Bioxid Soothing Gel has a very high antioxidant power in a cellular system. In fact, at 1.25 mg / ml concentration, the pre-treatment with Bioxid Soothing Gel is able to completely protect against oxidative damage caused by hydrogen peroxide (from 83% to 105%).

[0580] The antioxidant power of the soothing gel containing the composition of the invention on a cell line of human endotheliocytes is demonstrated and certified. In fact, the soothing gel of the cell model is able to completely protect against oxidative damage caused by hydrogen peroxide.

[0581] Example 11

[0582] The objective is the in vitro evaluation of the depigmenting activity on melanocyte cell cultures. The test allows to evaluate the depigmenting activity of a product in vitro on melanocyte cultures, measuring the amount of melanin produced. We initially performed a cell viability test to identify the concentrations of the product on which to practice the depigmenting test.

[0583] The test was performed on murine melanoma (B16) cells, a well-known cell line commonly used in melanogenesis studies. The cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2).A cell viability test was performed prior to the depigmenting test to assess the possible negative effect of the tested product on the cells and to select the concentrations to be used for the analysis of melanin content. This step is crucial to distinguish between an inhibition of the melanogenesis pathway and a toxic effect. Cell viability was assessed by the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a yellow compound that is bio-transformed by cells into a purple product (formazan). This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. The cells were treated with MTT (1 mg / ml) and incubated for 3 hours under standard culture conditions. At the end of the period, the MTT solution was removed and 100 pL of isopropanol were added to each well to dissolve the formazan crystals. Absorbance (optical density, OD) was determined spectrophotometrically at a wavelength of 540 nm.

[0584] B16 cells were seeded and kept in constant darkness under standard culture conditions. Once a semi-confluent single-celled layer was reached, the cells were treated with the chosen concentrations of the tested product. The negative control was represented by untreated cells, while a well-known depigmenting drug (PC) was used as a positive control. For melanin content analysis, cells were collected and lysed. Melanin was quantified by absolute absorbance at 490 nm using an MICroplate reader, and the values were interpolated on a standard synthetic melanin curve. Melanin concentrations were expressed as pg / ml melanin per 106cells.

[0585] Shortly before cell lysis, melanocytes were observed and photographed under a brightfield microscope. Absorbance measured at 540 nm is proportional to cell viability. The percentages were calculated based on the absorbance values at 540 nm, considering the absorbance of the negative control (untreated cells) as 100%. The cell viability of melanocytes treated with the tested product was comparable to that of untreated melanocytes for all concentrations tested. We chose to carry out the melanin dosage considering the concentrations of 1.0, 0.5 and 0.2 mg / ml. The absorbance measured at 490 nm is directly proportional to the amount of melanin extracted from the cells. The values wereinterpolated on a standard synthetic melanin curve and the results expressed as pg / ml melanin per 106cells. The sample is able to significantly inhibit melanin production at the tested concentration of 1.0 mg / ml (16.25% reduction).

[0586] In conclusion, the tested product significantly reduces melanin production in melanocyte cell cultures at the tested concentration of 1.0 mg / ml.

[0587] The tested cosmetic Deep Oxygenizing Lightening, containing the composition of the invention, showed a significant ability to reduce the production of melanin by B16 cells.

[0588] Deep Oxygenizing Lightening

[0589] Base Gel 73.70% (Water 90.76%, Carbomer 5.00%, Triethanolamine 1.64%, Glycerine 1.00%, Propylene Glycol 1.00%, Imidazolidinyl urea 0.30%, Potassium Sorbate 0.20%, Disodium EDTA 0.10%) Water + Oxygen 25.00%, ( Water 50.00%, Oxygen 50.00), Jojoba Oil dac 1.00%, Liposomes Hyaluronic Acid pf 0.10%, Kojic Acid 0.10%, Vitamin C (Ascorbic Acid) 0.10%

[0590] Example 12

[0591] The objective of this test is the in vitro evaluation of the protective and repairing action of a cosmetic product against external and internal environmental stress. The test was performed on human keratinocytes treated with three concentrations of the tested product (1.0, 0.5 and 0.25 mg / ml) chosen after a preliminary cytotoxicity test. To evaluate the repairing action of the cosmetic product against external stress, keratinocytes were stimulated with urban dust or UV rays and subsequently treated with the tested product. The test was practiced on human keratinocytes (Huker) cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2). The tested sample comprises water, oxygen, carbomer, triethanolamine, macadamia ternifolia seed oil, glycerine, propylene glycol, imidazolidinyl urea,potassium sorbate, tocopheryl acetate, ascorbic acid, disodium EDTA, BHA, BHT, retinyl palmitate, lecithin, pentylene glycol, hyaluronic acid. The sample was dissolved and then diluted in the culture medium to the desired final concentrations.

[0592] Cell viability was assessed via the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a yellow compound biotransformed by cells into a purple product (formazan). This conversion presumably occurs thanks to NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. The cells were treated with MTT (1 mg / ml) and incubated for 3 hours under standard culture conditions. At the end of the period, the MTT solution was removed and 100 pl of isopropanol were added to each well to dissolve the formazan crystals. Absorbance (optical density, OD) was determined spectrophotometrically at a wavelength of 540 nm.

[0593] Cells treated with the tested product and negative control were counted. The same number of cells for each treatment was detached, centrifuged, washed in PBS and lysed for total protein extraction. Subsequently, the total protein assay was performed with spectrophotometric reading at a wavelength of 570 nm.

[0594] To simulate a condition of environmental stress, the cells were stimulated with a standard sample of urban pollution (urban dust) and subjected to irradiation with UVA rays. Urban dust is a standard reference material containing polycyclic aromatic hydrocarbons (PAHs), PAHs nitrates, PCBs (polychlorinated biphenyls), chlorinated pesticides and inorganic heavy metals. This material was prepared from atmospheric particulate matter collected in the Washington DC area for over 12 months with a specially designed system. UV radiation was generated by a sunlight simulation lamp with a constant emission in the UVA range (315-400 nm) with an irradiance of 1.7 mW / cm2, while UVB emission was shielded to avoid direct cell mortality.

[0595] A preliminary MTT test was performed to select the non-cytotoxic concentrations of the tested product to be used. Untreated cells retained in growth medium represent the negative control. After 24 hours of contact, the cells were washed with PBS and subjected to MTTtesting. To assess the anti-pollution restorative activity, the cells were first exposed to the desired stimuli with such intensity as to cause significant damage and then treated with the tested product for 24, 48 and 72 hours. At the end of each contact time, cell viability was assessed via MTT and protein assay was performed.

[0596] Based on the results obtained from the preliminary MTT test, we chose to perform the anti-pollution test using the concentrations of 1.0, 0.5 and 0.25 mg / ml.

[0597] Regarding the evaluation of the anti-pollution reparative activity (cell viability), it was found that the viability of cells stimulated with urban dusts improved significantly after treatment with the tested product at 1.0 mg / ml (restorative effect of 10.65% and 18.78% respectively after 24 and 48 hours), at 0.5 mg / ml (effect of 19.89% and 11.92% respectively after 48 and 72 hours) and at 0.25 mg / ml (effect of 17.15% and 10.39% respectively after 48 and 72 hours). For UV-stimulated cells, viability improved with 1.0 mg / ml (22.98% after 48 hours), 0.5 mg / ml (23.43% and 15.02% after 48 and 72 hours), and 0.25 mg / ml (10.93% and 12.88% after 48 and 72 hours).

[0598] As regards protein assay, the protein content of cells stimulated with urban dust improved with 1.0 mg / ml (17.8%, 23.45% and 21.62% after 24, 48 and 72 hours, respectively), 0.5 mg / ml (16.78% and 26.68% after 48 and 72 hours) and 0.25 mg / ml (11.24% and 31.89% after 48 and 72 hours). For UV-stimulated cells, the protein content improved with 1.0 mg / ml (13.49% and 16.45% after 48 and 72 hours), 0.5 mg / ml (17.39% and 18.96% after 48 and 72 hours), and 0.25 mg / ml (15.74% and 15.20% after 48 and 72 hours).

[0599] In conclusion, the tested sample shows a restorative action against environmental stress (urban pollution and UV radiation) on keratinocytes in vitro.

[0600] The cosmetic Deep Oxygenizing Anti-aging gel, containing the composition of the invention, has a protective and restorative effect against "outdoor and indoor environmental stress" carried out in vitro on human keratinocytes.Example 13

[0601] The objective of this test is the in vitro evaluation of the soothing / anti-redness action of the cosmetic product. The soothing activity of the tested product was evaluated by measuring its ability to prevent cytokine formation in human keratinocyte cell cultures, in particular the synthesis of interleukin-8 (IL-8). The test was practiced on human keratinocytes (Huker) cultured in DMEM (Dulbecco's Modified Eagle's Medium) enriched with fetal bovine serum (10%) and glucose (4.5 g / l) and incubated under standard culture conditions (37°C, 5% CO2), following good cell culture practices. The tested sample comprises water, oxygen, carbomer, triethanolamine, Argania spinosa seed oil, glycerine, propylene glycol, imidazolidinyl urea, potassium sorbate, disodium EDTA, pentylene glycol, lecithin, hyaluronic acid.

[0602] A cell viability test was performed prior to cytokine assay to select concentrations to be used for cytokine analysis. The viability test was repeated (in the presence of lipopolysaccharide - LPS) to evaluate any negative effects of the treatments on the cells. Cell viability was assessed through an MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a yellow compound that is biotransformed by cells into a purple product (formazan). This conversion occurs thanks to NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. The cells were treated with MTT (1 mg / ml) and incubated for 3 hours under standard culture conditions. At the end, the MTT solution was removed and 100 pl of isopropanol were added to each well to dissolve the formazan crystals. Absorbance (optical density, OD) was determined spectrophotometrically at a wavelength of 540 nm.

[0603] The test was performed in parallel on two groups of cells: one untreated and the other treated with lipopolysaccharide (LPS) to stimulate the production of interleukins. Cells were treated with the selected concentrations of the tested product and a positive control (PC). Cells not treated with the sample represent negative controls. The content of IL-8 in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA), a plate-based technique designed todetect and quantify substances such as peptides, proteins, antibodies, and hormones.

[0604] In an ELISA test, an interleukin-specific antigen to be identified is immobilized on a solid surface (the bottom of a well) to which the culture medium is added for assay. Detection is via a biotinylated secondary antibody that subsequently reacts with streptavidin-HRP. The colorimetric reaction is proportional to the amount of cytokine present in the medium. The results are read with a spectrophotometer at 450 nm. The values obtained were then interpolated into a standard interleukin curve.

[0605] Concentrations of 1.0, 0.5 and 0.25 mg / ml were chosen to perform cytokine assay.

[0606] With regard to the control of cell viability in the presence of LPS, it was found that treatment with LPS and the tested sample did not significantly affect cell viability. Regarding cytokine assay, the tested product was shown to significantly reduce IL-8 at the tested concentrations of 0.5 and 0.25 mg / ml.

[0607] In conclusion, the tested product is able to reduce the synthesis of interleukins in vitro, in particular it reduces the production of interleukins in human keratinocyte cell cultures in vitro at the tested concentrations of 0.5 and 0.25 mg / ml.

[0608] Deep Oxygenizing Ultralight, containing the composition of the invention, is able to reduce the production of pro-inflammatory interleukins in in vitro tests on human keratinocytes treated with LPS.

[0609] Example 14

[0610] Evaluation of the antifungal efficacy of the gel on dermatophytes (Microsporum canis and Trichophyton mentagrophytes) and on Malassezia pachydermatis, fungi relevant for animal health, in particular for dermatitis and otitis.

[0611] The objective of this example is to carry out an in vitro test to evaluate the minimum inhibitory concentration (MIC) and the minimum lethalconcentration (MLC) of the product on strains of Microsporum canis and Trichophyton mentagrophytes isolated from animals.

[0612] Three strains of dermatophytes belonging to the species Microsporum canis and one strain of Trichophyton mentagrophytes isolated during routine diagnostic activities were tested. Specifically: strain 54 / 23 of M. canis isolated from a 3-year-old female European cat with lesions strain 69 / 23 of M. canis isolated from a male European cat 4 months old without lesions strain 70 / 23 of M. canis isolated from a male European cat 4 months old without lesions strain 90 / 23 of T. mentagrophytes isolated from a female European cat 4m old without lesions

[0613] The standard inoculum consisted of a 4x4 mm square of agar culture of each fungal strain tested taken from the most peripheral area of the colony maintained at 26°C for about 15 days on Sabouraud agar medium with chloramphenicol (SAB-CAF). Each fungal strain was tested in duplicate.

[0614] During a first step, two different products were tested, one called "booster" (hereinafter referred to as "product 1") and one marked "ionpet" lot 210 (product 2); secondly, a product called "xanthan oxygenbased gel" Lot H266 (product 3) was also tested.

[0615] Each product was diluted in Sabouraud broth, specifically various dilutions of the product (1:4; 1:8; 1:16; 1:32; 1:64) distributed in tubes in the amount of 4 ml each; Sabouraud broth without inhibitors was used as a positive control. The inoculum was then inserted into each tube.

[0616] The cultures thus prepared were incubated at 26°C for 10 days under continuous agitation and the presence or absence of growth at different dilutions was then observed, thus evaluating the minimum inhibitory concentration (MIC) defined as the lowest concentration capable of determining the absence of fungal growth within the broth.

[0617] Subsequently, the inoculums present in the broth were transferred to Petri dishes containing SAB-CAF and incubated at 26°C for 10 days to observe the presence or absence of growth and evaluate the minimumlethal concentration (MLC) defined as the minimum concentration capable of determining the absence of growth on clean medium and therefore the death of the fungus.

[0618] Product 1 resulted in growth inhibition on all three strains of M. canis tested at 1:4 and 1:8 dilutions (MIC = 1:8) while for T. mentagrophytes the MIC was 1:4.

[0619] Product 2 had no inhibitory effect on M. canis at any of the concentrations tested. With product 3 for M. canis, an MIC of 1:4 was shown for strains 54 / 23 and 69 / 23 and 1:8 for strain 70 / 23. For the strain of T. mentagrophytes (also tested in doubles) in one case there was an MIC = 1:4 and in one case no inhibition was observed.

[0620] Transplantation on agar medium allowed to determine an MLC of 1: 4 for product 1 on all M. canis strains. No fungicidal activity was observed at the tested concentrations for product 2; product 3 presented MLC at 1:4 only in one case (not in duplicate) for strains 69 and 70 and no fungicidal effect on strain 54.

[0621] As regards T. mentagrophytes, the absence of activity is confirmed at the tested concentrations of product 3, while for product 1 only in one case MLC was observed at 1:4.

[0622] In a second example, the aim was to evaluate the effect of the gel with different contact times on hair infected in vitro with the same dermatophytes. The test was performed on the three strains of M. canis in the example described above. For each strain, the inoculum was obtained starting from a culture on SAB-CAF, on the surface of which 10 ml of saline solution were added. The aerial mycelium was scraped, aspirated with the liquid, shaken in a test tube with sterile glass balls to fragment the mycelium, the largest pieces were left to settle, the remaining material was aspirated, transferred to another test tube and brought to opacities of about 2 on the McFarland scale by adding saline solution. 3 Petri dishes were set up (one for each strain) on the bottom of which untreated hair was placed, cut to a maximum length of 1 cm and sterilized in an autoclave; 25 ml sterile deionized water, 2-3 drops of 10% yeast extract and 300 microliters of suspension of each strainwere then added (Rebell and Taplin, 1974). The plates were left to incubate at 26°C with periodic microscopic evaluations of the hair, until a widespread presence of colonization of the same by the tested fungi was observed, with the presence of "perforation channels". Products 1 and 3 (described in the previous example) were tested, setting up 5 dilutions by doubling in Sabouraud broth starting from the product as it is (TQ), 1:2; 1:4; 1:8; 1:16; culture broth alone was used as a control. 24-well plates were set up. In each plate, three rows of wells containing 1 ml of the different product dilutions described above were prepared, the sixth well of each row was used as a control with culture broth only. Subsequently, tufts of hair colonized by the three previously prepared strains of M. canis were placed in each well. After 24, 48 and 110 hours of contact, some hair from each well was passed over plates of SAB-CAF to see the fungicidal effect at different contact times. After 24 hours of contact, product 1 resulted in complete inhibition of M. canis growth both on TQ and at 1:2 dilution on 2 of the tested strains, while in the third strain there was only a partial inhibition, with growth slowed down at the 1:2 concentration. After 48 hours the effect was reconfirmed (for one strain the 1:2 concentration was not readable due to contamination with other mold) while after 110 hours of contact, in addition to complete inhibition of growth on TQ and at the 1:2 concentration, a partial inhibition of growth was also observed at the 1:4 concentration. Product 3 after 24 hours of contact resulted in complete inhibition of growth even at the 1:4 concentration. This effect was also confirmed after 48 and 110 hours.

[0623] According to a third example, the aim is to evaluate MIC and MLC on a strain of Malassezia pachydermatis isolated from dogs with otitis in vitro. A diagnostic isolate of Malassezia pachydermatis from a 6-year-old female pointer dog was used. The culture was maintained for 4 days on mDixon agar, then some colonies were suspended in a tube containing 9 ml of saline and some sterile glass balls. The tube was vortexed to obtain a uniform suspension of the yeast, and the opacity of the suspension was brought to a level between 1 and 2 on the Me Farland scale. Products 1 and 3 (according to the two previous examples) were tested, setting up 8 dilutions via doubling in non-agarmDixon medium starting from the product as it is (TQ), 1:2; 1:4; 1:8; 1:16; 1:32; 1:64 and 1:128. Culture broth alone (mDixon) was used as both positive (inoculating the strain) and negative (broth only) control, while miconazole at the final concentration of 100 ppm was used as an efficacy control.

[0624] A 96-well plate was used, placing 100 pL of the different product concentrations along the rows. 100 pl of M. pachydermatis suspension was then placed in each well. Each product has been tested in quadruple.

[0625] The plate was placed to incubate at 26°C for 4 days and the presence or absence of growth at different dilutions was then observed, thus evaluating the minimum inhibitory concentration (MIC) defined as the lowest concentration capable of determining the absence of growth within the broth. Subsequently, 10 pl of the contents of the wells containing the TQ concentration and those with the 1:2 concentration were passed on agar mDixon medium, and incubated at 26°C for 4 days to observe the presence or absence of growth and evaluate the minimum lethal concentration (MLC) defined as the minimum concentration capable of determining the absence of growth on clean medium and therefore the death of the fungus.

[0626] After 4 days of incubation, complete inhibition of Malassezia growth (MIC) was observed only in the TQ well, both for product 1 and product 3, in all replicates. At the 1:2 concentration, partial inhibition was observed only for product 3. Transplantation on agar mDixon medium showed on TQ for product 3 (ABCD) the absence of growth from only three repeats, while in one very rare colonies grew; also, for product 1 (EFGH) the absence of growth was observed in three repetitions, while in one M. pachydermatis it increased. The 1:2 dilution was confirmed to have no fungicidal effect for either product, although for product 3 (ABCD wells) a smaller number of colonies was observed.

[0627] From the three examples, the following conclusions can be drawn: as far as dermatophytes are concerned, with the broth method under continuous agitation, product 1 presented an MIC equal to 1:8 for M. canis, and 1:4 for T. mentagrophytes, and MLC of 1:4 for M. canis andalways 1:4 (but not repeatable) for T. mentagrophytes. For product 2, MIC and MLC, evaluated only on M. canis, were not determined (ND) as the product did not even have an inhibiting effect at the tested concentrations.

[0628] Product 3 had 1:4 MIC for two strains of M. canis and 1:8 for the third, while on T. mentagrophytes the MIC was 1:4 (non-repeatable). MLC is not determined for one isolate of M. canis and T. mentagrophytes and 1:4 (but not repeatable) for the other isolates of dermatophytes. The 1:4 concentration therefore appears to be at the limit of efficacy, in particular for product 3, and T. mentagrophytes appears somewhat less sensitive than M . canis, although further tests on other isolates would be required to assess the different individual response. The culture on hair allows the growth of dermatophytes which, for some species, are able to pierce the hair with special arrangements of the hyphae (piercing organs). While not fully simulating the conditions that occur in the parasitic phase (during which arthropores are formed), in this way it is still possible to obtain hair with external and internal hyphae to be used for contact simulations with the products to be tested (Korshid et al., 2023). During the test practiced on hair infested with the three strains of M. canis, for which it was also possible to test the concentrations 1:2 and TQ, it was observed that product 1 seems to have a lower efficacy than product 3, which is confirmed to give complete inhibition at a concentration 1:4. This could be related to the composition of the product itself which allows greater or lesser penetration through the parasitized hair. Regarding M. pachydermatis, both products tested (product 1 and 3) had an inhibiting effect only if not diluted (TQ) and only the product used as such showed a lethal effect, although not in all repetitions.

Claims

CLAIMS1. A composition comprising:• an aqueous carrier comprising water and at least one rheological modifier; and• an aqueous solution comprising reactive oxygen species (ROS) resulting from the splitting of dissolved ozone in said aqueous solution, comprising at least superoxide anions (O2 ), singlet oxygen C ), hydroxyl radicals (OH*), oxidizing oxygen radical (0‘); wherein the composition, at a temperature < 30°C, has a viscosity between 300,000 and 600,000 mPa-s measured at 20-25°C by means of a rotational viscometer with R6 rotor.

2. The composition according to claim 1 wherein reactive oxygen species are obtained in the absence of electrolytic generation and in the absence of anodic oxidation of chloride ions.

3. The composition according to claims 1 or 2 wherein the reactive oxygen species are obtained by mechano-induced cleavage of ozone into an aqueous solution, caused by the forced passage of the solution through submicrometric passages under pressure difference.

4. The composition according to anyone of claims 1-3, wherein the aqueous carrier, prior to the addition of the ROS-based aqueous solution, has a viscosity between 300,000 and 600,000 mPa-s, measured at 20-25°C by rotational viscometer with rotor R6.

5. The composition according to anyone of claims 1-4, wherein the weight ratio of the ROS-based aqueous solution to the carrier is between 0.5 and 2.

6. The composition according to anyone of claims 1-5, wherein the rheological modifier is selected from carbomer, xanthan gum, and combinations thereof.

7. The composition according to the above claim, wherein saidrheological modifier is previously mixed with pharmaceutically and cosmetically acceptable additives.

8. The composition according to anyone of claims 1-7, further comprising at least one additive selected from:(i) pH regulators, preferably citric acid, malic acid, acetic acid, lactic acid, phosphoric acid, triethanolamine;(ii) preservatives, preferably potassium sorbate, monosodium glutamate, ozonated glycerine, calcium propionate, imidazolidinyl urea;(iii) humectants, preferably propylene glycol, sorbitol, glycerine; (iv) stabilizers, preferably tetrasodium glutamate diacetate (GLDA);(v) antioxidants, preferably tocopherol, ascorbic acid, disodium calcium EDTA.

9. The composition according to anyone of claims 1-8, which is formulated in the form of creams, lotions, ointments, capsules, ovules with gel inside, sprays, aerosols, liposomal formulations.

10. The composition according to anyone of claims 1-9, for cosmetic use.

11. The composition according to the previous claim for applications such as: after-shave or post-hair removal; peeling; laser treatments; anti-aging, moisturizing, anti-redness, anti-pollution, protective, repairing, depigmenting face creams; repairing, protective, depigmenting hand creams; toning, regenerating body creams; trichological compositions, to counteract hair loss and for hair thickening, to combat alopecia; face, body, hand, intimate cleansers; complete skin care, for the periocular microcirculation; cellulite; intimate lubricant; nail strengthening gel; slimming body cream; intimate cleanser; gel for nail regrowth; skin depigmentation, especially for applications in skin aging treatments; to stimulate the skin's natural antioxidant response;to counteract hair loss; to counteract or reduce the appearance of wrinkles and fine lines of the skin; to increase the skin's resistance to environmental insults; to reduce dark spots and hyperpigmentation of the skin; to improve skin texture and reduce visible signs of aging; as an adjuvant to electromedical therapies, for instance treatments such as shock waves - lasers, radio frequencies, pulsed light.

12. The composition according to anyone of claims 1-9 for pharmaceutical use.

13. The composition according to claim 12, for use in the treatment or prevention of skin or mucous membrane infections of a mammal, comprising humans, comprising newborns, infants and the elderly, preferably fungal infections, more preferably candidiasis.

14. The composition according to the previous claim for use in all diseases and pathologies that benefit from the administration of effective amounts of ROS, such as: Healing activity; Anti-fungal activity; Anti-inflammatory activity; Anti-bacterial activity against gram-positive, gram-negative and anaerobic bacteria; antiseptic activity; Anti-viral activity, in particular of DNA or RNA viruses and lipid enveloped viruses such as HIV, SARS-CoV-2, flu viruses, Respiratory syncytial virus, Herpes simplex (HSV-1, HSV-2); Antitumor activity; Immune system modulation activities; Immune stimulation: neuroprotection, tissue regeneration, wound healing; particularly for use in the treatment of inflammation, especially skin inflammation such as sunburn, light, medium or severe burns, inflammation of the mucous membranes and deep skin layers such as gastritis, haemorrhoids, rhinitis, conjunctivitis, muscle and muscular / skeletal inflammation such as muscle pain, tendinitis and bursitis, arthritis, comprising rheumatoid arthritis, chronic and degenerative inflammation, such as fibromyalgia; neurodegenerative diseases such as multiple sclerosis; to reduce dark spots and hyperpigmentation caused by photoaging; to improve the ability to neutralize harmful free radicals generated by exposure to UV rays or pollution; healing of skin wounds suchas cuts and lacerations, surgical wounds to speed up wound closure and reduce the formation of hypertrophic scars or keloids, post-surgical dental wounds, pressure sores; Inflammation and lesions of the mucous membranes, such as oral lesions, such as gingivitis, stomatitis or periodontitis, mouth ulcers, canker sores, vaginal or anal wounds; gastric ulcers; intestinal lesions: such as Crohn's disease or ulcerative colitis; fungal diseases such as candidiasis, in particular mucosal, cutaneous, systemic candidiasis; acne, bacterial acne, psoriasis, hair loss; atopic and contact dermatitis; onychomycosis; fungal infections affecting the nails, infections caused by dermatophytes such as Microsporum canis and Trichophyton mentagrophytes, Candida or non-dermatophytic molds; Dermatophytosis and superficial skin infections caused by dermatophytes such as Trichophyton, Microsporum and Epidermophyton; infections caused by Malassezia spp., deep and systemic mycosis such as pulmonary or systemic infections caused by Aspergillus spp.; urinary tract infections, systemic infections caused by Histoplasma capsulatum; infections caused by Cryptococcus neoformans; vaginal infections caused by Candida albicans, Candida glabrata, Candida parapsilosis, Candida kefir, Gardnerella vaginalis; Mycosis of the skin and scalp such as Tinea capitis and Tinea corporis in adult humans and children, fungal infections affecting skin folds, interdigital spaces or the nail and periungual areas; treatment of chronic ulcerations: such as diabetic or venous ulcers, skin abscesses, bacterial rhinitis, bacterial bronchitis, sinusitis; gastrointestinal infections, peptic ulcer disease caused by Helicobacter pylori, bacterial cystitis; bacterial prostatitis; antibiotic resistance, methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa diseases, viral infections such as HIV, SARS-CoV-2, flu virus infections, respiratory syncytial virus, herpes simplex (HSV-1, HSV-2); HPV skin or mucosal viral infections such as human papillomavirus, viral hepatitis such as hepatitis B (HBV) and hepatitis C (HCV), viral infections of the central nervous system such as neurotropic viruses (e.g. Herpes zoster, rabies virus), systemic viral infections such as HIV,infectious mononucleosis (Epstein-Barr virus, EBV), gastrointestinal infections by Rotavirus and Norovirus; solid tumors such as breast cancer, lung cancer, liver cancer; glioblastoma, hematological cancers such as leukemia: acute myeloid leukemia (AML), lymphoma; gastrointestinal cancers such as gastric and colorectal cancer, melanoma and skin cancers, gynaecological cancers such as ovarian and cervical carcinoma; vascular diseases such as ischemia and hypertension; Alzheimer's or Parkinson's, anti-aging therapies such as oxidative hormesis.

15. A method for preparing the composition according to anyone of claims 1-9 comprising the steps of:(i) preparing the carrier by mixing water and the rheological agent, and optionally one or more additives, obtaining an aqueous carrier;(ii) bringing the carrier to room temperature;(iii) adding, at a temperature < 30°C, the aqueous solution based on ROS deriving from the splitting of ozone to the carrier;(iv) mixing until homogenized while maintaining the temperature < 30°C;(v) using the resulting composition or transferring it to containers / dispensers for packaging.

16. A kit comprising the composition according to anyone of claims 1- 9 in single pack or predefined dosing units, optionally a reusable applicator or a disposable applicator containing effective doses of the composition, instructions for use optionally accessible remotely through an electronic means.

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