Formulation including ozonated oil for the prevention of aging and related damage

Description

FORMULATION INCLUDING OZONIZED OIL FOR THE PREVENTION OF AGING AND DAMAGE TO IT RELATED ------- 5 Scope of application The present invention relates to the use of a formulation including ozonated oil for the prevention of aging and damage related to it. 10 Known art It has long been proven that aging is due to the accumulation of oxidative damage to DNA (P. Alexander, The role of DNA lesions in leading processes to aging, Symp. Soc. Exp. Biol. 21, 1967. 29–50). In the 15 years of data have been provided to support the hypothesis that DNA is progressively damaged during aging. It has been shown that different types of DNA lesions are age-related, including increased of fragmentation (Holmes et al., Oxidative and other 20 DNA damages as the basis of aging: a review, Mutat. Res. 275 , 275 1992. 305–315). On this basis, the lesions of the DNA damage due to oxidative damage has been proposed as biomarkers of aging (Ames et al., Endogenous mutagens and the causes of aging Mutat. Res. 250 1991. 3–16). Some studies have shown that appreciable amounts of 8-hydroxy-DNA nitrogenous bases 2'-deoxyguanosine (or 8-OH-dG) has been detected in 5 liver, brain and heart with a progressive aging-related accumulation of these organs over the course of life. The greatest increase in damage oxidative stress on DNA related to aging has been observed in organs composed of cells 10 mostly perennial, namely heart and brain. These results have shown that the progressive accumulation of Oxidative damage to DNA is the basis of aging (Izzotti et al., Age-related increases of 8-hydroxy-2'- deoxyguanosine and DNA protein cross-link in mouse 15 organs. Mutat. Res., 446: 215-223, 1999). Aging is a process characterized by a progressive loss of physiological activities that occurs at different levels. At the cellular level it can be distinguish different characteristics including the most 20 peculiarities are cellular senescence, shortening telomere length and protein levels associated with aging (López-Otín et al., The hallmarks of aging. Cell 2013 Jun 6;153(6):1194-217). Aging can be characterized by progressive loss of physiological integrity, with resulting in impaired functions and susceptibility to disease and death. At the 5 mobile phone, there are two main features of the aging process: shortening of the Telomere length and cellular senescence. Telomeres are tandem nucleotide repeats located at the ends of chromosomes that maintain stability 10 genomics. Telomeres shorten during replication (mitosis) due to intrinsic inability to completely replicate the final part of the filament DNA retardation (Tsoukalas et al., Association of nutraceutical supplements with longer telomere length. 15 Int J Mol Med. 2019; 44:218–26). The length of the Telomeres measure between 4 and 15 kilobases and gradually shortens with speed proportional to the speed of the aging of the organism. Telomere shortening is also associated with 20 different environmental factors such as stress, lack of endurance physical activity, excess body weight, cigarette smoking, chronic inflammation, deficiency of vitamins and, above all, high levels of stress oxidative (Armanios M. Telomeres and age-related disease: how telomere biology informs clinical paradigms. J Clin Invest. 2013; 123:996–1002). There cellular senescence is an arrest of the cell cycle 5 which can be caused by shortening of telomeres, as well as other stimuli associated with aging such as non-telomeric DNA damage. The purpose of senescence is to prevent propagation of damaged cells triggering the 10 their elimination through the immune system. The accumulation of senescent cells with aging, in particular in the immune system, reflects an increase in the generation of these cells and / or a decrease in their clearance, which in turn aggravates 15 damage and contribute to aging. A growing body of research has found several pharmacological agents that can reduce biomarkers of aging and the rate of telomere shortening (Townsley et al. Danazol treatment for telomere 20 diseases. N Engl J Med. 2016; 374:1922–31). Different lifestyle interventions, including strength training resistance, diets and food supplements can modify the rate of telomere shortening targeting cellular metabolism and stress oxidative although the extent of this reduction is so far unknown relatively small, i.e. in the order of 2-5% compared to the untreated group (Richards et al., Higher 5 serum vitamin D concentrations are associated with longer leukocyte telomere length in women. Am J Clin Nutr. 2007; 86:1420–25). The possibility of activating the endogenous antioxidant defenses through the 10 oxygen administration, with a defined approach hormetic (Izzotti et al., The molecular mechanisms of adaptive response related to environmental stress. Int. J. Mol. Sci. 21: E7053. (2020)). A method of approach applied to medicine 15 anti-aging is hyperbaric oxygen therapy. Hyperbaric oxygen therapy (HBOT) uses oxygen at 100% at an ambient pressure above a atmosphere to increase the amount of oxygen dissolved in body tissues. Repeated exposures 20 intermittent hyperoxic may induce effects physiological such as increased tissue oxygen, the induction of antioxidant systems and the activation of gene-dependent tissue repair mechanisms regulated by nuclear erythroid factor 2 (Nrf2). Furthermore, It has recently been demonstrated that the administration hyperbaric oxygen can induce improvements cognitive in healthy subjects and reduce aging 5 through mechanisms involving changes regional changes in cerebral blood flow (Hadanny et al. Cognitive enhancement of healthy older adults using hyperbaric oxygen: a randomized controlled trial. Aging, 2020; 12:13740–61). 10 At the cellular level, the administration of hyperbaric oxygen can induce the expression of vascular endothelins and sirtuin, proliferation and stem cell differentiation, biogenesis mitochondrial (Hadanny et al., The hyperoxic-hypoxic 15 paradoxes. Biomolecules. 2020; 10:958). A recent study has demonstrated in humans that the administration of hyperbaric oxygen for 3 months in 60 sessions lasting of 1 hour each is able to decrease the biomarkers of aging with particular reference to 20 cellular immuno-senescence and shortening telomeric (Hachmo et al., Hyperbaric oxygen therapy increases telomere length and decreases immunosenescence in isolated blood cells: a prospective trial. Aging, 12: 2245, 2020). However, hyperbaric oxygen therapy is a very complex treatment that requires the availability of hyperbaric chambers and the stay 5 prolonged in such rooms of the treated subjects. There is therefore a need in the sector to put a formulation is available for the prevention or treatment slowing down of aging and damage to it related, in particular cellular senescence, 10 that overcomes the drawbacks and limitations of treatments of known art. Summary of the invention A similar problem has been solved according to the invention from the use of a formulation comprising 15 at least one ozonated oil for prevention or slowing down the aging process and / or to reduce or slow down one or more symptoms of aging and / or to reduce, slow down, or prevent cellular senescence in a subject. 20 It is also an object of the invention a formulation comprising at least one ozonated oil for the use for the prevention and / or treatment of a pathology due to or related to aging. Such pathology can be for example a metabolic disease, an inflammatory disease or disorder, including a autoimmune disease or disorder, a disease or disorder neurological or neurodegenerative disorder, a disease 5 or a lung or respiratory disorder, a disorder proliferative, a kidney disease or disorder, a liver disorder, a disease or disorder cardiovascular, a fibrotic disease or disorder, a viral infection, disorder, or disease 10 dermatological, or a geriatric disease or disorder. The non-use of the invention is also an object of the invention. therapeutic, particularly cosmetic use, of a formulation comprising at least one ozonated oil, preferably for the prevention of aging 15 and / or to reduce or slow down one or more symptoms of aging and / or to reduce, slow down or prevent cellular senescence in a subject. In a preferred embodiment, said use is cosmetic and is for the prevention and / or treatment of aging 20 skin, in particular to prevent, reduce or treat the signs of skin aging. By “prevention of aging” we mean the blocking or slowing down of aging subject. By “treatment of aging” we mean the regression of aging, especially signs and symptoms of aging, of the subject. 5 By “aging” we mean the set of changes that occur in cells and tissues cell phones as we age. This term includes each type of aging, for example includes the aging of any type of cell, tissue or 10 organ. “Senescence” is a synonym. To “reduce or slow down symptoms of aging” means to block, decrease or slow the development, progression and / or the progression of one or more symptoms or signs 15 of aging, such as degeneration of one or more tissues, skin aging, weakening of the immune system, slowing down or loss of one or more cognitive functions, for example attention span, concentration, memory, 20 language, balance, mood swings. By “cellular senescence” we mean a process biological in which the cell is no longer able to replicate and undergoes a progressive loss of physiological functions. By “skin aging” we mean the gradual decline and deterioration of the skin, due to cellular senescence and / or external factors, such as 5 environmental factors, pollution or lifestyle subject. In one implementation, prevention of aging and / or the reduction or slowing down of one or more symptoms of aging and / or the reduction 10 or the slowing of cellular senescence are assessed by measuring one or more biomarkers of aging. Biomarkers of aging are biological parameters that can be indicative and predictive, typically 15 together with other parameters, of the progression of aging. These biomarkers of aging are known in the industry. Preferably, said biomarker is one or more of cellular immuno-senescence of helper T lymphocytes and / or 20 cytotoxic T lymphocytes, telomere shortening, and expression of the Klotho alpha protein. These biomarkers can be assessed using common methodologies known in the sector. In particular, since the aging of T lymphocytes causes a progressive decreased expression of the CD28 receptor level of their outer membrane, the number of CD28 null lymphocytes compared to helper T lymphocytes (CD4+) 5 and cytotoxic (CD8+) represents a marker of aging and cellular immuno-senescence. Telomere length can be assessed in cells hematopoietic using common methods and compared with telomere length in cells of 10 control. Klotho protein expression can be assessed in blood plasma by solid-phase colorimetric antibody immunoreaction (enzyme linked immunosorbed assay, ELISA). The use of a 15 formulation comprising at least one ozonated oil for reduce at least one biomarker of aging in a subject, where said biomarker is preferably chosen between immuno-cellular senescence of T lymphocytes helper and cytotoxic T cells, telomere shortening, and 20 expression of the Klotho alpha protein. Preferably, such formulation comprising at least one ozonated oil is for use by oral administration. In another preferred embodiment, such formulation comprising at least one ozonated oil is for use by topical administration. Preferably, the ozonated oil has a 5 ozonide content from 500 to 1500 meq O / kg, plus preferably 600 to 1400 meq O / kg, even more preferably 700 to 1300 meq O / kg, even more preferably 1100 to 1300 meq O / kg, the most preferably 1200 meq O / kg. 10 The ozonide content can be measured according to techniques known in the sector, for example through the method for determining the peroxide number described in the Regulation (EEC) No. 2568 / 91 of 11 July 1991 on to the characteristics of olive oils and olive oils 15 olive pomace and related methods (Annex III). In accordance with the present invention and claims attached, with the expression “oil “ozonated” means: a vegetable oil that has been 20 subjected to an ozonation process; an acid unsaturated fat, preferably having a number of carbon atoms from 8 to 30, more preferably from 16 at 20, who was subjected to a trial of ozonation; an ester of an unsaturated fatty acid, preferably having a number of carbon atoms from 8 to 30, more preferably 16 to 20, which can be monoester, diester or triester, with an alcohol or a 5 polyol, especially glycerol, which has been subjected to an ozonation process; or their mixtures. Preferably, the at least one ozonated oil is chosen from: at least one vegetable oil that has been 10 subjected to an ozonation process; at least one unsaturated fatty acid that has been subjected to a ozonation process; at least one monoester of a unsaturated fatty acid, which has been subjected to a ozonation process; and their mixtures; more 15 preferably at least one vegetable oil that has been subjected to an ozonation process. Preferably, at least one vegetable oil that is been subjected to an ozonation process is chosen between: sunflower oil, peanut oil, argan oil, 20 olive oil, grapeseed oil, jojoba oil, oil soybean oil, corn oil, palm oil, sunflower oil cotton, rapeseed oil, coconut oil, castor oil, linseed oil, borage oil, evening primrose oil, and their mixtures; more preferably sunflower oil, peanut, argan oil, grapeseed oil, jojoba oil, soybean oil, corn oil, palm oil, olive oil cottonseed oil, rapeseed oil, coconut oil, 5 castor oil, linseed oil, borage oil, evening primrose oil, and their mixtures; even more preferably olive oil sunflower and / or peanut oil, most preferably sunflower oil. Preferably at least one unsaturated fatty acid 10 which has been subjected to an ozonation process It has a number of carbon atoms from 8 to 30, plus preferably 16 to 20, even more preferably 18. Preferably, at least one unsaturated fatty acid 15 has one to six double bonds in the chain carbonaceous, more preferably one to three double bonds in the carbon chain. Preferably the at least one fatty acid is monounsaturated (and therefore has only one double bond in the 20 carbon chain) or diunsaturated (i.e. it has two double bonds in the carbon chain). Preferably, the at least one fatty acid is a fatty acid oleic or linoleic acid. Preferably, the at least one monoester of an acid unsaturated fat, which has been subjected to a process of ozonation, is obtained by the esterification of the the aforementioned unsaturated fatty acid as described above, the 5 monoester having been subjected to a process of ozonation. Preferably, the at least one monoester of acid unsaturated fat is obtained by the esterification of the aforementioned unsaturated fatty acid with a primary alcohol 10 aliphatic having only one –OH group (monovalent), plus preferably ethanol. Preferably, the at least one monoester of acid unsaturated fat is ethyl oleate. Preferably, the at least one ozonated oil is 15 substantially free of antioxidants. Preferably, the at least one ozonated oil is obtained from an ozonation process that uses highly purified oxygen gas for introduction of oxygen. 20 Regarding oral administration, at least one ozonated oil can be in the form of ozonated oil as such or in a formulation for administration oral comprising at least one ozonated oil and at least a pharmaceutically acceptable excipient. The formulation for oral administration can be in a form chosen between oil, syrup, tablets and capsules, more preferably capsules. 5 Preferably, the capsules are gastro-resistant. Preferably, the capsules are chosen from capsules soft gel-based capsules, and gastro-resistant capsules, more preferably gastro-resistant capsules. Preferably, the composition of the capsules 10 gastro-resistant includes at least one agent coating, preferably hydroxypropyl- methylcellulose, and at least one gelling agent, preferably chosen from pectin, gellan gum, and their mixtures. 15 The formulation in capsules can be in the form liquid or in solid form, preferably solid, even more preferably in powder or granular form. Preferably, the capsules each contain 50 to 200 mg, more preferably 140 to 160 mg, the most 20 preferably 150 mg, of ozonated oil. The at least one pharmaceutically active excipient acceptable is selected from those in common use for formulations for oral administration. It is necessary note that such excipients do not include preferably no preservative or antioxidant. In fact, these agents interact with ozonated oil, as they are subject to oxidation 5 by the ozonide groups. Regarding the dosage of administration by route oral, it is variable, depending on the conditions of the patient and the progress of the disease. Preferably, oral administration 10 of the ozonated oil is performed by the administration of a quantity of ozonated oil from 75 to 1500 mg per day per 70 kg of body weight, plus preferably 150 to 1200 mg per day per 70 kg of body weight, even more preferably from 200 to 900 15 mg per day per 70 kg of body weight, the most preferably 450 to 700 mg per day per 70 kg of body weight. Preferably, oral administration is performed once or twice a day, more preferably 20 twice a day. Preferably, oral administration of ozonated oil is performed for a period of at least one month, more preferably from one month to 3 years, again more preferably from 2 months to 14 months, the most preferably 5 months to 12 months. According to a preferred embodiment, the the above formulation is for use by administration 5 topical. In particular, this administration is preferred for the treatment of aging skin, more specifically for cosmetic use of the formulation described here. The formulation for topical use can be 10 consisting of the above at least one ozonated oil or may include the aforementioned at least one ozonated oil, preferably in a concentration, in percentage in weight relative to the total weight of the formulation, from 1 to 50%, even more preferably 2 to 20%, the most 15 preferably from 3 to 15%. In a preferred embodiment, the formulation for topical use which is the subject of this The invention also includes a vehicle pharmaceutically acceptable, oil-compatible 20 ozonated. Preferably, the formulation for topical use of the invention is in a form selected from oil, gel, and emulsion, most preferably oil. The gel can be a hydrogel or a lipogel. Preferably, the hydrogel comprises, in percentage by weight of the total weight of the hydrogel, from 3 to 30%, more preferably from 5 to 20%, the most 5 preferably 8 to 15% of at least one oil ozonated. The term “hydrogel” here means that the formulation is in the form of a hydrophilic solution gelled in which the ozonated oil is dispersed in a 10 medium aqueous. The term “lipogel” here means that the formulation is in the form of a lipophilic solution gelled in which the ozonated oil is dispersed in a lipid medium. 15 According to a favorite aspect of the present invention, topical use is performed by topical application of the formulation on the tissue interested, preferably for a period longer than three days and less than sixty days, more 20 preferably from ten to thirty days. Preferably, topical application of the formulation is performed once or twice a day, more preferably twice a day. In some embodiments, the use according to this invention includes both topical administration and oral administration. Preferably, the formulation is to be administered 5 to an animal or human mammal over the age of 40 years. In one embodiment, the ozonated oil is under in the form of a food supplement or nutraceutical. Recent research has opened the possibility of 10 provide oxygen to the tissues not through the pulmonary respiration but directly through the tissue respiration by conveying a precursor of oxygen, ozone, appropriately conjugated with a lipid carrier (Izzotti et al., 15 Efficacy of high-ozonide oil in prevention of cancer relapses. Mechanisms and clinical evidence. Cancers, 14: 1174-1198 (2022); Izzotti et al., Prevention of Covid- 19 infection and related complications from ozonized oils. J pers. Med., 11: 226-242 (2021)). 20 It has now been found that the oil formulation ozonized with high ozonides of the invention administered orally prevents in treated subjects cellular aging and the progressive increase in Biomarkers of aging. Ozonated oil developed is formulated in gastro-resistant capsules and suitably adsorbed so that it can be taken orally. Oral administration of the 5 formulation according to the invention is much more practicable and manageable compared to exogenous treatments with hyperbaric oxygen, which is not without risks, however including accidental events such as fires and explosions. Clinical experience carried out in recent 10 years has also shown that this type of treatment It is very safe and does not induce any significant side effects. side effect. The effectiveness of this treatment in reducing the level of aging biomarkers was 15 determined in 28 subjects treated with ozonated oil orally according to common clinical practice practices of ozone therapy. The aim of this evaluation was determine if ozonated oil treatment is in able to slow down cellular aging and, in 20 if so, compare the magnitude of the effect obtained with that of hyperbaric oxygen therapy (Hachmo et al., Hyperbaric oxygen therapy increases Telomere length and decreases immunosenescence in isolated blood cells: a prospective trial. Aging, 12: 2245, 2020). Additional features and benefits of this invention will result from the following description 5 detailed. Brief description of the Figures Figure 1. Cellular immune senescence of T lymphocytes CD4+ helper T0 and T1. The vertical axis indicates the level of expression of the CD4 antigen common to all 10 T helper lymphocytes. The horizontal axis indicates the level of expression of the CD28 antigen is absent in Senescent lymphocytes. Treatment with ozonated oil. decrease this parameter by increasing the number of lymphocytes expressing CD28. 15 Figure 2. Cellular immuno-senescence of T lymphocytes cytotoxic CD8+ T0 and T1. The vertical axis indicates the expression level of the CD8 antigen common to all cytotoxic lymphocytes. The horizontal axis indicates the CD28 antigen expression level absent in 20 senescent lymphocytes. Treatment with ozonated oil. decrease this parameter by increasing the number of lymphocytes expressing CD28. Figure 3. Telomere length analysis by flow cytometry T0 and T1. The vertical axis indicates the level of expression of the probe fluorescent that identifies telomeres. The axis horizontal indicates the length of the telomeres for 5 each cell (single points) measured in number of nucleotide bases or kilobases. Treatment with oil ozonized increases this parameter. Figure 4. Cellular immunosenescence of lymphocytes T helper at T0 and T1. HOO = high ozonide ozonated oil 10 administered orally daily for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions for 3 months. Figure 5. Cellular immunosenescence of lymphocytes Cytotoxic T at T0 and T1. HOO = high ozonated oil 15 ozonides administered orally daily for 3 months. HBOT = hyperbaric oxygen therapy for 60 sessions daily for 3 months. Figure 6. Relative telomere length (RTL) at T0 and T1. HOO = ozonated oil with high ozonides 20 administered orally daily for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions for 3 months. Figure 7. Analysis of Klotho protein values plasma. HOO = high ozonized ozonated oil administered orally daily for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions for 3 months. 5 Detailed description of the invention The present invention will now be further illustrated through some examples of implementation such as reported below. EXAMPLE 1 10 METHODS TREATMENT Evaluation of biomarkers of aging It was carried out on 28 subjects who took under medical check-up for common clinical practice 15 ozone therapy oral administration of oil ozonated at a dose of 3 gastro-resistant capsules morning and 3 in the evening (size 00), for a content in each capsule equals 75 mg of adsorbed oil. subjects were in good health with ages ranging from 54 20 to 67 years old, 20 females and 8 males. The gastro-resistant capsule contained a solid formulation in granules comprising 75 mg of ozonated oleic acid having an ozonide content of 1200 meq O / kg and pharmaceutically excipients acceptable. The gastro-resistant capsule was a capsule conventional gastro-resistant based on hydroxypropyl- methylcellulose, pectin and gellan gum. 5 The treatment lasted for 3 months, that is, for a period of time similar to that normally practiced for the prevention of aging through oxygen therapy in a hyperbaric chamber (Hachmo et al., 2020). 10 Whole blood samples from thirty adults are were collected in EDTA tubes at baseline and after 60 days of treatment. The protocol was followed Ficoll to collect blood mononuclear cells peripheral blood (PBMC) and plasma: a 15 phosphate buffered saline (PBS) to dilute whole blood and density gradient separation was performed using Lympholyte Cell Separation Media (Cedarlane). The tubes were centrifuged at 2200 rpm for 20 minutes and the plasma was 20 moved to a new test tube and frozen immediately. The buffy coat was then transferred gently into a new test tube, washed twice in PBS and finally resuspended in RPMI-1640 medium 10% FBS at concentration of 2 x 106 cells / mL. Plasma was used for detection of the KLOTHO protein in the ELISA protocol while the PBMCs have been used both for the protocols of 5 immunophenotyping which for the determination of the telomere length. The express, free and unrestricted consent was obtained informed of the subjects to the collection and use of the blood collected. 10 ANALYSES PERFORMED Aging analyses were carried out cellular relative to 3 consolidated biomarkers scientific evidence for their correlation with the cellular aging phenomena: immuno-senescence 15 cell for helper T lymphocytes and cytotoxic T lymphocytes, telomere shortening, protein expression Klotho alpha. The integrated use of 4 biomarkers allows a overall assessment of the state of aging 20 of the subject under examination. The analyses were performed before (T0) and after (T1) treatment with ozonated oil administered orally for the purpose of evaluating the influence of such treatment on the dynamics of cellular aging and its ability to slow them down. CELLULAR IMMUNO-SENESCENCE T lymphocytes are the subpopulation of blood cells 5 white effectors of cell-mediated immunity. The maintaining their adequate functional state characterizes subjects with low levels of biological aging and is typical of subjects centenarians. The aging of T lymphocytes causes 10 a progressive decrease in the expression of the CD28 receptor at their outer membrane. Therefore, the presence of T lymphocytes lacking CD28 (CD28 null T lymphocytes) is an important marker of aging and cellular immuno-senescence. 15 The number of CD28 null lymphocytes compared to helper (CD4+) and cytotoxic (CD8+) T lymphocytes were evaluated by flow cytometry and labeling with antigen-specific fluorescent antibodies of T cells (CD4, CD8) and for the CD28 antigen. This 20 number is expressed as a percentage of CD28 T lymphocytes null senescent compared to T lymphocytes. A laboratory protocol was used for immunophenotyping of T subpopulations helper and cytotoxic T cells from PBMCs obtained by Ficoll separation. The aim of the method is identify senescent T cells by analysis flow cytometry and determine T cell levels 5 helper and cytotoxic T cells associated with aging. The levels of CD3+, CD4+ T cells are determined. CD28-null (senescent T media) and CD3+ T cells, CD8+, CD28-null (senescent cytotoxic T cells) and is then calculated the percentage of CD28-null T cells 10 within the CD4+ or CD8+ T cell populations. The immunophenotyping procedure was conducted to determine the percentage of senescent T helper and cytotoxic T cell subpopulations. PBMCs were stained with anti-CD3 antibodies 15 VioBlue conjugates, anti-CD4 PE-VIO 770A conjugates, anti- VioGreen-conjugated CD8 and anti-CD28 APC-VIO antibodies 770A (Miltenyi Biotec). Flow cytometric analysis was performed to detect the levels of T cells CD3+, CD4+, CD28-null (senescent T helper) and T cells 20 CD3+, CD8+, CD28-null (senescent cytotoxic T cells). Finally, the percentage of CD28- T cells was calculated. null between CD4+ and CD8+ T cell populations. TELOMERIC SHORTENING Telomeres are polynucleotide elements located at the end of the chromosomes where the non-repairable DNA alterations that can thus be eliminated at each cell duplication. 5 Telomere length therefore reflects the ability of the cell to repair DNA damage and its age biological. Aging causes a progressive shortening of telomere length. Telomere length was assessed by 10 fluorescent probe hybridization and cytometry flow. Relative telomere length (RTL) was determined by comparing the lymphocytes in the sample with control cells (tetraploid 1301 cell line with telomeres longer than 30 kilobases). This method is 15 much more specific and quantitatively reliable compared to the PCR assessments. The results are expressed as a percentage of telomere length compared to control cells. Therefore, greater the greater the percentage the telomere length 20 in the cells analyzed. A commercially available kit was used available for telomere detection in nucleated hematopoietic cells using a probe fluorescein-conjugated peptide nucleic acid (PNA). The results are evaluated by cytometry flow using a light source with excitation at 488 nm and the length is determined 5 relative telomere length (RTL) comparing cells sample (PBMC obtained by Ficoll separation) with control cells (cell line 1301, tetraploids and with telomeres longer than 30 kilobases). The method begins with a pretreatment of the cells 10 sample and control, which are mixed together and divided into separate test tubes. Denaturation then follows. and then overnight hybridization with the PNA probe telomere / FITC in a single mixture. On the second day washing steps are necessary before coloring 15 of the DNA in the dark. Finally, the samples are analyzed on a flow cytometer and the results expressed as RTL. Telomere length has been determined using a peptide nucleic acid probe 20 fluorescein conjugated according to the kit protocol Dako PNA / FITC (code K5327). A mixture of 2 x 106 PBMC (sample cells) and 2 x 106 TCL 1301 cells (control cells) was separated into four test tubes: two were added with a solution of hybridization containing the telomere probe of PNA conjugated with fluorescence, two without the probe and DNA was denatured at 82°C for 10 minutes. Hybridization 5 night in the dark at room temperature was followed from two washes in washing solution at 40°C. It was flow cytometry analysis and length were performed relative telomere length (RTL) was calculated as the ratio of the length of the telomeres of cells 10 sample and control cells with correction for the DNA index of G0 / 1 cells. KLOTHO ALPHA PROTEIN EXPRESSION The Klotho protein is involved in the expression of numerous growth factors necessary for the 15 maintenance of tissue integrity. The levels of This protein progressively decreases with age reflecting the progressive loss of capacity of the tissues to regenerate. Many studies have demonstrated the validity of the 20 Klotho protein as a biomarker of aging (Arking et al., Association of human aging with a functional variant of klotho. Proc Natl Acad Sci US A. 2002;99(2):856-61). Klotho protein expression in plasma blood was evaluated by immunoreaction solid-phase antibody colorimetric assay (enzyme linked) immunosorbed assay, ELISA). The Klotho protein is a 5 glucuronidase capable of hydrolyzing glucuronides steroids. The Klotho (KL) gene produces two molecules: one membrane-bound form and a circulating form (hormonal, soluble). Mutations of the KL gene are associated with human aging and the protein 10 circulating is a factor detectable in serum that decreases with age. The Klotho protein binds several FGF ligand types and regulation pathways suggest its correlation with anti-aging activity. The detection of circulating Klotho protein is 15 performed by quantitative ELISA Human secreted test Alpha Klotho RLISA IBL America (USA). RESULTS The evaluation was carried out on 28 subjects who took ozonated oil under medical supervision 20 which is common clinical practice. The following are reported the overall results of the analysis. OVERALL RESULTS AND COMPARISON WITH OXYGEN- HYPERBARIC THERAPY Cellular immunosenescence of helper T lymphocytes The overall results (average of the 28 subjects examined) of the cellular immuno-senescence values ​​of the Helper T lymphocytes at T0 and T1 are shown in Figure 4 5 (upper panel). The lower panel shows the percentage change of the parameter induced by 3 months of treatment with ozonated oil by mouth compared to to that induced by 3 months of oxygen treatment- hyperbaric oxygen therapy (HBOT) (60 sessions). 10 The results obtained indicate that the treatment with ozonated oil significantly decreases the percentage of senescent cells after 3 months of treatment (top panel). The extent of this decrease is comparable to that obtained by 15 HBOT treatment, a much more invasive treatment and complex of the simple daily administration of oral capsules (bottom panel). Cellular immunosenescence of T lymphocytes cytotoxic 20 The overall results (average of the 28 subjects examined) of the cellular immuno-senescence values ​​of the cytotoxic T lymphocytes at T0 and T1 are reported in Figure 5 (top panel). In the bottom panel is the percentage change of the parameter is reported induced by 3 months of treatment with ozonated oil for os compared to that induced by 3 months of treatment with hyperbaric oxygen therapy (HBOT) (60 sessions). 5 The results obtained indicate that the treatment with ozonated oil significantly decreases the percentage of senescent cells after 3 months of treatment (top panel). The extent of this decrease is higher than that obtained by 10 HBOT treatment (lower panel). Telomere shortening The overall results (average of the 28 subjects examined) of the relative length values ​​of telomeres are reported in Figure 6 (top panel). In the 15 The percentage change is shown in the lower panel of the parameter induced by 3 months of treatment with oil ozonized orally compared to that induced by 3 months of hyperbaric oxygen therapy (HBOT) treatment (60 sessions). 20 The results obtained indicate that the treatment with ozonated oil it decreases significantly induced telomere shortening from aging. In fact, after 3 months of treatment observes a significant increase in the length of the telomeres. The extent of this elongation is very (2.9 times) higher than that obtained from treatment with HBOT (bottom panel). 5 Plasma Klotho protein levels The overall results (average of the 28 subjects (examined) plasma Klotho protein values ​​are reported in Figure 7 (top panel). In the panel the percentage change is shown below 10 parameters induced by 3 months of treatment with oil ozonized orally compared to that induced by 3 months of hyperbaric oxygen therapy (HBOT) treatment (60 sessions). The results obtained indicate that the treatment 15 with ozonated oil significantly increases levels plasma Klotho protein with a 54.8% increase in just 3 months of treatment. Protein levels Klotho were not analyzed in the HBOT study DISCUSSION AND CONCLUSIONS 20 The results obtained indicate unequivocally that treatment with the particular oil formulation ozonated administered orally is effective in modulate biomarkers of aging. In in particular for all those analyzed we observe a significant reversal of aging phenomena typical of effective anti-aging medicine interventions. The reduction of related degenerative phenomena 5 in aging is detectable for immuno-senescence mobile phone, a parameter that directly impacts the functionality of the immune system and therefore on the susceptibility to many diseases. The effectiveness with which the degenerative phenomena of aging are prevented 10 from treatment with ozonated oil by mouth is still greater with respect to the variation in length of telomeres and plasma Klotho protein levels. In particular, for both of these biomarkers, it was a significant and quantitatively relevant finding was detected 15 increase after only 3 months of treatment. The anti-aging effect of ozonated oil administered orally has been shown to be greater for 3 biomarkers (immuno-senescence T lymphocytes cytotoxic, telomere elongation, increased levels 20 plasma Klotho protein) much higher than those obtained with 3 months of HBOT. In conclusion, the results obtained demonstrate that oral administration of ozonated oil in the specific formulation used it is an effective tool for the prevention of degenerative phenomena related to aging. This treatment represents thus an innovation in anti-aging medicine 5 particular relevance given the absence of effects collateral effects already demonstrated in several previous studies and the ease of administration compared to the state of art. 10 Barzanò & Zanardo Milano SpA

Claims

1. Use of a formulation comprising at least one ozonated oil for the prevention of aging and / or to reduce or slow one or more symptoms of aging and / or to reduce, slow or prevent cellular senescence in a subject.

2. A formulation comprising at least one ozonated oil for use in the prevention and / or treatment of a disease due to or related to aging.

3. The formulation for use according to claim 2, wherein said pathology due to or related to aging is selected from a metabolic disease, an inflammatory disease or disorder, including an autoimmune disease or disorder, a neurological or neurodegenerative disease or disorder, a pulmonary or respiratory disease or disorder, a proliferative disorder, a renal disease or disorder, a liver disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disease or disorder, or a geriatric disease or disorder. Barzanò & Zanardo 4. Use of the formulation according to claim 1 or the formulation for use according to claim 2 or 3, wherein said ozonated oil is for oral or topical administration or a combination thereof.

5. Use of the formulation according to claim 1 or the formulation for use according to any of claims 2-4, wherein said ozonated oil has an ozonide content of 500 to 1500 meq O2 / kg, preferably 600 to 1400 meq O2 / kg, more preferably 700 to 1300 meq O2 / kg, even more preferably 1100 to 1300 meq O2 / kg, most preferably 1200 meq O2 / kg.

6. Use of the formulation according to claim 1 or the formulation for use according to any of claims 2-5, wherein said at least one ozonated oil is selected from: at least one vegetable oil that has been subjected to an ozonation process; at least one unsaturated fatty acid that has been subjected to an ozonation process; at least one monoester of an unsaturated fatty acid, which has been subjected to an ozonation process; and mixtures thereof; preferably at least one vegetable oil that has been subjected to an ozonation process.

7. The formulation for use according to claim 6, wherein said at least one vegetable oil that has been subjected to an ozonation process is selected from: sunflower oil, peanut oil, argan oil, olive oil, grapeseed oil, jojoba oil, soybean oil, corn oil, palm oil, cottonseed oil, rapeseed oil, coconut oil, castor oil, linseed oil, borage oil, evening primrose oil, and mixtures thereof; preferably sunflower oil, peanut oil, argan oil, grapeseed oil, jojoba oil, soybean oil, corn oil, palm oil, cottonseed oil, rapeseed oil, coconut oil, castor oil, linseed oil, borage oil, evening primrose oil, and mixtures thereof; more preferably sunflower oil and / or peanut oil, most preferably sunflower oil.

8. The formulation for use according to claim 6 wherein said at least one ozonated oil is an unsaturated fatty acid that has been subjected to an ozonation process, wherein said unsaturated fatty acid is selected from oleic acid and linoleic acid or combinations thereof. Barzanò & Zanardo 9. Use of the formulation according to claim 1 or the formulation for use according to any of claims 2-8, wherein said at least one ozonated oil is in the form of ozonated oil as such or in a formulation for oral administration comprising said at least one ozonated oil and at least one pharmaceutically acceptable excipient.

10. The formulation for use according to claim 9, wherein said formulation for oral administration is in a form selected from oil, syrup, tablet and capsule, preferably capsule, even more preferably gastro-resistant capsule.

11. The formulation for use according to claim 10, wherein said capsule contains from 50 to 200 mg, preferably from 140 to 160 mg, more preferably 150 mg, of ozonated oil.

12. Use of the formulation according to claim 1 or the formulation for use according to any of claims 2 to 11, wherein the oral administration of the ozonated oil is performed by administering an amount of ozonated oil from 75 to 1500 mg per day per 70 kg of body weight, preferably from 150 to 1200 mg per day per 70 kg of body weight, more preferably from 200 to 900 mg per day per 70 kg of body weight, most preferably from 450 to 700 mg per day per 70 kg of body weight.

13. Use of the formulation according to claim 1 or the formulation for use according to any of claims 2 to 12, wherein the oral administration of the ozonated oil is performed for a period of at least one month, preferably from one month to 3 months, more preferably from 2 months to 14 months, most preferably from 5 months to 12 months.

14. Use of a formulation comprising at least one ozonated oil to reduce at least one biomarker of aging in a subject, wherein said biomarker is preferably selected from cellular immunosenescence of helper T lymphocytes and / or cytotoxic T lymphocytes, telomere shortening, and Klotho alpha protein expression.

15. Cosmetic use of a formulation comprising at least one ozonated oil as described in claims 4 to 13, for the prevention and / or treatment of skin aging, preferably to prevent, reduce or treat the signs of skin aging.

16. The cosmetic use according to claim 15, wherein said formulation is for topical administration.