Formulation comprising ozonized oil for the prevention of aging and of the damage linked thereto

Abstract

The present invention relates to the use of a formulation comprising ozonized oil for the prevention of aging and of the damage linked thereto. In particular, it relates to said formulation for use to prevent or treat pathologies related to aging. The cosmetic use of the formulation also falls within the scope of the present invention.

Description

[0001] FORMULATION COMPRISING OZONIZED OIL FOR THE PREVENTION OF AGING AND OF THE DAMAGE LINKED THERETO

[0002] Field of application

[0003] The present invention relates to the use of a formulation comprising ozonized oil for the prevention of aging and of the damage linked thereto.

[0004] Prior art

[0005] It has long been demonstrated that aging is due to the accumulation of oxidative DNA damage (P. Alexander, The role of DNA lesions in processes leading to aging. Symp. Soc. Exp. Biol. 21, 1967. 29-50). Over the years, data have been provided which support the hypothesis that DNA is progressively damaged during aging. It has been demonstrated that different types of DNA lesions are linked to age, including the increase in fragmentation (Holmes et al., Oxidative and other DNA damages as the basis of aging: a review. Mutat. Res. 275 1992. 305-315). On this basis, DNA lesions due to oxidative damage have been proposed as aging biomarkers (Ames et al., Endogenous mutagens and the causes of aging. Mutat. Res. 250 1991. 3-16). Some studies have demonstrated that substantial quantities of nitrogenous bases of DNA, 8-hydroxy-2'-deoxyguanosine (or 8-OH-dG), have been detected in the liver, brain and heart with a progressive accumulation correlated with the aging of these organs over the course of life. Most of the increase in age-related oxidative DNA damage has been observed in organs composed mainly of perennial cells, that is, the heart and brain. These results have demonstrated that the progressive accumulation of oxidative DNA damage is at the basis of aging (Izzotti et al., Age-related increases of 8-hydroxy-2'-deoxyguanosine and DNA protein cross-link in mouse organs. Mutat. Res. 446: 215-223, 1999).

[0006] Aging is a process characterised by a progressive loss of physiological activities which occurs on different levels. On a cellular level, one may distinguish various characteristics, among which the most peculiar are cellular senescence, telomere shortening and the level of proteins associated with aging (Lopez-Otin et al., The hallmarks of aging. Cell. 2013 Ja 6; 153(6): 1194-217). Aging may be characterised by a progressive loss of physiological integrity, with a consequent impairment of functions and susceptibility to disease and death. At the cellular level, there are two main characteristics of the aging process: shortening of telomere length and cellular senescence. Telomeres are tandem nucleotide repeats situated at the ends of the chromosomes that maintain genomic stability. Telomeres are shortened during replication (mitosis) due to the intrinsic inability to completely replicate the final part of the lagging strand of DNA (Tsoukalas et al., Association of nutraceutical supplements with longer telomere length. Int J Mol Med. 2019; 44:218-26). Telomeres measure between 4 and 15 kilobases in length and are gradually shortened at a rate proportional to the rate of body aging.

[0007] Telomere shortening is also associated with various environmental factors such as stress, lack of physical resistance activity, excess body weight, cigarette smoking, chronic inflammation, vitamin deficiencies and, above all, high levels of oxidative stress (Armanios M., Telomeres and age-related disease: how telomere biology informs clinical paradigms. J Clin Invest. 2013; 123:996-1002). Cellular senescence is a cell-cycle arrest which may be caused by telomere shortening, as well as other stimuli associated with aging, such as non-telomeric DNA damage. The purpose of senescence is to prevent the propagation of damaged cells by triggering their elimination through the immune system. The accumulation of senescent cells with aging, in particular precisely in the immune system, reflects an increase in the generation of these cells and / or a decrease in their clearance, which in turn exacerbates the damage and contributes to aging.

[0008] An increasing number of studies have found various pharmacological agents that can reduce aging biomarkers and the telomere shortening rate (Townsley et al., Danazol treatment for telomere diseases. N Engl J Med. 2016; 374:1922-31). Various lifestyle interventions, including resistance training, diets and food supplements can modify the telomere shortening rate by focusing on cell metabolism and oxidative stress, though to date the entity of this reduction has been relatively small, namely in the order of the 2-5% compared to the untreated group (Richards et al., Higher serum vitamin D concentrations are associated with longer leukocyte telomere length in women. Am J Clin Nutr. 2007; 86:1420-25).

[0009] The possibility of activating endogenous antioxidant defences by administering oxygen, with an approach defined as hormetic, has been postulated (Izzotti et al., The molecular mechanisms of adaptive response related to environmental stress. Int. J. Mol. Sci. 21 : E7053. (2020)).

[0010] One method of approach applied to anti-aging medicine is hyperbaric oxygen therapy. Hyperbaric oxygen therapy (HBOT) uses 100% oxygen at an ambient pressure above one atmosphere to increase the amount of oxygen dissolved in body tissues. Repeated intermittent hyperoxic exposures can induce physiological effects such as an increase in tissue oxygen, the induction of antioxidant systems and the activation of gene-dependent tissue repair mechanisms regulated by the nuclear erythroid factor 2 (Nrf2). Furthermore, it has recently been demonstrated that the administration of hyperbaric oxygen can induce cognitive improvements in healthy individuals and decrease aging through mechanisms involving 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).

[0011] At a cellular level, the administration of hyperbaric oxygen can induce the expression of vascular endothelins and sirtuin, proliferation and differentiation of stem cells, and mitochondrial biogenesis (Hadanny et al., The hyperoxic-hypoxic paradox. Biomolecules. 2020; 10:958). A recent study demonstrated that the administration of hyperbaric oxygen in humans for 3 months in 60 sessions lasting 1 hour each is capable of decreasing aging biomarkers, with particular reference to cellular immunosenescence and telomere shortening (Hachmo et al., Hyperbaric oxygen therapy increases telomere length and decreases immunosenescence in isolated blood cells: a prospective trial. Aging, 12: 2245, 2020).

[0012] However, hyperbaric oxygen therapy is a very complex treatment that requires the availability of hyperbaric chambers and a prolonged stay of the treated individuals in such chambers.

[0013] There is thus a felt need in the sector to provide a formulation for the prevention or slowing of aging and of the damage related thereto, in particular cellular senescence, which overcomes the drawbacks and limits of the treatments of the prior art.

[0014] Summary of the invention A similar problem has been solved according to the invention by the use of a formulation comprising at least one ozonized oil for the prevention or slowing of aging and / or to reduce or slow one or more symptoms of aging and / or to reduce, slow or prevent cellular senescence in an individual.

[0015] The subject matter of the invention also relates to a formulation comprising at least one ozonized oil for use for the prevention and / or treatment of a pathology due to or related to aging. This pathology may be, for example, 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 kidney disease or disorder, a liver disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disorder or disease, or a geriatric disease or disorder.

[0016] The subject matter of the invention also relates to the non-therapeutic use, in particular cosmetic use, of a formulation comprising at least one ozonized oil, preferably 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 an individual. In a preferred embodiment, said use is cosmetic and is for the prevention and / or treatment of skin aging, in particular to prevent, reduce or treat the signs of skin aging.

[0017] "Prevention of aging” means blocking or slowing the aging of an individual.

[0018] "Treatment of aging” means the regression of aging, in particular the signs and symptoms of aging, of an individual.

[0019] "Aging” means the set of changes that occur in cells and in cellular tissues with increasing age. This term includes every type of aging, for example it includes the aging of every type of cell, tissue or organ. "Senescence” is a synonym.

[0020] "Reduce or slow the symptoms of aging” means to block, decrease or slow the development, progression and / or advancement of one or more symptoms or signs of aging, such as, for example, degeneration of one or more tissues, skin aging, weakening of the immune system, slowing or loss of one or more cognitive functions, for example attention capacity, concentration, memory, language, and balance, or mood alterations.

[0021] "Cellular senescence” means a biological process in which the cell is no longer able to replicate and undergoes a progressive loss of physiological functions.

[0022] "Skin aging” means the gradual decline and deterioration of the skin, due to cellular senescence and / or to external factors, such as environmental factors, pollution or the individual's lifestyle.

[0023] In one embodiment, the prevention of aging and / or reduction or slowing of one or more symptoms of aging and / or the reduction or the slowing of cellular senescence are evaluated by measuring one or more aging biomarkers. Aging biomarkers are biological parameters that can be indicative and predictive, typically together with other parameters, of the progression of aging. Said aging biomarkers are known in the art. Preferably, said biomarker is one or more of cellular immunosenescence of helper T lymphocytes and / or cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein. These biomarkers can be evaluated by means of the common methods known in the art. In particular, since the aging of T cells causes a progressive decrease in the expression of the CD28 receptor at the level of their outer membrane, the number of CD28 null T lymphocytes relative to helper (CD4+) and cytotoxic (CD8+) T lymphocytes represents a marker of aging and cellular immunosenescence. The telomere length can be determined in hematopoietic cells by means of the common methods and compared with the length of telomeres in control cells. The expression of the Klotho protein can be evaluated in blood plasma by solid-phase antibody colorimetric immunoreaction (enzyme-linked immunosorbent assay - ELISA).

[0024] The subject matter of the invention also relates to the use of a formulation comprising at least one ozonized oil to reduce at least one aging biomarker in an individual, wherein said biomarker is preferably selected from cellular immunosenescence of helper T and cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein.

[0025] Preferably, the formulation comprising at least one ozonized oil is for use by oral administration.

[0026] In another preferred embodiment, the formulation comprising at least one ozonized oil is for use by topical administration.

[0027] Preferably, the ozonized oil has a content of ozonides from 500 to 1500 meq 02 / kg, more preferably from 600 to 1400 meq 02 / kg, even more preferably from 700 to 1300 meq 02 / kg, even more preferably from 1100 to 1300 meq 02 / kg, most preferably 1200 meq 02 / kg.

[0028] The content of ozonides can be measured according to techniques known in the art, for example by means of the method for the determination of the peroxide index described in Regulation (EEC) No 2568 / 91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis (Annex III).

[0029] In accordance with the present invention and appended claims, the expression "ozonized oil” means: a vegetable oil that has undergone an ozonation process; an unsaturated fatty acid, preferably having a number of carbon atoms from 8 to 30, more preferably from 16 to 20, which has undergone an ozonation process; an ester of an unsaturated fatty acid, preferably having a number of carbon atoms from 8 to 30, more preferably from 16 to 20, which can be monoester, diester or triester, with an alcohol or a polyol, in particular glycerol, which has undergone an ozonation process; or mixtures thereof.

[0030] Preferably, the at least one ozonized oil is selected from: at least one vegetable oil that has undergone an ozonation process; at least one unsaturated fatty acid that has undergone an ozonation process; at least one monoester of an unsaturated fatty acid that has undergone an ozonation process; and mixtures thereof; more preferably at least one vegetable oil that has undergone an ozonation process.

[0031] Preferably, the at least one vegetable oil that has undergone an ozonation process is selected from: sunflower oil, peanut oil, argan oil, olive oil, grapeseed oil, jojoba oil, soybean oil, maize 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, peanut oil, argan oil, grapeseed oil, jojoba oil, soybean oil, maize oil, palm oil, cottonseed oil, rapeseed oil, coconut oil, castor oil, linseed oil, borage oil, evening primrose oil, and mixtures thereof; even more preferably sunflower oil and / or peanut oil, most preferably sunflower oil.

[0032] Preferably the at least one unsaturated fatty acid that has undergone an ozonation process has a number of carbon atoms from 8 to 30, more preferably from 16 to 20, even more preferably 18. Preferably, the at least one unsaturated fatty acid has from one to six double bonds in the carbon chain, more preferably from one to three double bonds in the carbon chain.

[0033] Preferably, the at least one fatty acid is monounsaturated (and thus has a single double bond in the carbon chain) or diunsaturated (i.e. it has two double bonds in the carbon chain).

[0034] Preferably, the at least one fatty acid is oleic acid or linoleic acid.

[0035] Preferably, the at least one monoester of an unsaturated fatty acid that has undergone an ozonation process is obtained from the esterification of the aforesaid unsaturated fatty acid as described above, the monoester having undergone an ozonation process.

[0036] Preferably, the at least one monoester of an unsaturated fatty acid is obtained from the esterification of the aforesaid unsaturated fatty acid with an aliphatic primary alcohol having a single -OH group (monovalent), more preferably ethanol.

[0037] Preferably, the at least one monoester of an unsaturated fatty acid is ethyl oleate.

[0038] Preferably, the at least one ozonized oil is substantially devoid of antioxidant agents.

[0039] Preferably, the at least one ozonized oil is obtained from an ozonation process that uses highly purified gaseous oxygen for the introduction of oxygen.

[0040] As regards oral administration, the at least one ozonized oil can be in the form of an ozonized oil as such or in a formulation for oral administration comprising the at least one ozonized oil and at least one pharmaceutically acceptable excipient.

[0041] The formulation for oral administration can be in a form selected from oil, syrup, tablets and capsules, more preferably capsules.

[0042] Preferably, the capsules are gastro-resistant.

[0043] Preferably, the capsules are selected from soft gel-based capsules, and gastro-resistant capsules, more preferably gastro-resistant capsules.

[0044] Preferably, the composition of the gastro-resistant capsules comprises at least one coating agent, preferably hydroxypropyl methylcellulose, and at least one gelling agent, preferably selected from pectin, gellan gum, and mixtures thereof.

[0045] The formulation in capsules can be in liquid form or in solid form, preferably solid form, even more preferably in powder or granules.

[0046] Preferably, the capsules each contain from 50 to 200 mg, more preferably from 140 to 160 mg, most preferably 150 mg, of ozonized oil.

[0047] The at least one pharmaceutically acceptable excipient is selected from those commonly used in formulations for oral administration. It should be observed that such excipients preferably do not include any preservative or antioxidant agent. In fact, such agents interact with ozonized oil, as they are subject to oxidation by the ozonide groups.

[0048] As regards the dosage of oral administration, it varies according to the patient's conditions and the stage of the pathology. Preferably, the oral administration of the ozonized oil is carried out by administering a quantity of ozonized oil from 75 to 1500 mg per day per 70 kg of body weight, more preferably from 150 to 1200 mg per day per 70 kg of body weight, even 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.

[0049] Preferably, the oral administration is carried out once or twice a day, more preferably twice a day.

[0050] Preferably, the oral administration of the ozonized oil is carried out for a period of at least one month, more preferably from one month to 3 years, even more preferably from 2 months to 14 months, most preferably from 5 months to 12 months.

[0051] According to a preferred embodiment, the aforesaid formulation is for use by topical administration. In particular, said administration is preferred for the treatment of skin aging, more in particular for the cosmetic use of the formulation described herein.

[0052] The formulation for topical use can consist of the aforesaid at least one ozonized oil or it can comprise the aforesaid at least one ozonized oil, preferably in a concentration, expressed as a percentage by weight compared to the total weight of the formulation, from 1 to 50%, even more preferably from 2 to 20%, most preferably from 3 to 15%.

[0053] In a preferred embodiment, the formulation for topical use of the present invention further comprises a pharmaceutically acceptable vehicle, compatible with the ozonized oil.

[0054] Preferably, the formulation for topical use of the invention is in a form selected from oil, gel, and emulsion, more preferably oil.

[0055] The gel can be a hydrogel or a lipogel.

[0056] Preferably, the hydrogel comprises, in a percentage by weight out of the total weight of the hydrogel, from 3 to 30%, more preferably from 5 to 20%, most preferably from 8 to 15% of at least one ozonized oil.

[0057] The term "hydrogel” means here that the formulation is in the form of a gelled hydrophilic solution in which the ozonized oil is dispersed in an aqueous medium.

[0058] The term "lipogel” means here that the formulation is in the form of a gelled lipophilic solution in which the ozonized oil is dispersed in a lipid medium.

[0059] According to a preferred aspect of the present invention, the topical use is carried out by topically applying the formulation on the affected tissue, preferably for a period longer than three days and shorter than sixty days, more preferably from ten to thirty days.

[0060] Preferably, the topical application of the formulation is carried out once or twice a day, more preferably twice a day.

[0061] In some embodiments, the use according to the present invention comprises both topical administration and oral administration.

[0062] Preferably, the formulation is to be administered to a mammal, animal or human, aged over 40 years.

[0063] In one embodiment, the ozonized oil is in the form of a dietary supplement or nutraceutical.

[0064] Recent studies have opened the possibility of providing oxygen to tissues not through pulmonary respiration but directly through tissue respiration, by delivering to tissues a precursor of oxygen, ozone, suitably conjugated with a lipid carrier (Izzotti et al., 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 by ozonized oils. Jpers. Med., 11 : 226-242 (2021)).

[0065] It has now been found that the formulation of high-ozonide ozonized oil of the invention administered orally prevents, in treated subjects, cellular aging and the progressive increase of aging biomarkers. The ozonized oil developed is formulated in gastro-resistant capsules and duly adsorbed so that it can be taken by mouth. The oral administration of the formulation according to the invention is much more practicable and manageable compared to exogenous treatments with hyperbaric oxygen, which are moreover not devoid of risks, including accidental events such as fires and explosions. The clinical experience gained in recent years has also demonstrated that this type of treatment is very safe and does not induce any type of significant side effect.

[0066] The efficacy of this treatment in decreasing the level of aging biomarkers was determined in 28 subjects treated with ozonized oil orally according to the common clinical practice of ozone therapy. The aim of this evaluation was to determine whether the treatment with ozonized oil is capable of slowing cellular aging and, if so, to compare the entity 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).

[0067] The subject matter of the present invention relates to a use of a formulation comprising at least one ozonized 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 an individual.

[0068] Preferably, in said use of the formulation, the formulation comprises at least one ozonized oil for use for the prevention and / or treatment of a pathology due to or related to aging.

[0069] Preferably, in said use of the formulation, the formulation is for use in a pathology due to or related to aging selected from the group comprising or, alternatively, consisting of 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 kidney disease or disorder, a liver disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disorder or disease, or a geriatric disease or disorder.

[0070] Preferably, in said use of the formulation, said ozonized oil is for oral or topical administration or a combination thereof.

[0071] Preferably, in said use of the formulation, said ozonized oil has a content of ozonides from 500 to 1500 meq 02 / kg, preferably from 600 to 1400 meq 02 / kg, more preferably from 700 to 1300 meq 02 / kg, even more preferably from 1100 to 1300 meq 02 / kg, most preferably 1200 meq 02 / kg.

[0072] Preferably, in said use of the formulation, said at least one ozonized oil is selected from: at least one vegetable oil that has undergone an ozonation process; at least one unsaturated fatty acid that has undergone an ozonation process; at least one monoester of an unsaturated fatty acid that has undergone an ozonation process; and mixtures thereof; preferably at least one vegetable oil that has undergone an ozonation process.

[0073] Preferably, in said use of the formulation, said at least one vegetable oil that has undergone an ozonation process is selected from: sunflower oil, peanut oil, argan oil, olive oil, grapeseed oil, jojoba oil, soybean oil, maize 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, maize 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.

[0074] Preferably, in said use of the formulation, said at least one ozonized oil is an unsaturated fatty acid that has undergone an ozonation process, wherein said unsaturated fatty acid is selected from oleic acid and linoleic acid or combinations thereof.

[0075] Preferably, in said use of the formulation, said at least one ozonized oil is in the form of an ozonized oil as such or in a formulation for oral administration comprising said at least one ozonized oil and at least one pharmaceutically acceptable excipient.

[0076] Preferably, in said use of the formulation, said formulation for oral administration is in a form selected from oil, syrup, a tablet and a capsule, preferably a capsule, even more preferably a gastro-resistant capsule.

[0077] Preferably, in said use of the formulation, said capsule contains from 50 to 200 mg, preferably from 140 to 160 mg, more preferably 150 mg, of ozonized oil.

[0078] Preferably, in said use of the formulation, the oral administration of the ozonized oil is carried out by administering a quantity of ozonized 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.

[0079] Preferably, in said use of the formulation, the oral administration of the ozonized oil is carried out for a period of at least one month, preferably from one month to 3 years, more preferably from 2 months to 14 months, most preferably from 5 months to 12 months.

[0080] Preferably, in said use of the formulation, said formulation comprising at least one ozonized oil is for reducing at least one aging biomarker in an individual, wherein said biomarker is preferably selected from cellular immunosenescence of helper T lymphocytes and / or cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein.

[0081] The subject matter of the present invention relates to a formulation comprising at least one ozonized oil for use for the prevention and / or treatment of a pathology due to or related to aging, wherein said pathology due to or related to aging is selected from the group comprising or, alternatively, consisting of 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 kidney disease or disorder, a liver disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disorder or disease, or a geriatric disease or disorder. Preferably, said ozonized oil is for oral or topical administration, or a combination thereof.

[0082] Preferably, said ozonized oil has a content of ozonides from 500 to 1500 meq 02 / kg, preferably from 600 to 1400 meq 02 / kg, more preferably from 700 to 1300 meq 02 / kg, even more preferably from 1100 to 1300 meq 02 / kg, most preferably 1200 meq 02 / kg.

[0083] Preferably, said at least one ozonized oil is selected from: at least one vegetable oil that has undergone an ozonation process; at least one unsaturated fatty acid that has undergone an ozonation process; at least one monoester of an unsaturated fatty acid that has undergone an ozonation process; and mixtures thereof; preferably at least one vegetable oil that has undergone an ozonation process.

[0084] Preferably, said at least one vegetable oil that has undergone an ozonation process is selected from: sunflower oil, peanut oil, argan oil, olive oil, grapeseed oil, jojoba oil, soybean oil, maize 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, maize 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.

[0085] Preferably, said at least one ozonized oil is an unsaturated fatty acid that has undergone an ozonation process, wherein said unsaturated fatty acid is selected from oleic acid and linoleic acid or combinations thereof.

[0086] Preferably, said at least one ozonized oil is in the form of an ozonized oil as such or in a formulation for oral administration comprising said at least one ozonized oil and at least one pharmaceutically acceptable excipient. Preferably, said formulation for oral administration is in a form selected from oil, syrup, a tablet and a capsule, preferably a capsule, even more preferably a gastro-resistant capsule.

[0087] Preferably, said capsule contains from 50 to 200 mg, preferably from 140 to 160 mg, more preferably 150 mg, of ozonized oil.

[0088] Preferably, the oral administration of the ozonized oil is carried out by administering a quantity of ozonized 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.

[0089] Preferably, the oral administration of the ozonized oil is carried out for a period of at least one month, preferably from one month to 3 years, more preferably from 2 months to 14 months, most preferably from 5 months to 12 months.

[0090] Preferably, said formulation is for reducing at least one aging biomarker in an individual, wherein said biomarker is preferably selected from cellular immunosenescence of helper T lymphocytes and / or cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein.

[0091] The subject matter of the present invention relates to a cosmetic use of a formulation comprising at least one ozonized oil as described above, for the prevention and / or treatment of skin aging, preferably to prevent, reduce or treat the signs of skin aging.

[0092] Preferably, said formulation is for topical administration. Additional features and advantages of the present invention will be apparent from the following detailed description.

[0093] Brief description of the Figures

[0094] Figure 1. Cellular immunosenescence of CD4+ helper T lymphocytes at TO and T1. The vertical axis indicates the level of expression of the CD4 antigen common to all lymphocytes with helper T cell action. The horizontal axis indicates the level of expression of the CD28 antigen absent in senescent T lymphocytes. The treatment with ozonized oil decreases this parameter by increasing the number of T cells which express CD28.

[0095] Figure 2. Cellular immunosenescence of CD8+ cytotoxic T lymphocytes at TO and T1. The vertical axis indicates the level of expression of the CD8 antigen common to all cytotoxic T lymphocytes. The horizontal axis indicates the level of expression of the CD28 antigen absent in senescent T lymphocytes. The treatment with ozonized oil decreases this parameter by increasing the number of T lymphocytes which express CD28.

[0096] Figure 3. Analysis of telomere length by flow cytometry at TO and T1 . The vertical axis indicates the level of expression of the fluorescent probe that identifies the telomeres. The horizontal axis indicates the telomere length for each cell (single points) measured in number of nucleotide bases or kilobases. The treatment with ozonized oil increases this parameter.

[0097] Figure 4. Cellular immunosenescence of helper T lymphocytes at TO and T1. HOC = high-ozonide ozonized oil administered orally for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions over 3 months.

[0098] Figure 5. Cellular immunosenescence of cytotoxic T lymphocytes at TO and T1. HOC = high-ozonide ozonized oil administered orally for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions over 3 months.

[0099] Figure 6. Relative telomere length (RTL) at TO and T1. HOC = high-ozonide ozonized oil administered orally for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions over 3 months.

[0100] Figure 7. Analysis of plasma Klotho protein values. HOO = high-ozonide ozonized oil administered orally for 3 months. HBOT = hyperbaric oxygen therapy for 60 daily sessions over 3 months.

[0101] Detailed description of the invention

[0102] The present invention will now be further illustrated by means of some example embodiments as set forth here below.

[0103] EXAMPLE 1

[0104] METHODS

[0105] TREATMENT

[0106] The evaluation of aging biomarkers was carried out on 28 subjects who underwent, under medical supervision as per the common clinical practice of ozone therapy, oral administration of ozonized oil at a dose of 3 gastro-resistant capsules in the morning and 3 in the evening (size 00), the content in each capsule being equal to 75 mg of adsorbed oil. The subjects were in good health and aged between 54 and 67 years, 20 females and 8 males.

[0107] The gastro-resistant capsule contained a solid formulation in granules comprising 75 mg of ozonized oleic acid having a content of ozonides of 1200 meq 02 / kg and pharmaceutically acceptable excipients. The gastro- resistant capsule was a conventional gastro-resistant capsule based on hydroxypropyl methylcellulose, pectin and gellan gum.

[0108] The treatment lasted for 3 months, that is, for a period of time analogous to the one normally adopted for the prevention of aging by oxygen therapy in a hyperbaric chamber (Hachmo et al., 2020).

[0109] Whole blood samples of thirty adults were collected in EDTA test tubes at baseline and after 60 days of treatment. The Ficoll protocol was carried out to collect peripheral blood mononuclear cells (PBMCs) and plasma: a phosphate-buffered saline solution (PBS) was used to dilute the whole blood, and the separation in a density gradient was carried out using Lympholyte Cell Separation Media (Cedarlane). The test tubes were centrifuged at 2200 rpm for 20 minutes and the plasma was transferred into a new test tube and immediately frozen. The buffy coat was then gently transferred into a new test tube, washed twice in PBS and finally resuspended in RPM 1-1640 medium with 10% FBS at a concentration of 2 x 106 cells / mL.

[0110] The plasma was used for the detection of the KLOTHO protein in the ELISA protocol, whereas the PBMCs were used both for the immunophenotyping protocols and for the determination of telomere length.

[0111] The subjects gave their express free and informed consent to the collection and use of the collected blood.

[0112] ANALYSES PERFORMED

[0113] Cellular aging analyses were performed in relation to 3 biomarkers based on well-established scientific evidence of their correlation with cellular aging phenomena: cellular immunosenescence in the case of helper T and cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein.

[0114] The integrated use of 4 biomarkers enables an overall evaluation of the state of aging of the subject under examination. The analyses were carried out before (TO) and after (T1) treatment with ozonized oil administered orally with the aim of evaluating the influence of the treatment on the dynamics of cellular aging and its capacity to slow them.

[0115] CELLULAR IMMUNOSENESCENCE

[0116] T lymphocytes are a subpopulation of white blood cells that are effectors of cell-mediated immunity. The maintenance of an adequate functional state thereof characterises individuals with low levels of biological aging and is typical of individuals over a hundred years old. The aging of T lymphocytes brings about a progressive decrease in the expression of the CD28 receptor at the level of their outer membrane. Therefore, the presence of T lymphocytes devoid of CD28 (CD28 null T lymphocytes) is an important marker of aging and cellular immunosenescence.

[0117] The number of CD28 null T lymphocytes compared to helper T lymphocytes (CD4+) and cytotoxic T lymphocytes (CD8+) was determined by flow cytofluorometry and marking with fluorescent antibodies specific for the T cell antigens (CD4, CD8) and for the CD28 antigen. This number is expressed as a percentage of CD28 null senescent T lymphocytes compared to T lymphocytes. A laboratory protocol was used for immunophenotyping of the helper T and cytotoxic T cell subpopulations starting from PBMCs obtained by Ficoll separation. The purpose of the method is to identify senescent T lymphocytes by cytofluorometric analysis and to determine the levels of helper T and cytotoxic T lymphocytes associated with aging. The levels of CD3+, CD4+, CD28-null (senescent helper) T cells and CD3+, CD8+, CD28- null (senescent cytotoxic) T cells are determined and the percentage of CD28-nul I T cells within the populations of CD4+ or CD8+ T cells is then calculated.

[0118] The immunophenotyping procedure was conducted to determine the percentage of the senescent helper T and cytotoxic T subpopulations. The PBMCs were dyed with VioBlue anti-CD3 conjugated, PE-VIO 770A anti- CD4 conjugated, VioGreen anti-CD8 conjugated antibodies and APC-VIO 770A anti-CD28 antibodies (Miltenyi Biotec). The flow cytometry analysis was carried out to detect the levels of CD3+, CD4+, CD28-null (senescent helper) T cells and CD3+, CD8+, CD28-n ull (senescent cytotoxic) T cells. At the end, the percentage of cells CD28- null T cells among the populations of CD4+ and CD8+ T cells was calculated.

[0119] TELOMERE SHORTENING

[0120] Telomeres are polynucleotide elements located at the end of chromosomes in which irreparable DNA alterations are segregated and can thus be eliminated at every cell duplication. The telomere length therefore reflects the ability of a cell to repair DNA damage and its biological age. Aging brings about a progressive shortening of telomere length.

[0121] Telomere length was assessed by hybridisation with a fluorescent probe and flow cytometry. The relative telomere length (RTL) was determined by comparing the lymphocytes of the sample with control cells (tetrapioid 1301 cell line with telomeres longer than 30 kilobases). This method is much more specific and quantitatively reliable than determinations by PGR. The results are expressed as a percentage of telomere length versus the control cells. Therefore, the higher the percentage, the greater the telomere length in the cells analysed.

[0122] A commercially available kit was used to measure the telomeres in nucleated hematopoietic cells using a fluorescein-conjugated peptide nucleic acid (PNA) probe. The results are evaluated by flow cytometry using a light source with excitation at 488 nm and the relative telomere length (RTL) is determined by comparing the sample cells (PBMCs obtained by Ficoll separation) with the control cells (tetrapioid 1301 cell line with telomeres longer than 30 kilobases). The method starts with a pretreatment of the sample and control cells, which are mixed together and divided into separate test tubes. Then follows denaturation, and then overnight hybridisation with the telomere PNA probe / FITC in a single mixture. On the second day, washing steps are necessary before DNA staining in the dark. Finally, the samples are analysed with a flow cytometer, and the results expresses as RTL.

[0123] The telomere length was determined using a fluorescein-conjugated peptide nucleic acid probe according to the protocol of the Dako PNA kit / FITC (code K5327). A mixture of 2 x 106 PBMCs (sample cells) and 2 x 106 TCL 1301 cells (control cells) was separated into four test tubes: two were added with a hybridisation solution containing the fluorescein-conjugated telomere PNA probe, two without the probe, and the DNA was denatured at 82°C for 10 minutes. Overnight hybridisation in the dark at room temperature was followed by two washes with a wash solution at 40°C. Flow cytometry analysis was carried out, and the relative telomere length (RTL) was calculated as the ratio between the telomere length of the sample cells and control cells, with a correction for the DNA index of GO / 1 cells.

[0124] EXPRESSION OF THE ALPHA KLOTHO PROTEIN

[0125] The Klotho protein is involved in the expression of numerous growth factors necessary for maintaining tissue integrity. The levels of this protein decrease progressively with age, reflecting the progressive loss of the ability of tissues to regenerate.

[0126] Many studies have demonstrated the validity of the Klotho protein as an aging biomarker (Arking et al., Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A. 2002;99(2):856-61).

[0127] The expression of the Klotho protein in blood plasma was evaluated by solid-phase antibody colorimetric immunoreaction (enzyme-linked immunosorbent assay - ELISA). The Klotho protein is a glucuronidase capable of hydrolysing steroid glucuronides. The Klotho gene (KL) produces two molecules: one form linked to the membrane and a circulating form (hormonal, soluble). Mutations of the KL gene are associated with human aging, and the circulating protein is a factor detectable in serum which decreases with age. The Klotho protein binds different types of FGF ligands and regulates paths which suggest its correlation with anti-age activity. Measurement of the circulating Klotho protein is performed by means of a quantitative test, Human secreted Alpha Klotho ELISA, I BL America (USA).

[0128] RESULTS

[0129] The assessment was performed on 28 subjects who took ozonized oil under medical supervision as per common clinical practice. The overall results of the analysis are reported below.

[0130] OVERALL RESULTS AND COMPARISON WITH HYPERBARIC OXYGEN THERAPY

[0131] Cellular immunosenescence of helper T lymphocytes

[0132] The overall results (mean of the 28 subjects examined) of the values of helper T cell immunosenescence at TO and T 1 are shown in Figure 4 (top panel). The bottom panel shows the percentage variation in the parameter induced by 3 months of oral treatment with ozonized oil compared to that induced by 3 months of treatment with hyperbaric oxygen therapy (HBOT) (60 sessions).

[0133] The results obtained indicate that the treatment with ozonized oil significantly decreases the percentage of senescent cells after 3 months of treatment (top panel). The entity of this decrease is comparable to the one obtained by the treatment with HBOT, a much more invasive and complex treatment than the simple daily oral administration of capsules (bottom panel).

[0134] Cellular immunosenescence of cytotoxic T lymphocytes

[0135] The overall results (mean of the 28 subjects examined) of the values of cytotoxic T cell immunosenescence at TO and T 1 are shown in Figure 5 (top panel). The bottom panel shows the percentage variation in the parameter induced by 3 months of oral treatment with ozonized oil compared to that induced by 3 months of treatment with hyperbaric oxygen therapy (HBOT) (60 sessions).

[0136] The results obtained indicate that the treatment with ozonized oil significantly decreases the percentage of senescent cells after 3 months of treatment (top panel). The entity of this decrease is greater than the one obtained from the treatment with HBOT (bottom panel).

[0137] Telomere shortening

[0138] The overall results (mean of the 28 subjects examined) of the values of relative telomere length are shown in Figure 6 (top panel). The bottom panel shows the percentage variation in the parameter induced by 3 months of oral treatment with ozonized oil compared to that induced by 3 months of treatment with hyperbaric oxygen therapy (HBOT) (60 sessions).

[0139] The results obtained indicate that the treatment with ozonized oil significantly decreases the telomere shortening induced by aging. Indeed, after 3 months of treatment one observes a significant increase in the telomere length. The entity of this lengthening is much greater (2.9 times) than the one obtained from the treatment with HBOT (bottom panel).

[0140] Plasma levels of Klotho protein

[0141] The overall results (mean of the 28 subjects examined) of the plasma Klotho protein values are shown in Figure 7 (top panel). The bottom panel shows the percentage variation in the parameter induced by 3 months of oral treatment with ozonized oil compared to that induced by 3 months of treatment with hyperbaric oxygen therapy (HBOT) (60 sessions).

[0142] The results obtained indicate that the treatment with ozonized oil significantly increases the plasma levels of Klotho protein, with a 54.8% increase in only 3 months of treatment. The Klotho protein levels were not analysed in the study with HBOT.

[0143] DISCUSSION AND CONCLUSIONS

[0144] The results obtained clearly indicate that the treatment with the particular formulation of ozonized oil administered orally is effective in modulating aging biomarkers. In particular, for all those analysed one observes a significant reversal of aging phenomena, typical of effective interventions of anti-aging medicine. A decrease in the degenerative phenomena related to aging may be seen for cellular immunosenescence, a parameter that directly impacts on immune system function and thus on the susceptibility to many diseases. The effectiveness with which the degenerative phenomena of aging are prevented by the oral treatment with ozonized oil is even greater as regards the change in telomere length and the plasma Klotho protein levels. In particular, for both of these biomarkers, a significative and quantitatively relevant increase was found after only 3 months of treatment.

[0145] The anti-aging effect of orally administered ozonized oil was demonstrated to be greater for 3 biomarkers (immuno-senescence of cytotoxic T lymphocytes, telomere lengthening, increase in plasma Klotho protein levels), much greater than those obtained with 3 months of HBOT.

[0146] In conclusion, the results obtained demonstrate that oral administration of ozonized oil in the specific formulation used is an effective tool for the prevention of the degenerative phenomena linked to aging. This treatment thus represents an innovation of particular relevance in anti-aging medicine, given the absence of side effects, already demonstrated in several previous studies, and the simplicity of administration compared to the prior art.

Claims

CLAIMS1. A formulation comprising at least one ozonized oil for use for the prevention and / or treatment of a pathology due to or related to aging, wherein said pathology due to or related to aging is selected from the group comprising or, alternatively, consisting of 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 kidney disease or disorder, a liver disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disorder or disease, or a geriatric disease or disorder.

2. The formulation for use according to claim 1 , wherein said ozonized oil is for oral or topical administration, or a combination thereof.

3. The formulation for use according to claim 1 or 2, wherein said ozonized oil has a content of ozonides from 500 to 1500 meq 02 / kg, preferably from 600 to 1400 meq 02 / kg, more preferably from 700 to 1300 meq 02 / kg, even more preferably from 1100 to 1300 meq 02 / kg, most preferably 1200 meq 02 / kg.

4. The formulation for use according to any one of claims 1-3, wherein said at least one ozonized oil is selected from: at least one vegetable oil that has undergone an ozonation process; at least one unsaturated fatty acid that has undergone an ozonation process; at least one monoester of an unsaturated fatty acid that has undergone an ozonation process; and mixtures thereof; preferably at least one vegetable oil that has undergone an ozonation process.

5. The formulation for use according to claim 4, wherein said at least one vegetable oil that has undergone an ozonation process is selected from: sunflower oil, peanut oil, argan oil, olive oil, grapeseed oil, jojoba oil, soybean oil, maize 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, maize 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.

6. The formulation for use according to claim 4 or 5 wherein said at least one ozonized oil is an unsaturated fatty acid that has undergone an ozonation process, wherein said unsaturated fatty acid is selected from oleic acid and linoleic acid or combinations thereof.

7. The formulation for use according to any one of claims 1-6, wherein said at least one ozonized oil is in the form of an ozonized oil as such or in a formulation for oral administration comprising said at least one ozonized oil andat least one pharmaceutically acceptable excipient.

8. The formulation for use according to claim 7, wherein said formulation for oral administration is in a form selected from oil, syrup, a tablet and a capsule, preferably a capsule, even more preferably a gastro-resistant capsule.

9. The formulation for use according to claim 8, wherein said capsule contains from 50 to 200 mg, preferably from 140 to 160 mg, more preferably 150 mg, of ozonized oil.

10. The formulation for use according to any one of claims 1-9, wherein the oral administration of the ozonized oil is carried out by administering a quantity of ozonized 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.

11. The formulation for use according to any one of claims 1-10, wherein the oral administration of the ozonized oil is carried out for a period of at least one month, preferably from one month to 3 years, more preferably from 2 months to 14 months, most preferably from 5 months to 12 months.

12. The formulation for use according to any one of claims 1-11 to reduce at least one aging biomarker in an individual, wherein said biomarker is preferably selected from cellular immunosenescence of helper T lymphocytes and / or cytotoxic T lymphocytes, telomere shortening, and expression of the alpha Klotho protein.

13. A cosmetic use of a formulation comprising at least one ozonized oil in accordance with any one of claims 2- 11 , for the prevention and / or treatment of skin aging, preferably to prevent, reduce or treat the signs of skin aging.

14. The cosmetic use according to claim 13, wherein said formulation is for topical administration.

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