Formulations based on stable ozonides and use of such formulations for therapeutic applications in the human, veterinary, and agricultural fields

Abstract

The present invention relates to innovative formulations based on stable ozonides, developed for therapeutic applications in the human, veterinary, and agricultural fields. In particular, such stable ozonides comprised in the formulations are cis-methyl 8-(5-octyll,2,4- trioxolan-3-yl)octanoate, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis-3,5-dioctyl- 1,2,4-trioxolane, trans-3,5-dioctyl-l,2,4-trioxolane, cis-dimethyl 8,8'-(l,2,4-trioxolane-3,5- diyl)dioctanoate, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate. Such formulations, characterized by stable ozonides with specific cis / trans ratios, are designed for preventing and treating inflammatory diseases, for promoting tissue regeneration, and for antimicrobial and antiproliferative activities. They also find application in the agricultural field as enhancers of the endogenous defenses of plants. The invention offers advanced solutions for overcoming the limitations of the existing formulations, ensuring chemical stability, optimal delivery, and applicative versatility. Clinical applications include topical treatments for chronic wounds, inflammatory diseases, dermatitis, diabetic ulcers, and otitis, as well as oral and systemic administrations for inflammatory and cancerous conditions. Preclinical and clinical studies support the therapeutic efficacy, highlighting anti-inflammatory, regenerative, and antimicrobial properties. In the agricultural field, the formulation is used to improve the resistance of plants to biotic and abiotic stresses, with specific applications in the control of pests and in the preservation of the quality of crops.

Description

[0001] FORMULATIONS BASED ON STABLE OZONIDES AND USE OF SUCH FORMULATIONS FOR THERAPEUTIC APPLICATIONS IN THE HUMAN, VETERINARY, AND AGRICULTURAL FIELDS

[0002] The present invention relates to formulations based on stable ozonides developed for therapeutic applications in the human, veterinary, and agricultural fields. In particular, the present invention relates to formulations based on stable ozonide and the use of such formulations for the prevention and treatment of inflammatory diseases, for tissue regeneration, the promotion of antimicrobial and antiproliferative activities, as well as the use of such formulations in the human, animal, and plant fields.

[0003] Ozonated vegetable oils are widely used in the personal and animal care field and in the agricultural field, due to the several and recognized beneficial biological properties thereof, such as antimicrobial, anti-inflammatory, and regenerative properties. These products result from the reaction between ozone and the fatty acids present in vegetable oils, which leads to the formation of various chemical compounds including, primarily, ozonides.

[0004] To date, the scientific community agrees in supporting the thesis that the beneficial actions claimed by ozonated oils are to be assigned to oxidized substances resulting from the various ozonation processes and generally measured as peroxides. Although ozonides, among the families of oxidized compounds, are considered primarily responsible for the beneficial biological properties, there is no direct correlation, in the prior art, between the aforesaid activities and specific ozonides, but only with the class of oxidized compounds as a whole.

[0005] In the human and animal therapy field, there are known certain formulations which take advantage of the antimicrobial, anti-inflammatory, and tissue-regenerating activity of ozonated oils for topical applications. However, such solutions have significant problems and limitations, including:

[0006] • lack of a qualitative-quantitative characterization of the oxidized compounds, including ozonides;

[0007] • rapid degradation of ozonides under uncontrolled environmental conditions;

[0008] • lack of an effective delivery of active compounds into the target tissues;

[0009] • absence of scientific studies capable of demonstrating the correlation between ozonides and the related biological activities;

[0010] • absence of scientific studies capable of demonstrating the safety and toxicity aspects correlated with the content of ozonides in the various formulations.In the agricultural field, the use of ozonated vegetable oil formulations has been explored for enhancing the endogenous defenses of plants. However, the available formulations are often ineffective due to the low stability of the ozonides in liquid or solid matrices and the impossibility of quantitatively determining the ozonides in said formulations.

[0011] Many of the formulations based on ozonated oils containing ozonides are not supported by adequate preclinical and clinical studies demonstrating the actual therapeutic or protective capabilities thereof.

[0012] The present invention aims to solve the technical problems listed above, developing innovative formulations based on stable ozonides, with the following specific objectives:

[0013] • advanced chemical characterization, i.e., providing the complete chemical description of the individual stable ozonides and the biological activity associated thereto;

[0014] • chemical stability, i.e., ensuring the stability of the ozonides under different environmental conditions due to incorporation in vegetable oil matrices;

[0015] • optimal delivery, i.e., designing formulations which enable a controlled and targeted release of the active compounds, improving bio availability and thus therapeutic and / or protective efficacy;

[0016] • application versatility, i.e., creating formulations adapted to multiple routes of administration, including topical, oral, and systemic use in the human and veterinary fields, and specific methods of application for agriculture;

[0017] • adaptability to medical devices, i.e., integrating the formulations into creams, emulsions, lipogels, hydrogels, sprays, and other vehicles to broaden the therapeutic possibilities; • ecological compatibility and safety, i.e., using solvents and matrices compatible with safety and sustainability standards, minimizing the risks for the environment and the end consumers.

[0018] Furthermore, the present invention describes the use of such formulations in specific applications in different fields, providing adequate clinical and preclinical data supporting the different properties of the formulations described.

[0019] The present industrial invention relates to a formulation comprising cis and trans isomers of the following ozonides:

[0020] • cis-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate,

[0021] • trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate,• cis-3,5-dioctyl-l,2,4-trioxolane,

[0022] • trans-3,5-dioctyl-l,2,4-trioxolane,

[0023] • cis-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate,

[0024] • trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate.

[0025] In particular, said formulation comprises:

[0026] • cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate up to 55% w / w;

[0027] • cis,trans-3,5-dioctyl-l,2,4-trioxolane up to 20% w / w;

[0028] • cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate up to 25% w / w.

[0029] The molecules of the present invention are an innovation in the field of formulations comprising ozonides, offering several advantages.

[0030] Firstly, the complete characterization of the stable ozonides of the formulation allows assigning the observed biological activity to specific ozonides and not generally to the class of compounds as a whole.

[0031] The stable ozonides present in the formulation gradually release molecular oxygen, with the formation of oxidative species at low concentrations, which act as regulators of important cellular molecular pathways. Therefore, the moderate oxidative stress generated, acting on the RedOx balance, stimulates the activation of cellular antioxidant defense systems and the production of growth factors, so as to promote wound healing, tissue repair, and responses of anti-inflammatory, antimicrobial, and antiproliferative type.

[0032] Furthermore, the specific chemical features of these endoperoxide molecules, characterized by high stability, promote a low toxicity as compared to other highly reactive oxidized species (ROS), opening the way to new safe applications in the topical or systemic treatment of pathological conditions.

[0033] Advantageously, within each pair of diastereoisomers, the ratio of cis to trans forms is approximately 1:1.

[0034] In a preferred configuration, said formulation is further delivered in a vegetable or not, natural or synthetic, oil matrix.

[0035] The use of an oil matrix improves the bio availability of the formulation, facilitating a possible transdermal or systemic absorption in pharmaceutical applications. Furthermore, the oil matrix acts as a natural protectant against oxidation of the active substances, prolonging the shelf-life of the formulation itself.Said oil matrix is preferably extra virgin olive oil. The latter, being widely used in the food, cosmetic, and pharmaceutical industry, makes the formulation immediately usable in broad market contexts and fields.

[0036] Alternatively, said oil matrix is sunflower oil, with marked emollient, anti-inflammatory, and antioxidant properties.

[0037] Alternatively, said formulation is delivered in an organic solvent. The use of an organic solvent offers the advantage of rapid dissolution and a uniform application of the active ingredients. This choice allows a greater flexibility of use of the formulation, making it suited for applications in which a solubility of ozonides in aqueous biological means is required.

[0038] Preferably, but not exclusively, said organic solvent is selected from tetrahydrofuran, 2-methyltetrahydrofuran, DMSO, isopropanol, dichloromethane, chloroform, and diethyl ether. These organic solvents offer rapid evaporation, allowing a concentrated release of the active ingredient on the treated surface. Furthermore, said organic solvents are widely used in biological assays.

[0039] In a preferred configuration, in said formulation the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate are in an amount between 0.001 g and 100 g per 100g of oil matrix.

[0040] The formulation as described and in which the ozonides are in an amount between 0.001 g and 100 g per 100g of oil matrix finds use in the prevention and care of the health of humans, animals, and plants in the treatment of inflammatory diseases, support of tissue regeneration processes, and management of conditions characterized by infections or abnormal cellular proliferations.

[0041] In particular, the use of said formulation is particularly suitable for the prevention and / or treatment of issues related to inflammatory processes, in supporting tissue regeneration, in addition to antimicrobial activity.

[0042] Furthermore, MTT tests have confirmed an antiproliferative activity of the formulation of the dose-dependent type.

[0043] Again, the demonstrated antimicrobial, anti-inflammatory, regenerative, and antiproliferative activity makes the formulations based on stable ozonides particularly suitable for applications in the vulnological, surgical, dermatological, orthopedic, dental, proctological, gynecological, otorhinolaryngological, ophthalmic, geriatric, pediatric, oncological, and veterinary fields, and for the management of wounds and lesions, but also in agriculture.In fact, the use of said formulation is also particularly suitable in the treatment of plants, primarily as enhancers of plant defenses.

[0044] The indicated formulation based on stable ozonides is preferably administered topically, orally, or systemically for humans and animals, and in all the administration methods commonly used in the agricultural field, e.g., irrigation of soils, direct nebulization onto leaves, direct contact on branches and roots, or on pruning.

[0045] In particular, the described formulation is particularly suitable for topical administration in treating humans and animals when the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate contained therein are in an amount between 15 g and 40 g per 100 g of oil matrix.

[0046] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of humans and animals.

[0047] Ozonide formulations for topical use can be delivered as such or in creams, oleolites, emulsions, lipogels, hydrogels, alginates, on adsorbent physical supports such as sodium alginate fibers, polyurethanes, cotton gauze, viscose, processed with rheological additives, preservatives, surfactants, auxiliary active substances, fragrances and essential oils.

[0048] Such formulations defined for topical use are preferably used for manufacturing medical devices for the prevention and / or treatment of inflammatory diseases, support of tissue regeneration, and antimicrobial and antiproliferative activity.

[0049] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of balanitis xerotica obliterans in pediatric patients and animals.

[0050] Specifically, a formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 19 g of ozonides per 100g of plant matrix, was tested topically on preputial tissues taken from pediatric patients affected by balanitis xerotica obliterans(BXO), demonstrating inhibition of pro-inflammatory cytokines such as TNF-a, IL-ip and IFN-y, investigating the variation in the levels of gene transcripts, m-RNA, by means of Real-Time PCR in tissue fragments of patients treated with a formulation based on stable ozonides, with respect to untreated patients.

[0051] It has been demonstrated that stable ozonides induce the transcription factor HIF-la by activating the cellular response to hypoxia and angiogenesis. The analysis of the variation of the levels in gene transcripts, m-RNA, was carried out by Real-Time PCR. HIF-la induces the cascade expression of hundreds of genes, including several growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-a (TGF-a), and TGF-P, which are all involved in the complex multi-step tissue regeneration process. VEGF, one of the most potent pro-angiogenic growth factors of the skin, contributes to the restoration of blood flow, thereby providing oxygen, nutrients, and other mediators necessary to support the growth and function of reparative cells in damaged tissues. Patients treated with stable ozonides have shown a statistically significant increase in the gene expression of this factor. Furthermore, an increase in the levels of e-Cadherin, a transmembrane adhesion protein involved in cell-cell adhesion and necessary for the maintenance of epithelial integrity, has been shown.

[0052] Therefore, the topical treatment with stable ozonides induces the up-regulation of HIF-la, which in turn can trigger the re-epithelialization process by stimulating the first steps required for tissue regeneration, such as the reconstruction of angiogenesis and basement membrane.

[0053] The anti-inflammatory efficacy has also been further confirmed by comparative studies with topical corticosteroids: a comparison with mo metasone furoate (0.1%) showed that the action of stable ozonides is statistically significant and comparable to that of the corticosteroid, without side effects associated with conventional therapies.

[0054] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100 g of oil matrix, for the topical treatment of lichen sclerosus in pediatric patients and animals.

[0055] In particular, a formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 19 g of ozonides per 100 g of plant matrix, wastested on preputial tissues collected from pediatric patients affected by lichen sclerosus (LS). The results showed that the stable ozonides induce a controlled oxidative stress, capable of stimulating the endogenous antioxidant defenses of the cells without compromising essential cellular processes. Such a controlled stress contributes to maintaining the cellular redox balance, avoiding the accumulation of harmful free radicals.

[0056] The action mechanism of stable ozonides is based on the release of molecular oxygen with formation of oxidative species at low concentrations, which act as signaling molecules capable of regulating important molecular pathways. This modulation of the RedOx balance not only activates the cellular antioxidant defenses, but also promotes the production of growth factors, supporting wound healing, tissue repair, anti-inflammatory response, and antimicrobial activity.

[0057] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of children affected by phimosis associated with lichen sclerosus undergoing circumcision, exerting anti-inflammatory and antiproliferative activity.

[0058] In particular, a formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 19 g of ozonides per 100 of plant matrix, was tested in comparison with corticosteroids to evaluate the immune response and the proliferative pattern of lichen sclerosus in children undergoing circumcision. Through histological and immunohistochemical analyses of preputial samples, the infiltrate of immune cells (CD8+ and CD57+) and the markers of cell proliferation (Ki-67 and p53) were evaluated. The treatment with stable ozonides has shown to significantly reduce inflammation and cellular proliferation with respect to the untreated group. Furthermore, a greater reduction in proliferation markers (Ki-67 and p53) with respect to corticosteroids was observed, suggesting a more significant effect on tissue regeneration and epidermal integrity.

[0059] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are in an overall amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of phlebostatic ulcers in human patients.Specifically, a formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 30 g of ozonides per 100g of plant matrix, was tested on patients with phlebostatic ulcers located in the lower limbs.

[0060] The patients treated with stable ozonides exhibited a statistically significant reduction (P>0.007) in the mean re-epithelialization time with respect to the control group. Biopsies of the bed and margins of the ulcers topically treated with stable ozonides, then histologically analyzed, demonstrated stimulation of fibroblast proliferation and neoangiogenesis. Such results demonstrated a reduction in healing times and an improved quality of life.

[0061] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of cutaneous lesions in human and animal patients.

[0062] A formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane, and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 30 g of ozonides per 100 g of plant matrix, was tested on patients with chronic skin lesions located on legs and feet. The parameters analyzed were: area, infection, pain, WBP (Wound Bed Preparation), and occurrence of adverse events.

[0063] The results showed excellent efficacy of the formulation based on stable ozonides, with significant improvements in terms of reduction of the area of the lesions, control of the pain, and reduction of the infections, confirmed by the statistical analysis by means of linear ANOVA test (significant p). Furthermore, no adverse events were reported, highlighting the high safety profile of the product. The formulation has been shown to promote effective re-epithelization, attributed to the moderate oxidative stress induced by stable ozonides. This process contributes to a greater availability of oxygen at the local level, supporting the regeneration of tissue under optimal conditions, an effective control of inflammation, and a balanced bacterial bioburden. In addition, the reduction of pain was associated with the dual action of ozonides in the control of infection and inflammation, while the improvement in the conditions of the wound bed was confirmed by the significant reduction of the lesion area (p = 0.000034).

[0064] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of chronic diabetic ulcers in human and animal patients.

[0065] A formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 30 g of ozonides per 100 g of plant matrix, was tested on patients affected by chronic diabetic ulcers, in comparison with patients affected by the same disease treated with paraffin bandages and dressings. The analyzed parameters were: healing times, pain reduction, local infection control, and quality of tissue regeneration. The results demonstrated that the formulation based on stable ozonides is highly effective in treating chronic diabetic ulcers, providing several benefits, both clinical and qualitative. In the group treated with stable ozonides, significantly faster healing was observed with respect to the control group. In particular, the patients achieved a complete reduction of the ulcerative area within 90 days, with apparent progress already in the first 15 days. Another relevant aspect was the marked reduction in procedural pain, measured by means of the VAS scale. The mean score went from 7.3 at the beginning of the treatment to 1 after 45 days, highlighting a marked improvement in patient comfort during the healing process. On the histological plane, the analyses confirmed accelerated and superior tissue regeneration. During the treatment with stable ozonides, a significant proliferation of fibroblasts, an organized deposition of collagen, and the formation of new blood vessels were observed, attesting to an effective repair of tissues. Furthermore, the formulation based on stable ozonides showed a high capacity to reduce and prevent the formation of the bacterial bio film, improving the control of local infections. No patient reported adverse reactions or side effects during the study, highlighting the safety of the formulation. These results indicate that the approach based on stable ozonides is an innovative, safe, and effective solution for treating chronic diabetic ulcers, with significant advantages in terms of healing speed, pain reduction, and quality of the regenerated tissues.

[0066] Furthermore, the present invention relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the prevention and topical treatment of dermatitis associated with incontinence.A formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 30 g of ozonides per 100 g of plant matrix, was tested on elderly patients affected by incontinence-associated dermatitis, in comparison with patients affected by the same condition treated with zinc oxide paste and hyaluronate and colloidal silver spray powder. The products were applied at each diaper change, after proper hygiene with specific non-aggressive cleansing products. The healing times and costs were evaluated. Patients with neoplasms or cachexia or on therapy with immunosuppressive drugs or with a low life expectancy were excluded. The results demonstrated that patients treated with stable ozonides achieved complete healing within 29.7 days, with respect to patients treated with zinc oxide paste, achieved in 132.1 days, and patients treated with hyaluronate and colloidal silver spray powder, achieved in 87.3 days. These results suggest that the use of stable ozonides could form an innovative and advantageous therapeutic alternative for treating dermatitis associated with incontinence, improving clinical outcomes and the quality of life of patients.

[0067] Furthermore, the present invention relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of an oil matrix, for the topical treatment of chronic eczematous otitis externa or otomycosis in human and animal patients.

[0068] A formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 19 g of ozonides per 100g of plant matrix, was in fact tested for treating patients suffering from chronic eczematous otitis externa. Parameters such as itching, secretions, desquamation, and complications were evaluated.

[0069] After the treatment with the formulation based on stable ozonides, the results were: reduction in itching by 62.5%, reduction in desquamation by 72%, reduction in secretions by 80%. Total absence of side effects. It was concluded that the treatment with the formulation based on stable ozonides of the aforesaid invention showed an excellent result in eczema of the external auditory canal, even over prolonged periods without complications and particular usefulness for recurrent bacterial otitis externa non-responsive to topical antibiotic therapy and for otomycoses.The same formulation based on stable ozonides was tested on pediatric patients suffering from otomycosis. The use of stable ozonides led to complete recovery, confirming the efficacy thereof as a valid option for antifungal therapy, without side effects.

[0070] Furthermore, the present invention relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of genitourinary syndrome.

[0071] A formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, consisting of 19 g of ozonides per 100g of plant matrix, was tested for treating patients affected by genitourinary syndrome.

[0072] The results in the treated patients showed a significant reduction in pain (from 16.7% to 11.8%), average intensity of the symptoms (from 2.10 to 0.87), and dryness (from 85.5% to 53.8%). These results highlight the efficacy of formulations based on stable ozonides in treating the symptoms of genitourinary syndrome, contributing to improving the quality of life of the patients.

[0073] The present invention further relates to the topical use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for the topical treatment of infected wounds in human and animal patients.

[0074] In particular, a formulation containing the ozonides cis, trans-methyl 8-(5-octyl- 1,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, with a concentration of 19 g of ozonides per 100 g of plant matrix, was evaluated for the topical treatment of infected wounds in animals. In particular, male rats with infected wounds induced using clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus were treated, compared with an untreated control group.

[0075] Microbiological analyses were conducted to quantify the reduction in colony-forming units (CFU), the healing process was monitored, and histological studies were carried out to compare the treated and untreated wounds.The results showed an effective antimicrobial activity of the stable ozonides, with a significant reduction in the CFU of Candida albicans and Aspergillus fumigatus within 24-48 hours (p < 0.01) and a highly significant reduction for Pseudomonas aeruginosa and Staphylococcus aureus (p < 0.0001).

[0076] The wounds treated with stable ozonides showed a steady and progressive improvement in healing with respect to the control group. Within 10 days, the treated wounds were almost completely healed, while the untreated wounds exhibited persistent inflammation and the absence of significant healing.

[0077] Histological analysis confirmed tissue regeneration and reduction in inflammation and fibrosis in the wounds treated with stable ozonides.

[0078] These results indicate that stable ozonides form an innovative, effective, and safe topical treatment for veterinary use to accelerate the healing of infected wounds and significantly reduce the microbial load.

[0079] Furthermore, the described formulation is particularly suitable for oral administration in treating humans and animals when the ozonides contained therein are in an amount between 1 g and 50 g per 100g of oil matrix.

[0080] More specifically, the described formulation is particularly suitable for oral administration in the treatment of humans and animals when the ozonides contained therein are in an amount between 1 g and 10 g per 100g of oil matrix.

[0081] Even more specifically, the described formulation is particularly suitable for oral administration in the treatment of humans and animals, in a preferred configuration the ozonides are in an amount between 4 g and 10 g per 100g of oil matrix.

[0082] In such formulations for oral use, the stable ozonides are preferably, not exclusively, delivered in extra virgin olive oil.

[0083] The formulations based on stable ozonides for oral use can be provided as such or be delivered in dietary supplements, food, soft capsules, emulsions, nanoemulsions, solid oils on various supports, dehydrated nanoemulsions, colloidal hydrogels, clay microcapsules and gastro-resistant microspheres, sublingual patches, gastro-resistant capsules and tablets, orodispersible devices.

[0084] The invention is preferably formulated for the manufacture of dietary supplements or foods for special medical purposes, to enhance the endogenous defense system, to counteract issues affecting, for example, the gastrointestinal tract, the bladder tract, to exert an anti-inflammatory,microbicidal, tissue-regenerating action, to mitigate cellular aging processes, for the prevention and treatment of neurodegenerative diseases.

[0085] The present invention further relates to the oral use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are in an overall amount between 1 g and 10 g, more preferably between 4 g and 10 g, per 100g of oil matrix, for orally treating humans being and animals.

[0086] Advantageously, the invention further relates to the oral use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis, trans-3,5-dioctyl- 1,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are in an overall amount between 1 g and 10 g, more preferably between 4 g and 10 g, per 100g of oil matrix, for treating patients affected by inflammatory bowel diseases (IBD).

[0087] In support thereof, a formulation based on ozonides consisting of 4.5 g of ozonides per 100g of plant matrix was tested on a three-dimensional intestinal model from patients suffering from IBD. Patient-derived organoids replicate the complexity and function of the original tissue, maintaining patient- specific inflammatory features.

[0088] The results achieved demonstrated potent anti-inflammatory activity, reducing the levels of pro-inflammatory cytokines, such as IL-8 and TNF-a.

[0089] Furthermore, the results achieved highlighted a restoration of the integrity of the intestinal epithelial barrier, evidenced by an improvement in the cell junction complexes (e.g., proteins such as CLDN-1 and ZO-1) and an inhibition of the inflammatory cascade mediated by NF-kB / TBKl / IRF3 signaling.

[0090] Formulations particularly suitable for oral or systemic use for the treatment of humans and animals have been further defined.

[0091] Therefore, the present invention relates to the oral or systemic use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 15 g and 40 g per 100g of oil matrix, for treating HT-29 human colon epithelial cells, exerting anti-inflammatory activity.

[0092] In fact, the formulation based on stable ozonides was tested on HT-29 human colon epithelial cells, stimulated with lipopolysaccharide (LPS).The results demonstrated that the stable ozonides are capable of inducing the inhibition of pro-inflammatory cytokines, reducing the expression of TNF-a, IL-ip and the adhesion molecule ICAM-1, limiting the inflammatory response and preserving tissue integrity, suggesting the ability thereof to limit the response to inflammatory stimuli even in immune cells.

[0093] Furthermore, the present invention further relates to the oral or systemic use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 1 g and 10 g, more preferably between 4 g and 10 g, per 100g of oil matrix, for the treatment of brain ischemia.

[0094] In such a context, two formulations based on stable ozonides, containing 4.5 g and 29 g of ozonides per 100g of plant matrix, respectively, were tested in mice with brain ischemia induced by transient occlusion of the middle cerebral artery to evaluate the neuroprotective effects thereof. The first formulation showed to significantly reducing the infarct volume without altering the associated neurological deficits, while the second showed a reduction in brain edema induced by ischemia. Both formulations were administered intraperitoneally, which approach simulates the intravenous administration. Administered orally, both formulations did not affect the body weight of the mice, showing no acute toxicity.

[0095] The present invention further relates to the oral or systemic use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are overall in an amount between 1 g and 10 g, more preferably between 4 g and 10 g, per 100g of oil matrix, for treating cancer cells.

[0096] In such a context, a formulation consisting of 4.5 g of ozonides per 100g of plant matrix was subjected to tests on different cancer cell lines, including MCF-7 breast carcinoma, refractory MCF-7R breast carcinoma, and human leukemia HL-60R. The results obtained showed a dosedependent action.

[0097] In MCF-7, at concentrations of 1 mg / ml and 2 mg / ml, a reduction in cell growth of 30% and 65%, respectively, was observed. When delivered in a natural clay matrix, specifically halloysite, the same formulation resulted in a significantly greater reduction in cell viability, equal to 80% and 90%, respectively.On MCF-7R, the concentration of 2 mg / ml yielded an inhibition of cell growth of 15%. The concentrations of 1 mg / ml and 2 mg / ml delivered in halloysite, showed greater efficacy, with reductions of 25% and 35%, respectively.

[0098] In HL-60R, a concentration of 1 mg / ml resulted in an inhibition of cell growth equal to 25%. These results highlight the potential of formulations based on stable ozonides in anti-cancer efficacy, with a greater effect observed when delivered in a natural matrix such as halloysite.

[0099] As previously mentioned, the formulation of the present patent application for industrial invention is also suitable for use in the agricultural field.

[0100] In particular, the described formulation is particularly suitable for administration in the agricultural field when the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate contained therein are in an overall amount between 20 g and 75 g per 100g of oil matrix.

[0101] The formulations of the invention particularly suitable for administration in the agricultural field are preferably formulated in extra virgin olive oil, but also in sunflower oil or further combinations of vegetable oil matrices.

[0102] Such formulations of ozonides can be delivered as such, or combined with lecithins, emulsifiers, macro- and micronutrients, in natural and / or synthetic clays, to enhance the endogenous defense system of plant organisms, for the manufacture of plant protection products and / or strengtheners.

[0103] The present invention finally relates to the use of the formulation based on the ozonides cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate, when these are in an overall amount between 20 g and 75 g per 100g of oil matrix, for the preventive and curative treatment of plant species.

[0104] In the agricultural field, such a formulation, consisting of 20g of stable ozonides per 100g of oil matrix, was compared in the olive orchard in the management of the olive fruit fly (Baclrocera oleae) with respect to the use of systemic products. The specific objectives included the analysis of the repellent properties of the formulation on oviposition, the control of the symbiotic bacteria of the larvae, and the impact on the chemical and organoleptic features of the produced oil. Monitoring was conducted by using pheromone traps and weekly sampling the drupes to assess active and harmful infestations. The results showed an enhanced repellent action of the stableozonides on oviposition and a lower level of damaging infestation as compared to the controls. The quality of the oil produced from olives treated with stable ozonides showed lower free acidity, a better organoleptic profile (bitter, pungent, fruity) and a greater content in polyphenols as compared to the oil from olives treated with systemics. The olives treated with stable ozonides exhibited earlier ripening, greater pulp integrity, and fruit elasticity, thus facilitating harvesting without compromising the polyphenol content.

[0105] The results achieved highlight that the treatment with stable ozonides has proved to be effective in limiting olive fruit fly infestations and in improving some qualitative features of the oil.

Claims

AMENDED CLAIMSreceived by the International Bureau on 7 July 2026 (07.07.2026) 1. Formulation comprising the molecules:- cis-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate,- trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate,- cis-3,5-dioctyl-l,2,4-trioxolane,- trans-3,5-dioctyl-l,2,4-trioxolane,- cis-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate,- trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate.wherein:- cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate is present up to 55% w / w;- cis,trans-3,5-dioctyl-l,2,4-trioxolane is present up to 20% w / w;- cis, trans-dimethyl 8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate is present up to 25% w / w.

2. Formulation according to claim 1, wherein within each pair of diastereoisomers, the ratio of cis to trans forms is 1:1.

3. Formulation according to claim 1 or 2 carried in an oil matrix.

4. Formulation according to claim 3, wherein said oil matrix is extra virgin olive oil.

5. Formulation according to claim 3, wherein said oil matrix is sunflower oil.

6. Formulation according to claim 1 or 2 carried in an organic solvent.

7. Formulation according to claim 6, wherein said organic solvent is selected from tetrahydro furan, 2-methyltetrahydrofuran, DMSO, isopropanol, dichloromethane, chloroform and diethyl ether.

8. Formulation according to claims 3, 4 or 5, wherein the ozonides cis,trans-methyl-8-(5-octyl- l,2,4-trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis,trans-dimethyl- 8,8'-(l,2,4-trioxolan-3,5-diyl)dioctanoate are present in an amount between 0.001 g and 100 g per 100 g of oil matrix.

9. Formulation according to claim 8, wherein cis,trans-methyl-8-(5-octyl-l,2,4-trioxolan-3- yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis, trans-dimethyl-8,8'-( 1,2,4- trioxolane-3,5-diyl)dioctanoate are present in an amount between 15 g and 40 g per 100 g of oil matrix.

10. Formulation according to claim 8, wherein the ozonides cis, trans-methyl-8-(5-octyl- 1,2,4- trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis,trans-dimethyl-8,8'- (l,2,4-trioxolane-3,5-diyl)dioctanoate are present in an amount between 1 g and 10 g per 100 g of oil matrix.

11. Formulation according to claim 10, wherein the ozonides cis, trans-methyl-8-(5-octyl- 1,2,4- trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis,trans-dimethyl-8,8'-(l,2,4-trioxolane-3,5-diyl)dioctanoate are present in an amount between 4 g and 10 g per 100 g of oil matrix.

12. Formulation according to claim 8, wherein the ozonides cis, trans-methyl-8-(5-octyl- 1,2,4- trioxolan-3-yl)octanoate, cis,trans-3,5-dioctyl-l,2,4-trioxolane and cis,trans-dimethyl-8,8'- (l,2,4-trioxolane-3,5-diyl)dioctanoate are present in an amount between 20 g and 75 g per 100 g of oil matrix.

13. Formulation according to claim 9, for use in the topical treatment of humans and animals.

14. Formulation according to claim 9, for use in the topical treatment of balanitis xerotica obliterans in pediatric patients and animals.

15. Formulation according to claim 9, for use in the topical treatment of lichen sclerosus in pediatric patients and animals.

16. Formulation according to claim 9, for use in the topical anti-inflammatory and anti-proliferative treatment of children suffering from phimosis associated with lichen sclerosus undergoing circumcision.

17. Formulation according to claim 9, for use in the topical treatment of phlebostatic ulcers in human patients.

18. Formulation according to claim 9, for use in the topical treatment of chronic diabetic ulcers in human patients and animals.

19. Formulation according to claim 9, for use in the topical treatment of dermatitis associated with incontinence in human patients.

20. Formulation according to claim 9, for use in the topical treatment of chronic eczematous otitis externa or otomycosis in human patients and animals.

21. Formulation according to claim 9, for use in the topical treatment of genito-urinary syndrome in human patients.

22. Formulation according to claims 10 or 11, for use in the oral treatment in humans and animals.

23. Formulation according to claims 10 or 11, for use in the oral treatment of inflammatory bowel disease (IBD).

24. Formulation according to claims 10 or 11, for use in the oral or systemic treatment of conditions involving human colonic epithelial cells HT29.

25. Formulation according to claims 10 or 11, for use in the oral or systemic treatment of brain ischaemia.

26. Formulation according to claims 10 or 11, for use in the oral or systemic treatment of cancer cells.

27. Use of the formulation according to claim 13, for enhancing the endogenous defense mechanisms of plants.

28. Use of the formulation according to claim 13, for controlling oviposition and harmful infestations caused by Bactrocera oleae in agricultural fields.