Synergistic compositions and methods for inhibiting collagen loss

Abstract

The present invention relates to compositions comprising: (a) a carotenoid selected from the group consisting of octahydrolycopene, hexahydrolycopene, zeta carotene, or any combination thereof; and (b) a ceramide, said compositions having a synergistic effect in inhibiting or reducing collagen loss, and methods of use thereof.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 539,129, filed September 19, 2023, entitled “Synergic Compositions and Methods for Inhibiting Collagen Loss” (the contents of which are incorporated herein by reference in their entirety). Technical Field

[0002] This invention generally relates to the field of carotenoids and ceramides and methods of using them (such as for treating skin-related conditions, including collagen loss). Background Technology

[0003] Dermal collagen represents the most abundant extracellular matrix (ECM) protein in human skin. The main components of the dermis are fibroblasts, which produce, organize, and maintain collagen and elastin fibers. Collagen-1 is a major component of the dermal ECM, accounting for approximately 85-90% of total skin collagen and playing a crucial role in skin elasticity. Its synthesis decreases in aging and photodamaged skin, leading to wrinkles.

[0004] Inflammation and the accumulation of reactive oxygen species (ROS) are considered to be causes of skin aging and photodamage. Several studies have linked the immune system and inflammatory genes to photoaging. UV radiation induces a series of events that can cause inflammation: the release of inflammatory cytokines such as interleukin-1, ROS generation, the production of inflammatory mediators, peroxidation of membrane lipids, and skin cell death.

[0005] Neutrophils have been reported to infiltrate the epidermis and dermis of the skin after exposure to UVB natural sunlight and simulated solar radiation (SSR), infrared radiation, and heat. These neutrophils are rich in potent proteolytic enzymes that clear UV-induced apoptotic cells and kill skin cells using oxidized surface lipids, but they are also capable of degrading collagen and elastin fibers. Furthermore, infiltrating neutrophils, rather than keratinocytes and fibroblasts, have been reported to be the primary source of proteolytic enzymes and free radicals after exposure to erythematous doses of SSR, thus suggesting that neutrophils may be a key factor in photoaging. Collagen is the only protein susceptible to superoxide anion cleavage, as evidenced by the release of small peptides containing 4-hydroxyproline. In the presence of oxygen or hypochlorous acid, hydroxyl radicals cleave collagen into small peptides, and this cleavage appears to be specific to proline or 4-hydroxyproline residues.

[0006] There remains a great need for compositions and methods for preventing collagen damage induced by activated neutrophils. Summary of the Invention

[0007] In some embodiments, the present invention is at least partly based on the following surprising discovery that the composition provides a synergistic effect in preventing collagen loss and inhibiting myeloperoxidase (MPO) activity compared to either the carotenoid mixture or the ceramide alone, said composition comprising a combination of a carotenoid mixture and a ceramide, said carotenoid mixture comprising phytoene, phytohexene and zeta-carotene.

[0008] In some embodiments, the invention is further based, at least in part, on the surprising discovery that subjecting cells to the claimed composition prevents more than 80% of collagen loss induced by isolated neutrophils.

[0009] According to a first aspect, a composition is provided comprising: (a) a mixture of carotenoids including phytoene, phytohexene and zeta-carotene; and (b) a ceramide; wherein the weight / weight ratio (w / w) of (a) to (b) in the composition is between 25:1 (w / w) and 500:1 (w / w).

[0010] According to another aspect, a pharmaceutical composition is provided comprising an effective amount of the composition of the present invention and a pharmaceutically acceptable carrier.

[0011] According to another aspect, a method is provided for preventing or treating collagen loss-related diseases or disorders in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject, thereby preventing or treating collagen loss-related diseases or disorders in the subject.

[0012] According to another aspect, a method is provided for reducing the amount, activity, or both of any one of MPO, NO, superoxide, and any combination thereof in a subject in need, the method comprising administering a therapeutically effective amount of the composition of the present invention to the subject.

[0013] According to another aspect, a kit is provided comprising: (a) at least one carotenoid selected from phytoene, hexahydrolycopene, zetacarotene, and any combination thereof; and (b) ceramide.

[0014] In some embodiments, the carotenoid mixture comprises: 55-65% (w / w) of phytoene in the total carotenoids of the carotenoid mixture; 10-20% (w / w) of phytoene in the total carotenoids of the carotenoid mixture; and 15-25% (w / w) of zetacarotene in the total carotenoids of the carotenoid mixture.

[0015] In some embodiments, the w / w ratio of the combined phytoene and phytohexene to the ζ-carotene ranges from 15:1 (w / w) to 2:1 (w / w).

[0016] In some embodiments, the carotenoid mixture further includes additional carotenoids selected from the following: lycopene, β-carotene, γ-carotene, and any combination thereof.

[0017] In some embodiments, the composition comprises less than 5% (w / w) of the lycopene in the total carotenoids of the carotenoid mixture, less than 5% (w / w) of the β-carotene in the total carotenoids of the carotenoid mixture, and 0.2-1.5% (w / w) of the γ-carotene or a combination thereof in the total carotenoids of the carotenoid mixture.

[0018] In some embodiments, the carotenoid mixture comprises 10-15% (w / w) of the total carotenoid content of the carotenoid mixture.

[0019] In some embodiments, the carotenoid mixture further comprises tocopherol in an amount of 10-30% (w / w) of the total carotenoids in the carotenoid mixture.

[0020] In some embodiments, the carotenoid mixture further comprises 5-15% (w / w) of phytosterols in the total carotenoids of the carotenoid mixture.

[0021] In some embodiments, the ceramide includes multiple types of ceramides.

[0022] In some embodiments, the ceramide and any of the various types thereof are selected from: C2-ceramide, C1-ceramide, C3-ceramide, C6-ceramide, and ceramide 9.

[0023] In some embodiments, the composition has a synergistic effect in inhibiting: (i) collagen loss; (ii) the production, secretion, activity, or any combination thereof of nitric oxide (NO), myeloperoxidase (MPO), superoxide (SO), and any combination thereof; and (iii) both of (i) and (ii).

[0024] In some embodiments, the collagen is collagen-1 (collagen-1 type 1).

[0025] In some embodiments, the pharmaceutical composition is used to prevent or treat collagen loss-related diseases or disorders in subjects in need.

[0026] In some embodiments, the collagen loss-related diseases or disorders include collagen loss in the subject's skin.

[0027] In some embodiments, the subject suffers from a collagen loss-related disease or disorder.

[0028] In some embodiments, the collagen loss-related diseases are selected from age-related diseases, skin diseases, and inflammatory diseases.

[0029] In some embodiments, the dermatitis includes skin damage induced by any of the following: radiation, oxidative stress, DNA damage, telomere shortening, inflammation, smoking, and any combination thereof.

[0030] In some embodiments, the radiation includes UV radiation, visible light radiation, infrared radiation, or any combination thereof.

[0031] In some embodiments, the UV radiation includes UVA, UVB, UVC, or any combination thereof.

[0032] In some embodiments, compared to a control, the object has an increased amount, increased activity, or both of any of the following in the skin, systemically, or both: MPO, NO, SO, and any combination thereof.

[0033] In some embodiments, the method further includes determining in a sample obtained from or derived from the object the amount, activity, or both of the following: MPO, NO, SO, and any combination thereof.

[0034] In some embodiments, the application includes oral administration, topical administration, or both.

[0035] In some embodiments, the at least one carotenoid includes phytoene, hexahydrolycopene, and ζ-carotene.

[0036] In some embodiments, the kit further includes instructions for mixing (a) and (b) w / w at ratios from 25:1 (w / w) to 500:1 (w / w).

[0037] In some embodiments, the kit is used to prepare compositions suitable for preventing or treating collagen loss-related diseases in subjects in need.

[0038] The following implementation methods and aspects thereof are described and illustrated in conjunction with systems, tools and methods. These implementation methods and aspects are exemplary and illustrative, and not intended to limit the scope.

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, this specification (including definitions) shall prevail. Furthermore, these materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0040] Further embodiments of the invention and its full scope of application will become apparent from the detailed description given below. However, it should be understood that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description.

[0041] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent from reference to the accompanying drawings and from studying the following detailed description. Attached Figure Description

[0042] Figures 1A-1D Includes bar graphs showing dose-dependent inhibition of nitric oxide (NO) production and cell viability in macrophages induced by a mixture of carotenoids including phytoene, hexaphytoene, and ζ-carotene (hereinafter referred to as "Lumenato"; 1A and 1B, respectively) or ceramides (1C and 1D, respectively). In (1A and 1C), values ​​representing % inhibition are the mean ± standard error of the mean (SEM) of three independent experiments, each repeated three times; significance: * - p < 0.05, ** - p < 0.01, *** - p < 0.001, indicating significant inhibition compared to stimulated macrophages.

[0043] Figures 2A-2D Includes bar graphs showing dose-dependent inhibition of myeloperoxidase (MPO) secretion and superoxide production in neutrophils induced by Lumenato (2A and 2B, respectively) and ceramide (2C and 2D, respectively). In (2A–2D), values ​​representing % inhibition are the mean ± SEM of three independent experiments, each repeated three times; significance: * - p < 0.05, ** - p < 0.01, *** - p < 0.001, significantly inhibited compared to stimulated neutrophils.

[0044] Figures 3A-3DIncludes bar graphs showing synergistic inhibition of NO. Inhibition of nitric oxide (NO) was induced by (3A) Lumenato; (3B) ceramide; (3C) a combination of 6.5 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramide; and (3D) a combination of 13 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramide. In (3A-3D), values ​​representing % inhibition are the mean ± SEM of three independent experiments, each repeated three times. In (3C-3D), calculated additive (“additive”) and synergistic (“combined”) effects are provided; significance: * - p < 0.05, ** - p < 0.01, *** - p < 0.001, significantly increased compared to the additive effect.

[0045] Figures 4A-4D Immunofluorescence staining and quantitative bar graphs are included, showing the synergistic prevention of collagen-1 damage induced by the combination of 6.5 μg / ml Lumenato and ceramide, as detected by immunofluorescence analysis. Immunofluorescence staining for collagen-1, density determination of the staining (left bar graph), and cell counting in culture by DAPI staining are provided (right bar graph). Cells were co-cultured with neutrophils pre-incubated with (4A) Lumenato; (4B) different concentrations of ceramide; and (4C) a combination of Lumenato and ceramide. (4D) Vertical bar graphs show the percentage of prevention against collagen-1 damage calculated after scanning the density determination of the immunofluorescence staining for collagen-1. A control is also provided in (4A), where cells were co-cultured with unincubated neutrophils. In (4A-4C), the values ​​shown are the mean ± SEM of three different experiments. Significance: * - p < 0.05, *** - p < 0.001. In (4D), the horizontal line represents the calculated additive effect, and the effect above this line represents synergistic inhibition; Significance: ** - p < 0.01, a significant increase compared to the additive effect. I did not use tracking change. Please use this (version) or a similar approach in subsequent figures.

[0046] Figures 5A-5BIncludes photographs and bar graphs showing the synergistic prevention of collagen-1 damage induced by the combination of 6.5 μg / ml Lumenato and ceramide, as detected by Western blot analysis. (5A) Representative Western blot analysis of collagen-1 and actin in cell lysates after 24 h of incubation under different treatments. The intensity of each actin band after quantification by density assay is divided by the intensity of each band and expressed in arbitrary units in the bar graph; and (5B) the calculated % prevention of collagen-1 damage. The values ​​shown in (5A) are the mean ± SEM of three different experiments. In (5B), the horizontal line represents the calculated additive effect, and the effect above this line represents synergistic inhibition; significance: *** - p < 0.001, significantly increased compared to the additive effect.

[0047] Figures 6A-6D Includes fluorescence micrographs and bar graphs showing the synergistic prevention of collagen-1 damage induced by the combination of 13 μg / ml Lumenato and ceramide, as detected by immunofluorescence analysis. Immunofluorescence staining for collagen-1, density assays of the staining (left bar graph), and cell counting in culture by DAPI staining are provided (right bar graph). Cells were co-cultured with neutrophils pre-incubated with (6A) Lumenato; (6B) different concentrations of ceramide; and (6C) a combination of Lumenato and ceramide. (6D) Vertical bar graphs show the percentage of prevention against collagen-1 damage calculated after scanning the density assays of the immunofluorescence staining for collagen-1. A control is also provided in (6A), in which cells were co-cultured with unincubated neutrophils. In (6A-6C), the values ​​shown are the mean ± SEM of three different experiments. Significance: * - p < 0.05, *** - p < 0.001. In (6D), the horizontal line represents the calculated additive effect, and the effect above this line represents synergistic inhibition; significance: ** - p < 0.01, significantly increased compared to the additive effect.

[0048] Figures 7A-7BIncludes bar graphs showing the synergistic prevention of collagen I damage induced by the combination of 13 μg / ml Lumenato and ceramide, as detected by Western blot analysis. (7A) Representative Western blot analysis of collagen I and actin in cell lysates after 24 h of incubation under different treatments; and (7B) calculated protection against collagen I damage. The intensity of each actin band after quantification by densitometrics was divided by the intensity of each band in (7A) and expressed in arbitrary units in the bar graph. In (7B), the values ​​shown are the mean ± SEM of three different experiments, and the horizontal line represents the calculated additive effect, with the effect above this line representing synergistic inhibition; significance: ** - p < 0.01, significantly increased compared to the additive effect.

[0049] Figures 8A-8D Includes bar graphs showing synergistic inhibition of MPO secretion. (8A) Lumenato; (8B) Ceramide; (8C) Combinations of 6.5 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramide; and (8D) MPO inhibition induced by combinations of 13 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramide. In (8C and 8D), calculated additive (“additive”) and synergistic (“combination”) effects are presented; significance: * p < 0.05, significantly increased compared to the additive effect.

[0050] Figures 9A-9D Includes bar graphs and fluorescence micrographs showing the effects of Lumenato or ceramides and their combinations on fibroblasts. Immunofluorescence staining for collagen-1, density determination of the staining (left bar graph), and cell counting in culture by DAPI staining are provided (right bar graph). Fibroblasts were incubated with (9A) Lumenato; (9B) different concentrations of ceramides; (9C) a combination of 6.5 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramides; and (9D) a combination of 13 μg / ml Lumenato with 0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml ceramides. Detailed Implementation

[0051] According to one aspect of the invention, a composition is provided comprising: (a) at least one carotenoid selected from phytoene, phytohexene, zetacarotene or any combination thereof; and (b) a ceramide, wherein the weight / weight ratio of (a) to (b) is between 25:1 (w / w) and 500:1 (w / w).

[0052] In some embodiments, the compositions of the present invention comprise: (a) a mixture of carotenoids; and (b) ceramides. In some embodiments, the compositions of the present invention comprise: (a) a mixture of carotenoids; and (b) ceramides, having a w / w ratio between 25:1 (w / w) and 500:1 (w / w). In some embodiments, the carotenoid mixture comprises phytoene, phytohexene, zeta-carotene, or any combination thereof. In some embodiments, the carotenoid mixture comprises: phytoene, phytohexene, and zeta-carotene.

[0053] In some embodiments, the w / w ratio of the carotenoid mixture to ceramide in the composition is between 30:1 (w / w) and 400:1 (w / w), between 40:1 (w / w) and 450:1 (w / w), between 50:1 (w / w) and 200:1 (w / w), between 80:1 (w / w) and 240:1 (w / w), between 4:1 (w / w) and 350:1 (w / w), or between 200:1 (w / w) and 500:1 (w / w). Each possibility represents a separate embodiment of the invention.

[0054] In some embodiments, the compositions of the present invention include an active agent and an acceptable carrier.

[0055] In some embodiments, the carotenoid mixtures disclosed herein are or consist of Lumenato. In some embodiments, the carotenoid mixtures disclosed herein are derived from Lumenato. In some embodiments, Lumenato is the source of the carotenoid mixtures disclosed herein. In some embodiments, the carotenoid mixtures disclosed herein are extracted or obtained from Lumenato. In some embodiments, Lumenato is processed, extracted, fractionated, or any combination thereof to obtain the carotenoid mixtures disclosed herein.

[0056] In some embodiments, the active agent comprises primarily the following: (a) at least one carotenoid selected from phytoene, hexahydrolycopene or ζ-carotene; and (b) ceramide.

[0057] In some embodiments, the active agent comprises primarily the following: (i) phytoene and ceramide; (ii) ζ-carotene and ceramide; (iii) hexahydrolycopene and ceramide; (iv) phytoene, ζ-carotene and ceramide; (v) phytoene, hexahydrolycopene and ceramide; (vi) ζ-carotene, phytoene and ceramide; or (vii) phytoene, hexahydrolycopene, ζ-carotene and ceramide.

[0058] In some embodiments, the composition further includes an acceptable carrier.

[0059] As used herein, the term “mainly composed of” means that a given compound or substance constitutes the vast majority of the active ingredient portion or fraction of a composition.

[0060] In some embodiments, the composition is primarily defined as: (a) a mixture of carotenoids including phytoene, phytohexene, zeta-carotene, or any combination thereof; and (b) ceramides, comprising at least 95% by weight, at least 98% by weight, at least 99% by weight, or at least 99.9% by weight, or any value and range thereof, of the active ingredients (one or more) of the composition. Each possibility represents a separate embodiment of the invention.

[0061] In some embodiments, the ceramide concentration in the composition is at least 0.025 µg / ml, at least 0.03 µg / ml, at least 0.05 µg / ml, at least 0.075 µg / ml, at least 0.1 µg / ml, at least 0.15 µg / ml, at least 0.20 µg / ml, at least 0.25 µg / ml, at least 0.30 µg / ml, at least 0.35 µg / ml, at least 0.40 µg / ml, at least 0.45 µg / ml, at least 0.5 µg / ml, at least 1 µg / ml, at least 5 µg / ml, at least 8 µg / ml, at least 10 µg / ml, at least 15 µg / ml, or any value and range thereof. In some embodiments, the ceramide concentration in the composition is between 0.025 μg / ml and 0.5 μg / ml, between 0.03 μg / ml and 0.4 μg / ml, between 0.034 μg / ml and 0.34 μg / ml, or between 0.03 μg / ml and 0.45 μg / ml. Each possibility represents a separate embodiment of the invention.

[0062] In some embodiments, the composition comprises a mixture of carotenoids at concentrations of at least 1.5 µg / ml, at least 3.5 µg / ml, at least 4 µg / ml, at least 5 µg / ml, at least 6 µg / ml, at least 6.5 µg / ml, at least 8.5 µg / ml, at least 10 µg / ml, at least 15 µg / ml, at least 50 µg / ml, at least 75 µg / ml, at least 100 µg / ml, at least 150 µg / ml, at least 250 µg / ml, or any values ​​and ranges thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the amount of carotenoid mixture in the composition is between 1.5 μg / ml and 75 μg / ml, between 1.5 μg / ml and 50 μg / ml, between 1.5 μg / ml and 30 μg / ml, between 3.5 μg / ml and 30 μg / ml, between 3.5 μg / ml and 25 μg / ml, between 5 μg / ml and 20 μg / ml, between 6 μg / ml and 20 μg / ml, between 5 μg / ml and 15 μg / ml, between 6.5 μg / ml and 21.5 μg / ml, between 50 μg / ml and 250 μg / ml, between 75 μg / ml and 150 μg / ml, and between 75 μg / ml and 150 μg / ml. Each possibility represents a separate embodiment of the invention.

[0063] In some embodiments, the carotenoid is a natural carotenoid extracted, isolated, or purified from fruits, vegetables, or plants (including plant parts) – including any combination thereof. In another embodiment, the carotenoid is a carotenoid extracted from tomato plants. In another embodiment, the carotenoid is a carotenoid extracted from tomato fruit. In another embodiment, tomato carotenoid is a tomato extract rich in carotenoids. In another embodiment, tomato carotenoid is an all-natural tomato extract rich in carotenoids. In another embodiment, tomato carotenoid is a tomato carotenoid complex. In another embodiment, the tomato carotenoid complex comprises a complex of phytonutrients, including various carotenoids (such as phytoene, hexahydrolycopene, ζ-carotene, β-carotene, etc.), tocopherols, and phytosterols. In some embodiments, the carotenoid is a synthetic carotenoid.

[0064] In some embodiments, the carotenoid mixture comprises at least one natural carotenoid or a combination thereof, a synthetic carotenoid or a combination thereof, or both.

[0065] In some embodiments, the carotenoid mixture comprising at least one carotenoid selected from the group consisting of tomato extract: phytoene, phytohexene, zeta-carotene, or any combination thereof (in amounts specified below). In some embodiments, the carotenoid mixture exhibits reduced cytotoxicity. In some embodiments, the reduced cytotoxicity is compared to other tomato extracts. In some embodiments, the reduced toxicity allows for the administration of the composition of the invention to a subject in need at a higher dose without diminishing the subject's survival, health, or both. In some embodiments, administration of the composition of the invention to a subject in need allows for increased therapeutic efficacy by providing the active ingredient (such as phytoene, phytohexene, and zeta-carotene) in a higher amount that enhances therapeutic effect, without diminishing the subject's survival, health, or both due to high cytotoxicity.

[0066] In some implementations, reduced cytotoxicity is reduced toxicity to fibroblasts.

[0067] In some embodiments, the carotenoid mixture includes 10-40% (w / w), 15-35% (w / w), 20-45% (w / w), 25-35% (w / w), 20-30% (w / w), 35-65% (w / w), 45-65% (w / w), or 30-50% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0068] In some embodiments, the carotenoid mixture includes 10-20% (w / w), 5-15% (w / w), 1-10% (w / w), 3-12% (w / w), 4-14% (w / w), 5-10% (w / w), 8-15% (w / w), or 2-9% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0069] In some embodiments, the carotenoid mixture includes ζ-carotene in amounts of 15-25% (w / w), 4-20% (w / w), 6-18% (w / w), 5-15% (w / w), 6-12% (w / w), 9-17% (w / w), or 10-17% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0070] In some embodiments, the weight ratio of the combined phytoene and phytohexene to ζ-carotene ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 15:1 (w / w) to 2:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w). Each possibility represents a separate embodiment of the invention.

[0071] In some embodiments, the carotenoid mixture further includes additional carotenoids. As used herein, the term "additional carotenoid" means any carotenoid or its metabolite other than or different from phytolycopene, hexahydrolycopene, zetacarotene or any combination thereof.

[0072] In some embodiments, the carotenoid mixture includes additional carotenoids in amounts of 0.1-3% (w / w), 0.2-3.5% (w / w), 0.5-2.5% (w / w), 0.15-1.75% (w / w), 0.35-2.75% (w / w), 0.8-4% (w / w), 1-5% (w / w), or 1.5-4.75% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0073] In some embodiments, the additional carotenoids are selected from lycopene, beta-carotene, gamma-carotene, or any combination thereof.

[0074] In some embodiments, the carotenoid mixture includes lycopene in amounts of less than 10% (w / w), less than 7% (w / w), less than 5% (w / w), less than 3% (w / w), less than 2% (w / w), or less than 1% (w / w) of the total carotenoids in the carotenoid mixture, or any value and range therebetween. In some embodiments, the amount of lycopene is less than 5% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0075] In some embodiments, the carotenoid mixture includes lycopene in amounts of 1-3% (w / w), 1-5% (w / w), 2-6% (w / w), 0.5-4.5% (w / w), 0.1-3% (w / w), 0.6-4.8% (w / w), or 2.5-4% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0076] In some embodiments, the carotenoid mixture includes less than 10% (w / w), less than 7% (w / w), less than 5% (w / w), less than 3% (w / w), less than 2% (w / w), or less than 1% (w / w) of the total carotenoids in the carotenoid mixture, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0077] In some embodiments, the carotenoid mixture includes β-carotene in amounts of 1-3% (w / w), 1-5% (w / w), 2-6% (w / w), 0.5-4.5% (w / w), 0.1-3% (w / w), 0.6-4.8% (w / w), or 2.5-4% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0078] In some embodiments, the carotenoid mixture includes gamma-carotene in amounts of at least 0.15% (w / w), at least 0.18% (w / w), at least 0.2% (w / w), at least 0.25% (w / w), at least 0.35% (w / w), at least 0.5% (w / w), at least 0.75% (w / w), at least 0.9% (w / w), at least 1% (w / w), at least 1.2% (w / w), at least 1.35% (w / w), or at least 1.7% (w / w) of the total carotenoids in the carotenoid mixture, or any values ​​and ranges thereto. Each possibility represents a separate embodiment of the invention.

[0079] In some embodiments, the carotenoid mixture includes γ-carotene in amounts of 0.15-3% (w / w), 0.2-2% (w / w), 0.2-1.5% (w / w), 0.5-3% (w / w), 0.7-1.6% (w / w), 0.4-2.8% (w / w), or 1.2-3.2% (w / w) of the total carotenoids in the carotenoid mixture. Each possibility represents a separate embodiment of the invention.

[0080] In some embodiments, the carotenoid mixture includes lycopene in an amount of less than 5% (w / w) of the total carotenoids in the carotenoid mixture, β-carotene in an amount of less than 5% (w / w) of the total carotenoids in the carotenoid mixture, γ-carotene in an amount of 0.2-1.5% (w / w) of the total carotenoids in the carotenoid mixture, and any combination thereof.

[0081] In some embodiments, the carotenoid mixture comprises 5-25% (w / w), 10-15% (w / w), 12-35% (w / w), 3-17% (w / w), 2-20% (w / w), or 1-30% (w / w) of the total carotenoid mixture. Each possibility represents a single embodiment of the invention.

[0082] In some embodiments, the composition comprising a mixture of carotenoids further comprises tocopherol (e.g., vitamin E). In some embodiments, the composition comprises a mixture of tocopherol and carotenoids in amounts of 1-30% (w / w), 3-35% (w / w), 10-30% (w / w), 5-25% (w / w), 2-20% (w / w), 4-41% (w / w), 8-32% (w / w), or 13-39% (w / w). Each possibility represents a separate embodiment of the invention. In some embodiments, the carotenoid mixture further comprises, by weight, 1-30% (w / w), 3-35% (w / w), 10-30% (w / w), 5-25% (w / w), 2-20% (w / w), 4-41% (w / w), 8-32% (w / w), or 13-39% (w / w). Each possibility represents a separate embodiment of the invention.

[0083] In some embodiments, the weight ratio of the combined phytoene and hexahydrolycopene to tocopherol ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w). Each possibility represents a separate embodiment of the invention.

[0084] In some embodiments, the weight ratio of ζ-carotene to tocopherol ranges from 3:1 (w / w) to 1:3 (w / w), 3:1 (w / w) to 1:2 (w / w), 3:1 (w / w) to 1:1 (w / w), 2:1 (w / w) to 1:1 (w / w), 2:1 (w / w) to 1:2 (w / w), 2:1 (w / w) to 1:3 (w / w), 1:1 (w / w) to 1:2 (w / w), or 1:1 (w / w) to 1:3 (w / w). Each possibility represents a single embodiment of the invention.

[0085] In some embodiments, the composition comprising a mixture of carotenoids further comprises phytosterols.

[0086] In some embodiments, the composition comprises a mixture of phytosterols and carotenoids in amounts of 1-20% (w / w), 2-19% (w / w), 10-25% (w / w), 5-25% (w / w), 8-16% (w / w), 6-18% (w / w), 3-20% (w / w), 4-17% (w / w), or 5-15% (w / w). Each possibility represents a separate embodiment of the invention. In some embodiments, the carotenoid mixture further comprises phytosterols in amounts of 1-20% (w / w), 2-19% (w / w), 10-25% (w / w), 5-25% (w / w), 8-16% (w / w), 6-18% (w / w), 3-20% (w / w), 4-17% (w / w), or 5-15% (w / w). Each possibility represents a separate embodiment of the invention.

[0087] In some embodiments, the weight ratio of the combined phytosterols and hexahydrolycopene to phytosterols ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w). Each possibility represents a separate embodiment of the invention.

[0088] In some embodiments, the weight ratio of ζ-carotene to phytosterols ranges from 6:1 (w / w) to 2:1 (w / w), 5:1 (w / w) to 2:1 (w / w), 4:1 (w / w) to 2:1 (w / w), 3:1 (w / w) to 2:1 (w / w), 6:1 (w / w) to 3:1 (w / w), 5:1 (w / w) to 3:1 (w / w), 4:1 (w / w) to 3:2 (w / w), or 6:1 (w / w) to 3:1 (w / w). Each possibility represents a separate embodiment of the invention.

[0089] Methods for determining the amount of phytonutrients (such as carotenoids) are common and will be readily apparent to those skilled in the art. Non-limiting examples of such methods include, but are not limited to, gas chromatography, liquid chromatography, and mass spectrometry.

[0090] In some embodiments, the ceramide is a natural product. In some embodiments, the ceramide is derived from a natural product. In some embodiments, the term "derived from" includes any industrial processing, such as purification, separation, fractionation, fermentation, chemical modification, etc. In some embodiments, the ceramide is a synthetic product.

[0091] Non-limiting examples of ceramides include, but are not limited to: ceramide 1 (e.g., EOS), ceramide 2 (e.g., ceramide NS), ceramide 3 (e.g., ceramide NP), ceramides 6-11 (e.g., ceramide AP), ceramide 9 (e.g., ceramide EOP), phytosphingosine, sphongosine, or any combination thereof.

[0092] In some embodiments, the ceramide includes ceramides selected from the following: ceramide 1, ceramide 2, ceramide 3, ceramide 6-11, ceramide 9, or any combination thereof.

[0093] In some embodiments, the ceramide is or includes ceramide 2.

[0094] In some embodiments, an acceptable carrier is or includes oil. In some embodiments, the oil is or includes vegetable oil. In some embodiments, the vegetable oil is or includes seed oil. In some embodiments, the seed oil is or includes tomato seed oil.

[0095] In some embodiments, the w / w percentage of an acceptable carrier in the composition is 0.1% to 99.9%, 10% to 99.9%, 20% to 99.9%, 1% to 50%, 20% to 70%, or 50% to 99.9%, 30% to 90%. Each possibility represents a separate embodiment.

[0096] In some embodiments, the active agent mainly consists of the following components: ζ-carotene, phytosterols, 8-hydrolycopene, 6-hydrolycopene, lycopene, tocopherol, β-carotene and γ-carotene, and ceramide.

[0097] In some embodiments, the compositions of the present invention consist primarily of the following: ζ-carotene, phytosterols, 8-hydrolycopene, 6-hydrolycopene, lycopene, tocopherol, β-carotene and γ-carotene, ceramides, and an acceptable carrier.

[0098] In some embodiments, the compositions of the present invention are synergistic compositions. In some embodiments, the compositions of the present invention provide synergistic effects of the expression, activity, or inhibition, reduction, or any combination thereof of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), superoxide (SO), elastase, nitric oxide (NO), reactive oxygen species (ROS), or any combination thereof.

[0099] In some implementations, the synergistic effect occurs within cells or objects. In some implementations, the cell is the cell of the object.

[0100] In some implementations, the synergistic effect is compared with a control (or the effect exerted by it).

[0101] In some embodiments, the compositions of the present invention provide at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100% inhibition, prevention, or both, compared to a control. Each possibility represents a separate embodiment.

[0102] In some embodiments, a control refers to inhibition obtained by a single compound constituting the composition of the invention (e.g., (a) or (b)). In some embodiments, a control refers to inhibition obtained by either a mixture of carotenoids or a ceramide alone. In some embodiments, a control refers to inhibition obtained by either a mixture of carotenoids or a ceramide alone, wherein the mixture of carotenoids or the ceramide is present in the control at the same concentration as it is in the composition of the invention.

[0103] In some embodiments, the compositions of the present invention provide a synergistic effect in inhibiting, reducing, or preventing collagen loss. In some embodiments, the compositions of the present invention provide greater inhibition, reduction, or prevention of collagen loss compared to a control.

[0104] In some embodiments, collagen loss includes collagen degradation, collagen breakdown, or both. In some embodiments, prevention of collagen loss includes increasing collagen production, increasing collagen stability, reducing collagen degradation or breakdown, or any combination thereof.

[0105] In some embodiments, the compositions of the present invention provide a synergistic effect or are characterized by synergistic activity, wherein the activity includes inhibition of NO production, secretion, or both. In some embodiments, the compositions of the present invention provide greater inhibition of NO production, secretion, or both compared to a control.

[0106] In some embodiments, the compositions of the present invention provide a synergistic effect or are characterized by synergistic activity, wherein the activity includes inhibition of SO production, secretion, or both. In some embodiments, the compositions of the present invention provide greater inhibition of SO production, secretion, or both compared to a control.

[0107] In some embodiments, the compositions of the present invention provide a synergistic effect or are characterized by synergistic activity, wherein the activity includes inhibition of MPO production, secretion, activity, or any combination thereof. In some embodiments, the compositions of the present invention provide greater inhibition of MPO production, secretion, activity, or any combination thereof compared to a control. In some embodiments, MPO activity includes peroxidase enzymatic activity, which includes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ions (Cl⁻). In some embodiments, MPO activity includes peroxidase enzymatic activity, which includes the production of hypobromoic acid (HOBr) from H₂O₂ and bromide anions. In some embodiments, the MPO activity disclosed herein is during the respiratory burst of neutrophils. In some embodiments, MPO activity requires a heme group (e.g., as a cofactor). In some embodiments, MPO activity includes the oxidation of tyrosine to tyrosine radicals.

[0108] In some embodiments, the compositions of the present invention provide a synergistic effect or are characterized by synergistic activity, wherein the activity includes inhibition of ROS production, secretion, or both. In some embodiments, the compositions of the present invention provide greater inhibition of ROS production, secretion, or both compared to a control.

[0109] In some embodiments, the composition includes synergistically effective amounts of: (a) a mixture of carotenoids, including at least one of phytoene, hexahydrolycopene, or ζ-carotene; and (b) ceramide.

[0110] In some embodiments, the synergistic effective amounts include (a) and (b) of the following compositions of the invention, w / w: 10:1 (w / w) to 1:10 (w / w), 10:1 (w / w) to 1:5 (w / w), 5:1 (w / w) to 1:10 (w / w), 5:1 (w / w) to 1:5 (w / w), 4:1 (w / w) to 1:5 (w / w), 3:1 (w / w) to 1:5 (w / w), 2:1 (w / w) to 1:5 (w / w), 1:1 (w / w) to 1:5 (w / w), 1:1 (w / w) to 1:4 (w / w), or 2:1 (w / w) to 1:2 (w / w). Each possibility represents a separate embodiment of the invention.

[0111] As used herein, the term "synergistically effective amount" refers to an amount of the composition of the present invention characterized by inhibition of at least one of collagen loss, NO, MPO, SO, ROS, MMP, or any combination thereof, or prevention of collagen loss-related diseases or disorders, or both, at least 5%, at least 10%, at least 25%, at least 50%, or at least 75% more than the inhibition of at least one of collagen loss, NO, MPO, SO, ROS, MMP, or any combination thereof, or prevention of collagen loss-related diseases or disorders by either of (a) and (b) of the composition of the present invention alone. Each embodiment represents a single embodiment.

[0112] In some embodiments, the compositions of the present invention are used to reduce collagen loss, increase collagen levels, or both in subjects in need.

[0113] In some embodiments, the compositions of the present invention have the activity of increasing collagen levels. In some embodiments, the activity of increasing collagen levels includes any activity selected from the following: increasing collagen synthesis, increasing the weight of collagen per tissue weight, increasing the number, length, or both of collagen fibrils, increasing the amount of natural or properly folded collagen, reducing the amount of structurally damaged collagen, increasing collagen stability, reducing collagen degradation or breakdown, and any combination thereof.

[0114] In some embodiments, the collagen is selected from: collagen I, collagen II, collagen III, collagen IV, collagen XI, collagen X, collagen XI, collagen V, or any combination thereof. In some embodiments, the collagen is or includes collagen I.

[0115] Methods for measuring collagen levels are common and will be apparent to those skilled in the art. Non-limiting examples of methods for determining or measuring collagen levels include immunofluorescence microscopy, electron microscopy, Western blotting, and QrT-PCR, some of which are illustrated below. For example, collagen loss, increased collagen levels, or both can be determined by comparing collagen levels measured in the presence and absence of the compositions of the present invention.

[0116] In one embodiment, the composition of the present invention may be provided to an individual on its own. In some embodiments, the composition of the present invention is a pharmaceutical or nutraceutical composition. In some embodiments, the pharmaceutical or nutraceutical composition comprises a pharmaceutically or nutraceutical-acceptable carrier or excipient, respectively. In some embodiments, the composition of the present invention is a cosmeceutical composition. In some embodiments, the cosmeceutical composition comprises a cosmeceutical-acceptable excipient. In some embodiments, the composition described herein is an ingestible skin composition.

[0117] In some embodiments, the ingestible skin composition includes a composition that is administered orally or consumed, thereby exerting the activity of the active agent in or on the skin of the subject to which the composition is administered or consumed.

[0118] In one embodiment, "pharmaceutical composition," "cosmeceutical composition," or "nutritional supplement composition" means a formulation of the composition described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition, cosmeceutical composition, or nutritional supplement composition is to facilitate the administration of the composition to a living organism. In one embodiment, the phrase "physiologically acceptable carrier" means a carrier or diluent that does not cause significant irritation to mammals and does not eliminate the biological activity and properties of the applied composition. Adjuvants are included under these phrases.

[0119] According to another aspect of the invention, a cosmeceutical composition is provided, comprising an effective amount of the composition of the invention, an acceptable salt thereof, or both, and a cosmeceutical-acceptable carrier.

[0120] In some embodiments, the cosmeceutical composition of the present invention is a topical composition.

[0121] According to another aspect of the invention, a pharmaceutical composition is provided comprising an effective amount of the composition of the invention, an acceptable salt thereof, or both, and a pharmaceutically acceptable carrier.

[0122] In some implementations, the effective therapeutic dose is sufficient to significantly reduce collagen loss.

[0123] In some embodiments, the pharmaceutical compositions of the present invention are used to prevent or treat diseases or disorders related to collagen loss in subjects in need.

[0124] In some implementations, the terms "pharmaceutical acceptable" and "cosmeceutical acceptable" refer to carriers approved by regulatory agencies (such as federal or state governments) for use in animals, and more specifically for use in humans.

[0125] In some embodiments, pharmaceutical and cosmeceutical compositions include incorporating the compositions of the present invention into or onto particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or into liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte shadows, or protoplasts. Those skilled in the art will understand that such incorporation can affect physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0126] In some embodiments, the pharmaceutical composition is an oral composition or a topical composition. In some embodiments, the pharmaceutical composition described herein is a topical composition. In some embodiments, the pharmaceutical composition described herein is an oral composition.

[0127] In some embodiments, the oral composition is in the form of a soft gel capsule. In some embodiments, the oral composition is in the form of a beverage, a shot, a gummy, or a powder. In some embodiments, the oral composition is mixed with or assimilated into a food, such as chocolate, ice cream, or other food.

[0128] In some embodiments, the pharmaceutical composition is an injectable composition. In some embodiments, the pharmaceutical composition is for systemic use.

[0129] In some embodiments, the compositions of the present invention are referred to herein as the active ingredient of a pharmaceutical composition. In some embodiments, the compositions of the present invention are the sole active ingredient in a pharmaceutical composition.

[0130] In some embodiments, the pharmaceutical composition present in the present invention is of pharmaceutical grade purity.

[0131] In some embodiments, pharmaceutical-grade purity is characterized by a chemical purity of at least about 90%, at least about 95%, greater than 95%, greater than 99%, or any value and range thereof. In some embodiments, pharmaceutical-grade purity is characterized by a chemical purity between 90% and 99.999%, between 90% and 95%, between 90% and 97%, or between 95% and 99%. Each possibility represents a separate embodiment of the invention.

[0132] In one embodiment, "excipient" refers to an inert substance added to the composition to further facilitate the administration of the active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. In some embodiments, excipients and / or carriers improve the stability of the active ingredient in living organisms. In some embodiments, carriers improve the stability of the active ingredient in the pharmaceutical composition. In some embodiments, carriers improve the bioavailability of the active ingredient.

[0133] The formulation and administration techniques for the drug are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition (which is incorporated herein by reference in its entirety). In one embodiment, suitable routes of administration include, for example, oral, rectal, transmucosal, nasal, enteral, or parenteral delivery—including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intracardiac, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0134] According to another aspect, a kit is provided comprising: (a) a mixture of carotenoids, including at least one carotenoid selected from phytoene, hexahydrolycopene, zetacarotene, or any combination thereof; and (b) ceramide.

[0135] In some embodiments, the carotenoid mixture comprises at least one natural carotenoid or a combination thereof, a synthetic carotenoid or a combination thereof, or both.

[0136] In some embodiments, the carotenoid mixture and ceramide are stored in separate compartments. In some embodiments, the carotenoid mixture and ceramide are stored in the same compartment. In some embodiments, the carotenoid mixture and ceramide are in powder or liquid (e.g., solution) form.

[0137] In some embodiments, the kit further includes mixing a mixture of carotenoids and ceramides under appropriate conditions to obtain the composition of the present invention.

[0138] In some implementations, suitable conditions refer to a final w / w ratio of the carotenoid mixture and ceramide between 1:5 and 5:1.

[0139] In some embodiments, suitable conditions refer to mixing temperatures between 20°C and 40°C, between 22°C and 35°C, between 20°C and 35°C, between 20°C and 30°C, between 22°C and 27°C, or between 20°C and 27°C. Each possibility represents a separate embodiment.

[0140] In some embodiments, the kit further includes an acceptable carrier. In some embodiments, the acceptable carrier includes or is acceptable for pharmaceuticals, cosmetics, or nutritional supplements. In some embodiments, the acceptable carrier is or includes a liquid carrier.

[0141] In some embodiments, any one of the carotenoid mixture, ceramide, and acceptable carrier is stored in different compartments. In some embodiments, any one of the carotenoid mixture, ceramide, and acceptable carrier is stored in the same compartment.

[0142] In some embodiments, the kit further includes instructions for mixing an acceptable carrier with a mixture of carotenoids and ceramides under suitable conditions.

[0143] In some embodiments, mixing includes obtaining a composition wherein the final w / w percentage of an acceptable carrier is 0.1% to 99.9%, 10% to 99.9%, 20% to 99.9%, 1% to 50%, 20% to 70%, 50% to 99.9%, or 30% to 90%. Each possibility represents a separate embodiment.

[0144] In some implementations, the instructions for mixing can be varied, for example, to meet the needs of a subgroup of patients to be treated or the needs of a single patient who may have different needs due to a specific disease, the severity of the disease, age, sex or weight, and those skilled in the art can easily meet those needs.

[0145] According to another aspect of the invention, a method for preventing or treating collagen loss in a subject in need is provided, comprising administering a therapeutically effective amount of the composition of the invention to the subject, thereby preventing or treating collagen loss-related diseases or disorders in the subject.

[0146] According to another aspect of the invention, a method for preventing or treating collagen loss in a subject in need is provided, comprising administering to the subject a synergistically effective amount of a composition comprising: (a) a carotenoid mixture including at least one carotenoid selected from phytoene, hexahydrophytoene, or zetacarotene; and (b) a ceramide, thereby preventing or treating collagen loss-related diseases or disorders in the subject.

[0147] In some implementations, the object is a mammal. In some implementations, the mammal is a human.

[0148] In some implementations, the subject suffers from a collagen loss-related disease.

[0149] In some embodiments, the object is characterized by an increase in the expression, activity, or both of the following: MMP, MPO, superoxide (SO), elastase, nitric oxide (NO), or any combination thereof.

[0150] In some embodiments, the method further includes determining the expression level, amount, abundance, activity, or any combination thereof in a sample obtained from or derived from the object.

[0151] In some implementations, it is determined whether the procedure is performed in vitro or outside the body.

[0152] In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from collagen loss induced by: aging, exposure to environmental factors, diet, lack of sleep, stress, or any combination thereof.

[0153] In some implementations, the environmental factor is selected from radiation, pollution, smog, or any combination thereof.

[0154] In some embodiments, a subject suffering from a collagen loss-related disease is characterized by: decreased skin elasticity, decreased epidermal thickness, increased skin susceptibility to damage (such as mechanical damage), increased skin wrinkles, sagging, or both, and any combination thereof.

[0155] Methods for determining either skin elasticity or epidermal thickness are common and will be readily apparent to those skilled in the art, and non-limiting examples of such methods include, but are not limited to, skin aspiration or indentation followed by detection of skin displacement by optical measurements, and histological analysis of skin biopsies.

[0156] According to some embodiments, a method is provided for inhibiting or reducing in a subject any amount, activity, or both of the following: MMP, MPO, superoxide (SO), elastase, nitric oxide (NO), and any combination thereof, the method comprising administering to the subject a synergistic (as above) / therapeuticly effective amount of the composition of the present invention.

[0157] In one implementation, MMP is MMP-9, MMP-8, MMP-1, or any combination thereof.

[0158] In some implementations, the method further includes reducing ROS production, secretion, or both in cells or objects.

[0159] Methods for determining the amount, activity, or both of any one of MMP, MPO, SO, elastase, NO, and ROS are common and will be apparent to those skilled in the art. Non-limiting examples of such methods include, but are not limited to, ELISA, immunohistochemistry, oxidative assays and enzymatic assays (such as those illustrated below) and / or other methods.

[0160] In some embodiments, the inhibition or reduction compared to the control is 20-90%, 30-90%, 40-95%, 50-99%, 50-95%, 70-90%, or 75-99%. Each possibility represents a separate embodiment of the invention.

[0161] As used herein, a control includes skin from a healthy subject. In some embodiments, a control is a healthy skin sample derived, isolated, or obtained from the same subject (e.g., a subject suffering from a collagen loss-related disease). In some embodiments, a control includes a skin sample derived, isolated, or obtained from the same subject suffering from collagen loss treated only with a mixture of carotenoids or ceramides (but not both simultaneously).

[0162] In some embodiments, the control includes untreated cell cultures. In some embodiments, the control includes cell cultures treated only with a mixture of carotenoids or ceramides (but not both at the same time).

[0163] In some embodiments, administration includes oral administration. In some embodiments, administration includes topical administration. In some embodiments, administration includes a combination of oral and topical administration.

[0164] In some implementations, collagen loss-related diseases are selected from: osteogenesis imperfecta, osteoporosis, E.D.-D. syndrome, infantile extraosseous hypertrophy (also known as Kafir disease), collagenopathy (types II and XI), Dupuitren contracture, Alport syndrome, pulmonary hemorrhage-nephritis syndrome, Ulrich myopathy, Bethlem myopathy, atopic dermatitis, dystrophic bullous epidermolysis, posterior polymorphic corneal dystrophy 2, EDM2, EDM3, Schmid metaphyseal dysplasia, bullous pemphigoid, psoriasis, eczema, and solar radiation-induced hyperplasia.

[0165] In some implementations, collagen loss-related diseases include the loss of collagen-1.

[0166] As used in this article, the phrase “collagen loss-related disease” refers to any disease, disorder, or associated symptom that is induced by, involves, is promoted by, propagated by, or is enhanced by collagen loss, any equivalent of, or any combination thereof.

[0167] In some embodiments, the method includes treating or preventing, in a subject with a condition selected from: osteogenesis imperfecta, osteoporosis, E.D.-D. syndrome, infantile extraosseous hypertrophy (also known as Kafir disease), collagen diseases (types II and XI), Dupuytren contracture, Allport syndrome, pulmonary hemorrhage-nephritis syndrome, Ulrich myopathy, Bethlem myopathy, atopic dermatitis, dystrophic bullous epidermolysis, posterior polymorphic keratodystrophy type 2, EDM2, EDM3, Schmid metaphyseal dysplasia, bullous pemphigoid, psoriasis, eczema, solar radiation-induced hyperplasia, or any combination thereof, the method comprising administering a therapeutically effective amount of the composition of the present invention to the subject.

[0168] In some embodiments, collagen loss includes decreased collagen expression (transcription and / or translation), assembly, stability, or any combination thereof, increased degradation and / or breakdown, or any combination thereof.

[0169] In some implementations, collagen loss-related diseases are selected from age-related diseases, skin diseases, and inflammatory diseases.

[0170] As used in this article, “age-related disease” refers to a disease or condition whose incidence increases rapidly with age. As used in this article, the term “rapidly” means exponentially.

[0171] Non-limiting examples of age-related conditions—which may be collectively referred to as 'lesser ailments of aging' (LAA)—include, but are not limited to, general muscle weakness, hypothermia, slow healing of wrinkles and bruises in the skin, emaciation (overall weight loss), muscle loss, and decreased bone density.

[0172] As used herein, the phrase "dermatitis" refers to any skin pathological condition or its symptoms. In one embodiment, collagen loss is a pathogenic factor leading to the development of a skin disease. In one embodiment, collagen loss is a pathophysiological factor resulting from the onset, initiation, progression, or any combination thereof of a skin disease. In one embodiment, collagen loss is a characteristic feature of a developing or ongoing skin disease.

[0173] In some implementations, the skin disease is an immune-related skin disease. In one implementation, an immune-related skin disease is an inflammatory skin disease, an autoinflammatory skin disease, or an autoimmune skin disease.

[0174] As used herein, the phrase “inflammatory disease” refers to any disease involving a multicomponent response of an organism (e.g., cells of the immune system, molecular signaling mediators such as cytokines) to a harmful exogenous entity (e.g., bacteria, fungi, viruses, protozoa, allergens, etc.).

[0175] In some embodiments, the dermatitis is characterized by or determined based on skin lesions. In some embodiments, the skin lesions are induced by any of the following: radiation, oxidative stress, DNA damage, telomere shortening, inflammation, tobacco use, and any combination thereof.

[0176] In some implementations, radiation includes any wavelength of radiation within the spectrum. As used herein, the term "spectrum" covers a range from 10... -9 m to 10 -3 The wavelength of m. In some embodiments, the radiation wavelengths within the spectrum include UV radiation, visible light radiation, infrared radiation, or combinations thereof. In some embodiments, exposure to radiation includes exposure to sunlight.

[0177] As used herein, the term "ultraviolet (UV)" encompasses any wavelength within the UV range. In some embodiments, UV is UV radiation. In some embodiments, radiation is UV radiation. In some embodiments, UV radiation is UVA radiation, UVB radiation, UVC, or any combination thereof.

[0178] As used herein, the term “infrared” covers wavelengths in the range of 700 nm to 1,000 nm (with frequencies from 430 THz to 300 GHz).

[0179] In some embodiments, compared to a control, the subject has an increased amount, increased activity, or both of any one of MMP, MPO, SO, elastase, NO, ROS, and any combination thereof in the skin, systemically, or both.

[0180] In some embodiments, the increase compared to the control is 1-10%, 5-30%, 15-50%, 25-75%, 70-150%, 100-350%, 250-550%, 500-750%, or 700-1,000%. Each possibility represents a separate embodiment of the invention.

[0181] As used herein, the terms "treatment" or "treating" encompass the reduction of at least one symptom, decrease in severity, or inhibition of progression of a disease, disorder, or condition. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a composition useful herein to be considered an effective treatment, it need only reduce the severity of the disease, disorder, or condition, decrease the severity of associated symptoms, or provide an improvement in the quality of life of the patient or subject.

[0182] As used herein, the term “prevention” for a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the subject matter currently described, the term “prevention” refers to a preventative process in which an object is exposed to the composition or preparation currently described prior to the induction or onset of a disease / disorder process. This can be done when an individual has a genetic lineage indicating a predisposition to the disease / disorder to be prevented. For example, this might apply to individuals whose ancestors showed a predisposition to certain types, such as inflammatory disorders. The term “suppression” is used to describe a situation in which the disease / disorder process has begun but has not yet manifested as a condition with obvious symptoms. Thus, an individual’s cells may have a disease / disorder, but external signs of the disease / disorder have not yet been clinically identified. In either case, the term prevention may be used to encompass both prevention and suppression. Conversely, the term “treatment” refers to the clinical application of an active agent to counteract a pre-existing condition in which clinical manifestations have already been realized in a patient.

[0183] In some implementations, prevention includes reducing disease severity, delaying disease onset, reducing cumulative disease incidence, or any combination thereof.

[0184] As used herein, the terms “object” or “individual” or “animal” or “patient” or “mammal” refer to any object for which treatment is desired, particularly mammalian objects such as humans.

[0185] In this discussion, unless otherwise stated, adjectives such as “substantially” and “about” modifying the conditional or relational characteristics of one or more features of embodiments of the invention should be understood as meaning that the condition or characteristic is defined as being within acceptable tolerances for the operation of the embodiment when it is intended to be applied. Unless otherwise indicated, the word “or” in the specification and claims is considered an inclusive “or” rather than an exclusive “or”, and means at least one or any combination of the items it combines.

[0186] It should be understood that the terms “a” and “an” as used above and elsewhere in this document refer to “one or more” of the listed components. Unless otherwise specifically stated, it will be clear to those skilled in the art that the use of the singular includes the plural. Therefore, the terms “a”, “an”, and “at least one” are used interchangeably in this application.

[0187] To better understand this teaching and in no way limit its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should be understood to be modified by the term "about" in all cases, unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based on the number of significant figures reported and the application of common rounding techniques.

[0188] In the descriptive claims of this application, the verbs “comprise,” “include,” and “have,” and their inflectional forms, are used to indicate that one or more objects of the verb are not necessarily a complete enumeration of the components, elements, or parts of one or more subjects of the verb.

[0189] Other terms used herein are intended to be defined by their meanings as known in the art.

[0190] Unless specifically stated or obvious from the context, as used herein, the term "or" is to be understood as inclusive.

[0191] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" mean that any integer or group of integers described herein is included, but do not exclude any other integer or group of integers.

[0192] As used herein, the terms “comprises,” “comprising,” “containing,” “having,” etc., can mean “includes,” “including,” etc.; “consisting essentially of,” “consistses essentially of,” etc., also have the meanings given to them by U.S. patent law, and the terminology is open-ended, allowing for the presence of content other than that described, provided that the essential or novel characteristics of the described content are not altered by the presence of content other than that described, but excluding prior art implementations. In one implementation, the terms “comprises,” “comprising,” “having,” etc., can be used interchangeably with “consisting.”

[0193] Further objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following embodiments, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the invention as described above and claimed in the claims section finds experimental support in the following embodiments.

[0194] Example In general, the nomenclature used herein and the laboratory procedures used in this invention encompass chemical, molecular, biochemical, and cell biological techniques. These techniques are described in detail in the literature. See, for example, "Molecular Cloning: Alaboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); "Cell Biology: A Laboratory Handbook" Volumes I-III Cellis, JE, ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry by Leroy "Skip" G Wade (6th edition); Organic Chemistry by TW Graham Solomons and Craig Fryhle.

[0195] Materials and Methods Macrophage isolation and culture – peritoneum As previously described by Hadad et al. (2012), macrophages were collected from the peritoneal cavity of 6-8 week old male ICR mice (Harlan, Israel) after intraperitoneal injection of 1.5 ml thioglycolate broth (4%) 4 days prior to harvest. Peritoneal macrophages were washed three times with PBS, and hypotonic lysis of erythrocytes was performed as appropriate to produce a highly enriched (90-95%) macrophage population. Peritoneal macrophages (1×10⁶ cells / well) were cultured in 96-well plates at 37°C under a 5% CO₂ atmosphere in RPMI 1640 medium containing 10% FCS, 2 MM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Beit-Haemek, Israel). 6 Cells / well). Using serum from Salmonella enterocolitica typhus serology (…). Salmonela enteric a serotype typhimuriumCells were stimulated with 1 μg / ml LPS. This study was conducted with the approval of the Ben-Gurion University of the Negev committee for ethical care and use of animals in experiments (approval number IL-33-06-2020) and in accordance with the Guide for Care and Use of Laboratory Animal (National Research Council, 1996) in accordance with the Israel Animal Welfare Act.

[0196] Neutrophil purification Forty (40) ml of neutrophil counts were drawn from healthy volunteers who had given their written consent, with a count between 3 and 7 × 10⁻⁶. 6 Blood samples were collected at a concentration of neutrophils per ml. Neutrophils with a purity of 95% were obtained by Ficoll / Histopaque centrifugation, dextran precipitation, and hypotonic lysis of erythrocytes. Cell counts were performed, and viability was determined using the trypan blue exclusion method. This study was approved by the Institutional Human Research Committee of the Soroka University Medical Center (No. 0370-16-SOR). Neutrophils were stimulated with TNFα (Peprotech, Rocky Hill, NJ, USA).

[0197] Element Lumenato—composed of 0.55% lycopene, 24.06% phytopene, 6.75% hexahydrolycopene, 11.27% tocopherol, 0.61% β-carotene, 3.15% phytosterols, 7.48% ζ-carotene, 0.16% γ-carotene, and tomato seed oil—was supplied by LycoRed Natural Products Industries Ltd. (Beer-Sheva, Israel). C2-ceramide (d18:1 / 2:0) was purchased from Avanti Polar lipids Inc. (Alabaster, Alabama, USA). Working concentrations of phytonutrients were prepared from stock solutions dissolved in DMSO (final concentration of ceramide 5 mmol / mL, Lumenato 200 mg / mL) by adding appropriate volumes of the stock solutions to warm culture medium.

[0198] Fibroblast culture Normal human dermal fibroblasts (NHFD) from adult donors were cultured in Promocell (Promocell, Heidelberg, Germany) and supplemented with Promocell to reach the final concentrations in the medium: 0.02 ml / ml fetal bovine serum, 1 mg / ml basic fibroblast recombinant human growth factor and 5 µg / ml recombinant human insulin, 2 MM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Beit-Haemek, Israel). Cells were cultured at 37°C in a humid atmosphere containing 5% CO2. When the fibroblasts reached more than 80% confluence, the cells were seeded into 24-well plates.

[0199] Cell survival The cell count was performed using the trypan blue exclusion method or by colorimetric MTT assay ([3,4,5-dimethylthiazol-2-yl]-2,5-biphenyl-tetrazole). Metabolic activity assays were used to assess cell viability, as previously done by Solomonov et al. (2021). For MTT measurements, cells were cultured in 96-well plates. MTT was dissolved in medium (5 mg / ml) and added to each sample at 10% of the medium volume. After incubation for 30 min, formazan crystals were dissolved in 100 MM HCl and 10% Triton X-100 (completely dissolved in isopropanol, in an equal volume to the medium). The medium served only as background. Absorbance intensity was measured at 570 nm using a Versamax Microplate Reader (Molecular Devices, Menlo Park, CA, USA), with a reference wavelength of 690 nm.

[0200] Nitric oxide (NO) production determination The NO level in the cell culture supernatant was determined by measuring the nitrite concentration using Griess reagent and sodium nitrite as a standard.

[0201] Immunofluorescence analysis For immunofluorescence assays, fibroblasts or co-cultures of fibroblasts and neutrophils were fixed with methanol at -20°C for 3 min, followed by washing in PBS. The fixed co-cultures were incubated with anti-collagen I antibody (Southern Biotech Birmingham, Alabama, USA) at a dilution of 1:500 in 5% BSA / PBS for 90 min at room temperature, as previously described by Solomonov et al. (2018). Cells were washed three times in PBS and incubated with Cy3 anti-mouse (1:100, in 5% BSA / PBS; Jackson Immuno Research Laboratories, Inc., PA, USA) for 60 min at room temperature. Cells were washed three times in PBS and the nuclei were stained with DAPI. A final wash was then performed, and cells were analyzed by fluorescence microscopy (Olympus, BX60, Hamburg, Germany). The fluorescence intensity of collagen III was determined using the CellProfiler program. Total cell lysate Total cell lysates were prepared using 1% Triton X-100, 50 MM HEPES (pH 7.5), 150 MM NaCl, 1 MM EDTA, 1 MM EGTA, 10% glycerol, 25 MM NaF, 10 μM ZnCl2, 1 MM PMSF and 100 μM leucopeptide, as described earlier by Hadad et al. in 2012.

[0202] Immunoblotting analysis The lysate proteins (10 μg) were separated by electrophoresis on a 7.5% polyacrylamide SDS gel. The resolved proteins were electrophoretically transferred to nitrocellulose, and the protein bands were detected by staining with Ponceau S and then blocked in 5% emulsion in TBS (10 MM Tris, 135 MM NaCl, pH 7.4). Immunoblot determination was performed as previously described by Hadad et al. (2012)—incubated overnight at 4°C with a primary antibody against collagen I (Southern Biotech Birmingham, Alabama, USA) and a primary antibody against actin (MP Biomedicals, 9 Goddard Irvine, CA, USA), and then incubated at room temperature for 1 hour with a secondary antibody (peroxidase-conjugated goat anti-goat or anti-mouse) (Amersham Biosciences, Buckinghamshire, United Kingdom), followed by development using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences).

[0203] Superoxide formation Horseradish peroxidase (HRP)-dependent oxidation of Amplex Red was performed using a highly sensitive fluorescent biosensor. The oxidation of Amplex Red occurs extracellularly via HRP, once H2O2 is produced by the NADPH oxidase, the first product of which is O2. - Spontaneous dismutation occurs, and HRP captures it. Resting and activated neutrophils (2 × 10⁻⁶) are then collected. 5 Cells / well were suspended in KRPG buffer (phosphate buffer, 145 mM NaCl, 4.86 mM KCl, 1.22 mM MgSO4, 5.5 mM D-glucose and 0.54 mM CaCl2, pH 7.35) containing HRP (0.1 units / ml) and Amplex Red (50 µM). Fluorescence was recorded using a microplate reader with excitation at 535 nm and emission at 595 nm. Background fluorescence was measured in the absence of neutrophils.

[0204] Myeloperoxidase (MPO) activity One hundred (100) µl of 37°C o-anisidine hydrochloride solution (1 mg o-anisidine, 10 ml phosphate buffer pH 6.0 + 0.0015% H2O2) was added to 100 µl of supernatant in a 96-well plate, and the optical density was measured immediately. Then, the change in absorbance at 450 nm was measured at 2 min intervals on a Thermomax microplate reader (Molecular Devices, Menlo Park, CA).

[0205] Statistical analysis Data are presented as mean ± SEM. Significant differences from the control were determined using one-way or two-way ANOVA followed by a post-hoc Bonferroni multiple comparison test provided by GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

[0206] result The dose-response effect of Lumenato or ceramide on NO production was investigated—assessed by NO secretion levels in the culture supernatant of peritoneal macrophages. Lumenato or ceramide was added 1 h before the addition of 1 mg / ml LPS to peritoneal macrophages cultured at 37°C for 24 h. Secreted NO in the cell culture supernatant was determined. Lumenato (in the range of 6.5–208 μg / ml) or ceramide (in the range of 0.034–17 μg / ml) caused a dose-dependent inhibition of NO production. Figure 1A and Figure 1C ), without affecting cell viability as measured by MTT assay ( Figure 1B and Figure 1D ).

[0207] Similarly, the effects of Lumenato or ceramide in these ranges on neutrophil activation were investigated. Lumenato or ceramide was added to neutrophils at 37°C for 15 min, followed by overnight activation with TNFα, and the concentration of MPO in the supernatant was determined. Figure 2A and Figure 2C Immediately after activation via cytochrome C reduction, the effects of Lumenato or ceramide on the production of short-lived superoxide were determined. Figure 2B and Figure 2D ).like Figures 2A-2DAs shown, the addition of Lumenato or ceramide caused dose-dependent inhibition of MPO release and superoxide production.

[0208] To investigate whether the combination resulted in synergistic inhibition, Lumenato (6.5 or 13 μg / ml) or ceramide (0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml) were added, alone or in combination, to macrophages 1 h prior to overnight LPS addition. Secreted NO in the cell culture supernatant was determined. These combinations caused approximately twice the additive effect of synergistic inhibition of NO production. Figures 3A-3D ).

[0209] To investigate whether these combinations could synergistically protect against collagen-1 damage induced by TNFα-activated neutrophils, the inventors employed the optimal conditions for co-culturing fibroblasts and neutrophils as determined by Solomonov et al. (2021). Neutrophils were stimulated with TNFα because the upregulation of this cytokine is a key early response of keratinocytes in the skin to sunlight and UVB and represents an important component of the inflammatory cascade in the skin. During overnight incubation, short-lived neutrophils were not resistant and therefore did not interfere with collagen staining of the co-culture. For co-culture, 1 × 10⁶ cells were used. 5 Fibroblasts were plated at 100 ng / ml and cultured overnight to obtain confluence cultures. Before activation with 100 ng / ml TNFα, 2 × 10⁶ cells were cultured. 5 Add Lumenato, ceramide, or a combination thereof to neutrophils / ml for 15 min. Add activated neutrophils with or without these components to fibroblasts overnight. As shown by immunofluorescence staining and density assay, 2 × 10⁶ cells / ml. 5 The addition of TNFα-activated neutrophils / ml caused significant (p<0.001) collagen-1 damage, reducing collagen-1 from 53±3.2% to 38.3±1.9%, without affecting fibroblast numbers. Figure 4A As shown by immunofluorescence staining, the addition of 6.5 μg / ml Lumenato had no effect on preventing collagen-1 damage. Figure 4A As shown by immunofluorescence staining and density assays, the presence of ceramides (0.034 μg / ml, 0.17 μg / ml, or 0.34 μg / ml) resulted in low and similar protection against collagen-1 damage (p < 0.05). Figure 4B As shown by immunofluorescence staining and density assays, combinations of Lumenato with various ceramide concentrations resulted in dose-dependent protection against collagen-1 damage. Figure 4C Cell counts were unaffected in all cultures. Figures 4A-4CThe calculated percentage of collagen-1 loss prevention resulting from each component and its combination is presented in [the table / data]. Figure 4D In the study, the combination of 6.5 μg / ml Lumenato and 0.34 μg / ml ceramide achieved the best and most significant (p<0.01**) prevention of collagen-1 damage compared to the additive effect (79.3% + 13.2%, compared to 43%). Similar results were obtained by Western blot analysis, such as... Figures 5A-5B As shown. Immunoblot analysis revealed that the addition of TNFα-activated neutrophils significantly reduced collagen-1 (approximately 50%). Figure 5A The combination of 6.5 μg / ml Lumenato and 0.34 μg / ml ceramide yielded a significantly (p<0.001) synergistic prevention of collagen-1 loss compared to the additive effect—96 ± 10.8%, compared to 46.4% with Lumenato alone. Figure 5B ).

[0210] The effect of 13 μg / ml Lumenato was also investigated in the same experiments described in Figures 4-5. Pretreatment of neutrophils with 13 μg / ml Lumenato before supplementation into fibroblasts showed low and insignificant protection against collagen damage. Figure 6A Different doses of ceramides alone offer low protection against collagen loss. Figure 6B ,and Figure 4B (Same as shown). In stark contrast, the combination of 13 μg / ml Lumenato with different ceramide concentrations provided significant (p<0.001) protection against collagen loss. Figure 6C Cell count remained unaffected in all cultures. Figure 6DThe calculated percentage of protection against collagen-1 loss resulting from these combinations is presented in Figure 7. The results showed that the combinations produced significant (p<0.01) synergistic protection, on the order of magnitude of approximately twice the additive effect. The combination of 13 μg / ml Lumenato with (i) 0.034 μg / ml ceramide provided a synergistic effect, preventing collagen-1 loss by 83.7 ± 6.4% compared to the additive effect (39%); the combination with (ii) 0.17 μg / ml ceramide provided 89.4 ± 7.9% protection, compared to an additive effect of 47.7%; and the combination with (iii) 0.34 μg / ml ceramide provided 92.2 ± 13.2% protection, compared to an additive effect of 52%. Similar results were obtained using Western blot analysis, indicating that different combinations of Lumenato and ceramide provided significant synergistic (p<0.01) protection against activated neutrophil-induced collagen-1 loss (Figure 7). MPO activity measured in the supernatant of the co-cultures showed synergistic activity consistent with the synergistic protection against collagen damage induced by activated neutrophils in the combination of Lumenato and ceramide (Figure 8). Addition of Lumenato and ceramide to fibroblast cultures—single or in combination—did not affect collagen-1 expression or fibroblast number. Figures 9A-9D ).

[0211] discuss In this study, the inventors demonstrated that collagen-1 damage induced by activated neutrophils in fibroblast cultures can be prevented in a dose-dependent manner by Lumenato or ceramide. The inventors previously reported an 80% prevention of neutrophil-induced collagen loss in the presence of 208 μg / ml Lumenato. In the current study, however, the inventors used much lower concentrations of Lumenato (6.5 μg / ml or 13 μg / ml) and ceramide (0.034–0.34 μg / ml), neither of which showed protection against collagen-1 damage individually, or only low protection; however, when combined, they achieved synergistic protection against collagen-1 damage—greater than 80%. By using the exemplary composition of the present invention (a combination of low concentrations of Lumenato and ceramide), the inventors demonstrated a surprisingly significant synergistic prevention of collagen-1 loss.

[0212] The effects of Lumenato and ceramides are likely mediated by inhibiting neutrophil activation rather than by increasing collagen-1 synthesis, as their addition, either individually or in combination, to fibroblast cultures does not affect collagen-1 expression or cell count. This proposal is supported by the dose-dependent inhibition of superoxide production and MPO release in stimulated neutrophils by each of these components, as well as the inhibition of MPO activity detected in the supernatant of the co-culture.

[0213] The inventors have also demonstrated that ceramides or lumenato inhibit NO production induced by LPS-stimulated macrophages in a dose-dependent manner. Furthermore, the inventors have clearly demonstrated herein that a combination of low concentrations of lumenato and ceramides results in synergistic inhibition of NO production induced by stimulated macrophages.

[0214] The inventors demonstrate that ceramides block ROS release from neutrophils and prevent collagen-1 damage induced by activated neutrophils, thus possessing valuable potential in protecting against collagen damage—such as after exposure to natural sunlight, erythematous doses of UVB, or solar irritant radiation (which induces ROS release by infiltrating neutrophils into the skin).

[0215] Furthermore, the inventors have demonstrated that the combination of low concentrations of Lumenato with ceramides induces significant synergistic protection against collagen-1 damage induced by activated neutrophils, thus indicating the potential of this combination to protect the skin from photoaging.

[0216] Although the invention has been specifically described, those skilled in the art will understand that various modifications and variations are possible. Therefore, the invention should not be construed as limited to the specifically described embodiments, and the scope and concept of the invention will be more readily understood by referring to the appended claims.

Claims

1. A composition comprising: (a) A mixture of carotenoids, including phytoene, phytoene and zeta-carotene; and (b) ceramides; The weight / weight ratio (w / w) of (a) to (b) in the composition is between 25:1 (w / w) and 500:1 (w / w).

2. The composition according to claim 1, wherein the carotenoid mixture comprises: The amount of the 8-hydrolycopene is 55-65% (w / w) of the total carotenoids in the carotenoid mixture; The amount of the hexahydrolycopene is 10-20% (w / w) of the total carotenoids in the carotenoid mixture; and the amount of the ζ-carotene is 15-25% (w / w) of the total carotenoids in the carotenoid mixture.

3. The composition according to claim 2, wherein the w / w ratio of the combined phytoene and phytohexene to the ζ-carotene ranges from 15:1 (w / w) to 2:1 (w / w).

4. The composition according to any one of claims 1 to 3, wherein the carotenoid mixture further comprises other carotenoids selected from the following: lycopene, β-carotene, γ-carotene, and any combination thereof.

5. The composition according to claim 4, comprising less than 5% (w / w) of the lycopene in the total carotenoids of the carotenoid mixture, less than 5% (w / w) of the β-carotene in the total carotenoids of the carotenoid mixture, and 0.2-1.5% (w / w) of the γ-carotene or a combination thereof in the total carotenoids of the carotenoid mixture.

6. The composition according to any one of claims 1 to 5, wherein the carotenoid mixture comprises 10-15% (w / w) of the total carotenoid content of the carotenoid mixture.

7. The composition according to any one of claims 1 to 6, wherein the carotenoid mixture further comprises tocopherol in an amount of 10-30% (w / w) of the total carotenoids in the carotenoid mixture.

8. The composition according to any one of claims 1 to 7, wherein the carotenoid mixture further comprises 5-15% (w / w) of phytosterols in the total carotenoids of the carotenoid mixture.

9. The composition according to any one of claims 1 to 8, wherein the ceramide comprises multiple types of ceramides.

10. The composition according to any one of claims 1 to 9, wherein the ceramide and any one of the plurality of types thereof are selected from: C2-ceramide, C1-ceramide, C3-ceramide, C6-ceramide, and ceramide 9.

11. The composition according to any one of claims 1 to 10, wherein the composition has a synergistic effect in inhibiting: (i) collagen loss; (ii) the production, secretion, activity, or any combination thereof of nitric oxide (NO), myeloperoxidase (MPO), superoxide (SO), and (iii) both of (i) and (ii).

12. The composition of claim 11, wherein the collagen is collagen-1.

13. A pharmaceutical composition comprising an effective amount of the composition according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, for use in preventing or treating collagen loss-related diseases or disorders in subjects in need.

15. The pharmaceutical composition for use according to claim 14, wherein the collagen is collagen-1.

16. The pharmaceutical composition for use according to claim 14 or 15, wherein the collagen loss-related disease or disorder includes collagen loss in the skin of the subject.

17. A method for preventing or treating collagen loss-related diseases or disorders in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 13 to the subject, thereby preventing or treating collagen loss-related diseases or disorders in the subject.

18. A method for reducing the amount, activity, or both of any one of MPO, NO, superoxide, and any combination thereof in a subject in need, said method comprising administering to said subject a therapeutically effective amount of the composition according to any one of claims 1 to 12.

19. The method of claim 18, wherein the subject suffers from a collagen loss-related disease or disorder.

20. The method of claim 19, wherein the collagen loss-related diseases are selected from: age-related diseases, skin diseases, and inflammatory diseases.

21. The method of claim 20, wherein the dermatological condition comprises skin damage induced by any of the following: radiation, oxidative stress, DNA damage, telomere shortening, inflammation, smoking, and any combination thereof.

22. The method of claim 21, wherein the radiation includes UV radiation, visible light radiation, infrared radiation, or any combination thereof.

23. The method of claim 22, wherein the UV radiation comprises UVA, UVB, UVC, or any combination thereof.

24. The method according to any one of claims 18 to 23, wherein, compared with a control, the object has an increased amount, increased activity, or both of any one of the following in the skin, systemically, or both: MPO, NO, SO, and any combination thereof.

25. The method according to any one of claims 18 to 24, further comprising determining in a sample obtained from or derived from the object an amount, activity, or both of the following: MPO, NO, SO, and any combination thereof.

26. The method according to any one of claims 18 to 25, wherein the application comprises oral administration, topical administration, or both.

27. A set comprising: (a) At least one carotenoid selected from phytoene, phytoene, zeta-carotene and any combination thereof; and (b) ceramides.

28. The kit according to claim 27, wherein the at least one carotenoid comprises phytoene, phytohexene, and zeta-carotene.

29. The kit according to claim 27 or 28, further comprising a specification for mixing (a) and (b) w / w at a ratio of 25:1 (w / w) to 500:1 (w / w).

30. The kit according to any one of claims 27 to 29, for preparing a composition suitable for preventing or treating collagen loss-related diseases in subjects in need.

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