WO2025003218A1 - Liposomal-based composition - Google Patents

Liposomal-based composition Download PDF

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Publication number
WO2025003218A1
WO2025003218A1 PCT/EP2024/067943 EP2024067943W WO2025003218A1 WO 2025003218 A1 WO2025003218 A1 WO 2025003218A1 EP 2024067943 W EP2024067943 W EP 2024067943W WO 2025003218 A1 WO2025003218 A1 WO 2025003218A1
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WIPO (PCT)
Prior art keywords
composition
ceramide
acid
cholesterol
mucosa
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Ceased
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PCT/EP2024/067943
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French (fr)
Inventor
Cristina ALONSO MERINO
Meritxell MARTÍ GELABERT
Mª Luisa CODERCH NEGRA
Ana Cristina CALPENA CAMPMANY
Mª Luisa PÉREZ GARCÍA
Beatriz CLARES NAVEROS
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Granada
Universitat de Barcelona UB
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Granada
Universitat de Barcelona UB
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Priority to EP24734917.8A priority Critical patent/EP4731197A1/en
Publication of WO2025003218A1 publication Critical patent/WO2025003218A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents

Definitions

  • the present invention relates to a liposomal-based composition and to the use thereof as an agent for the impermeabilization of oral mucosae, therefore, said composition also relates to the use thereof as a medical product, particularly for preventing viral infections, such as SARS-CoV-2, and to the use thereof for preventing contaminants, such as biocides, from penetrating through the oral mucosae. Therefore, the invention could be comprised in the field of pharmaceutical technology.
  • SARS-CoV-2 permeation of SARS-CoV-2 through the nasal or oral mucosa in contrast to its low penetration through the keratinized tissue of the stratum corneum of the skin is widely known. This is mainly due to the different lipid composition and packaging structures formed by this virus. It has been shown that, for the skin, the main determining factor of the barrier function is the lipid content of the epidermal stratum corneum, rather than the thickness or number of corneocyte layers present (S.H. White, D. Mirejovsky, G.l. King, Structure of Lamellar Lipid Domains and Corneocyte Envelopes of Murine Stratum Corneum.
  • the oral mucosa consists of connective tissue known as lamina intestinal which is covered by a stratified squamous epithelium.
  • the oral mucosa is covered by a stratified epithelium, the maturation pattern of which is very similar to that of the skin, which provides a barrier against the aggression of endogenous or exogenous substances present in the oral cavity and also prevents the loss of material from the underlying tissue. Morphological diversity ranging from regions of orthokeratinized mucosa to non-keratinized mucosa can be observed.
  • the resistance of the oral/nasal mucosa to diffusion is mainly associated with the intercellular lipids of the outer layers of the tissue.
  • the nature of the intercellular material is therefore an important determining factor in oral epithelial permeability.
  • the lipid components present are similar to those of the epidermis; the main components are neutral lipids which consist mainly of ceramides and acylceramides and are derived from the lamellae of membrane-coating granules.
  • the epithelium of non-keratinized oral regions does not contain acylceramides or acylglycosylceramides, only small amounts of ceramide, and relatively large amounts of glycosylceramide. Ceramides are present only in small amounts in non-keratinized epithelia, there is no mechanism to convert glycosylceramide to ceramide, as occurs in keratinized epithelium. Other physiological characteristics that distinguish mucosal tissues from the skin such as an extensive vasculature, their moist surface, and the presence of mucus, should also be taken into account. Mucosal tissues are covered with negatively charged mucus which contains large glycoproteins called mucins.
  • compositions capable of reinforcing the mucosa increasing its barrier effect, decreasing its permeability in order to reduce or prevent penetration of viruses, such as SARS-CoV-2, and contaminants, such as biocides, among others.
  • a first aspect of the present invention relates to a composition
  • a composition comprising a liposomal base composed of: one or more ceramides, between 30% and 60%; one or more free fatty acids, between 10 and 30%; and one or more sterols, between 20 and 40%, wherein the percentages refer to weight with respect to total composition.
  • the invention relates to the composition defined above, wherein the ceramides are selected from the ceramides present in the stratum corneum of human skin and from a mixture thereof, and preferably wherein the ceramides are selected from ceramide 3, ceramide 6, and a mixture thereof.
  • the invention relates to the composition defined above, wherein the free fatty acids are selected from myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, octacosanoic acid, and mixtures thereof, and preferably wherein the free fatty acid is palmitic acid.
  • the invention relates to the composition defined above, wherein the sterols are selected cholesterol, cholesterol esters, and cholesterol sulfate, and preferably wherein the sterols are cholesterol.
  • the invention relates to the composition defined above, wherein: the ceramides are selected from ceramide 3, ceramide 6, and a mixture thereof, and preferably the ceramides are selected from ceramide 3 and ceramide 6; the free fatty acids are selected from myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, octacosanoic acid, and mixtures thereof, and preferably wherein the free fatty acid is palmitic acid.
  • the invention relates to the composition defined above comprising: one or more ceramides selected from ceramide 3 or ceramide 6; one or more free fatty acids, wherein the free fatty acid is palmitic acid; and cholesterol.
  • the invention relates to the composition defined above comprising:
  • the invention relates to the composition defined above comprising:
  • the invention relates to the composition defined above comprising:
  • the invention relates to the composition defined above comprising:
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the composition defined above and one or more pharmaceutically acceptable excipients.
  • Excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition and of not being harmful to those applying said composition.
  • compositions of the present invention may be administered in the form of any pharmaceutical formulation, whose nature, as is well known, will depend on the nature of the active ingredient and on its administration route.
  • any administration route may be used, for example, oral, parenteral, nasal, ocular, rectal, and topical.
  • Another aspect of the invention relates to the composition defined above, for use as a medical product.
  • compositions comprising a liposomal base composed of: one or more ceramides, between 30% and 60%; one or more free fatty acids, between 10 and 30%; and one or more sterols, between 20 and 40%, wherein the percentages refer to weight with respect to total composition, for use in preventing a contaminant from penetrating through the mucosae.
  • the invention relates to the composition for the use as defined above, wherein the contaminant is selected a virus, bacteria, dust mites, bromated flame retardants such as polybromated biphenyls (PBBs), dioxins and biocides.
  • the contaminant is selected a virus, bacteria, dust mites, bromated flame retardants such as polybromated biphenyls (PBBs), dioxins and biocides.
  • the invention relates to the composition for the use as defined above, wherein the contaminant is a virus, preferably wherein the virus is selected from the influenza virus, adenovirus, rotavirus, and SARS-CoV-2, more preferably for preventing the SARS-CoV-2.
  • the invention relates to the composition for the use as defined above, wherein the contaminant is a biocide, and preferably wherein the biocide is selected from fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
  • Another aspect of the invention relates to the use of the composition defined above, for manufacturing a medical product.
  • Another aspect of the invention relates to the use of the composition defined above, for manufacturing a medical product for preventing infection from a virus selected from influenza, adenovirus, rotavirus, and SARS-CoV-2, preferably for preventing infection from SARS-CoV-2 virus.
  • Another aspect of the present invention relates to a method for preventing a disease in a subject in need thereof, particularly in humans, which comprises administering to said subject an effective amount of the composition defined above.
  • Another aspect of the present invention relates to a method for preventing infection from a virus, preferably wherein the virus is selected from influenza, adenovirus, rotavirus, or SARS-CoV-2, more preferably wherein the virus is SARS-CoV-2, in a subject in need thereof, particularly in humans, which comprises administering to said subject an effective amount of the composition defined above.
  • Another aspect of the invention relates to the use of the composition defined above for preventing a contaminant from penetrating through the mucosa, preferably through the oral mucosa or nasal mucosa.
  • the invention relates to the use of the composition defined above for preventing a virus from penetrating through the mucosae, wherein the virus is SARS-CoV-2.
  • the invention relates to the use of the composition defined above for preventing a contaminant from penetrating through the mucosae, preferably wherein the contaminant is a toxic active component, and more preferably wherein the toxic active component is a biocide.
  • Another aspect of the invention relates to a method for preventing a contaminant from penetrating through the mucosae, comprising the administration of the composition defined above.
  • the invention relates to the prevention method defined above, wherein the virus is SARS-CoV-2.
  • the invention relates to the prevention method defined above, wherein the contaminant is a virus, and preferably wherein the virus is selected from the influenza virus, adenovirus, rotavirus, and SARS-CoV-2, more preferably for preventing the SARS-CoV-2.
  • the invention relates to the prevention method defined above, wherein the contaminant is a biocide, and preferably wherein the biocide is selected from fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
  • FUN fungitrol
  • PRO propiconazole
  • PER permethrin
  • ceramides refers to a family of molecules composed of N-acetylsphingosine and a fatty acid linked by an amide bond. It is the base molecule of sphingolipids, that are very abundant in the lipid bilayer of cell membranes.
  • CER present in the stratum corneum of human skin, derived from the aforementioned types of sphingoid bases and fatty acids, which differ from one another depending on the composition of the head group or on the esterification of the fatty acids (ceramide 1 , ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6, ceramide 7, ceramide 8, ceramide 9, ceramide 10, ceramide 11 , and ceramide 12.
  • the invention relates to ceramide 3 and ceramide 6.
  • free fatty acids refers to long-chain carboxylic acids, and they can be, according to the nature of the carbon chain, saturated, unsaturated, linear, branched, and can have hydroxyl groups as substituents.
  • Free fatty acids FFAs
  • FFAs Free fatty acids
  • the free fatty acids of the stratum corneum of human skin are mainly straight-chain saturated derivatives having between 14 and 28 carbons in length. Most of these fatty acids have 20 carbons or more, with fatty acids having 22 and 24 carbons being the most abundant. Free fatty acids participate in the structure of CERs, linked with long-chain sphingoids.
  • Examples include, among others, myristic acid (C14), palmitic acid (C16), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24), cerotic acid (C26), and octacosanoic acid (C28).
  • sterols refers to cyclopentanoperhydrophenanthrene derivatives that are characterized by having alcohol as the organic oxygen function.
  • examples of sterols include, among others, cholesterol, cholesterol esters, and cholesterol sulfate.
  • cholesterol refers to a highly hydrophobic sterol-type lipid derived from cyclopentanoperhydrophenanthrene (or sterane), made up of four condensed or fused carbocycles with the following chemical formula C27H45OH.
  • “Cholesterol esters” refer to cholesterol derivatives in which a fatty acid esterifies the hydroxyl group of cholesterol.
  • “Cholesterol sulfate” refers to a multifunctional cholesterol precursor steroid metabolite that is produced in large amounts in keratinizing squamous epithelia.
  • prevention is used to refer to preventing the onset of the disease that occurs in a patient who is predisposed or has risk factors, but still has no symptoms of the disease. Prevention also includes preventing the recurrence of a disease in a subject who has previously suffered from said disease.
  • contaminant refers to a substance that is either present in an environment where it does not belong or is present at levels that might cause harmful effects to humans or the environment.
  • examples of contaminants are, without limitation viruses, bacteria, dust mites, bromated flame retardants such as polybromated biphenyls (PBBs), dioxins and biocides.
  • biocide refers to any substance or mixture, in the form in which it is supplied to the user, consisting of, containing or generating one or more active substances, with the intention of destroying, deterring, rendering harmless, preventing the action of, or otherwise exerting a controlling effect on, any harmful organism by any means other than mere physical or mechanical action.
  • the US Environmental Protection Agency (EPA) uses a slightly different definition for biocides as "a diverse group of poisonous substances including preservatives, insecticides, disinfectants, and pesticides used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products". In the context of the present invention, both definitions are applicable, unless it is not meaningful in view of context.
  • biocides are, without limitation, pesticides (this includes fungicides, herbicides, insecticides, algicides, miticides, piscicides, rodenticides, repellents and attractant, and slimicides); a preservative; a disinfectant. Particularly, example includes, among others, glyphosate, fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
  • pesticides this includes fungicides, herbicides, insecticides, algicides, miticides, piscicides, rodenticides, repellents and attractant, and slimicides
  • a preservative a disinfectant.
  • glyphosate fungitrol
  • PRO propiconazole
  • PER permethrin
  • Example 1 Liposomal-based compositions
  • All liposomes are formed using the thin film hydration method. Lipids are dissolved in an organic solvent using 3 ml of a mixture of CHCI3: MeOH (2: 1 ) (v/v) (Chloroform (Merck), Methanol LiChrosolv® Reag. (Merck)). Then, the solvent is evaporated in a rotary evaporator at 50 °C and 100 rpm until a thin lipid film is formed on the walls of the flask. Next, the dry lipid film is hydrated using an aqueous PBS 10% urea solution (Probus, >99%) dissolved in PBS (Sigma Aldrich) and heated repeatedly until a smooth white liposome mixture is formed. The heating temperature depends on the phase transition temperature of the components. Composition F1. 1% Cer3.- Total lipid concentration (Ceramide+cholesterol+palmitic acid) 1%.
  • Palmitic Acid >99% Sigma Aldrich
  • Composition F2 10% Cer3 - Total lipid concentration
  • Palmitic Acid >99% Sigma Aldrich
  • Palmitic Acid >99% Sigma Aldrich
  • Composition F4 10% Cer3Cer6. Total lipid concentration (Ceramide3+Ceramide 6+cholesterol+palmitic acid) 10%
  • Palmitic Acid >99% Sigma Aldrich
  • Example 2 Evaluation of the barrier function by means of transmucosal water loss
  • the compositions described in Example 1 were evaluated for transmucosal water loss in the sublingual buccal mucosa and in a synthetic Nuclepore membrane by means of Tewameter TM300.
  • FDC transepidermal water loss
  • FDC Franz static diffusion cells
  • the cells are placed in a thermostatic bath (Julabo) for acclimatization until reaching a temperature of 32 ⁇ 1 °C.
  • TEWL measurements can be performed with a Tewameter®.
  • the device is placed in the opening of the FDC, therefore, it gives a continuous response of the transepidermal water loss values. Furthermore, it contains two vertically located sensors that indicate two values of temperature and relative humidity (% RH), respectively. The measurements are performed before the application, 70 pl of composition are deposited and re-evaluated 1 hour after application. Furthermore, a control measure must be performed (without any type of application), measurements are performed in duplicate.
  • Table 1 describes the results of the compositions of the invention with which TEWL is also evaluated with porcine sublingual mucosa (Table 1 ).
  • Drugs were selected based on their different solubility and permeability characteristics. These two factors are directly related to the absorption process. Each of them belonging to a Biopharmaceutical Classification System (BCS) group. Additionally, this selection was performed based on its pharmacological characteristics and prophylactic use in preventing the treatment of COVID, preventing the replication of the virus.
  • BCS Biopharmaceutical Classification System
  • the evaluated drugs were caffeine (CAF), ibuprofen (IBU), dexamethasone (DEX), and ivermectin (IVE) dissolved in methanol at a concentration of 1% for each active ingredient.
  • CAF caffeine
  • IBU ibuprofen
  • DEX dexamethasone
  • IVE ivermectin
  • Class I high solubility (hydrophilic)-high permeability
  • Class II low solubility (hydrophobic)-high permeability, ibuprofen
  • Class III high solubility (hydrophilic) - low permeability, dexamethasone
  • Class IV low solubility (hydrophobic)-low permeability, ivermectin
  • the drugs are deposited to determine their kinetics.
  • 300 pl infinite dose
  • 300 pl infinite dose
  • 1% solution of the 4 drugs in methanol were applied to each Franz cell, in triplicate. Aliquots of 0.2 ml were collected at different times (30 min, 1 h, 2 h, 4 h).
  • the active ingredients were diluted in suitable graduated flasks and filtered through a 0.22 gm nylon filter (Cameo, Sigma-Aldrich, St Louis, II. S.A.). They were then further analyzed with a high performance liquid chromatography diode array detector (HPLC-DAD).
  • HPLC-DAD high performance liquid chromatography diode array detector
  • the release of the active pharmaceutical ingredient (API) was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which corresponds to the cumulative amount of API quantified in the receiver liquid per surface area of the sample (Thakker, K.D.; Chern, W.H. Development and validation of in vitro release tests for semisolid dosage forms — case study. Dissolution Technol. 2003, 10, 10-15).
  • the equation is as follows (1 ):
  • Qn is the cumulative amount of active ingredient released at time n (pg/cm 2 ); Cn is the concentration of active ingredient in the sample (pg/ml) ; Vc is the volume of the vertical diffusion cell (7 ml); X'/T? Ci is the sum of the API concentrations (pg/ml) determined at sampling intervals 1 to n-1 ; Vs is the volume of the sample, and A is the surface area of the sample (1 .77 cm 2 ).
  • the kinetic permeation assay was performed in triplicate for caffeine on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 5.
  • composition F4 gives the mucosa an impermeability that is very similar to that of the skin. Therefore, for a low molecular weight hydrophilic compound indicating a high permeability such as caffeine, it exhibits an impermeability similar to that of the skin when composition F4 is applied to the mucosae.
  • the kinetic permeation assay was performed in triplicate for ibuprofen on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 6.
  • F4 confers to the mucosa an impermeability that is very similar to that of the skin. Therefore, for a low molecular weight hydrophobic compound indicating a high permeability such as ibuprofen, it exhibits an impermeability similar to that of the skin when formulation F4 is applied to the mucosae.
  • the kinetic permeation assay was performed in triplicate for dexamethasone on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 7.
  • F4 confers to the mucosa an impermeability that is 5 times less than that of virgin mucosa, although it does not reach the values of the skin. Therefore, for a high molecular weight hydrophilic compound indicating a low skin permeability such as dexamethasone, it exhibits a very pronounced impermeability with respect to mucosae when formulation F4 is applied thereto, although it does not reach the impermeabilization of the skin.
  • the kinetic permeation assay was performed in triplicate for ivermectin on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 8.
  • Table 8 Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax), and) for ivermectin through the skin, sublingual mucosa, and sublingual mucosa with F4.
  • Ivermectin is the active ingredient with the lowest permeability both in the skin and in the mucosa. Furthermore, the difference between the skin and mucosa is not as pronounced as in the case of dexamethasone. In this case, the permeability of ivermectin through the unmodified mucosa is 20 times higher compared to the skin. The application of formulations for impermeabilization seems to have less effect, although it should be highlighted that F4 confers to the mucosa an impermeability that is 1 .5 times less than that of the virgin mucosa, without ever reaching the values of the skin. Therefore, for a very high molecular weight hydrophobic compound indicating a low skin permeability such as ivermectin, it presents a slight decrease in permeability with respect to mucosae when formulation F4 is applied thereto.
  • the biocides evaluated were fungitrol (FUN), propiconazole (PRO), and permethrin (PER) dissolved in ethanol at a concentration of 1% for each active ingredient.
  • FUN fungitrol
  • PRO propiconazole
  • PER permethrin
  • the size is very similar between 300 and 400 MW, and the hydrophilicity would be similar for fungitrol and propiconazole, with permethrin being the most hydrophobic active ingredient.
  • the kinetic permeation assay was performed in triplicate for fungitrol on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 11 .
  • F4 confers to the mucosa an impermeability that is very similar to that of the skin.
  • the kinetic permeation assay was performed in triplicate for propiconazole on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 12.
  • F4 confers to the mucosa an impermeability that is very similar to that of the skin.
  • the kinetic permeation assay was performed in triplicate for permethrin on porcine skin, mucosa, and modified mucosa after depositing composition F4.
  • the release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm 2 ), which is equivalent to the total amount of API quantified in the receiver liquid per unit area.
  • Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section.
  • the percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 13.
  • F4 confers to the mucosa an impermeability that is very similar to that of the skin.
  • Example 5 Kinetic permeation of a SARS-CoV-2 virus model using vertical Franz diffusion cells
  • Metallic nanoparticles coated on their surface with a bilayer of cationic gemini surfactants have been used.
  • gold nanoparticles have been chosen, since they have a characteristic strong absorption band in the visible part of the spectrum that can be easily characterized by UV-vis absorption spectroscopy, from which the concentration of particles, their size and polydispersity can be calculated.
  • the amphiphilic compound 1 ,3-bis[(3-octadecyl-1 -imidazolium)methyl]benzene 1 -2Br was chosen as a coating surfactant, with the capacity to stabilize the gold surface, forming a monolayer around the gold core in which the positive charges derived from the imidazolium salts are close to the metallic surface and the hydrophobic chains (of 18 carbon atoms) are arranged towards the outside of the nanoparticle.
  • This amphiphilic compound is also capable of assembling forming bilayers around the gold core, resulting in a cationic surface of the nanoparticle, and generating the virus model. For the synthesis of these nanoparticles, a procedure was followed that is briefly summarized below.
  • NP-Au UV-vis absorption spectroscopy and Dynamic Light Scattering
  • the experiment was performed with a similar design as the kinetic drug permeation assay of Example 3.
  • the receiver fluid (RF) was H 2 O (HPLC degree).
  • 70pL of liposomal formulation F4 10% Cer3Cer6 is deposited in the mucosa.
  • 300pL (infinite dose) of 27.2pg/g NP-Au were applied in occlusion with Parafilm® to each Franz cell, in triplicate. Aliquots of 0.3 mL were collected at different times (30 min, 1 h, 2h, 4h). The volume that has been removed is immediately added, with the replenishing medium.
  • the active ingredients were filtered (0.45 pm of size, nylon filter). They were further prepared with acid digestion treatment and analysed by ICP- MS.
  • the membrane was removed from the Franz cells and the excess composition was removed from the membrane surface. This was cleaned 3 times with distilled water (Washing, W) and gently dried with a cotton swab. The pieces of mucosa were placed in glass vials and the total receptor fluid was recovered. The washing and mucosa samples as well as receptor fluid were treated with an acid digestion process in order to obtain the amount of virus model retained in the samples. Extractions are placed in chromatography vials until their analysis by plasma mass spectrometry. Their determination is carried out according to the following methodology: in Teflon capsules (cleaned with Agua Regia), are tared and later, the sample is weighed and 0.5 ml of Aqua Regia is added.
  • Table 14 Mean values of amount of retained and permeated model virus on mucosa membrane and mucosa membrane modified with the impermeabilization composition F4, expressed in percentage.
  • Table 14 shows the surface washing virus model amount, the mucosa (epidermis and dermis) retained virus model and permeated virus model (amount present in the medium of the acceptor compartment of the cell at different times) after its deposition on the membranes.
  • the results of the F4 modified membrane correspond to the values of the virus on membranes previously treated with Cer3Cer6 F4 formulation.
  • compositions of the invention protect people in general and a health worker in particular from being infected by a virus, such as, for example, SARS-CoV-2 and being protected from contaminants, such as, for example, biocides.

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Abstract

The present invention relates to a liposomal-based composition and to the use thereof as an agent for the impermeabilization of oral mucosae, therefore, said composition also relates to the use thereof as a medicinal product, particularly for preventing viral infections, such as SARS-CoV-2, and to the use thereof for preventing contaminants, such as biocides, from penetrating through the mucosae.

Description

LIPOSOMAL-BASED COMPOSITION
The present invention relates to a liposomal-based composition and to the use thereof as an agent for the impermeabilization of oral mucosae, therefore, said composition also relates to the use thereof as a medical product, particularly for preventing viral infections, such as SARS-CoV-2, and to the use thereof for preventing contaminants, such as biocides, from penetrating through the oral mucosae. Therefore, the invention could be comprised in the field of pharmaceutical technology.
STATE OF THE ART
The permeation of SARS-CoV-2 through the nasal or oral mucosa in contrast to its low penetration through the keratinized tissue of the stratum corneum of the skin is widely known. This is mainly due to the different lipid composition and packaging structures formed by this virus. It has been shown that, for the skin, the main determining factor of the barrier function is the lipid content of the epidermal stratum corneum, rather than the thickness or number of corneocyte layers present (S.H. White, D. Mirejovsky, G.l. King, Structure of Lamellar Lipid Domains and Corneocyte Envelopes of Murine Stratum Corneum. An X-ray Diffraction Study, Biochemistry 1988, 27, 3725-3732; Bouwstra J, Pilgram G, Gooris G, Koerten H, Ponec M. New aspects of the skin barrier organization. Skin Pharmacol Appl Skin Physiol. 2001 ; 14 Suppl 1, 52-62). In the stratum corneum of the skin, ceramides, fatty acids, and cholesterol are the main lipids that determine the permeability barrier (B. Janusova, J. Zbytovska, P. Lorenc, H. Vavrysova, K. Palat, A. Hrabalek, K. Vavrova, Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes, Biochim. Biophys. Acta - Mol. Cell Biol. Lipids. 2011 , 1811, 129-137; D. Kessner, A. Ruettinger, M.A. Kiselev, S. Wartewig, R.H.H. Neubert, Properties of ceramides and their impact on the stratum corneum structure: A review - Part 2: Stratum corneum lipid model systems, Skin Pharmacol. Physiol. 2008, 21, 58-74).
The oral mucosa consists of connective tissue known as lamina propria which is covered by a stratified squamous epithelium. The oral mucosa is covered by a stratified epithelium, the maturation pattern of which is very similar to that of the skin, which provides a barrier against the aggression of endogenous or exogenous substances present in the oral cavity and also prevents the loss of material from the underlying tissue. Morphological diversity ranging from regions of orthokeratinized mucosa to non-keratinized mucosa can be observed.
In general, it is accepted that the resistance of the oral/nasal mucosa to diffusion is mainly associated with the intercellular lipids of the outer layers of the tissue. The nature of the intercellular material is therefore an important determining factor in oral epithelial permeability. In keratinized oral epithelia, the lipid components present are similar to those of the epidermis; the main components are neutral lipids which consist mainly of ceramides and acylceramides and are derived from the lamellae of membrane-coating granules. The epithelium of non-keratinized oral regions does not contain acylceramides or acylglycosylceramides, only small amounts of ceramide, and relatively large amounts of glycosylceramide. Ceramides are present only in small amounts in non-keratinized epithelia, there is no mechanism to convert glycosylceramide to ceramide, as occurs in keratinized epithelium. Other physiological characteristics that distinguish mucosal tissues from the skin such as an extensive vasculature, their moist surface, and the presence of mucus, should also be taken into account. Mucosal tissues are covered with negatively charged mucus which contains large glycoproteins called mucins. Mucus and saliva play an important role during penetration and can contribute to the barrier layer of mucosal tissues (Beste Kinikoglu, Odile Damour, Vasif Hasirci, Tissue engineering of oral mucosa: a shared concept with skin J Artif Organs 2015, 18, 8-19; Amir H Shojael. Buccal Mucosa As A Route For Systemic Drug Delivery: A Review. J. Pharm. Pharmaceut. Sci. 1988, 1 (1), 15-30; Winning TA, Townsend GC. Oral mucosal embryology and histology. Clin Dermatol. 2000; 18, 499-51 1 ).
Therefore, it would be desirable to have a composition capable of reinforcing the mucosa, increasing its barrier effect, decreasing its permeability in order to reduce or prevent penetration of viruses, such as SARS-CoV-2, and contaminants, such as biocides, among others.
DESCRIPTION OF THE INVENTION A first aspect of the present invention relates to a composition comprising a liposomal base composed of: one or more ceramides, between 30% and 60%; one or more free fatty acids, between 10 and 30%; and one or more sterols, between 20 and 40%, wherein the percentages refer to weight with respect to total composition.
In another embodiment, the invention relates to the composition defined above, wherein the ceramides are selected from the ceramides present in the stratum corneum of human skin and from a mixture thereof, and preferably wherein the ceramides are selected from ceramide 3, ceramide 6, and a mixture thereof.
In another embodiment, the invention relates to the composition defined above, wherein the free fatty acids are selected from myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, octacosanoic acid, and mixtures thereof, and preferably wherein the free fatty acid is palmitic acid.
In another embodiment, the invention relates to the composition defined above, wherein the sterols are selected cholesterol, cholesterol esters, and cholesterol sulfate, and preferably wherein the sterols are cholesterol.
In another embodiment, the invention relates to the composition defined above, wherein: the ceramides are selected from ceramide 3, ceramide 6, and a mixture thereof, and preferably the ceramides are selected from ceramide 3 and ceramide 6; the free fatty acids are selected from myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, octacosanoic acid, and mixtures thereof, and preferably wherein the free fatty acid is palmitic acid.
In another embodiment, the invention relates to the composition defined above comprising: one or more ceramides selected from ceramide 3 or ceramide 6; one or more free fatty acids, wherein the free fatty acid is palmitic acid; and cholesterol. In another embodiment, the invention relates to the composition defined above comprising:
46.0% of ceramide 3;
31 .0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 1 % in water and the percentages refer to weight with respect to total composition.
In another embodiment, the invention relates to the composition defined above comprising:
46.0% of ceramide 3;
31 .0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 10% in water and the percentages refer to weight with respect to total composition.
In another embodiment, the invention relates to the composition defined above comprising:
22.0% of ceramide 3;
23.0% of ceramide 6;
32.0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 1 % in water and the percentages refer to weight with respect to total composition.
In another embodiment, the invention relates to the composition defined above comprising:
22.0% of ceramide 3;
23.0% of ceramide 6;
32.0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 10% in water and the percentages refer to weight with respect to total composition. Another aspect of the invention relates to a pharmaceutical composition comprising the composition defined above and one or more pharmaceutically acceptable excipients.
Excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition and of not being harmful to those applying said composition.
The compositions of the present invention may be administered in the form of any pharmaceutical formulation, whose nature, as is well known, will depend on the nature of the active ingredient and on its administration route. In principle, any administration route may be used, for example, oral, parenteral, nasal, ocular, rectal, and topical.
Another aspect of the invention relates to the composition defined above, for use as a medical product.
Another aspect of the present invention relates to a composition comprising a liposomal base composed of: one or more ceramides, between 30% and 60%; one or more free fatty acids, between 10 and 30%; and one or more sterols, between 20 and 40%, wherein the percentages refer to weight with respect to total composition, for use in preventing a contaminant from penetrating through the mucosae.
In another embodiment the invention relates to the composition for the use as defined above, wherein the contaminant is selected a virus, bacteria, dust mites, bromated flame retardants such as polybromated biphenyls (PBBs), dioxins and biocides.
In another embodiment the invention relates to the composition for the use as defined above, wherein the contaminant is a virus, preferably wherein the virus is selected from the influenza virus, adenovirus, rotavirus, and SARS-CoV-2, more preferably for preventing the SARS-CoV-2. In another embodiment the invention relates to the composition for the use as defined above, wherein the contaminant is a biocide, and preferably wherein the biocide is selected from fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
Another aspect of the invention relates to the use of the composition defined above, for manufacturing a medical product.
Another aspect of the invention relates to the use of the composition defined above, for manufacturing a medical product for preventing infection from a virus selected from influenza, adenovirus, rotavirus, and SARS-CoV-2, preferably for preventing infection from SARS-CoV-2 virus.
Another aspect of the present invention relates to a method for preventing a disease in a subject in need thereof, particularly in humans, which comprises administering to said subject an effective amount of the composition defined above.
Another aspect of the present invention relates to a method for preventing infection from a virus, preferably wherein the virus is selected from influenza, adenovirus, rotavirus, or SARS-CoV-2, more preferably wherein the virus is SARS-CoV-2, in a subject in need thereof, particularly in humans, which comprises administering to said subject an effective amount of the composition defined above.
Another aspect of the invention relates to the use of the composition defined above for preventing a contaminant from penetrating through the mucosa, preferably through the oral mucosa or nasal mucosa.
In another embodiment, the invention relates to the use of the composition defined above for preventing a virus from penetrating through the mucosae, wherein the virus is SARS-CoV-2.
In another embodiment, the invention relates to the use of the composition defined above for preventing a contaminant from penetrating through the mucosae, preferably wherein the contaminant is a toxic active component, and more preferably wherein the toxic active component is a biocide. Another aspect of the invention relates to a method for preventing a contaminant from penetrating through the mucosae, comprising the administration of the composition defined above.
In another embodiment, the invention relates to the prevention method defined above, wherein the virus is SARS-CoV-2.
In another embodiment the invention relates to the prevention method defined above, wherein the contaminant is a virus, and preferably wherein the virus is selected from the influenza virus, adenovirus, rotavirus, and SARS-CoV-2, more preferably for preventing the SARS-CoV-2.
In another embodiment, the invention relates to the prevention method defined above, wherein the contaminant is a biocide, and preferably wherein the biocide is selected from fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
Throughout the invention, the term "ceramides" refers to a family of molecules composed of N-acetylsphingosine and a fatty acid linked by an amide bond. It is the base molecule of sphingolipids, that are very abundant in the lipid bilayer of cell membranes. There are 12 types of CER present in the stratum corneum of human skin, derived from the aforementioned types of sphingoid bases and fatty acids, which differ from one another depending on the composition of the head group or on the esterification of the fatty acids (ceramide 1 , ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6, ceramide 7, ceramide 8, ceramide 9, ceramide 10, ceramide 11 , and ceramide 12. Particularly, the invention relates to ceramide 3 and ceramide 6.
The term "free fatty acids" refers to long-chain carboxylic acids, and they can be, according to the nature of the carbon chain, saturated, unsaturated, linear, branched, and can have hydroxyl groups as substituents. Free fatty acids (FFAs) are produced by the hydrolysis of oils and fats. The free fatty acids of the stratum corneum of human skin are mainly straight-chain saturated derivatives having between 14 and 28 carbons in length. Most of these fatty acids have 20 carbons or more, with fatty acids having 22 and 24 carbons being the most abundant. Free fatty acids participate in the structure of CERs, linked with long-chain sphingoids. Examples include, among others, myristic acid (C14), palmitic acid (C16), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24), cerotic acid (C26), and octacosanoic acid (C28).
Throughout the invention, the term "sterols" refers to cyclopentanoperhydrophenanthrene derivatives that are characterized by having alcohol as the organic oxygen function. Examples of sterols include, among others, cholesterol, cholesterol esters, and cholesterol sulfate.
The term "cholesterol" refers to a highly hydrophobic sterol-type lipid derived from cyclopentanoperhydrophenanthrene (or sterane), made up of four condensed or fused carbocycles with the following chemical formula C27H45OH.
"Cholesterol esters" refer to cholesterol derivatives in which a fatty acid esterifies the hydroxyl group of cholesterol.
"Cholesterol sulfate" refers to a multifunctional cholesterol precursor steroid metabolite that is produced in large amounts in keratinizing squamous epithelia.
Throughout the present invention, the term "prevention" is used to refer to preventing the onset of the disease that occurs in a patient who is predisposed or has risk factors, but still has no symptoms of the disease. Prevention also includes preventing the recurrence of a disease in a subject who has previously suffered from said disease.
The term “contaminant” refers to a substance that is either present in an environment where it does not belong or is present at levels that might cause harmful effects to humans or the environment. In the context of the present invention, examples of contaminants are, without limitation viruses, bacteria, dust mites, bromated flame retardants such as polybromated biphenyls (PBBs), dioxins and biocides.
The term "biocide" refers to any substance or mixture, in the form in which it is supplied to the user, consisting of, containing or generating one or more active substances, with the intention of destroying, deterring, rendering harmless, preventing the action of, or otherwise exerting a controlling effect on, any harmful organism by any means other than mere physical or mechanical action. The US Environmental Protection Agency (EPA) uses a slightly different definition for biocides as "a diverse group of poisonous substances including preservatives, insecticides, disinfectants, and pesticides used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products". In the context of the present invention, both definitions are applicable, unless it is not meaningful in view of context. Examples of biocides are, without limitation, pesticides (this includes fungicides, herbicides, insecticides, algicides, miticides, piscicides, rodenticides, repellents and attractant, and slimicides); a preservative; a disinfectant. Particularly, example includes, among others, glyphosate, fungitrol (FUN), propiconazole (PRO), and permethrin (PER).
Throughout the description and the claims, the word “comprises” and its variants are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention may be partially deduced from both the description and the embodiment of the invention. The following examples are provided by way of illustration and are not intended to limit the present invention.
EXAMPLES
Next, the invention will be illustrated by means of assays carried out by the inventors.
Example 1 : Liposomal-based compositions
All liposomes are formed using the thin film hydration method. Lipids are dissolved in an organic solvent using 3 ml of a mixture of CHCI3: MeOH (2: 1 ) (v/v) (Chloroform (Merck), Methanol LiChrosolv® Reag. (Merck)). Then, the solvent is evaporated in a rotary evaporator at 50 °C and 100 rpm until a thin lipid film is formed on the walls of the flask. Next, the dry lipid film is hydrated using an aqueous PBS 10% urea solution (Probus, >99%) dissolved in PBS (Sigma Aldrich) and heated repeatedly until a smooth white liposome mixture is formed. The heating temperature depends on the phase transition temperature of the components. Composition F1. 1% Cer3.- Total lipid concentration (Ceramide+cholesterol+palmitic acid) 1%.
Ceramide 3 (Xingrui Industry CO., Limited) 46.0 wt%
Cholesterol (>99% Sigma Aldrich) 31.0%
Palmitic Acid (>99% Sigma Aldrich) 23.0%
Composition F2. 10% Cer3 - Total lipid concentration
(Ceramide+cholesterol+palmitic acid) 10%
Ceramide 3 (Xingrui Industry CO., Limited) 46.0 wt%
Cholesterol (>99% Sigma Aldrich) 31.0%
Palmitic Acid (>99% Sigma Aldrich) 23.0%
Composition F3.-1% Cer3Cer6. Total lipid concentration (Ceramide 3+Ceramide 6+cholesterol+palmitic acid) 1%
Ceramide 3 (Evonik) 22.0 wt%
Ceramide 6 (Evonik) 23.0%
Cholesterol (>99% Sigma Aldrich) 32.0 %
Palmitic Acid (>99% Sigma Aldrich) 23.0%
Composition F4. 10% Cer3Cer6. Total lipid concentration (Ceramide3+Ceramide 6+cholesterol+palmitic acid) 10%
Ceramide 3 (Evonik) 22.0 wt%
Ceramide 6 (Evonik) 23.0%
Cholesterol (>99% Sigma Aldrich) 32.0 %
Palmitic Acid (>99% Sigma Aldrich) 23.0%
Example 2: Evaluation of the barrier function by means of transmucosal water loss The compositions described in Example 1 were evaluated for transmucosal water loss in the sublingual buccal mucosa and in a synthetic Nuclepore membrane by means of Tewameter TM300.
The use of both animal and human biological membranes is essential to increase knowledge concerning the skin barrier, or the oral and nasal mucosa. However, ethical reasons or the complex methods for their attainment, preservation, and reproducibility, as well as their high cost, make it necessary to find synthetic membranes with a behavior similar to that of biological membranes and which, therefore, remove the limitations of previous membranes. Furthermore, the use of artificial membranes will obviate the great intra- and inter-individual variability.
Based on the above, two types of membranes, i.e., artificial and biological, have been used in order to verify the similarity or differences between them. Specifically, the following membranes are used:
-Whatman® Nuclepore™ artificial membranes, made of polycarbonate and with a pore size of 0.05 pm, which have been shown to have a permeability similar to that of human mucosae.
-porcine sublingual mucosae. Pig tongues were obtained from the Faculty of Pharmacy of the University of Barcelona, from the Hospital de Bellvitge campus with the protocols of the ethics committee and the supervision of said facility. Samples were dermatomized to a thickness of 500-700 pm (Dermatome GA630, Aesculap) and portions of the sublingual oral mucosa were obtained such that they fit into the Franz diffusion cells. Furthermore, in order to know the specific thickness, each portion of mucosa was measured with a digital micrometer (40X MAHR).
For the study of the barrier function of the mucosae, transmucosal water loss by means of Tewameter TM300 has initially been used in both Nuclepore synthetic membrane and sublingual oral mucosa.
Measurements of transepidermal water loss (TEWL) are carried out in Franz static diffusion cells (FDC) (3 ml, 1 .86 cm2, Lara-Spiral). These consist of a donor chamber and a receiver chamber (3 ml in volume) which are separated by a membrane for example, skin, mucosa, or artificial membrane. The receiver compartment of the lower chamber contains the receiver fluid containing 1 % bovine serum albumin (Sigma Aldrich) and 0.04% gentamicin sulfate (Sigma Aldrich) in phosphate-buffered saline (pH = 7.6). It is filled with a syringe with a needle and making sure that there are no bubbles in the liquid or at the lower part of the membrane. The cells are placed in a thermostatic bath (Julabo) for acclimatization until reaching a temperature of 32 ± 1 °C.
Once the cells have been stabilized for 1 hour and have reached the optimum temperature, TEWL measurements can be performed with a Tewameter®. The device is placed in the opening of the FDC, therefore, it gives a continuous response of the transepidermal water loss values. Furthermore, it contains two vertically located sensors that indicate two values of temperature and relative humidity (% RH), respectively. The measurements are performed before the application, 70 pl of composition are deposited and re-evaluated 1 hour after application. Furthermore, a control measure must be performed (without any type of application), measurements are performed in duplicate.
Table 1 describes the results of the compositions of the invention with which TEWL is also evaluated with porcine sublingual mucosa (Table 1 ).
Table 1. Transepidermal water loss from Nuclepore synthetic membrane, from porcine sublingual mucosa, and from the same mucosae modified by applying the different compositions
Figure imgf000013_0001
The significant permeability of both the artificial membrane 80 g/hm2 and sublingual mucosa 72 g/hm2 with respect to the permeability of the skin, which is usually between approximately 5 and 10 g/hm2, should be highlighted. The compositions of the invention greatly reduce water permeability (over 90%) both through the synthetic membrane and through the mucosa.
Example 3: Kinetic drug permeation using vertical Franz diffusion cells
Four drugs are evaluated to study the permeation characteristics of the composition F4-treated and untreated membrane to the passage of active ingredients. Furthermore, porcine skin permeation is studied. As mentioned in the introduction, the barrier structure of the skin differs greatly from that of the mucosae. In this sense, it will be evaluated whether the mucosae modified with the compositions that mimic the stratum corneum could have a barrier effect similar to that of the skin.
Drugs were selected based on their different solubility and permeability characteristics. These two factors are directly related to the absorption process. Each of them belonging to a Biopharmaceutical Classification System (BCS) group. Additionally, this selection was performed based on its pharmacological characteristics and prophylactic use in preventing the treatment of COVID, preventing the replication of the virus.
The evaluated drugs were caffeine (CAF), ibuprofen (IBU), dexamethasone (DEX), and ivermectin (IVE) dissolved in methanol at a concentration of 1% for each active ingredient. Next, the main physicochemical properties which are important in permeability through keratin tissues, such as the skin and mucosae, and the classification to which they belong are described in detail. One drug from each class has been chosen:
Class I: high solubility (hydrophilic)-high permeability, caffeine
Class II: low solubility (hydrophobic)-high permeability, ibuprofen
Class III: high solubility (hydrophilic) - low permeability, dexamethasone
Class IV: low solubility (hydrophobic)-low permeability, ivermectin
Table 2. Compounds to be evaluated, physicochemical properties, and classification.
Figure imgf000015_0001
As in the previous TEWL study, sublingual mucosa and porcine skin dermatomized to a thickness of 500-700 pm are used. Kinetic diffusion studies were performed using a vertical diffusion cell (Lara Spiral, Couternon, France (1.86 cm2 and 3 ml). The receiver fluid (RF) used was PBS:EtOH (1 :1 ). A water bath was set at 43°C to obtain a membrane surface temperature of 32 ± 1 °C.
Parameters such as TEWL, humidity, and temperature for the skin and mucosae were determined before the start of the test with Tewameter TM 300 (Courage + Khazaka, Cologne, Germany). 70 pl of composition F4 is deposited in each mucosa. After 1 h, the TEWL is measured again (Table 3).
Table 3. Evaluation of transepidermal water loss from the skin, from porcine sublingual mucosa, and from the same mucosa modified by applying composition F4 before applying the drugs.
Figure imgf000015_0002
Numbered composition according to Table 1
These TEWL results corroborate the previously obtained results expressed in Table 1.
Subsequently, the drugs are deposited to determine their kinetics. To that end, 300 pl (infinite dose) of the 1% solution of the 4 drugs in methanol were applied to each Franz cell, in triplicate. Aliquots of 0.2 ml were collected at different times (30 min, 1 h, 2 h, 4 h). The active ingredients were diluted in suitable graduated flasks and filtered through a 0.22 gm nylon filter (Cameo, Sigma-Aldrich, St Louis, II. S.A.). They were then further analyzed with a high performance liquid chromatography diode array detector (HPLC-DAD).
All analyses were performed with reverse phase HPLC, using HPLC Agilent 1620 Infinity II LC System (Waldbronn, Germany) equipped with a quaternary pump (G711 1 B), multisampler (G7167A), multi-column thermostat (G71 16A), and WR diode-array detector (G71 15A). The software used was OpenLab. The validation of the analytical procedures followed the guidelines developed by the International Conference on Harmonization (ICH) (Ich, ICH Topic Q2 (R1 ) Validation of Analytical Procedures: Text and Methodology. Int. Conf. Harmon., 2005; 1994, (November 1996), 1 -17). The ICH guidelines were followed to obtain the calibration curve, the limit of quantification (LoQ), and the limit of detection (LoD). The HPLC-DAD analytical conditions and the method for the four active ingredients are described in detail in Table 4.
Table 4. Conditions of the method which are used in HPLC/DAD analysis for caffeine, ibuprofen, dexamethasone, and ivermectin.
Figure imgf000016_0001
Figure imgf000017_0002
The release of the active pharmaceutical ingredient (API) was evaluated through the released cumulative amount (Qn, pg/cm2), which corresponds to the cumulative amount of API quantified in the receiver liquid per surface area of the sample (Thakker, K.D.; Chern, W.H. Development and validation of in vitro release tests for semisolid dosage forms — case study. Dissolution Technol. 2003, 10, 10-15). The equation is as follows (1 ):
Figure imgf000017_0001
Wherein: Qn is the cumulative amount of active ingredient released at time n (pg/cm2); Cn is the concentration of active ingredient in the sample (pg/ml) ; Vc is the volume of the vertical diffusion cell (7 ml); X'/T? Ci is the sum of the API concentrations (pg/ml) determined at sampling intervals 1 to n-1 ; Vs is the volume of the sample, and A is the surface area of the sample (1 .77 cm2).
Two parameters, Qn and % API release, were used to obtain graphs showing absorption and penetration kinetics (Y-axis). Time or its square root (- t) is indicated on the X-axis. The percentage of drug released over time best fit the equation, which represents the absorption kinetics, as described by Mallandrich et. al. (Mallandrich, M.; Fernandez-Campos, F.; Clares, B.; Halbaut, L.; Alonso, C.; Coderch, L.; Garduno- Ramirez, M.L.; Andrade, B.; Del Pozo, A.; Lane, M.E.; et al. Developing Transdermal Applications of Ketorolac Tromethamine Entrapped in Stimuli Sensitive Block Copolymer Hydrogels. Pharm. Res. 2017, 34, 1728-1740). This step allowed determining the best absorption model to represent the penetration kinetics of the API through the different membranes. The model was obtained with the non-linear regression software STATGRAPHICS plus 5 (Statgraphics Technologies, Inc., Virginia, U. S.A.), and the best equation was selected based on the highest correlation coefficient corrected by the number of degrees of freedom (R2 DoF). Once having defined the model, it was possible to calculate other parameters, such as the flow (J), permeability coefficient (Kp), delay time (Tl), maximum concentration (Cmax), maximum time (tmax), and area under the curve (AUC). All results are expressed as mean ± standard deviation (SD). 3.1 Caffeine permeation
The kinetic permeation assay was performed in triplicate for caffeine on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 5.
Table 5. Mean values of area under the curve (AUC), flow (J), permeability coefficient (Kp), maximum concentration (Cmax), and) for caffeine through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000018_0001
The application of composition F4 gives the mucosa an impermeability that is very similar to that of the skin. Therefore, for a low molecular weight hydrophilic compound indicating a high permeability such as caffeine, it exhibits an impermeability similar to that of the skin when composition F4 is applied to the mucosae.
3.2 Ibuprofen permeation
The kinetic permeation assay was performed in triplicate for ibuprofen on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 6.
Table 6. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax), and) for ibuprofen through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000019_0001
It should be highlighted that F4 confers to the mucosa an impermeability that is very similar to that of the skin. Therefore, for a low molecular weight hydrophobic compound indicating a high permeability such as ibuprofen, it exhibits an impermeability similar to that of the skin when formulation F4 is applied to the mucosae.
3.3. Dexamethasone permeation
The kinetic permeation assay was performed in triplicate for dexamethasone on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 7.
Table 7. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax), and) for dexamethasone through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000020_0001
It should be highlighted that F4 confers to the mucosa an impermeability that is 5 times less than that of virgin mucosa, although it does not reach the values of the skin. Therefore, for a high molecular weight hydrophilic compound indicating a low skin permeability such as dexamethasone, it exhibits a very pronounced impermeability with respect to mucosae when formulation F4 is applied thereto, although it does not reach the impermeabilization of the skin.
3.4. Ivermectin permeation
The kinetic permeation assay was performed in triplicate for ivermectin on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 8.
Table 8. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax), and) for ivermectin through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000021_0001
Ivermectin, as expected, is the active ingredient with the lowest permeability both in the skin and in the mucosa. Furthermore, the difference between the skin and mucosa is not as pronounced as in the case of dexamethasone. In this case, the permeability of ivermectin through the unmodified mucosa is 20 times higher compared to the skin. The application of formulations for impermeabilization seems to have less effect, although it should be highlighted that F4 confers to the mucosa an impermeability that is 1 .5 times less than that of the virgin mucosa, without ever reaching the values of the skin. Therefore, for a very high molecular weight hydrophobic compound indicating a low skin permeability such as ivermectin, it presents a slight decrease in permeability with respect to mucosae when formulation F4 is applied thereto.
Example 4: Kinetic permeation of biocides using vertical Franz diffusion cells
Three biocides are also evaluated to study the permeation characteristics of the treated and untreated membranes to the passage of active ingredients. Furthermore, porcine skin permeation is studied. As mentioned in the introduction, the barrier structure of the skin differs greatly from that of the mucosae. In this sense, whether the mucosae modified by the compositions of the invention could have a barrier effect similar to that of the skin will be evaluated.
The biocides evaluated were fungitrol (FUN), propiconazole (PRO), and permethrin (PER) dissolved in ethanol at a concentration of 1% for each active ingredient. In this case, the size is very similar between 300 and 400 MW, and the hydrophilicity would be similar for fungitrol and propiconazole, with permethrin being the most hydrophobic active ingredient.
Table 9. Biocides and their physicochemical properties.
Figure imgf000022_0001
Figure imgf000023_0001
Table 10. Conditions of the method which are used in HPLC/DAD analysis for fungitrol, propiconazole, and permethrin
Figure imgf000023_0002
4.1. Fungitrol permeation
The kinetic permeation assay was performed in triplicate for fungitrol on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 11 .
Table 11. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax) y) for fungitrol through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000024_0001
The application of F4 confers to the mucosa an impermeability that is very similar to that of the skin.
4.2. Propiconazole permeation
The kinetic permeation assay was performed in triplicate for propiconazole on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 12.
Table 12. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax) and) for propiconazole through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000024_0002
Figure imgf000025_0001
The application of F4 confers to the mucosa an impermeability that is very similar to that of the skin.
4.3. Permethrin permeation
The kinetic permeation assay was performed in triplicate for permethrin on porcine skin, mucosa, and modified mucosa after depositing composition F4. The release of the active pharmaceutical ingredient was evaluated through the released cumulative amount (Qn, pg/cm2), which is equivalent to the total amount of API quantified in the receiver liquid per unit area. Other kinetic parameters were determined (flux, Cmax) as described in detail in the experimental section. The percentage of drug released over time was measured for the different four active ingredients, obtaining permeation properties of each one. The results are shown in Table 13.
Table 13. Mean values of area under the curve (AUC) flow (J), permeability coefficient (Kp), maximum concentration (Cmax) and) for permethrin through the skin, sublingual mucosa, and sublingual mucosa with F4.
Figure imgf000025_0002
Figure imgf000026_0001
The application of F4 confers to the mucosa an impermeability that is very similar to that of the skin.
Example 5: Kinetic permeation of a SARS-CoV-2 virus model using vertical Franz diffusion cells
5.1 Synthesis of the virus model
In order to analyze the potential impermeabilization effect against SARS-CoV-2 exerted on the mucosa by the compositions of the invention, a chemical model of the virus was required. For its design, fundamental aspects that characterize it such as its spherical morphology, its approximate size of 120 nm in diameter, and the cationic and hydrophobic nature of its surface, were taken into account.
Metallic nanoparticles coated on their surface with a bilayer of cationic gemini surfactants have been used. Specifically, gold nanoparticles have been chosen, since they have a characteristic strong absorption band in the visible part of the spectrum that can be easily characterized by UV-vis absorption spectroscopy, from which the concentration of particles, their size and polydispersity can be calculated. The amphiphilic compound 1 ,3-bis[(3-octadecyl-1 -imidazolium)methyl]benzene 1 -2Br was chosen as a coating surfactant, with the capacity to stabilize the gold surface, forming a monolayer around the gold core in which the positive charges derived from the imidazolium salts are close to the metallic surface and the hydrophobic chains (of 18 carbon atoms) are arranged towards the outside of the nanoparticle. This amphiphilic compound is also capable of assembling forming bilayers around the gold core, resulting in a cationic surface of the nanoparticle, and generating the virus model. For the synthesis of these nanoparticles, a procedure was followed that is briefly summarized below. Commercial citrate-stabilized gold nanoparticles with a concentration of ~3.8 109 particles/ml and diameter of 100 nm were used. These nanoparticles were functionalized with 1 -2Br by treatment with a 0.55 mM aqueous solution of the amphiphilic compound, using sonication followed by stirring at room temperature for 12 hours. The obtained nanoparticles (NP-Au) were characterized by UV-vis absorption spectroscopy and Dynamic Light Scattering (DLS). This allowed knowing their concentration (6.4- 1 O'12 M), as well as their size, which corresponds to a diameter of 120 nm (PDI 0.15) and its Z potential is ca. 45 mV, which indicates its cationic nature and its high stability.
5.2 Virus model permeation
Lastly, another technological challenge has been the production of new artificial virus models that make it possible to evaluate all those compositions with the potential to protect mucosae from SARS-CoV-2 or other contaminants. For this, the viability of the gold nanoparticles (NP-Au) previously described and synthesized as a model of the SARS-CoV-2 virus was studied, both with virgin mucosa and liposome applied mucosa.
The experiment was performed with a similar design as the kinetic drug permeation assay of Example 3. The receiver fluid (RF) was H2O (HPLC degree). 70pL of liposomal formulation F4 10% Cer3Cer6 is deposited in the mucosa. To that end 300pL (infinite dose) of 27.2pg/g NP-Au were applied in occlusion with Parafilm® to each Franz cell, in triplicate. Aliquots of 0.3 mL were collected at different times (30 min, 1 h, 2h, 4h). The volume that has been removed is immediately added, with the replenishing medium. The active ingredients were filtered (0.45 pm of size, nylon filter). They were further prepared with acid digestion treatment and analysed by ICP- MS.
After the permeation test is complete, the membrane was removed from the Franz cells and the excess composition was removed from the membrane surface. This was cleaned 3 times with distilled water (Washing, W) and gently dried with a cotton swab. The pieces of mucosa were placed in glass vials and the total receptor fluid was recovered. The washing and mucosa samples as well as receptor fluid were treated with an acid digestion process in order to obtain the amount of virus model retained in the samples. Extractions are placed in chromatography vials until their analysis by plasma mass spectrometry. Their determination is carried out according to the following methodology: in Teflon capsules (cleaned with Agua Regia), are tared and later, the sample is weighed and 0.5 ml of Aqua Regia is added. It is left 2 days at 90°C. After cooling down, 10 ml of thiourea (200 ppm 1 % HCI) are added. Finally, it is covered, homogenized and weighed, in order to pass to glass tubes, washing the tubes with the etching solution. The analysis was performed by inductively coupled plasma mass spectrometry with the Agilent Technologies Inc. - 7500ce ICP-MS equipment.
Table 14. Mean values of amount of retained and permeated model virus on mucosa membrane and mucosa membrane modified with the impermeabilization composition F4, expressed in percentage.
Figure imgf000028_0001
Table 14 shows the surface washing virus model amount, the mucosa (epidermis and dermis) retained virus model and permeated virus model (amount present in the medium of the acceptor compartment of the cell at different times) after its deposition on the membranes. The results of the F4 modified membrane correspond to the values of the virus on membranes previously treated with Cer3Cer6 F4 formulation.
It is important to highlight the highest retention of the model virus on the mucosa submitted to the impermeabilizing composition F4. Most of the model virus amount (94%) is in the surface washing, this means that the model virus does not even penetrate on the mucosa. While in the case of the virgin mucosa there is a virus amount of about 67%. This means great protection against the passage of the virus model.
With these results, it can be expected that the compositions of the invention protect people in general and a health worker in particular from being infected by a virus, such as, for example, SARS-CoV-2 and being protected from contaminants, such as, for example, biocides.

Claims

1 . A composition comprising a liposomal base composed of: one or more ceramides, between 30% and 60%; one or more free fatty acids, between 10 and 30%; and one or more sterols, between 20 and 40%, wherein the percentages refer to weight with respect to total composition, for use in preventing a contaminant from penetrating through the mucosae.
2. The composition for the use according to claim 1 , wherein the ceramides are selected from the ceramides present in the stratum corneum of human skin and from a mixture thereof.
3. The composition for the use according to claim 2, wherein the ceramides are selected from ceramide 3, ceramide 6, and a mixture thereof.
4. The composition for the use according to any of claims 1 to 3, wherein the free fatty acids are selected from myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, octacosanoic acid, and mixtures thereof.
5. The composition for the use according to claim 4, wherein the free fatty acid is palmitic acid.
6. The composition for the use according to any of claims 1 to 5, wherein the sterols are selected from cholesterol, cholesterol esters, and cholesterol sulfate.
7. The composition for the use according to claim 6, wherein the sterols are cholesterol.
8. A composition comprising a liposomal base composed of:
46.0% of ceramide 3;
31 .0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 1 % in water and the percentages refer to weight with respect to total composition. 9. A composition comprising a liposomal base composed of:
46.0% of ceramide 3;
31 .0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 10% in water and the percentages refer to weight with respect to total composition.
10. A composition comprising a liposomal base composed of:
22.0% of ceramide 3;
23.0% of ceramide 6;
32.0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 1 % in water and the percentages refer to weight with respect to total composition.
11 . A composition comprising a liposomal base composed of:
22.0% of ceramide 3;
23.0% of ceramide 6;
32.0% of cholesterol; and
23.0% of palmitic acid, wherein the total lipid concentration is 10% in water and the percentages refer to weight with respect to total composition.
12. A pharmaceutical composition comprising the composition according to any of claims 8 to 11 and one or more pharmaceutically acceptable excipients.
13. The composition according to any of claims 8 to 12, for use as a medical product.
14. The composition according to any of claims 8 to 12, for use for use in preventing a contaminant from penetrating through the mucosae.
15. The composition for the use according to any of claims 1 to 12, wherein the contaminant is a virus. 16. The composition for the use according to claim 15, wherein the virus is selected from influenza, adenovirus, rotavirus and SARS-CoV-2.
17. The composition for the use according to any of claims 1 to 12, wherein the contaminant is a biocide.
18. The composition for the use according to claim 17, wherein the biocide is selected from fungitrol, propiconazole, and permethrin.
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