WO2020250195A1 - Complex between a lactoferrin hydrolysate and a silica, production process thereof, and related uses. - Google Patents

Complex between a lactoferrin hydrolysate and a silica, production process thereof, and related uses. Download PDF

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Publication number
WO2020250195A1
WO2020250195A1 PCT/IB2020/055537 IB2020055537W WO2020250195A1 WO 2020250195 A1 WO2020250195 A1 WO 2020250195A1 IB 2020055537 W IB2020055537 W IB 2020055537W WO 2020250195 A1 WO2020250195 A1 WO 2020250195A1
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silica
complex
treatment
complex according
lactoferrin hydrolysate
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PCT/IB2020/055537
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French (fr)
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Lucio Lenzi
Morena Restani
Mirco GROSSI
Claudio ANGELINETTA
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L.I.Co.Rice S.R.L.
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Publication of WO2020250195A1 publication Critical patent/WO2020250195A1/en

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    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/25—Silicon; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • the present invention relates to a complex between a lactoferrin hydrolysate and a silica, a process for the preparation thereof, and relative uses.
  • oral hygiene is a determining factor in the prevention of diseases of the dental apparatus, such as caries, gingivitis, periodontitis, peri-implantitis , etc.
  • Periodontitis is known to be a chronic inflammatory disease characterized by the destruction of the tooth's support structures (periodontal ligament, alveolar bone) . Periodontitis has a very high prevalence in the general population (around 10-15% of adults) and induces numerous negative effects on quality of life.
  • Peri-implantitis is an inflammatory process that affects the tissue surrounding an osseo- integratedimplant leading to a loss in the supporting bone tissue and therefore in the implant itself.
  • the cause can be infectious, or it can be due to incorrect implant-prosthetic passivation (occlusal trauma) , although both causes can be clinically found.
  • Radiographic examination makes it possible to easily distinguish peri-implantitis from occlusal trauma caused by bacteria.
  • diabetes mellitus is a clinically and genetically heterogeneous group of metabolic disorders characterized by high levels of glucose in the blood (hyperglycaemia) .
  • Hyperglycaemia can be a direct consequence of a deficit of insulin secretion caused by the dysfunction of the pancreatic b cells or by resistance to the same hormone by the hepato-muscular tissues, or a combination of the aforementioned phenomena.
  • This metabolic disorder is frequently associated with an alteration of the metabolism of adipocytes and can by all means be considered a syndrome.
  • chronic hyperglycaemia causes multiple damage to different organs such as the heart, eyes, kidneys, nerves, vascular system.
  • Diabetes mellitus and periodontal disease are both characterized by a state of systemic inflammation.
  • Chronic hyperglycaemia, as well as periodontitis, induce the hyper-activation of the immune system, resulting in the hyper-production of pro-inflammatory molecules (TNF-a, IL6, IL1) . Entering the systemic circle, these can negatively affect the organism.
  • TNF-a, IL6, IL1 pro-inflammatory molecules
  • Entering the systemic circle these can negatively affect the organism.
  • chronic inflammation of the gingival tissues can worsen the glycaemic and clinical profile of a patient suffering from diabetes mellitus. This relationship must therefore be considered with a bidirectional nature, in light of the foregoing.
  • Another aspect related to chronic periodontitis is the alteration of the immune system in terms of the balance between antioxidant agents and those reacting to oxygen, in favour of the latter.
  • Oxidative stress can aggravate the clinical manifestations of a diabetic patient.
  • diabetes is in turn a cause of oxidative stress and this can justify the fact that the clinical scenarios deriving from periodontitis are particularly aggressive in diabetic patients.
  • the high prevalence and severity of periodontal disease in diabetic patients can be due to a state of chronic hyperglycaemia and an accumulation of advanced glycation end-products (AGEs) .
  • AGEs advanced glycation end-products
  • the protracted oxidative stress in the gingival tissues could justify the aggressive destruction of periodontal tissues found in diabetic patients.
  • the interaction between AGEs and their cell receptors induces an increase in oxidative stress that can contribute to chronic monocyte hyper activation, activation of the NF-Kb system resulting in the expression of pro-inflammatory mRNAs .
  • Lactoferrin is an antimicrobial and iron-carrying glycoprotein, naturally present in various biological fluids of mammals, such as milk, saliva, tears. It is also present in neutrophil granulocytes, which are immune cells with defence functions against bacterial and fungal infections. Lactoferrin is considered a particularly useful product for its antioxidant, immunomodulatory and anti-infective properties.
  • lactoferrin Through pepsinic digestion of lactoferrin, it is possible to obtain peptides, which are commonly indicated as lactoferrin hydrolysates or even, more simply, as lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in fact, in some cases such activity is reinforced.
  • lactoferrin hydrolysates or even, more simply, as lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in fact, in some cases such activity is reinforced.
  • lactoferrin hydrolysates or even, more simply, as lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in fact, in some cases such activity is reinforced.
  • lactoferricin and its antimicrobial, antiviral, antitumor and immunological properties are described .
  • Patent application WO 2016/046108 describes a composition containing a lactoferrin hydrolysate and glycerophosphoinositol or salts thereof and/or verbascoside or extracts that contain it, and the use thereof for the prevention and/or treatment of dermatological, otologic and stomatological inflammations and infections.
  • Such composition can be applied for the treatment of different infections, especially in the veterinary field, which can cause diseases of the ears, skin or of the stomatological type.
  • Lactoferrin hydrolysates have not yet been widely used, mainly because of their poor stability and compatibility with the excipients normally used in the pharmaceutical or para-pharmaceutical field (e.g. toothpaste, mouthwash, dietary supplements and the like) . Lactoferrin hydrolysates also have relatively low bioavailability when applied topically and are subject to oxidative degradation phenomena.
  • the Applicant therefore set out to solve the problem of obtaining a composition obtaining a lactoferrin hydrolysate which has high stability and bioavailability, so as to perform an antibacterial and antioxidant action which is effective and long-lasting over time.
  • the present invention therefore relates to a complex of at least one lactoferrin hydrolysate and at least one silica having a specific surface area comprised between 100 m 2 /g and 1000 m 2 /g, preferably between 150 m 2 /g and 700 m 2 /g (BET area, measured according to standard ISO 9277-2010), said complex being obtainable through the addition of said lactoferrin hydrolysate to said silica in the form of a sol-gel with an aqueous solvent, and subsequent drying of the sol-gel.
  • the drying is carried out through treatment with supercritical CO2 or by lyophilization, as better illustrated below.
  • the present invention relates to a pharmaceutical or nutraceutical composition, which comprises said complex and at least one physiologically acceptable excipient.
  • the pharmaceutical composition is for oral use or for topical use.
  • the present invention relates to a composition for oral hygiene, in particular for the oral hygiene of diabetic patients, which comprises said complex and at least one physiologically acceptable excipient.
  • the present invention relates to a complex as defined above for use, through topical application, in the prevention or treatment of diseases of the dental apparatus, in particular for use in the prevention or treatment of periodontitis or peri-implantitis , preferably in patients with diabetes.
  • the present invention relates to a complex as defined above for use, through topical application or oral administration, in the prevention or treatment of inflammations of the urogenital apparatus, in particular of the vagina, such as vaginosis or vaginitis of bacterial or viral origin, or yeast infections (candidiasis) .
  • the present invention relates to a complex as defined above for use, through oral administration, in the prevention or treatment of intestinal dismicrobism, i.e. for restoring the balance of the intestinal microbiome.
  • the present invention relates to a complex as defined above for use, through topical application, in the treatment of skin lesions, in particular skin sores, ulcers or erosions.
  • lactoferrin hydrolysates they are products well known in the state of the art, which can be prepared according to known techniques, in particular through treatment of a lactoferrin with a proteolitic enzyme, e.g. pepsin.
  • the starting lactoferrin can be of natural or synthetic origin, and can include different forms, both monomeric and oligomeric, possibly already bound to iron (ololactoferrin) or not bound to iron (apolactoferrin) . Further details on lactoferrin hydrolysates and the preparation thereof are reported, for example, in the following articles: Wakabayashi H., et al, Current Pharm. Design, 9(16) (2003), 1277-87; Theolier J., et al, Dairy Science & Technology, 94(2) (2013), 181-193;
  • the peptides resulting from the proteolysis of lactoferrin generally have a number of amino acid units from 5 to 30, preferably from 5 to 15.
  • silica this can be obtained through synthesis or through the processing of materials of natural origin, as better illustrated below.
  • synthetic silicas particularly preferred forms are fumed silicas or precipitated silicas.
  • the silica is a hydrophilic silica.
  • particularly preferred silicas are in the form of aerogel. These are amorphous silicas characterized by both a high specific surface area, as well as very high porosity and very low density. The porosity is generally greater than 85% and can even reach values of 95-99%, and the density is typically from 0.003 g/cm 3 to 0.8 g/cm 3 , preferably from 0.005 g/cm 3 to 0.3 g/cm 3 .
  • a particularly preferred silica is that in the form of aerogel obtained from vegetable waste, in particular from the husk resulting from the processing of grains such as rice, oats, wheat, rye, spelt.
  • This silica which is obtained from renewable sources, features a high level of bio-compatibility and has a high surface area and a highly porous, open-pored structure, which favours the interaction with the lactoferrin hydrolysate.
  • the silica obtained from rice husk is particularly preferred.
  • the silica in the form of aerogel from vegetable waste can be obtained through a multi-stage process that comprises: combustion of vegetable waste, processing of the ashes thus obtained with an alkaline solution (NaOH) to obtain a silicate (of sodium) , processing of the latter with an acid solution (HC1) with the formation of a wet sol-gel, and finally drying of the wet sol-gel, preferably by treatment with supercritical CO2. Further details on the preparation of such silica forms are reported, for example, in patent application WO 2016/193877 and in the article by R.S. Kumar et al . Int. J. Chem. Eng. and Appls . , Vol. 4, No. 5, October 2013.
  • said at least one lactoferrin hydrolysate and said at least one silica are present in a weight ratio of 0.05:1 to 10:1, more preferably from 0.1:1 to 5:1.
  • composition for oral hygiene according to the present invention comprises:
  • composition according to the present invention comprises:
  • silica amounts indicated above refer to the amount of overall silica present in the composition, whether it is complexed or not complexed with lactoferrin hydrolysate.
  • the composition according to the present invention comprises the complex of at least one lactoferrin hydrolysate and at least one silica as defined above in mixture with a hydrophilic silica that has not been complexed with lactoferrin hydrolysate, in particular a synthetic silica, preferably a hydrophilic fumed silica.
  • a non-complexed silica has the main purpose of further stabilizing complex between silica and lactoferrin hydrolysate, substantially acting as a barrier to the interaction with other ingredients of the composition that could destabilize the complex itself.
  • the ratio by weight between silica complexed with the lactoferrin hydrolysate and hydrophilic silica not complexed with the lactoferrin hydrolysate is preferably comprised between 1:200 and 1:1, more preferably between 1:150 and 1:5.
  • this preferably comprises a mixture of water and a polyol, more preferably glycerol or sorbitol.
  • the weight ratio of water to polyol is preferably comprised between 1:20 and 1:1, more preferably between 1:10 and 1:2.
  • lactoferrin hydrolysate and silica complex according to the present invention makes it possible to obtain an improved antibacterial and antioxidant effect with respect to the lactoferrin hydrolysate used alone with the same concentrations, which is carried out for longer times, so as to prolong the effect without having to repeat the application of the composition too often.
  • the complex according to the present invention is preferably prepared through a process which comprises: preparing a silica having a specific surface area ranging from 100 m 2 /g to 1, 000 m 2 /g, preferably from
  • lactoferrin hydrolysate to the silica in the form of a sol-gel can be achieved through the gradual addition of the peptide to the sol-gel and mechanical mixing until a homogeneous dispersion is obtained.
  • the mixing temperature is generally maintained between 15°C and 50°C.
  • the drying is carried out by treatment with supercritical CO2 or by lyophilization .
  • the complexation between silica and the lactoferrin hydrolysate is guaranteed, without the latter undergoing any structural modifications that would compromise the functionality thereof.
  • the complex between lactoferrin hydrolysate and silica according to the present invention can be treated with at least one polyol, preferably glycerol or sorbitol.
  • the polyol has the function of covering the silica particles loaded with the lactoferrin hydrolysate, further stabilizing the structure.
  • Water can be used as an aqueous solvent, optionally in a mixture with at least one soluble organic solvent, for example ethyl acetate or acetone.
  • the drying step with supercritical CO2 is carried out in an autoclave, at a temperature from 15°C to 60°C and a pressure from 80 bars to 150 bars.
  • the treatment is carried out for a time preferably ranging from 2 hours to 10 hours, more preferably from 4 hours to 8 hours.
  • the drying step by means of lyophilization is carried out through cooling under vacuum at a temperature between -80 °C and -30 °C and subsequent heating, always under vacuum, at a temperature no greater than 60°C, preferably between 20°C and 50°C.
  • the pressure during lyophilization is preferably maintained between 0.1 bar and 0.8 bar.
  • the oral hygiene composition according to the present invention comprises, in addition to the complex, at least one physiologically acceptable excipient. This is in order to obtain a product for oral hygiene in different forms, for example toothpaste, mouthwash, gel, chewing gum, tablets for oral use, gingival dyeing, and the like.
  • composition according to the present invention can comprise ingredients commonly used in the field of dental hygiene products, such as for example: abrasive agents in particulate form, humectants, binders, thickeners, viscosity regulators, surfactants, sweeteners, flavourings, preservatives, anti-plaque agents, dyes, etc.
  • ingredients commonly used in the field of dental hygiene products such as for example: abrasive agents in particulate form, humectants, binders, thickeners, viscosity regulators, surfactants, sweeteners, flavourings, preservatives, anti-plaque agents, dyes, etc.
  • compositions which include the complex according to the present invention, they may be presented in different forms, such as tablets, capsules, aqueous suspensions, syrups, suppositories, and contain the common excipients used for such products.
  • nutraceutical compositions which include the complex according to the present invention, they may be in the form of capsules, tablets, liquid formulations, bars, and the like.
  • complex water and other products with nutritional value, they can include other products such as carbohydrates, lipids, proteins, vitamins, minerals and the like.
  • the complex according to the present invention may be combined with other biologically active products, e.g. glycerophosphoinositol or salts thereof, which has the main function of increasing the antibacterial effect.
  • biologically active products e.g. glycerophosphoinositol or salts thereof, which has the main function of increasing the antibacterial effect.
  • nutraceutical compositions particularly suitable in the prevention or treatment of intestinal dismicrobism, they may contain, in addition to the complex, one or more strains of microorganisms useful for the intestinal microbiome, such as Lactobacilli and the like (e.g. Lactobacillus easel) .
  • the complex may be formulated in the form of a suspension, gel, tincture, cream and the like, with the excipients commonly used for such products.
  • the formulation may possibly contain a pH adjuster, in order to guarantee a physiological pH, e.g. lactic acid which provides a pH around 3.4/4, typical of the vaginal environment.
  • compositions shown in Table 1 were prepared (% by weight of the total composition) : TABLE 1
  • comparative - hydrophilic silica hydrophilic fumed silica having a specific surface area of about 200 m 2 /g (BET area, measured according to standard ISO 9277-2010) (commercial product AerosilTM 200 by Evonik) ;
  • silica in the form of an aerogel having a specific surface area of about 550 m 2 /g (BET area, measured according to standard ISO 9277-2010) and a density of about 0.050 g/cm 3 .
  • lactoferrin hydrolysate peptide having a number of amino acid units from 5 to 10, obtained through the treatment of bovine lactoferrin with pepsin.
  • the rice husk silica and lactoferrin hydrolysate were previously complexed through the following process.
  • the rice husk silica was prepared as described in Example 1 of WO 2016/193877, with the difference that, after obtaining the silica sol-gel, this was added to the lactoferrin hydrolysate and then subjected to drying with supercritical CO2 as described in the same Example 1, with the exception that the silica sol- gel with added lactoferrin hydrolysate was autoclaved at a temperature of 50°C and a pressure of 100 bar, for a time equal to 6 hours, in order to obtain the drying of the sol-gel without causing the degradation of the peptide.
  • Antioxidant activity (scavenger) .
  • the antioxidant activity (scavenger for free radicals) of the above compositions was evaluated by measuring the ability to counteract the formation of free radicals (ROS) in cell cultures of human keratinocytes .
  • ROS free radicals
  • keratinocytes were treated with scalar concentrations of the product (1:2 dilutions starting from 1.0 mg/ml) and then exposed to UVA rays at room temperature to stimulate ROS production.
  • the untreated cells represent the negative control.
  • a neutral red uptake (NRU) assay was performed before and after irradiation in order to verify that the cell viability did not significantly decrease under the experimental conditions.
  • the results of the NRU test before exposure to UVA rays demonstrated the absence of cell viability inhibition at all concentrations used. Therefore all the concentrations were taken into consideration for the ROS dosing.
  • Human keratinocytes (Huker) grown in DMEM (Dulbecco's Modified Eagle medium) containing 10% of foetal bovine serum (FBS) and 1% of antibiotics (penicillin and streptomycin) , incubated under standard culture conditions (37°C, 5% CO2) . Good cell cultivation practices were applied.
  • DMEM Dulbecco's Modified Eagle medium
  • FBS foetal bovine serum
  • antibiotics penicillin and streptomycin
  • UVA rays The lamp used in the experiments was a sunlight simulator that reproduces the solar spectrum with a constant UVA emission range comprised between 315 nm and 400 nm and with a radiance of 1.7 mW/cm 2 .
  • the emission of UVB is appropriately screened to avoid cytotoxic damage directed to cell cultures.
  • test compositions under examination were dissolved in water and then diluted in a growth medium to the desired final concentrations comprised between 0.0156 and 1.0 mg/ml (1:2 dilutions) .
  • DCFDA dimethylsulfoxide
  • DMSO dimethylsulfoxide
  • Each cell sample (human keratinocytes ) was seeded in 96-well plates. Once a semi-confluent monolayer was reached, the cells were washed with phosphate buffer (PBS) and then incubated with the DCFDA solution for 20 min under standard culture conditions. The DCFDA was then removed, the cells were washed in PBS, treated with the different concentrations of the test composition and with the positive control, incubated for 20 min at standard conditions and then irradiated with UVA for 4, 8, 12, 16 and 20 min. At the end of each irradiation period a fluoride reading was performed at the excitation wavelength of 485 nm and emission wavelength of 530 nm.
  • PBS phosphate buffer
  • Neutral red (NR) is a weakly cationic probe that passes through the cell membrane by nonionic passive diffusion and is concentrated in lysosomes, where electrostatic interactions bind it to the anionic sites of the matrix.
  • the uptake of NR depends on the ability of the cells to create pH gradients, determined in turn by the production of ATP.
  • the NR thus only accumulates in living cells, from which it is extracted with an acidic solution of ethanol: the absorbance of the solution, measured at the spectrophotometer, is proportional to the cell viability.
  • the cells were treated with neutral red (NR) (50 pg/ml) and incubated for 3 hours under standard conditions. The cells were subsequently washed with PBS to remove the dye residues and the NR was extracted. The plates were gently agitated for at least 10 min (to facilitate the dissolution of NR) and the absorbance (optical density, OD) was determined through spectrophotometric reading at 540 nm. The absorbance measured at 540 nm is proportional to the cell viability. The percentages were calculated based on the absorbance values at 540 nm and considering the absorbance of the negative control (untreated cells) as 100%.
  • NR neutral red
  • compositions were tested to verify the antimicrobial activity with respect to E. coli.
  • An amount equal to 9 g of the composition was added to 1 ml of inoculum of E. coli (ATCC 8739) so as to obtain a final charge of about 10 5 CFU.
  • 0.5 ml of the suspension was taken to carry out the plate count (surface spatulation method) . The % variation of the bacterial load was therefore determined with respect to the initial one.

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Abstract

Complex between at least one lactoferrin hydrolysate and at least one silica having a specific surface area ranging between 100 m2/g and 1000 m2/g, preferably between 150 m2/g and 700 m2/g (BET area, measured according to ISO 9277-2010), said complex being obtainable by adding said lactoferrin hydrolysate to said silica in a sol-gel form with an aqueous solvent, and subsequent drying of the sol-gel. The complex is characterized by high stability and bioavailability, so as to exert an effective antibacterial and antioxidant action over time. The complex is particularly suitable for use in products for oral hygiene. It is also suitable in the prevention or treatment of inflammations of the urogenital apparatus, in particular of the vagina, in the prevention or treatment of intestinal dismicrobism, in the treatment of skin lesions, in particular skin sores, ulcers or erosions.

Description

COMPLEX BETWEEN A LACTOFERRIN HYDROLYSATE AND A SILICA, PRODUCTION PROCESS THEREOF, AND RELATED USES.
The present invention relates to a complex between a lactoferrin hydrolysate and a silica, a process for the preparation thereof, and relative uses.
It is well known that oral hygiene is a determining factor in the prevention of diseases of the dental apparatus, such as caries, gingivitis, periodontitis, peri-implantitis , etc.
Periodontitis is known to be a chronic inflammatory disease characterized by the destruction of the tooth's support structures (periodontal ligament, alveolar bone) . Periodontitis has a very high prevalence in the general population (around 10-15% of adults) and induces numerous negative effects on quality of life.
Peri-implantitis is an inflammatory process that affects the tissue surrounding an osseo- integratedimplant leading to a loss in the supporting bone tissue and therefore in the implant itself. The cause can be infectious, or it can be due to incorrect implant-prosthetic passivation (occlusal trauma) , although both causes can be clinically found. Radiographic examination makes it possible to easily distinguish peri-implantitis from occlusal trauma caused by bacteria.
To prevent or slow down the development of these pathologies, particularly accurate oral hygiene becomes critical in people suffering from dysfunctions or diseases, in particular diabetes, which increases oxidative stress in the oral cavity.
From a clinical point of view, diabetes mellitus is a clinically and genetically heterogeneous group of metabolic disorders characterized by high levels of glucose in the blood (hyperglycaemia) . Hyperglycaemia can be a direct consequence of a deficit of insulin secretion caused by the dysfunction of the pancreatic b cells or by resistance to the same hormone by the hepato-muscular tissues, or a combination of the aforementioned phenomena. This metabolic disorder is frequently associated with an alteration of the metabolism of adipocytes and can by all means be considered a syndrome. In the long-term, chronic hyperglycaemia causes multiple damage to different organs such as the heart, eyes, kidneys, nerves, vascular system.
Numerous recent epidemiological studies confirm that diabetes can be considered an important risk factor for the development of periodontal disease. The scientific evidence of the last 15-20 years supports a significant independent association between periodontal inflammation and glycaemic state. The susceptibility to the onset of periodontitis is three times greater in patients with diabetes mellitus.
Recent data provided by the World Health Organization shows that the incidence of the diabetic pathology is dramatically increasing. A direct consequence of this data is the parallel increase in patients suffering from periodontal disease, since diabetes is a risk factor for the development of periodontitis, regardless of the reduction in smoking habits and improved oral hygiene.
Diabetes mellitus and periodontal disease are both characterized by a state of systemic inflammation. Chronic hyperglycaemia, as well as periodontitis, induce the hyper-activation of the immune system, resulting in the hyper-production of pro-inflammatory molecules (TNF-a, IL6, IL1) . Entering the systemic circle, these can negatively affect the organism. In this sense, chronic inflammation of the gingival tissues can worsen the glycaemic and clinical profile of a patient suffering from diabetes mellitus. This relationship must therefore be considered with a bidirectional nature, in light of the foregoing.
Another aspect related to chronic periodontitis is the alteration of the immune system in terms of the balance between antioxidant agents and those reacting to oxygen, in favour of the latter. Oxidative stress can aggravate the clinical manifestations of a diabetic patient. On the other hand, diabetes is in turn a cause of oxidative stress and this can justify the fact that the clinical scenarios deriving from periodontitis are particularly aggressive in diabetic patients. The high prevalence and severity of periodontal disease in diabetic patients can be due to a state of chronic hyperglycaemia and an accumulation of advanced glycation end-products (AGEs) . The protracted oxidative stress in the gingival tissues could justify the aggressive destruction of periodontal tissues found in diabetic patients. The interaction between AGEs and their cell receptors induces an increase in oxidative stress that can contribute to chronic monocyte hyper activation, activation of the NF-Kb system resulting in the expression of pro-inflammatory mRNAs .
Lactoferrin is an antimicrobial and iron-carrying glycoprotein, naturally present in various biological fluids of mammals, such as milk, saliva, tears. It is also present in neutrophil granulocytes, which are immune cells with defence functions against bacterial and fungal infections. Lactoferrin is considered a particularly useful product for its antioxidant, immunomodulatory and anti-infective properties.
Through pepsinic digestion of lactoferrin, it is possible to obtain peptides, which are commonly indicated as lactoferrin hydrolysates or even, more simply, as lactoferricin which, although having a rather lower molecular weight with respect to the starting protein, contain the N-terminal sequence of lactoferrin and maintain the biological activity thereof; in fact, in some cases such activity is reinforced. On this point see for example the article of J. L. Gifford et al, Cell. Mol. Life Sci . 62 (2005),
2599-2598, where lactoferricin and its antimicrobial, antiviral, antitumor and immunological properties are described .
Patent application WO 2016/046108 describes a composition containing a lactoferrin hydrolysate and glycerophosphoinositol or salts thereof and/or verbascoside or extracts that contain it, and the use thereof for the prevention and/or treatment of dermatological, otologic and stomatological inflammations and infections. Such composition can be applied for the treatment of different infections, especially in the veterinary field, which can cause diseases of the ears, skin or of the stomatological type.
Lactoferrin hydrolysates have not yet been widely used, mainly because of their poor stability and compatibility with the excipients normally used in the pharmaceutical or para-pharmaceutical field (e.g. toothpaste, mouthwash, dietary supplements and the like) . Lactoferrin hydrolysates also have relatively low bioavailability when applied topically and are subject to oxidative degradation phenomena.
The Applicant therefore set out to solve the problem of obtaining a composition obtaining a lactoferrin hydrolysate which has high stability and bioavailability, so as to perform an antibacterial and antioxidant action which is effective and long-lasting over time.
This problem and others which will be better illustrated below have been solved by the association of a lactoferrin hydrolysate with at least one silica having a high specific surface area, which is able to complex with the peptide to form a stable complex which increases and preserves the long antioxidant and antibacterial activity of the lactoferrin hydrolysate.
According to a first aspect, the present invention therefore relates to a complex of at least one lactoferrin hydrolysate and at least one silica having a specific surface area comprised between 100 m2/g and 1000 m2/g, preferably between 150 m2/g and 700 m2/g (BET area, measured according to standard ISO 9277-2010), said complex being obtainable through the addition of said lactoferrin hydrolysate to said silica in the form of a sol-gel with an aqueous solvent, and subsequent drying of the sol-gel.
Preferably, the drying is carried out through treatment with supercritical CO2 or by lyophilization, as better illustrated below. According to another aspect, the present invention relates to a pharmaceutical or nutraceutical composition, which comprises said complex and at least one physiologically acceptable excipient. Preferably, the pharmaceutical composition is for oral use or for topical use.
According to another aspect, the present invention relates to a composition for oral hygiene, in particular for the oral hygiene of diabetic patients, which comprises said complex and at least one physiologically acceptable excipient.
According to another aspect, the present invention relates to a complex as defined above for use, through topical application, in the prevention or treatment of diseases of the dental apparatus, in particular for use in the prevention or treatment of periodontitis or peri-implantitis , preferably in patients with diabetes.
According to another aspect, the present invention relates to a complex as defined above for use, through topical application or oral administration, in the prevention or treatment of inflammations of the urogenital apparatus, in particular of the vagina, such as vaginosis or vaginitis of bacterial or viral origin, or yeast infections (candidiasis) .
According to another aspect, the present invention relates to a complex as defined above for use, through oral administration, in the prevention or treatment of intestinal dismicrobism, i.e. for restoring the balance of the intestinal microbiome.
According to another aspect, the present invention relates to a complex as defined above for use, through topical application, in the treatment of skin lesions, in particular skin sores, ulcers or erosions.
With regard to lactoferrin hydrolysates, they are products well known in the state of the art, which can be prepared according to known techniques, in particular through treatment of a lactoferrin with a proteolitic enzyme, e.g. pepsin. The starting lactoferrin can be of natural or synthetic origin, and can include different forms, both monomeric and oligomeric, possibly already bound to iron (ololactoferrin) or not bound to iron (apolactoferrin) . Further details on lactoferrin hydrolysates and the preparation thereof are reported, for example, in the following articles: Wakabayashi H., et al, Current Pharm. Design, 9(16) (2003), 1277-87; Theolier J., et al, Dairy Science & Technology, 94(2) (2013), 181-193;
Eliassen L.T., et al, Anticancer Res., 22(5) (2002 Sep-
Oct), 2703-10. The peptides resulting from the proteolysis of lactoferrin generally have a number of amino acid units from 5 to 30, preferably from 5 to 15.
As regards silica, this can be obtained through synthesis or through the processing of materials of natural origin, as better illustrated below. Among the synthetic silicas, particularly preferred forms are fumed silicas or precipitated silicas. Preferably, the silica is a hydrophilic silica.
For the purposes of the present invention, particularly preferred silicas are in the form of aerogel. These are amorphous silicas characterized by both a high specific surface area, as well as very high porosity and very low density. The porosity is generally greater than 85% and can even reach values of 95-99%, and the density is typically from 0.003 g/cm3 to 0.8 g/cm3, preferably from 0.005 g/cm3 to 0.3 g/cm3.
A particularly preferred silica is that in the form of aerogel obtained from vegetable waste, in particular from the husk resulting from the processing of grains such as rice, oats, wheat, rye, spelt. This silica, which is obtained from renewable sources, features a high level of bio-compatibility and has a high surface area and a highly porous, open-pored structure, which favours the interaction with the lactoferrin hydrolysate. The silica obtained from rice husk is particularly preferred.
The silica in the form of aerogel from vegetable waste can be obtained through a multi-stage process that comprises: combustion of vegetable waste, processing of the ashes thus obtained with an alkaline solution (NaOH) to obtain a silicate (of sodium) , processing of the latter with an acid solution (HC1) with the formation of a wet sol-gel, and finally drying of the wet sol-gel, preferably by treatment with supercritical CO2. Further details on the preparation of such silica forms are reported, for example, in patent application WO 2016/193877 and in the article by R.S. Kumar et al . Int. J. Chem. Eng. and Appls . , Vol. 4, No. 5, October 2013.
Preferably, in the complex according to the present invention, said at least one lactoferrin hydrolysate and said at least one silica are present in a weight ratio of 0.05:1 to 10:1, more preferably from 0.1:1 to 5:1.
Preferably, the composition for oral hygiene according to the present invention comprises:
from 10% to 70% by weight of at least one silica; from 0.05% to 15% by weight of at least one lactoferrin hydrolysate;
from 25% to 80% by weight of an aqueous phase.
More preferably, the composition according to the present invention comprises:
from 20% to 50% by weight of at least one silica; from 0.2% to 10% by weight of at least one lactoferrin hydrolysate;
from 40% to 75% by weight of an aqueous phase.
It should be noted that the silica amounts indicated above refer to the amount of overall silica present in the composition, whether it is complexed or not complexed with lactoferrin hydrolysate.
According to a preferred embodiment, the composition according to the present invention comprises the complex of at least one lactoferrin hydrolysate and at least one silica as defined above in mixture with a hydrophilic silica that has not been complexed with lactoferrin hydrolysate, in particular a synthetic silica, preferably a hydrophilic fumed silica. The addition of a non-complexed silica has the main purpose of further stabilizing complex between silica and lactoferrin hydrolysate, substantially acting as a barrier to the interaction with other ingredients of the composition that could destabilize the complex itself.
The ratio by weight between silica complexed with the lactoferrin hydrolysate and hydrophilic silica not complexed with the lactoferrin hydrolysate is preferably comprised between 1:200 and 1:1, more preferably between 1:150 and 1:5.
As for the aqueous phase, this preferably comprises a mixture of water and a polyol, more preferably glycerol or sorbitol. The weight ratio of water to polyol is preferably comprised between 1:20 and 1:1, more preferably between 1:10 and 1:2.
The Applicant has found that the lactoferrin hydrolysate and silica complex according to the present invention makes it possible to obtain an improved antibacterial and antioxidant effect with respect to the lactoferrin hydrolysate used alone with the same concentrations, which is carried out for longer times, so as to prolong the effect without having to repeat the application of the composition too often.
The complex according to the present invention is preferably prepared through a process which comprises: preparing a silica having a specific surface area ranging from 100 m2/g to 1, 000 m2/g, preferably from
150 m2/g to 700 m2/g (BET area, measured according to the standard ISO 9277-2010) in sol-gel form with an aqueous solvent;
adding at least one lactoferrin hydrolysate to the silica in a sol-gel form;
subjecting the so obtained mixture to a drying phase .
The addition of the lactoferrin hydrolysate to the silica in the form of a sol-gel can be achieved through the gradual addition of the peptide to the sol-gel and mechanical mixing until a homogeneous dispersion is obtained. The mixing temperature is generally maintained between 15°C and 50°C.
Preferably, the drying is carried out by treatment with supercritical CO2 or by lyophilization . In this way, the complexation between silica and the lactoferrin hydrolysate is guaranteed, without the latter undergoing any structural modifications that would compromise the functionality thereof.
The complex between lactoferrin hydrolysate and silica according to the present invention can be treated with at least one polyol, preferably glycerol or sorbitol. The polyol has the function of covering the silica particles loaded with the lactoferrin hydrolysate, further stabilizing the structure.
Water can be used as an aqueous solvent, optionally in a mixture with at least one soluble organic solvent, for example ethyl acetate or acetone.
Preferably, the drying step with supercritical CO2 is carried out in an autoclave, at a temperature from 15°C to 60°C and a pressure from 80 bars to 150 bars.
In order to avoid the degradation of the lactoferrin hydrolysate, using a temperature no greater than 60°C is preferable. The treatment is carried out for a time preferably ranging from 2 hours to 10 hours, more preferably from 4 hours to 8 hours.
Preferably, the drying step by means of lyophilization ( cryo-drying) is carried out through cooling under vacuum at a temperature between -80 °C and -30 °C and subsequent heating, always under vacuum, at a temperature no greater than 60°C, preferably between 20°C and 50°C. The pressure during lyophilization is preferably maintained between 0.1 bar and 0.8 bar.
The oral hygiene composition according to the present invention comprises, in addition to the complex, at least one physiologically acceptable excipient. This is in order to obtain a product for oral hygiene in different forms, for example toothpaste, mouthwash, gel, chewing gum, tablets for oral use, gingival dyeing, and the like.
For this purpose, the composition according to the present invention can comprise ingredients commonly used in the field of dental hygiene products, such as for example: abrasive agents in particulate form, humectants, binders, thickeners, viscosity regulators, surfactants, sweeteners, flavourings, preservatives, anti-plaque agents, dyes, etc.
With regard to pharmaceutical compositions which include the complex according to the present invention, they may be presented in different forms, such as tablets, capsules, aqueous suspensions, syrups, suppositories, and contain the common excipients used for such products.
With regard to nutraceutical compositions which include the complex according to the present invention, they may be in the form of capsules, tablets, liquid formulations, bars, and the like. As well as the complex, water and other products with nutritional value, they can include other products such as carbohydrates, lipids, proteins, vitamins, minerals and the like.
In the aforesaid composition, the complex according to the present invention may be combined with other biologically active products, e.g. glycerophosphoinositol or salts thereof, which has the main function of increasing the antibacterial effect.
In the case of nutraceutical compositions, particularly suitable in the prevention or treatment of intestinal dismicrobism, they may contain, in addition to the complex, one or more strains of microorganisms useful for the intestinal microbiome, such as Lactobacilli and the like (e.g. Lactobacillus easel) .
With regard to the use of the complex according to the present invention for topical applications, the complex may be formulated in the form of a suspension, gel, tincture, cream and the like, with the excipients commonly used for such products. The formulation may possibly contain a pH adjuster, in order to guarantee a physiological pH, e.g. lactic acid which provides a pH around 3.4/4, typical of the vaginal environment.
The following examples of embodiment are provided for the sole purpose of illustrating the present invention and are not to be understood in a sense limiting the scope of protection defined by the appended claims.
EXAMPLES 1-3.
The compositions shown in Table 1 were prepared (% by weight of the total composition) : TABLE 1
Figure imgf000015_0001
(*) comparative - hydrophilic silica: hydrophilic fumed silica having a specific surface area of about 200 m2/g (BET area, measured according to standard ISO 9277-2010) (commercial product Aerosil™ 200 by Evonik) ;
- rice husk silica: silica in the form of an aerogel having a specific surface area of about 550 m2/g (BET area, measured according to standard ISO 9277-2010) and a density of about 0.050 g/cm3.
- lactoferrin hydrolysate: peptide having a number of amino acid units from 5 to 10, obtained through the treatment of bovine lactoferrin with pepsin.
The rice husk silica and lactoferrin hydrolysate were previously complexed through the following process. The rice husk silica was prepared as described in Example 1 of WO 2016/193877, with the difference that, after obtaining the silica sol-gel, this was added to the lactoferrin hydrolysate and then subjected to drying with supercritical CO2 as described in the same Example 1, with the exception that the silica sol- gel with added lactoferrin hydrolysate was autoclaved at a temperature of 50°C and a pressure of 100 bar, for a time equal to 6 hours, in order to obtain the drying of the sol-gel without causing the degradation of the peptide.
The complex thus obtained and the other ingredients of the composition were mixed in a flask with the aid of a magnetic anchor, until a homogeneous formulation was obtained. It is to be noted that the hydrophilic silica was added as such, i.e. not complexed with the lactoferrin hydrolysate.
The antioxidant and antibacterial properties of these compositions were verified, evaluating the variation of the efficacy over time.
Antioxidant activity (scavenger) .
The antioxidant activity (scavenger for free radicals) of the above compositions was evaluated by measuring the ability to counteract the formation of free radicals (ROS) in cell cultures of human keratinocytes . For this purpose keratinocytes were treated with scalar concentrations of the product (1:2 dilutions starting from 1.0 mg/ml) and then exposed to UVA rays at room temperature to stimulate ROS production. The untreated cells represent the negative control. At the same time, a neutral red uptake (NRU) assay was performed before and after irradiation in order to verify that the cell viability did not significantly decrease under the experimental conditions. The results of the NRU test before exposure to UVA rays demonstrated the absence of cell viability inhibition at all concentrations used. Therefore all the concentrations were taken into consideration for the ROS dosing.
The results obtained by the ROS dosing demonstrated the antioxidant action, expressed as % reduction of ROS with respect to the negative control.
More in detail, the materials and operating conditions used are shown below.
Human keratinocytes (Huker) grown in DMEM (Dulbecco's Modified Eagle medium) containing 10% of foetal bovine serum (FBS) and 1% of antibiotics (penicillin and streptomycin) , incubated under standard culture conditions (37°C, 5% CO2) . Good cell cultivation practices were applied. In order to simulate a condition of environmental stress capable of inducing the formation of ROS, the cells were subjected to irradiation with UVA rays. The lamp used in the experiments was a sunlight simulator that reproduces the solar spectrum with a constant UVA emission range comprised between 315 nm and 400 nm and with a radiance of 1.7 mW/cm2. The emission of UVB is appropriately screened to avoid cytotoxic damage directed to cell cultures.
The effects on ROS production at different irradiation times were evaluated: 4, 8, 12, 16 and 20 min .
Each sample of the test compositions under examination was dissolved in water and then diluted in a growth medium to the desired final concentrations comprised between 0.0156 and 1.0 mg/ml (1:2 dilutions) . 2 ' , 7 ' -dichlorofluorescine diacetate (DCFDA) was used as a fluorimetric tracer, dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mM and then diluted in the appropriate buffer up to a concentration of use of 250 mM .
Each cell sample (human keratinocytes ) was seeded in 96-well plates. Once a semi-confluent monolayer was reached, the cells were washed with phosphate buffer (PBS) and then incubated with the DCFDA solution for 20 min under standard culture conditions. The DCFDA was then removed, the cells were washed in PBS, treated with the different concentrations of the test composition and with the positive control, incubated for 20 min at standard conditions and then irradiated with UVA for 4, 8, 12, 16 and 20 min. At the end of each irradiation period a fluoride reading was performed at the excitation wavelength of 485 nm and emission wavelength of 530 nm.
In order to verify that the cell viability did not vary significantly in the presence of the composition under examination and in the experimental conditions applied, the neutral red uptake (NRU) assay was performed before and after irradiation of the cells. Neutral red (NR) is a weakly cationic probe that passes through the cell membrane by nonionic passive diffusion and is concentrated in lysosomes, where electrostatic interactions bind it to the anionic sites of the matrix. The uptake of NR depends on the ability of the cells to create pH gradients, determined in turn by the production of ATP. The NR thus only accumulates in living cells, from which it is extracted with an acidic solution of ethanol: the absorbance of the solution, measured at the spectrophotometer, is proportional to the cell viability.
After gently removing the medium, the cells were treated with neutral red (NR) (50 pg/ml) and incubated for 3 hours under standard conditions. The cells were subsequently washed with PBS to remove the dye residues and the NR was extracted. The plates were gently agitated for at least 10 min (to facilitate the dissolution of NR) and the absorbance (optical density, OD) was determined through spectrophotometric reading at 540 nm. The absorbance measured at 540 nm is proportional to the cell viability. The percentages were calculated based on the absorbance values at 540 nm and considering the absorbance of the negative control (untreated cells) as 100%. In the absence of UVA irradiation, the cell viability of the keratinocytes treated with the product was comparable to that of the untreated keratinocytes for all the concentrations tested, therefore all the concentrations were taken into account in the ROS dosages.
Antimicrobial activity.
Each composition was tested to verify the antimicrobial activity with respect to E. coli. An amount equal to 9 g of the composition was added to 1 ml of inoculum of E. coli (ATCC 8739) so as to obtain a final charge of about 105 CFU. After increasing contact times (1, 4, 8 and 24 hours), 0.5 ml of the suspension was taken to carry out the plate count (surface spatulation method) . The % variation of the bacterial load was therefore determined with respect to the initial one.
The results are shown in the following tables, where the left column shows the times (in minutes for Table 2 and in hours for Table 3) . The same results are shown in the attached graphs Fig. 1 and Fig. 2.
TABLE 2
(antioxidant effect :% ROS reduction)
Figure imgf000020_0001
(*) comparative TABLE 3
(antibacterial effect: % bacterial load reduction)
Figure imgf000021_0001
(*) comparative
From the results obtained, it appears evident that the complex comprising lactoferrin hydrolysate and silica makes it possible to increase the efficacy both in terms of antioxidant and antibacterial effect in a more gradual way and maintain them over time, compared to the lactoferrin hydrolysate used alone.

Claims

1. Complex between at least one lactoferrin hydrolysate and at least one silica having a specific surface area ranging between 100 m2/g and 1000 m2/g, preferably between 150 m2/g and 700 m2/g (BET area, measured according to ISO 9277-2010), said complex being obtainable by adding said lactoferrin hydrolysate to said silica in a sol-gel form with an aqueous solvent, and subsequent drying of the sol-gel.
2. Complex according to any one of the preceding claims, wherein said at least one silica is a synthetic silica, selected from fumed silica and precipitated silica.
3. Complex according to any one of the preceding claims, wherein said at least one silica is in the aerogel form.
4. Complex according to claim 3, wherein said at least one silica in the aerogel form has a porosity greater than 85% and a density between 0.003 g/cm3 and 0.8 g/cm3, preferably between 0.005 g/cm3 and 0.3 g/cm3.
5. Complex according to claim 3 or 4, wherein said at least one silica is obtained from vegetable waste, particularly from husk deriving from processing of cereals, preferably of rice.
6. Complex according to any one of the preceding claims, wherein said at least one lactoferrin hydrolysate and said at least one silica are present in a weight ratio between 0.05:1 and 10:1, preferably between 0.1:1 and 5:1.
7. Complex according to any one of the preceding claims, in the form of particles surface treated with at least one polyol, preferably glycerol or sorbitol .
8. Process for preparing a complex according to any one of claims from 1 to 7, said process comprising :
preparing a silica having a specific surface area ranging between 100 m2/g and 1000 m2/g, preferably between 150 m2/g and 700 m2/g (BET area, measured according to ISO 9277-2010) in a sol-gel form with an aqueous solvent;
adding at least one lactoferrin hydrolysate to the silica in a sol-gel form;
subjecting the so obtained mixture to a drying phase .
9. Process according to claim 8, further comprising treating the dried mixture by adding at least one polyol, preferably glycerol or sorbitol.
10. Process according to claim 8 or 9, wherein the drying phase is carried out by treatment with supercritical CO2.
11. Process according to claim 8 or 9, wherein the drying phase is carried out by lyophilization .
12. Process according to claim 10, wherein the drying phase by supercritical CO2 is carried out in an autoclave, at a temperature between 15°C and 60°C and a pressure between 80 bar and 150 bar.
13. Process according to claim 11, wherein the drying phase by lyophilization is carried out by under vacuum cooling at a temperature between -80°C and -30°C and subsequent heating, also under vacuum, at a temperature not higher than 60 °C, preferably between 20°C and 50°C.
14. Process according to claim 13, wherein the lyophilization is carried out at a pressure between 0.1 bar and 0.8 bar .
15. Oral care composition, comprising at least one complex according any one of claims from 1 to 7, and at least one physiologically acceptable excipient.
16. Composition according to claim 15, wherein said at least one complex is in admixture with at least one hydrophilic silica not complexed with a lactoferrin hydrolysate, preferably a fume hydrophilic silica.
17. Composition according to claim 16, comprising :
from 10% to 70% by weight, preferably from 20% to 50% in weight, of at least one silica;
from 0.05% to 15% by weight, preferably from 0.2% and 10% by weight, of at least one lactoferrin hydrolysate;
from 25% to 80% by weight, preferably from 40% to 75% by weight, of an aqueous phase.
18. Composition according to any one of claims from 15 to 17, wherein the aqueous phase comprises a mixture of water and a polyol, preferably glycerol or sorbitol .
19. Complex according to any one of claims from 1 to 7 for use, by topical application, in the prevention or treatment of teeth diseases, particularly for use in the prevention or treatment of parodontosis or periimplantitis , preferably in patients affected by diabetes .
20. Pharmaceutical or nutraceutical composition, comprising at least one complex according to any one of claims from 1 to 7, and at least one physiologically acceptable excipient.
21. Complex according to any one of claims from 1 to 7 for use, by topical application or oral administration, in the prevention or treatment of inflammations of the urogenital apparatus, in particular of the vagina, such as vaginosis or vaginitis of bacterial or viral origin, or infections by yeasts (candidiasis) .
22. Complex according to any one of claims from 1 to 7 for use, by oral administration, in the prevention or treatment of intestinal dismicrobism .
23. Complex according to any one of claims from 1 to 7 for use, by topical application, in the treatment of skin lesions, particularly sores, ulcers or skin erosions.
PCT/IB2020/055537 2019-06-13 2020-06-12 Complex between a lactoferrin hydrolysate and a silica, production process thereof, and related uses. WO2020250195A1 (en)

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IT202100018008A1 (en) * 2021-07-08 2023-01-08 Claudio Angelinetta COMPLEX BASED ON LACTOFERRIN AND/OR A HYDROLYSATE OF ITS PRODUCTION PROCESS, AND RELATED USES.
WO2023281430A1 (en) * 2021-07-08 2023-01-12 Angelinetta Claudio Complex based on lactoferrin and/or a hydrolyzate thereof, process for producing the same, and uses thereof

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