Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/42—Phosphorus; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/0241—Containing particulates characterized by their shape and/or structure
• • 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
  • A61K8/24—Phosphorous; 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
  • 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
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
  • C01B25/00—Phosphorus; Compounds thereof
  • C01B25/16—Oxyacids of phosphorus; Salts thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/60—Particulates further characterized by their structure or composition
  • A61K2800/61—Surface treated
  • A61K2800/612—By organic compounds
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
  • C01P2002/00—Crystal-structural characteristics
  • C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
  • C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
  • C01P2002/00—Crystal-structural characteristics
  • C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
  • C01P2002/82—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
  • C01P2004/00—Particle morphology
  • C01P2004/01—Particle morphology depicted by an image
  • C01P2004/03—Particle morphology depicted by an image obtained by SEM
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
  • C01P2004/00—Particle morphology
  • C01P2004/01—Particle morphology depicted by an image
  • C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM

Definitions

• the invention relates to dental care products containing biomimetic carbonate- substituted strontium-hydroxyapatite particles having a protein-functionalized surface, and the process for their preparation.
• the invention relates to dental care products for dental hygiene containing biomimetic carbonate-substituted strontium-hydroxyapatite particles having a protein-functionalized surface such as, for example, solutions, suspensions, oils, gels, pastes, dentifrices, or other solid products.
• the invention relates to a process for preparing a suspension for dental hygiene, to a process for manufacturing a dental care product containing the aforementioned biomimetic carbonate-substituted strontium-hydroxyapatite particles having a protein-functionalized surface, as well as to a method of providing at the teeth outer surface a source of Sr 2+ ions and antibacterial proteins which may be locally released at an acidic pH by means of the biomimetic carbonate-substituted strontium- hydroxyapatite particles having a protein-functionalized surface.
• Enamel is the hardest material in vertebrates and is the most highly mineralized skeletal tissue present in the body.
• Mature enamel considered the most resistant and tough material in the biological world, is composed of carbonate hydroxyapatite (CHA) (95- 97% wt) and less, about 1% wt, of organic material.
• CHA carbonate hydroxyapatite
• mature enamel does not contain cells and therefore cannot be regenerate itself and therefore cannot be biologically remodelled. Consequently, enamel regeneration cannot take place in vivo. There is no biological process that can repair degraded or damaged enamel, evidencing the need for synthetic enamel biocompatible materials able to repair teeth decay.
• Enamel makes up the outermost layer of the tooth crown having a thickness of about 1 - 2 mm and containing a high mineral content which imparts to the enamel a high modulus, but also making it susceptible to cracking.
• Dentine lies below the enamel and is tougher, forming the bulk of the tooth and absorbing stresses from enamel, preventing its fracture.
• the mechanisms involved in the damage of dental hard tissue are related to the acid attacks on the outer few micrometers of the enamel, with the consequent demineralization and dissolution of the minerals.
• the oral cavity contains a numerous amount of microorganisms that adhere to the surface of teeth for long periods of time. These multiple species of bacteria become dental biofilm, more commonly referred to as dental plaque, which is composed of bacteria that take part in the complex ecosystems of the mouth.
• dental plaque is composed of bacteria that take part in the complex ecosystems of the mouth.
• the natural, non- frequent regulation of tooth shedding plays a large role in making dental biofilm the most diverse biofilm in the human body despite the relatively small size of the teeth.
• the biofilm is usually a pale yellow that develops naturally on the teeth. Initially, the biofilm is soft enough to come off easily. However, it starts to harden within 48 hours, and in about 10 days the plaque becomes dental calculus (tartar), rock-hard and difficult to remove without the professional intervention with ultrasonic tools and specialized sharp instruments.
• Lactoferrin (formerly known as lactotransferrin) is a glycoprotein with a molecular weight of about 80 kDa, and a member of a transferrin family, thus belonging to those proteins capable of binding and transferring Fe 3+ ions.
• Lactoferrin is comprised of a single polypeptide chain containing 703 amino acids folded into two globular lobes. These lobes, also called C - (carboxy) and N - (amino) terminal regions, are connected with a a-helix.
• Lactoferrin was found in mucosal secretions, including tears, saliva, vaginal fluids, semen, nasal and bronchial secretions, bile, gastrointestinal fluids, urine, amniotic fluid and at higher concentrations in milk and colostrums, which makes it the second most abundant protein in milk, after caseins. In blood plasma, neutrophils after degranulation were observed to be the main source of lactoferrin.
• Lactoferrin is involved in several physiological functions, many of which do not appear to be connected with its iron binding ability: regulation of iron absorption in the bowel, immune response, antioxidant, cariostatic, anticarcinogenic and anti-inflammatory properties and protection against microbial infection.
• the antimicrobial activity of lactoferrin is mostly due to its iron-binding properties, which deprive the bacterial flora from an element necessary for its growth.
• lactoferrin may serve as iron donor, and in this manner support the growth of some bacteria with lower iron demands such as Lactobacillus sp. or Bifidobacterium sp., generally considered as beneficial.
• lactoferrin has also a bactericidal activity due to its direct interaction with the infectious agent.
• This bactericidal activity is not iron-dependent and may be mediated through more than one pathway such as the binding of lactoferrin to specific receptors on the surface of some microorganisms which induces cell-death in Gram-negative bacteria due to a disruption in the cell wall.
• LPS lipopolysacharide
• LPS lipopolysacharide
• the bactericidal activity affecting Gram-positive bacteria is mediated by electrostatic interactions between the negatively charged lipid layer and the positively charged lactoferrin surface that cause changes in the permeability of the membrane.
• the teeth bone tissue is primarily constituted by non-stoichiometric hydroxyapatite containing specific substituting ions at both the cationic and anionic reticular sites
• the use of products comprising hydroxyapatite has been proposed for the treatment of bone defects in the fields of reconstructive bone surgery, surgical stomatology, traumatology, orthopedics and dentistry.
• lactoferrin exerts several beneficial effects on the health of humans and animals, including anti- infective, anticancer, and anti-inflammatory effects. This has enlarged the potential application of lactoferrin as a food additive and for oral care applications.
• this reference discloses an apatite-based nanostructured material obtained by lattice destabilisation treatment under high energy.
• European Patent Application EP 2039342 discloses an agent for maintaining hardness of tooth substances comprising lactoferrin and iron-lactoferrin, particularly for use in food or drink.
• German Patent Application DE 102010063720 discloses dental care composition comprising silver particles, at least one calcium phosphate compound, and lactoferrin.
• International Patent Application PCT/EP2011/002606 in the name of the same Applicant discloses dental care products comprising carbonate-substituted fluoro- hydroxyapatite particles and a process for their preparation..
• the carbonate-substituted strontium-hydroxyapatite particles of the invention are biomimetic for composition, structure and morphology allowing a strong chemical affinity with natural enamel and dentine hydroxyapatite crystals and inducing the formation of a rimineralizing and protective surface coating on enamel and dentine.
• the Applicant has observed that carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface show an increased antibacterial effect with respect to the lactoferrin alone of the aforementioned prior art.
• the Applicant has also surprisingly discovered that said strontium hydroxyapatite functionalized by lactoferrin presents a greater degree of crystallinity with respect to a zinc carbonate-substituted hydroxyapatite without strontium and lactoferrin.
• having a protein-functionalized surface or having a lactoferrin- functionalized surface is used to indicate that the carbonate-substituted strontium- hydroxyapatite surface is functionalised by lactoferrin which is bonded to the surface itself.
• the Applicant deems, without however wishing to be bound by any interpretative theory, that such a bond may derive from several kinds of interactions which can take place between the lactoferrin and the carbonate-substituted strontium- hydroxyapatite surface, for example covalent or electrostatic chemical bonds, hydrogen and van der Waals bonds, and hydrophobic and hydrophilic interactions.
• the present invention provides improved products for dental hygiene having both remineralization and cariostatic effects, comprising carbonate-substituted strontium-hydroxyapatite particles capable to convey, directly to the surface of the enamel, Sr 2+ ions only when this is necessary, i.e.
• products for dental hygiene comprising said substantially insoluble hydroxyapatite-type carrier is capable of forming a thin film on the outer surface of the enamel even in the limited time available during the normal routine of dental hygiene.
• Said film solubilizes when the pH of the oral cavity becomes acidic (pH ⁇ 5) locally releasing strontium ions and lactoferrin, only when this is necessary, effectively improving teeth remineralisation and showing a cariostatic and antibacterial effect.
• carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface of the invention have the formula:
• x is a number comprised between 0.0010 and 0.015; and y is a number comprised between 0.0010 and 0.010; and wherein said carbonate-substituted strontium-hydroxyapatite particles have a crystallinity degree CD comprised between 55 and 85%.
• the crystallinity degree being defined as
• CD (1-X/Y) ⁇ 100
• particles is intended to indicate nanoparticles or microparticles.
• nanometric size When particles ere considered of nanometric size, their dimension range from few nanometers up to some hundreds of nanometers, but for biomedical applications the dimension range is usually reduced from 30 nra to 200 nm because only nanoparticles of these dimensions can go across the cellular membrane. In order to avoid any possible consequence related to the presence of nanoparticles inside the cells it is preferred to use nanoparticles larger than 100 nm for biomedical applications which do not require an intracellular action. On the other hand nanoparticles aggregated in a cluster can realize particles of micrometric dimension.
• nanoparticle is used to indicate a particle having a size generally below 0.1 ⁇ , preferably between 0.01 ⁇ and 0.1 ⁇ .
• microparticle is used to indicate aggregates or "clusters" of inorganic nanoparticles mentioned above and having a size comprised between 0.2 ⁇ and 10 ⁇ , preferably between 0.5 ⁇ and 2 ⁇ .
• Single nanoparticles can have quite different morphology, but preferably, the nanoparticles of the invention have a flat and round shape mimicking the morphology of bone hydroxyapatite nanoparticles.
• the Applicant considers the nanoparticles biomimetic morphology the most adapted to interact with the dentine and enamel surface.
• crystallinity degree is intended to indicate the percentage of the hydroxyapatite compound present in the crystalline state.
• the crystallinity degree can be measured according to known methods, such as, for example, by using x-ray diffraction analysis.
• the crystallinity degree CD is measured according to the method described in: Landi, E., Tampieri, A., Celotti, G., Sprio, S., " Densification behaviour and mechanisms of synthetic hydroxyapatites” , J. Eur. Ceram. Soc, 2000, 20, 2377-2387 (hereinafter in short: the Landi et al. method).
• Landi E., Tampieri, A., Celotti, G., Sprio, S.
• the expression: lower than, as used before any numerical value is meant to exclude such a numerical value and used to encompass only a range of lower values.
• the aforementioned lactoferrin which is incorporated in the apatite structure, effectively exploit an antibacterial activity capable of preventing generation of carious tooth and periodontal diseases such as alveolar blennorrhoea and reducing halitosis phenomena. Therefore, the dental composition of the invention is also advantageously capable of effectively exploiting an antibacterial effect and, accordingly, effectively treating teeth and gums disorders and in general increasing oral hygiene even in the limited time available during the normal routine of dental hygiene.
• the particles of the invention are carbonate-substituted hydroxyapatite particles having a lactoferrin-functionalized surface which incorporate carbonate ions in the apatite structure.
• This feature advantageously enhances the biological activity of the particles of the invention, since the carbonate ion is also found in the structure of natural hydroxyapatite.
• the carbonate ion can occupy in two different sites in the natural hydroxyapatite structure: namely, it can partially substitute the OH-ion (site A) and/or the P0 4 3" ion (site B).
• site A the OH-ion
• site B the P0 4 3" ion
• Both the total carbonate content (in the range of 3-8 wt. %) and the relative quantities of type A and type B carbonation (A/B in the range of 0.7-0. 9) found in the natural carbonate-substituted hydroxyapatite depend on the age of the individual and on the biological localization of the calcified tissue.
• the carbonation preferably takes place at site B.
• the hydroxyapatite particles comprise from 1 to 15% by weight and, more preferably, from 1 to 10% by weight based on the total weight of the particles of carbonate substituted into the hydroxyapatite structure.
• the biological activity of the particles of the invention is advantageously enhanced, since their structure more closely resembles the structure of the natural apatite present in the teeth tissues.
• the ratio A/B between the carbonate substitution at the hydroxyl site (A) and the carbonate substitution at the phosphate site (B) of the hydroxyapatite is comprised between 0.05 and 0.5 and, still more preferably, comprised between 0.18 and 0.33.
• the carbonate substitution at the phosphate site (B) of the hydroxyapatite is greater than or equal to 65% by weight and, still more preferably, comprised between 90% and 100% by weight, of the total carbonate present in the hydroxyapatite.
• the carbonate substitution at the phosphate site (B) advantageously induces a higher affinity of the hydroxyapatite particles for the osteoblast cell, increasing cellular adhesion and collagen production.
• the total amount of lactoferrin in the carbonate-substituted strontium- hydroxyapatite particles is comprised between 0.01% and 5.0% by weight based on the total weight of the particles.
• dental care products of the invention further comprise an effective amount of a metal M ion.
• the metal M is selected from the group comprising: Mg, Se, K and mixtures thereof.
• dental care products of the invention comprise from 0.1% to 20% by weight with respect of the total Ca content of a metal M ion substituted into the hydroxyapatite structure.
• the dental care product including the particles of the invention may be in any physical form suitable for oral hygiene such as suspension, oil, gel or other solid product.
• the dental care product is in the form of a suspension including from 1% to 40% by weight, more preferably from 10% to 20% by weight, of carbonate-substituted hydroxyapatite particles.
• the suspension has pH comprised between 6 and 13.
• the suspension may be advantageously directly used as such or mixed with other ingredients in the formulation of effective dental care products.
• this suspension may be produced by means of a quite simple and economic method, as will be described in more detail hereinbelow, and may be directly used, for example as a gargle or mouthwash, to treat the teeth and gums or may be mixed with other ingredients when formulating a solid or liquid product such as a toothpaste or a mouthwash.
• the inventors have surprisingly observed that the suspension of the invention is stable for an extended period of time even if no stabilizing agents are added thereto.
• the suspension of the invention is stable for at least 30 days and, more generally, for about two-three months, without using any stabilizing agent.
• the dental care product is selected from the group consisting of: toothpaste, tooth powder, chewing gum for oral and dental hygiene, ointment for the gums, mouthwash and mouth bath concentrate and gargle.
• the dental care products of this invention will, of course, also preferably contain other ingredients commonly used and known in the art to formulate such products, depending on the form of the oral product.
• the product in the case of an oral product in the form of a dentifrice cream or paste, the product will preferably comprise a particulate abrasive agent, a humectant-containing liquid phase and a binder or thickener which acts to maintain the abrasive agent in stable suspension in the liquid phase.
• a surfactant and a flavoring agent are also usual preferred ingredients of commercially acceptable dentifrices.
• a suitable particulate abrasive agent is preferably selected from the group comprising: silica, alumina, hydrated alumina, calcium carbonate, anhydrous dicalcium phosphate, dicalcium phosphate dihydrate and water- insoluble sodium metaphosphate.
• the amount of particulate abrasive agent will generally range from 0.5% to 40% by weight of the toothpaste.
• Humectants of preferred use are glycerol and sorbitol syrup (usually comprising an approximately 70% solution). However, other humectants are known to those in the art including propylene glycol, lactitol, and hydrogenated corn syrup. The amount of humectant will generally range from 10% to 85% by weight of the toothpaste.
• the liquid phase can be aqueous or nonaqueous.
• binding or thickening agents have been indicated for use in dentifrices, preferred ones being sodium carboxymethylcellulose and xanthan gum.
• Others include natural gum binders such as gum tragacanth, gum karaya and gum arabic, alginates and carrageenans.
• Silica thickening agents include the silica aerogels and various precipitated silicas. Mixtures of binders may be used.
• the amount of binder included in a dentifrice is generally between 0.1% and 5% by weight.
• surfactant in a dentifrice and again the literature discloses a wide variety of suitable materials.
• Surfactants which have found wide use in practice are sodium lauryl sulfate and sodium lauroylsarcosinate.
• Other anionic surfactants may be used as well as other types such cationic, amphoteric and non-ionic surfactants.
• Surfactants are usually present in an amount comprised between 0.5% and 5% by weight of the dentifrice.
• Flavors of possible use are those usually used in dentifrices, for example those based on oils of spearmint and peppermint. Examples of other flavoring materials which may be used are menthol, clove, wintergreen, eucalyptus and aniseed. An amount comprised between 0.1% and 5% by weight is a suitable amount of flavor to incorporate in a dentifrice.
• the dental care products of the invention may include a wide variety of other optional ingredients.
• these optional ingredients may include an anti-plaque agent such as moss extract, an anti-tartar ingredient, such as a condensed phosphate, e.g. an alkali metal pyrophosphate, hexametaphosphate or polyphosphate; a sweetening agent, such as saccharine and salts thereof; an opacifying agent, such as titanium dioxide; a preservative, such as formalin; a coloring agent; a pH controlling agent, such as an acid, base or buffer, such as citric acid.
• Suitable amounts of these optional ingredients may be easily selectable by those skilled in the art as a function of the specific characteristics to be imparted to the toothpaste.
• the composition will comprise in addition to the ingredients mentioned above a suitable gum base which may be easily selectable by those skilled in the art.
• the composition will comprise suitable ingredients in liquid or soluble form easily selectable by those skilled in the art, such as sorbitol, glycerol, oils and flavoring materials, solubilizing agents such as hydrogenated and ethoxylated ricin oil, surfactants, such as sodium lauryl sulfate and sodium lauroylsarcosinate, preserving agents, viscosity regulators and other suitable ingredients which may be easily selectable by those skilled in the art.
• suitable ingredients in liquid or soluble form easily selectable by those skilled in the art such as sorbitol, glycerol, oils and flavoring materials, solubilizing agents such as hydrogenated and ethoxylated ricin oil, surfactants, such as sodium lauryl sulfate and sodium lauroylsarcosinate, preserving agents, viscosity regulators and other suitable ingredients which may be easily selectable by those skilled in the art.
• the present invention relates to dental care compositions comprising the aforementioned carbonate-substituted strontium- hydroxyapatite particles having a lactoferrin-functionalized surface.
• the present invention relates to the aforementioned carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin- functionalized surface.
• the present invention relates to an improved process for manufacturing a dental care product comprising carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface which requires low investment and operating costs.
• a first process for manufacturing a dental care product selected from the group consisting of: toothpaste, tooth powder, chewing gum, ointment for the gums, mouthwash and mouth bath concentrate and gargle, according to the invention is defined in attached claim 12 and comprises the steps of:
• this process advantageously allows to readily incorporate the particles in the dental care product in a quite simple and convenient manner exploiting the useful properties, in particular stability and pH characteristics, of the suspension of particles produced in accordance with the invention.
• the process for manufacturing a dental care product of the invention does not require any separation or drying of the particles, with a notable reduction of the manufacturing plant complexity, of the related investment and operating costs, of product losses during the manufacture and of production rejects.
• the mixing step of the aqueous suspension of particles with other ingredients of the dental care product may be carried out with a better temperature control since the aqueous suspension reduces the friction and helps in removing the heat generated in the mixing apparatus.
• An alternative second process for manufacturing a dental care product selected from the group consisting of: toothpaste, tooth powder, chewing gum, ointment for the gums, mouthwash and mouth bath concentrate and gargle, according to the invention is defined in attached claim 13 and comprises the steps of:
• This alternative process allows to manufacture a dental care product in all those instances in which the use of the above-described suspension of particles may not be desirable for logistic or other reasons.
• step a) comprises the steps of:
• step c ⁇ di) adding lactoferrin to the aqueous suspension of step c ⁇ );
• step d ⁇ agitating a suspension of particles obtained from step d ⁇ ) over a time comprised between 1 and 48 hours at a temperature comprised between 10°C and 60°C.
• these steps allow to prepare in a fairly quick and economical way a suspension of carbonate-substituted strontium-hydroxyapatite particles having a lactofemn-functionalized surface, which may be readily used as such as a composition for oral hygiene or used in admixture with other ingredients to yield dental care products for oral hygiene.
• these steps allow to prepare a suspension of carbonate-substituted strontium-hydroxyapatite particles which is stable for an extended period of time even if no stabilizing agents are added thereto.
• the suspension thus prepared is stable for at least 30 days and, more generally, for about two-three months, without using any stabilizing agent.
• step ci) is preferably carried out in order to achieve an aqueous suspension having a pH comprised between 6 and 13.
• the aforementioned step aj) of preparing an aqueous solution or suspension comprising a Ca compound may be carried out in any conventional manner, such as by dissolving or suspending the Ca compound in water.
• the Ca compound is a calcium salt selected from the group comprising: calcium hydroxide, calcium carbonate, calcium acetate, calcium oxalate, calcium nitrate, and mixtures thereof. In this way, the cost of the process may advantageously be reduced since these Ca compounds are commodities readily available from the marked at a very low cost.
• the aforementioned Sr compound is a strontium salt selected from the group comprising: strontium carbonate, strontium oxide, and strontium hydroxide, and mixtures thereof.
• the aqueous solution or suspension of step bi) may further comprise a metal M compound.
• the aforementioned M compound is a metal salt selected from the group comprising: Mg compound, Se compound, K compound and mixtures thereof.
• the aforementioned Mg compound is a magnesium salt selected from the group comprising: magnesium carbonate, magnesium oxide, and magnesium hydroxide, magnesium acetate, and mixtures thereof.
• the aforementioned Se compound is a selenium salt selected from the group comprising: selenium dioxide, selenium trioxide, selenious acid, selenic acid, and mixtures thereof.
• the aforementioned K compound is a potassium salt selected from the group comprising: potassium carbonate, potassium oxide, potassium hydroxide, potassium nitrate, potassium hydrogen carbonate, potassium acetate, and mixtures thereof.
• the carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface are formed in step c ⁇ ) by adding P0 4 3" agitating this solution or suspension in order to capture the carbon dioxide present in the atmosphere and achieve the desired carbonate substitution at the phosphate site (B) of the hydroxyapatite compound being formed.
• the carbonate substitution may be advantageously carried out by simply agitating the solution or suspension for example by means of a mechanical stirrer.
• the required agitation of the solution or suspension may be achieved by bubbling air, a C0 2 " containing gas or a mixture thereof into the liquid phase or by combining a mechanical stirring with a gas bubbling.
• step ci) is carried out by adding, preferably dropwise, an aqueous solution including P0 4 " ions to the aqueous solution or suspension of step aj) and bi).
• the aqueous solution including P0 4 3" ions added in step ci) may further comprise HC0 3 " ions.
• the aforementioned aqueous solution including HC0 3 " , P0 4 " ions may be prepared by bubbling air, C0 2 or a mixture thereof through water to obtain a solution of carbonic acid and then adding H 3 P0 4 .
• step ci) may be carried out by simultaneously adding a first solution containing C0 3 " ions and a second solution containing P0 4 " ions to the aqueous solution or suspension of step aj) and bj).
• lactoferrin can be added during the step dj) after the formation of hydroxyapatite nanocrystals. In this way is possible to obtained a hydroxyapatite nanocrystals superficial functionalization by milk protein.
• lactoferrin in the aqueous suspension comprising a Ca compound, Ca carbonate and Sr compound. In this way, it may be possible to obtain to the desired functionalization of the hydroxyapatite compound formed.
• the aforementioned step a) comprises the steps of: a 2 ) preparing an aqueous solution or suspension comprising a Ca compound, Ca carbonate;
• step a 2 adding a Sr compound and lactoferrin to the aqueous solution or suspension of step a 2 );
• step d 2 agitating a suspension of particles obtained from step c 2 ) over a time comprised between 1 and 48 hours at a temperature comprised between 10°C and 60°C.
• the aqueous solution or suspension of step b 2 may further comprise a metal M compound.
• Suitable amounts of the ingredients may be easily selected by those skilled in the art so as to obtain the desired chemical constitution of carbonate-substituted strontium- hydroxyapatite solid particles having a lactoferrin-functionalized surface.
• the aforementioned step a') comprises the steps of:
• the separation step b 3 ) is carried out by decantation, centrifugation or filtration using apparatuses and techniques well known to those skilled in the art.
• the drying step c 3 ) is carried out by freezing the wet solid particles at a temperature lower than 0°C until reaching a constant weight.
• the drying step c 3 ) is preferably carried out by freeze-drying the wet solid particles at a temperature comprised between -20° and -50°C, most preferably at about -40°C.
• the process may also comprise the additional step d 3 ) of washing the separated solid particles with water or a basic solution prior to effecting the drying step c 3 ).
• this additional washing step d 3 serves the useful function of removing any acid residues possibly absorbed or trapped by the particles.
• the mixing step b) and b') is carried out in a mixing apparatus maintained under a predetermined vacuum degree, easily selectable by those skilled in the art in order to obtain a uniform mixture of ingredients, reached by using conventional vacuum pumps.
• the mixing step b) is carried out by
• step b 1 mixing the aqueous suspension of step a) with other ingredients of the toothpaste except for any surfactant;
• the incorporation step b 2 is preferably carried out under vacuum using a conventional equipment in order to minimize the undesired formation of foam.
• the present invention relates to a method of providing at a teeth outer surface a source of Sr* " ions and lactoferrin which may be locally released at an acidic pH by means of hydroxyapatite-type carrier particles, the method comprising contacting the teeth with a dental care product as described herein so as to form on the teeth outer surface a film including said carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface.
• the contacting step may be carried out in a number of ways depending upon the dental care product. For example, if the dental care product is a toothpaste, the contacting step may be simply carried out by washing the teeth, while if the dental care product is a mouthwash, the contacting step is carried out by maintaining the mouthwash in the oral cavity for a suitable time, for example few minutes.
• the carbonate- substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface of the invention have a triple effect on tooth surface.
• the dental care products of the invention are advantageously capable of restoring the enamel surface because the particles of the invention bind to the enamel surfaces, mimicking the natural bone hidroxyapatite, at any erosion area due for example, to the acid foods and drinks as will be shown in greater detail in the Examples below.
• the dental care products of the invention comprising strontium ions into the structure of hydroxyapatite functionalized by protein, advantageously form a thin biomimetic coating on the outer surface of the enamel even during the usual tooth brushing.
• the hydroxyapatite functionalized by lactoferrin forming the thin coating present on the outer surface of the enamel is capable of effectively exploiting an antibacterial effect and therefore, this protein protects the tooth surface from attack by bacteria and plaque.
• the Applicant has surprisingly and unexpectedly found that said strontium hydroxyapatite functionalized by lactoferrin shows an increased antibacterial effect with respect to the lactoferrin alone.
• the plaque, the acid food and drink partially dissolve this coating, releasing strontium ions that only in this moment are free in the mouth and are present in the physiologic percentage in which these ions are present in the tooth hydroxyapatite.
• Said free ions showing a cariostatic effect, are capable of binding with the tooth hydroxyapatite forming in situ a strontium hydroxyapatite.
• said strontium hydroxyapatite functionalized by lactoferrin presents a greater degree of crystallinity with respect to a zinc carbonate-substituted hydroxyapatite without strontium and lactoferrin.
• this hydroxyapatite having higher degree of crystallinity, is more resistant to the plaque formation and to the action of food and acid drinks.
• the dissolution of the hydroxyapatite biomimetic coating in acid conditions determine the release of lactoferrin protein capable of effectively exploiting also an antibacterial effect and, accordingly, of effectively treating teeth and gums disorders and in general increasing oral hygiene even in the limited time available during the normal routine of dental hygiene.
• lactoferrin is capable of exploiting its antibacterial effect for a long time after the normal routine of dental hygiene.
• Fig. 1 shows an X-Ray diffraction pattern of comparative zinc carbonate- substituted hydroxyapatite particles (Zn-CHA), without strontium substitution and lactoferrin functionalization, which displays a cristallinity degree CD of 60%
• Fig. 2 shows an X-Ray diffraction pattern of one example of carbonate- substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface (LF-SrCHA) according to the invention (Example 2), with molar % of strontium substitution comprised between 0.0010 and 0.015, which displays a cristallinity degree CD of 67%;
• Fig. 3 shows an X-Ray diffraction pattern of one example of carbonate- substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface (LF-SrCHA) according to the invention (Example 3), with the aforementioned molar % of strontium, which displays a cristallinity degree CD of 66% ;
• LF-SrCHA lactoferrin-functionalized surface
• Fig. 4 shows a FTIR spectrum of one example of carbonate-substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface, according to the invention, which displays an apatitic phase;
• Figs. 5a and 5b show TEM and SEM images of some examples of carbonate- substituted strontium-hydroxyapatite particles having a lactoferrin-functionalized surface according to the invention which display the micrometric size and a flat and round shape of the single nanoparticles;
• Figs. 6a and 6b show respectively SEM image (6a) and EDX spectrum (6b) of enamel brushed with a toothpaste containing LF-SrCHA according to the invention (in vitro test).
• Figs. 7a and 7b show respectively SEM image and EDX spectrum of enamel brushed with a common toothpaste.
• aqueous suspension of comparative zinc carbonate-substituted hydroxyapatite particles was prepared according to the following method. In a first step, 550 g of water was mixed with 55 g CaC0 3i , 22g of ZnC0 3 and 175 g Ca(OH) 2 .
• the pH was maintained between 3 and 8, more preferably between 3 and 5.
• the resulting aqueous suspension was maintained under agitation between 0.5 and 9 hours, more preferably between 1.5 and 4.5 hours.
• the resulting suspension was brought between a temperature of 23° and 45°C, more preferably between 27° and 38°C.
• the suspension was maintained under agitation between 1 and 12 hours, more preferably between 3 and 7 hours.
• the final pH of suspension is maintained between 6 and 13, more preferably between 7 and 12.
• 550 g of water was mixed with 77 g CaC0 3 , 10 g SrC0 3 and 175 g Ca(OH) 2 .
• the pH was maintained between 3 and 8, more preferably between 3 and 5.
• the resulting aqueous suspension was maintained under agitation between 0.5 and 9 hours, more preferably between 1.5 and 4.5 hours. During this step, the resulting suspension was brought between a temperature of 23° and 45°C, more preferably between 27° and 38°C.
• the suspension was maintained under agitation between 1 and 1 1 hours, more preferably between 2 and 7 hours.
• the final pH of suspension is maintained between 6 and 13, more preferably between 7 and 12.
• a first step 540 g of water was mixed with 70 g CaC0 3i 5 g SrC0 3 and 168 g Ca(OH) 2 .
• the pH was maintained between 3 and 8, more preferably between 3 and 5.
• the resulting aqueous suspension was maintained under agitation between 0.5 and 8 hours, more preferably between 1 and 4 hours.
• the resulting suspension was brought between a temperature of 23° and 40°C, more preferably between 27° and 35°C.
• the suspension was maintained under agitation between 1 and 12 hours, more preferably between 3 and 9 hours.
• the final pH of suspension is maintained between 6 and 13, more preferably between 7 and 12.
• this hydroxyapatite having higher degree of crystallinity, is more resistant to the plaque formation and to the action of food and acid drinks.
• a toothpaste including carbonate-substituted hydroxyapatite particles having a lactoferrin-functionalized surface according to the invention was prepared according to the following method and from the following ingredients.
• an aqueous suspension including carbonate-substituted strontium- hydroxyapatite particles having a lactoferrin-functionalized surface (LF-SrCHA) (total solid content: 30% by weight) was prepared in the same manner and using the same ingredients and quantities described in Example 2.
• the aqueous suspension thus obtained was then mixed with the other ingredients of the toothpaste as shown in the table below except for the surfactant.
• the mixing was carried out in a conventional mixing apparatus maintained under a suitable vacuum degree selected among the usual values known to those skilled in the art.
• the surfactant was incorporated in the mixing apparatus while maintaining a predetermined vacuum degree selected among the usual values known to those skilled in the art.
• Slabs of enamel (3x3mm) were obtained from interproximal surfaces of premolars extracted for orthodontic reasons. After the extraction, the teeth were cut with diamond disks and the slabs obtained were sonicated for 10 min in 50% by weight of ethanol in order to removed any debris. The slabs were etched with 37% by weight of orthophosphoric acid for 1 min, then repeatedly washed with distilled water using an electric toothbrush and air dried. The test was then carried out by treating various slabs of enamel with a common toothpaste and an toothpaste containing 10% wt of the active agent LF-SrCHA according to the present invention.
• Each enamel slab was brushed three times a day for a period of 21 days.
• the intervals between brushing sessions were more than 5 hours. Any washing process has been performed for 30 sec using an electric toothbrush submitted at constant pressure and using a bean sized aliquot of toothpaste wetted with tap water, closely resembling the in vivo usual tooth-brushing procedure.
• the single enamel slab was washed with tap water using a cleaned tooth-brush in order to remove residual toothpaste. Toothbrushes were repeatedly washed with tap water after every utilization.
• each enamel slab After the brushing treatment 21 days long each enamel slab have been characterized by X-Ray diffraction technique (DRX) , Scanning Electro microscopy (SEM) with EDX probe and Infrared Fourier Transformed Spectroscopy (FTIR).
• DRX X-Ray diffraction technique
• SEM Scanning Electro microscopy
• FTIR Infrared Fourier Transformed Spectroscopy
• the Ca / P molar ratio of 1.9 is the characteristic value of natural enamel.
• Figures 6a and 7a show the presence of a crack on the enamel surface normally due to acid foods and drinks.
• the dental care products of the invention are capable of restoring the enamel surface because the particles of the invention bind to the enamel surfaces, mimicking the natural bone hydroxyapatite, at any erosion area.
• a mouthwash including LF-SrCHA particles according to the invention was prepared by mixing a suspension produced in accordance with the preceding Example 2 in a conventional way with conventional ingredients.
• a mouthwash was obtained having the composition reported in the following Table 2.
• a chewing gum including LF-SrCHA particles according to the invention was prepared by mixing a suspension produced in accordance with the preceding Example 2 in a conventional way with conventional ingredients.
• Streptococcus Mutans is considered one of the most important cariogenic species of the human oral microbial flora.
• Antimicrobial activity was determined by the agar diffusion method and the zones of growth inhibition were measured.
• test product 1.8 ml of the test product were inoculated with 200 ⁇ of Streptococcus Mutans bacterial suspension of known concentration (2xl0 5 CFU, colony-forming units/ml).
• Streptococcus Mutans ATCC 35668 was also inoculated in equal volume of sterile water as a control. After the contact time of 1 hour, an appropriately diluted aliquot was taken from each treated sample and then sown on the steril agar plates to determine the number of live microorganisms expressed as CFU/ml.
• the inoculated plates were incubated at 37 °C for 24-48 h and then bacterial colonies were counted.
• Table 4 shows that lactoferrin functionalization into substituted hydroxyapatite advantageously increases the antibacterial activity (85% of microorganisms reduction) with respect to the comparative lactoferrin which displays a microorganisms reduction of 35%.

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