IT202100010802A1 - SOLID MUCOADHESIVE FORMULATION INCLUDING PROBIOTICS FOR USE IN THE PREVENTION AND TREATMENT OF ORAL DYSBIOSIS. - Google Patents

Description

Description of the patent for an invention entitled:

?MUCOADHESIVE SOLID FORMULATION INCLUDING PROBIOTICS FOR USE IN THE PREVENTION AND TREATMENT OF ORAL DYSBIOSIS?

State of art

Compositions useful for the release of active principles in a controlled way in the sites of action are described in the literature in order to ensure greater efficacy in the site of interest and a reduction of systemic effects.

Pathologies and disorders affecting the oral mucosa are due to colonization of pathogenic bacteria in the mouth (oral dysbiosis) which can lead to dental caries, periodontal problems, peri-implantitis, mucositis and bad breath (Badet C. et al., Open Microbiol J. 2008; 2:38-48).

Periodontal problems, also called periodontitis, are associated with the growth of bacterial species such as Porphyromonas gingivalis, Treponema denticola, Prevotella denticola, Tannerella forsythia, Aggregatibacter actynomycetecomitans and Fusobacterium nucleatum which together form the red complex (Rinkee Mohanty, et al. al J Family Med Prim Care 2019, 8: 3480?3486).

WO 2012/022773A1 discloses an oral composition, in the form of orodispersible tablets and toothpaste, comprising Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480, useful for the reduction of pathogens in the oral cavity. Oral. WO 2018/169296 describes a medical device in the form of a mucoadhesive film useful for the release of probiotics. The film ? obtained with acrylate polymers, polymers derived from cellulose, or polyethylene glycols, xanthan gum, hydroxypropyl methylcellulose and propylene glycol, vinyl polymers, glucans. The mucoadhesive agents are selected from polyethylene oxide, glycerol, glycerol monoacetate, propylene glycol, triacetin, PEG, polysorbate, cetyl alcohol (1-hexadecanol). The film ? characterized by the ability to release probiotics in the cavity? oral, in a controlled way.

CN 107865891 discloses compositions in the form of gels or buccal tablets containing L. acidophilus, S. salivarius K12, S. salivarius M18. The gel can be prepared with a polymeric matrix selected from an acrylate, polyoxyethylene, hydroxypropylcellulose, hydroxypropylmethylcellulose and chitosan polymer; a bioadhesive agent, selected from sodium hyaluronate, hydroxypropyl methylcellulose, sodium alginate; a humectant selected from glycerol, propylene glycol, polyethylene glycol and sorbitol. The preparation of the gel ? described using only an acrylic polymer (Carbomer); triethanolamine ? used to bring the pH to values greater than 4 and the probiotic? dispersed in the gel obtained.

The bacterial biofilm that forms in the dental plaque and in the mucous membranes surrounding the tooth? difficult to remove with daily cleaning of the teeth, it has a pathogenic action on the health of the mouth and for this reason its formation should be prevented.

CN 110404010A discloses a gel comprising probiotics and plant extracts selected from acnebusters hazelnut extract, activonol, aloe extract, ocaline and menthol oil for use in the treatment of inflammation of the skin cavity. Oral.

CN 107080731A describes a process comprising four steps for the production of a castor oil-based dental gel.

US 2017232048A1 discloses a formulation for the treatment of disorders of the cavity oral in the form of tablets, which, when dissolved, form a film that adheres to the mucous membrane. The formulation includes a prebiotic, selected from: inulin, red Acacia, Konjac paste, xanthan gum and a probiotic together with sodium alginate, glycerol dibehenate and calcium phosphate.

WO 2020/106591 describes a medical device in the form of a strip, consisting of pullulan and a probiotic, which, adhering to the oral mucosa, releases probiotics.

WO 2017/25447 describes the use of a mixture of probiotics, L. paracasei, L. plantarum and L. delbrueckii subsp. lactis for the treatment of inflammation of the cavity? Oral. WO 2017/125453 describes the use of probiotics L. paracasei LPc-G110, in compositions in the form of emulsions, tablets and granules, for the treatment and prevention of periodontal pathologies.

WO 2018/165576 describes a composition comprising L. rhamnosus LB20, Bifidobacterium brevis SP46 and L. acidophilus RP 32 in lyophilic form, in capsules, for the prevention of oral bacterial biofilm in humans.

Probiotics are widely used to prevent and treat bacterial infections in humans and animals, but there is still a need for effective formulations that are well accepted by subjects, capable of releasing probiotics in a controlled manner over time.

Does the person skilled in the art know the complexity? of the release of probiotics from solid or gel compositions, that ? influenced by many factors such as the properties? and the concentrations of the polymers used, the pH, the contact time and the capacity? of the polymers to hydrate without collapsing.

(Int. J. Pharm. 2010, 389, 24) describes wafer and film preparations of alginate from 1 to 5% by weight, with carboxymethylcellulose (CMC) from 0.5 to 3% by weight, comprising paracetamol in lyophilic form .

(Marine Drugs 2019, 17, 112) describes an in situ-forming gel formulation consisting of carrageenan, hydroxypropyl cellulose and calcium, potassium or sodium salts which can be used in the cavity Oral.

There is still a need to have alternative formulations with controlled release capable of releasing probiotics, useful for the treatment and/or prevention of pathologies and discomforts related to inflammation of the cavity. Oral.

? been found, and ? object of the invention, a solid mucoadhesive formulation obtained by drying a solution comprising probiotics and natural agents, in which the probiotics are released in a controlled manner into the oral mucosa.

The wording? stable at room temperature, ? well accepted and ? prepared with an easily scalable process for industrial preparations.

The wording? useful for releasing probiotics, but also selected compounds among antibiotics, antiseptics, antifungals, anti-inflammatory and/or soothing agents, which can be included in the formulation, for use in the treatment/prevention of disorders and pathologies of the cavity Oral.

Summary of the invention

A mucoadhesive formulation obtained by a process of drying a solution comprising a natural thickening/viscosifying agent at a concentration of 0.1 to 5.0% (w/w), a natural mucoadhesive agent at a concentration of 0.1 to 2 % (w/w) and a calcium salt at a concentration of 0.02 to 0.1% (w/w), useful for the controlled release of probiotics.

The formulation obtained by drying a solution comprising alginate at a concentration of 0.5 to 1% (w/w), xyloglucan at 0.2 to 0.5% (w/w), calcium chloride at a concentration of 0.02 to 0.1% (w/w) and glycerol at a concentration of 1 to 2% (w/w) with respect to the finished formulation.

The composition comprising probiotics, selected from the group comprising Lactobacillus salivarus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus animalis, Lactobacillus reuteri, Bifidobacterium longum, Bifidobacterium infantis, and mixtures thereof in an amount from 1x10<8>-1x10<13 >(CFU/100g).

A process for preparing a mucoadhesive formulation comprises the steps of:

a) adding to an aqueous solution a natural thickening agent at a concentration of 0.5 to 1.0% (w/w), a bioadhesive agent (mucoadhesive) at a concentration of 0.1 to 1.0% (w/w p), and glycerol from 1.0 to 2.0% with respect to the final solution;

b) add a probiotic chosen from lactobacilli or bifidobacteria or mixtures thereof in a quantity? from 1x10<8>-1x10<13 >(CFU/100g);

c) drying the partitioned aliquots of the solution or the whole solution and then partitioning the solid.

The process for the preparation of the mucoadhesive formulation including the steps of:

a) add an alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 1.0% (w/w), and calcium chloride at a concentration of 0.02 to 0.1%, to an aqueous solution of maltodextrin at a concentration of 6.0% to 10.0% (w/w);

b) add to the homogeneous solution a probiotic selected from among Lactobacillus salivarus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus animalis, Lactobacillus reuteri, and bifidobacteria, selected from the group comprising Bifidobacterium longum, Bifidobacterium infantis, and mixtures thereof in a quantity ? from 1x10<8>-1x10<13 >(CFU/100g); c) drying by freeze-drying or spray-drying or wet granulation the divided aliquots of the solution or the solution as a whole and then dividing the solid. A mucoadhesive formulation obtained by a drying process of a solution comprising a natural thickening/viscosifying agent, at a concentration of 0.1 to 5.0% (w/w); a natural mucoadhesive agent, at a concentration of 0.1 to 2.0% (w/w); a calcium salt, at a concentration of 0.02 to 0.1% (w/w); and glycerol, 1.0 to 2.0% (w/w), and probiotics in an amount from 1x10<8>-1x10<13 >CFU/100g, for use in the treatment and/or prevention of pathologies and/or disorders of the cavity Oral.

Detailed description of the invention

The present invention refers to a mucoadhesive formulation in solid form comprising probiotics useful for the prevention and/or treatment of disorders related to inflammation of the cavity. oral and under conditions of oral dysbiosis obtained through drying processes.

The mucoadhesive formulation? characterized by the ability to adhere to the oral mucosa, hydrate and release the probiotics at the site in a controlled way.

The formulation can be a medical device.

The wording? obtained through a drying process of a solution characterized by comprising a natural thickening/viscosifying agent, at a concentration of 0.1 to 5.0% (w/w), a natural mucoadhesive agent, at a concentration of 0.1 to 2% (w/w), and a calcium salt at a concentration of 0.02 to 0.1% (w/w) and probiotics in an amount? from 1x10<8>-1x10<13 >CFU/100g.

The natural thickening/viscosifying agent of natural origin ? selected from the group comprising guar flour, gum tragacanth, corn starch, wheat starch, potato starch, rice starch, gelatin, isinglass, guar gum, xanthan gum, Kuzu root, agar-agar, sodium alginate , potassium alginate, ammonium alginate, propylene glycol alginate, acacia gum or gum arabic, karaya gum, tara gum, sorbitol, mannitol, sucrose acetate isobutyrate, cellulose derivatives and mixtures thereof.

The natural bioadhesive (mucoadhesive) agent? selected from the group comprising propolis, pectins, zeins, casein, gelatin, albumin, collagen, chitosan, oligosaccharides and polysaccharides such as cellulose, dextran, polysaccharides from tamarind seeds, xanthan gum, gum arabic, hyaluronic acid and mixtures thereof.

The preferred natural thickening/viscosifying agent according to the invention is? selected from the group comprising alginic acid, sodium alginate, calcium alginate and potassium alginate. The term alginate refers to the anionic polymer, a polysaccharide widely present in the cell walls of brown algae. The alginate ? a linear copolymer containing blocks of (1,4) ?-mannuronate (M) conjugated with ?-L-guluronate (G). Conjugated or alternating M and G residues form the alginate polymers, and G residues are responsible for hydrogel formation when binding to potassium, calcium, or sodium cations.

The natural bioadhesive/mucoadhesive agent according to the invention is xyloglucan from tamarind seeds, prepared as described in EP 1898876. By xyloglucan is meant a class of polysaccharides structurally related to cellulose and intimately associated with it in the cell wall of higher plants. They also constitute one of the main components, probably with an energy reserve function, of seeds of plants such as Tamarindus indica, native to India and South-East Asia, Detarium senegalense and Afzelia africana, widespread both in central and eastern Africa, and Hymenaea courbaril widespread in Central and South America. Xyloglucans are characterized by a (1,4)-?-D-glucan backbone substituted with ?-D-xylopyranose and ?-D-galactopyranosyl-(1,2)-?-D-xylopyranose side chains ?-(1,6) binding to glucan residues. The distribution of residues in the side chains ? different in xyloglucans of different species.

? it has been surprisingly found that a solid formulation obtained by freeze-drying or drying an aqueous solution comprising alginate at a concentration of 0.5 to 2% (w/w), xyloglucan at a concentration of 0.2% to 1% (w/w ) and a calcium salt at a concentration of 0.03 to 0.1% (w/w), releases the active compounds in a controlled way, such as probiotics for example.

Favorite calcium salt? the calcium chloride, in a weight ratio with the alginate from 1:10 to 1:20.

The solid formulations are characterized by being obtained through a drying process of a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan of 0.25 to 1.0% (w/ p) with calcium chloride at a concentration of 0.025 to 0.1% (w/w) with respect to the finished formulation and probiotics in an amount? from 1x10<8>-1x10<13>CFU/100g. [A+B]

In a particular aspect, the solid formulations are characterized by being obtained through a drying process of a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan of 0.25 to 1, 0% (w/w), calcium chloride at a concentration of 0.02 to 0.1% (w/w), compared to the finished formulation and probiotics in an amount from 1x10<8>-1x10<13 >CFU/100g. [TO]

The formulations are characterized by being obtained through a drying process of a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 0.5% ( w/w), calcium chloride at a concentration of 0.025 to 0.1% (w/w) with respect to the finished formulation and probiotics in an amount from 1x10<8>-1x10<13 >CFU/100g. [B1-B9]

In another aspect, the formulations are characterized by being obtained through a process of drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 0.5% (w/w), calcium chloride at a concentration of 0.020 to 0.10% (w/w), and glycerol at a concentration of 1.0 to 2.0% (w/w), compared to the finished formulation and probiotics in a quantity? from 1x10<8>-1x10<13 >CFU/100g. [B10-B17]

The formulations are characterized by being obtained through a drying process of a solution comprising alginate at a concentration of 0.5 to 1% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration from 0.02 to 0.05% (w/w), and glycerol at a concentration from 1 to 2% (w/w), compared to the finished formulation and probiotics in a quantity? from 1x10<8>-1x10<13>CFU/100g.

In another aspect, the formulations are characterized by being obtained through a process of drying a solution comprising alginate at a concentration of 0.5 to 1% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration of 0.02 to 0.05% (w/w), and glycerol 2% (w/w), compared to the finished formulation and probiotics in an amount from 1x108-1x1013 CFU/100g . [B10-B17]

In a particular aspect, the formulations are characterized by being obtained through a drying process of a solution comprising alginate at a concentration of 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration of 0.05% (w/w) and glycerol 2.0% (w/w), compared to the finished formulation and probiotics in a quantity? from 1x10<8>-1x10<13 >CFU/100g. [B13]

In a particular aspect, the formulations are characterized by being obtained through a drying process of an alginate solution at a concentration of 0.5% (w/w), 0.25% (w/w) xyloglucan, calcium chloride at a concentration of 0.05% (w/w), and glycerol 2.0% (w/w), compared to the finished formulation and probiotics in a quantity? from 1x10<8>-1x10<13 >CFU/100g. [B15]

The drying processes are chosen from freeze-drying and spray-drying, wet granulation and fluid bed drying.

Solutions useful to be dried include alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at 0.25 to 1.0% (w/w), calcium chloride at a concentration of 0.025 at 0.1% (w / w), compared to the finished solution, are characterized by viscosity values? from 20 to 0.2 Pa?sec when applied a share rate from 10 to 300 sec<-1>.

Solutions useful for drying include alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.25% (w/w), calcium chloride at a concentration of 0.037 at 0.05% (w/w) and glycerol at a concentration of 1.0 to 2.0% (w/w), with respect to the finished solution, are characterized by viscosity values? from 6 to 0.1 Pa?sec when subjected to a share rate from 10 to 300 sec<-1>. [B10-B17].

The solutions comprising glycerol at concentrations from 1.0 to 2.0% (w/w) are characterized by viscosity values? smaller than those without; in particular the solutions with glycerol at concentrations greater than 1.0% (w/w) have a lower viscosity.

The solid formulations of the invention are characterized by particular properties? mechanisms that make them suitable for including active ingredients and releasing them in the site of interest.

Solid formulations comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.25% (w/w), calcium chloride at a concentration of 0.037 to 0, 05 % (w/w), and glycerol at a concentration of 1.0 to 2.0% (w/w), are characterized by a strain force of 4500 to 16000 mN and a strain work of 4000 to 10000 mN?mm, compared to glycerol-free formulations, which are characterized by a strain force from 500 to 10000 mN and a strain work from 500 to 15000 mN?mm.

Glycerol-containing formulations exhibit higher strain forces than glycerol-free frameworks demonstrating that the glycerol-containing framework resists higher strain forces without permanently changing its appearance. The solid formulations obtained by drying solutions containing glycerol at concentrations from 1.0 to 2.0% are characterized by greater elasticity compared to glycerol-free compositions.

The formulations characterized by being obtained by drying from a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.25% (w/w), calcium chloride at a concentration of 0.02 to 0.05% (w/w), and glycerol of 1.0 to 2.0% (w/w), with respect to the finished formulation, are characterized by absorbing an aqueous solution in a percentage by weight from 250 to 550% of the weight before hydration, in a time of three to eight hours, in which such absorption is constant in all formulations. [B10Lio-B17Lio]

The glycerol-free formulations are characterized by absorbing an aqueous solution in a percentage by weight from 120 to 550% in a time of three to eight hours, but the absorption is not achieved. constant; with small variations of one of the components, the absorption changes disproportionately.

The formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration of 0.02 to 0.05% (w/w), and glycerol at a concentration of 1.0 to 2.0% (w/w), compared to the finished formulation (B10Lio-B17Lio), are characterized by a more? homogeneous compared to the compositions without glycerol and the absorption remains in the range of 250-500%. [B10Lio-B17Lio]

Furthermore, the formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration of 0, 02 at 0.05% (w/w), and glycerol at a concentration of 1.0 to 2.0% (w/w), compared to the finished formulation, when absorbing an aqueous solution, in a time of four to eight hours, do not collapse the solid structure. [B10Lio-B17Lio]

The formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration between 0.02 and 0.05% (w/w), and glycerol from 1.0 to 2.0% (w/w), with respect to the finished formulation (B10-lio-B17-lio), are characterized by retaining a polymeric structure with an adequate consistency to keep them on the application site, without collapse of the solid structure. [B10Lio-B17Lio]

The formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration of 0.020 to 0, 05% (w/w), and 1.0 to 2.0% (w/w) glycerol, relative to the finished formulation (B10-lio-B17-lio) may comprise active compounds to be released at the site of interest, as for example in the oral or vaginal mucous membranes. [B10Lio-B17Lio]

The active compounds which can be included in the formulations of the invention are selected from the group comprising antibiotics, anti-inflammatories, disinfectants, antimicrobials, soothing, healing, probiotics.

In a particular aspect, the formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration from 0.02 to 0.05% (w/w), and glycerol from 1.0 to 2.0% (w/w), with respect to the finished formulation include probiotics, selected from the group comprising lactobacilli or bifidobacteria in an amount? from 1x10<8>-1x10<13 >CFU/100g. [B10Lio-B17Lio]

The formulations of the invention obtained through drying processes of a solution comprising alginate at a concentration of from 0.5 to 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride at a concentration from 0.02 to 0.05% (w/w), and glycerol from 1.0 to 2.0% (w/w) with respect to the finished formulation (B10Lio-B17Lio) together with probiotics selected from the group comprising Lactobacillius salivarus, Lactobacillius brevis, Lactobacillius helveticus, Lactobacillius paracasei, Lactobacillius rhamnosus, Lactobacillius animalis, Lactobacillius reuteri, and bifidobacteria, selected from the group comprising Bifidobacterium longum, Bifidobacterium infantis, and mixtures thereof in a quantity from 1x10<8>-1x10<13 >(CFU/100g).

The formulations obtained by drying a solution comprising alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 0.5% (w/w), calcium chloride at a concentration of 0.02 to 0.10% (w/w), and glycerol at a concentration of 1.0 to 2.0% (w/w), compared to the finished formulation and probiotics in an amount? from 1x10<8>-1x10<13>CFU/100g, are characterized by the capacity? to release the loaded bacteria for at least eight hours with a percentage of vitality? greater than 90%.

Another object of the invention? a process for the preparation of a mucoadhesive formulation, for the delivery of probiotics comprising the steps of:

a) add to an aqueous solution a natural thickening agent at a concentration of 0.5 to 1.0% (w/w), a bioadhesive agent (mucoadhesive) at a concentration of 0.1 to 1.0% (w/w ), and glycerol from 1.0 to 2.0% with respect to the final solution;

b) add a probiotic chosen from lactobacilli or bifidobacteria or mixtures thereof in a quantity? from 1x10<8>-1x10<13 >(CFU/100g);

c) drying the partitioned aliquots of the solution or the whole solution and then partitioning the solid.

The process for the preparation of the mucoadhesive formulation for the controlled release of probiotics including the steps of:

a) add an alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 1.0% (w/w), and calcium chloride at a concentration of 0.02 to 0.1%, to an aqueous solution of cryoprotectant at a concentration of 6.0% to 10.0% (w/w);

b) add to the homogeneous solution a probiotic selected from among Lactobacillus salivarus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus animalis, Lactobacillus reuteri, and bifidobacteria, selected from the group comprising Bifidobacterium longum, Bifidobacterium infantis, and mixtures thereof in a quantity ? from 1x10<8>-1x10<13 >(CFU/100g); c) drying by freeze-drying or spray-drying or wet granulation the divided aliquots of the solution or the solution as a whole and then dividing the solid.

In a particular aspect, the process for preparing the mucoadhesive formulation for the controlled release of probiotics comprises the steps of:

- add alginate at a concentration of 0.5 to 1% (w/w), xyloglucan at a concentration of 0.2 to 1% (w/w) and calcium chloride at a concentration of 0.02 to 0.1 %, to an aqueous solution of maltodextrins at a concentration of 6% to 10.0% (w/w),

- add to the homogeneous solution a probiotic selected from among Lactobacillus salivarus, Lactobacillusbrevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus animalis, Lactobacillus reuteri, and bifidobacteria, selected from the group comprising Bifidobacterium longum, Bifidobacterium infantis, and their mixtures in a quantity from 1x10<8 >-1x10<13 >(CFU/100g), - divide the solid into single or multi-dose applicators, dry by freeze-drying;

In the freeze-drying process, the cryoprotectant ? selected from sucrose, glycine, maltrodextrin, trehalose, mannitol and mixtures thereof.

The formulation including probiotics ? useful for the treatment and/or prevention of disorders associated with inflammatory states of the mucous membranes, such as the oral mucosa.

The wording? useful for the release of probiotic microorganisms with activity? beneficial and can it can also be used in combination with antibiotics, chemotherapy, radiotherapy, immunosuppressants and be useful for alleviating problems and uncomfortable situations in patients receiving treatments that may give rise to adverse events affecting the mucosa.

The formulation including probiotics ? useful for the treatment and/or prevention of pathologies or disorders related to dysbiosis and inflammation of the cavity oral, such as, for example, gingivitis, periodontitis, stomatitis, mucositis, peri-implant mucositis, and caries.

The mucoadhesive formulation comprising probiotics, when placed in the oral mucosa, is characterized by being hydrated in 10 to 30 minutes, forming a structure that releases the probiotics in 10 minutes to at least eight hours, in which the probiotics maintain their vitality.

Does the mucoadhesive formulation object of the invention adhere to the mucous membranes of the cavity? oral by releasing the probiotics included in the oral mucosa and saliva.

The formulation can be in the form of a single-dose disk, lozenge or tablet to be placed in the cavity? oral, such as in the dental arch or over the teeth or between the teeth.

The unit dosage can understand a quantity of probiotics from 10<6 > to 1x10<11>, in a weight of 0.1 to 2 grams of the solid formulation.

Examples

Example 1 ? Preparation of solutions

The three groups of solutions were prepared: A1-A9; B1-B9; B10-B17.

Solutions A1-A9 and solutions B1-B9 were prepared by adding alginate (Giusto Faravelli-Milan, Italy), xyloglucan (Indena, Milan, Italy) and calcium chloride (Sigma Aldrich, Milan, Italy), at the reported percentage concentrations in Tables 1 and 2, to aqueous solutions of maltodextrin (Giusto Faravelli-Milan, Italy) at a concentration of 8% (w/w).

Solutions B10-B17 were prepared by adding alginate, xyloglucan and calcium chloride, at the percentage concentrations shown in Table 3, to aqueous solutions of maltodextrin at a concentration of 8% (w/w). To homogeneous solutions? glycerol was added (Carlo Erba, Milan, Italy) at the concentrations shown in Table 3.

Table 1 ? Solutions A1-A9.

Table 2 ? Solutions B1-B9.

Table 3 ? Solutions B10-B17.

Example 2 ? Determination of viscosity? of solutions A1-A9, B1-B17

The viscosity values? some solutions, prepared according to Example 1, were obtained using a rotational rheometer (MCR 102, Anton Paar, Turin, Italy) using a plate-cone measuring system (diameter = 50 mm, angle =1? ), at a temperature of 37?C, applying increasing flow gradients (from 10 to 300 s<-1>). Three replicas were performed for each solution. Tables 4A and 4B show the viscosity values? of solutions A1-A9; Tables 5A and 5B show the viscosity values? of the compositions solutions B1-B9; Tables 6A and 6B show the viscosity values? of the B10-B17 solutions.

Example 3 ? Freeze-drying process of solutions A1-A9, B1-B17

A quantity? equal to 2 grams of each solution A1-A9, B1-B9 and B10-B17, prepared according to Example 1, ? was placed in a well of a 12-well plate and lyophilized with a Heto Drier lyophilizer (Analitica De Mori, Milan, Italy), with the parameters:

- Freezing temperature: -80 ?C;

- Freezing time: 48 hours

- Primary drying: time 48 hours; Temperature < 0?C; Pressure = 4mmHg. - Secondary drying: time 12 hours; Temperature 25?C; Pressure = 4mmHg. The samples A1Lio - A7Lio, B1Lio - B9Lio and B10Lio - B17Lio were obtained.

Example 4 ? Determination of properties? mechanics of freeze-dryers A1Lio - A7Lio and B1Lio -B17Lio - Penetration resistance

The mechanical resistance to penetration of the lyophilis A1Lio - A7Lio and B1Lio - B17Lio, prepared according to Example 3, ? was determined with a structure analyzer, using the TA.XT plus Texture Analyzer equipment (Stable Micro Systems, Goldaming, UK) equipped with a 1 kg load cell and a P/10 measuring system, consisting of a cylindrical probe diameter 10mm. Before the analysis, each lyophile ? been placed on the instrument base below the probe. The measurement system? been lowered to a speed? of 1.00 mm/s for a distance of 1.5 mm in order to compress the sample. For each lyophile the measurement ? was done in triplicate.

Table 7 shows the strength and strain work values of A1Lio -A9Lio lyophilis.

Table 8 shows the force and strain work values of the B1Lio -B9Lio lyophiles.

Table 9 shows the force and deformation work values of the lyophilized B10Lio -B17Lio.

Table 7 ? Freeze dryers A1Lio - A9Lio.

Table 8 ? Freeze dryers B1Lio - B9Lio.

Table 9 ? Freeze-philic B10Lio - B17Lio

The freeze-dried B10Lio-B17Lio have a uniform and spongy structure.

Example 5 ? Capacity determination? d?hydration of the lyophiles of the lyophilic compositions A1Lio - A7Lio and B1Lio - B17Lio

The capacity? d?hydration of the lyophilic compositions A1Lio - A7Lio and B1Lio - B17Lio, prepared according to Example 3, ? was determined by the use of Franz diffusion cells (PermeGear, Bethlehem, Palestine) at 37 ?C.

A quantity? corresponding to about 200 mg of each lyophile ? was placed on a dialysis membrane placed between the donor and recipient chambers of a diffusion cell. The receiving room? was filled with a saliva-mimicking solution prepared in distilled water according to the following composition: KCl 0.15% w/v, NaCl 0.043% w/v, CaCl2 0.022% w/v, NaHCO3 0.042% w/v, NaH2PO40, 091% w/v. After 6 hours, the weight of the solution in the receiving chamber of each lyophile? been measured and the % of artificial saliva absorbed by each sample ? been calculated as a percentage (%) using the equation:

(%) artificial saliva absorbed = [(Wf-Wi)/Wi]?100 where: Wf ? the weight of the sample after hydration; wi? the weight of the dry sample. The measurement ? was carried out on each sample in triplicate.

Tables 10-12 show the values of dry weight, weight after 6 hours of hydration and percentages of absorbed artificial saliva of the lyophiles.

Table 10 ? A1Lio, A3Lio, A7Lio and A9Lio freeze dryers.

Table 11 ? Freeze dryers B1Lio - B9Lio

Table 12 ? Freeze-philic B11Lio, B13Lio, B15Lio and B17Lio

Example 6 ? Determination of properties? mechanics of lyophiles after hydration - Penetration resistance

The strength and strain work of the selected lyophilized samples were measured as described in Example 4, after a hydration time of 6 hours.

Tables 13-15 show the strength and deformation work values of the lyophiles after 6 hours of hydration

Table 13 ? A1Lio, A3Lio, A7Lio and A9Lio freeze dryers

Table 14 ? Freeze dryers B1Lio - B9Lio

Table 15 ? Freeze-philic B10Lio - B17Lio

Example 7 - Preparation of lyophilic formulations containing probiotics

At a quantity equal to 100 g of the B13 and B15 solutions, prepared as described in Example 1, ? was added a quantity? of freeze-dried probiotics of the L. reuteri KP-1E1-100B strain, Danisco, at a concentration of approximately 10<9 >CFU/100 g. The percent composition of the solutions? shown in Table 16.

Table 16 ? percentage composition of the freeze-dried solutions B13Lio and B15Lio

Solutions containing L. reuteri KP-1E1 were lyophilized as described in Example 3.

The unitary composition of the buccal tablet? shown in Table 17.

Table 17

Example 8 ? Determination of properties? mechanics of lyophilis containing probiotics - Resistance to penetration

The lyophilis B13Lio and B15Lio, containing L. reuteri KP-1E1, -100B, Danisco prepared according to Example 7, were subjected to measurement of force and strain work, as described in Example 4. The obtained values are shown in Table 18.

Table 18 ? Lyophilized B13Lio and B15Lio containing probiotics

The results have shown a greater capacity? of resistance to the penetration of the lyophilized B13Lio L. reuteri KP-1E1 (AL0008).

Example 9 ? Capacity determination? d?hydration formulations of lyophiles containing probiotics

The capacity? d?hydration of lyophiles B13Lio and B15Lio containing L. reuteri KP-1E1-100B, Danisco, prepared according to Example 7, ? was determined according to the procedure described in Example 5, after 3 and 6 hours of hydration. The dry weight and weight values after 3 and 6 hours of hydration are shown in Table 19, while the values of % of artificial saliva absorbed after 3 and 6 hours of hydration are shown in Table 20.

Table 19 ? Lyophilized B13Lio and B15Lio containing probiotics.

Table 20 ? Lyophilized B13Lio and B15Lio containing probiotics.

The lyophilic formulations B13Lio and B15Lio containing probiotics are able to absorb a large amount of of artificial saliva, with a similar hydration profile after both 3 hours and 6 hours.

Example 10 ? Determination of properties? mechanics of lyophilic formulations containing probiotics after hydration: resistance to penetration

The lyophiles B13Lio and B15Lio comprising containing L. reuteri KP-1E1-100B, Danisco, prepared according to Example 7, were subjected to force and strain work measurement after being hydrated for 6 hours as described in Example 6 .

The force and strain work values after 6 hours of hydration, shown in Table 21, show that the lyophilized B13Lio L. reuteri KP-1E1 ? characterized by a greater capacity? of resistance to penetration.

Table 21 ? Lyophilized B13Lio and B15Lio containing probiotics

Example 11?Determination of properties? mucoadhesive of lyophilis containing probiotics after hydration

The properties? mucoadhesives of the lyophilic formulations B13Lio and B15Lio containing L. reuteri KP-1E1-100B, Danisco, prepared according to Example 7, were evaluated after 6 hours of hydration with artificial saliva (Example 9), at 37 ?C, using the ?TA.XT plus Texture Analyzer equipment (Stable Micro Systems, Goldaming, UK) equipped with a 1 kg load cell. At the vertical arm of the instrument ? a cylindrical sample holder probe (P/10, with a diameter of 10 mm) was fixed. A quantity? equal to 40 ?L of hydrated lyophile (according to Example 9) ? layered on a 10 mm diameter filter paper disk and fixed on the sample holder probe. A dispersion of porcine gastric mucin type II (Sigma Aldrich, Milan, I) at 8% w/w ? was prepared in artificial saliva solution (0.15% w/v KCl, 0.043% w/v NaCl, 0.022% w/v CaCl2, 0.042% w/v NaHCO3, 0.091% w/v NaH2PO4 in distilled water), and then used as a biological substrate. Based on the tool ? been placed a beaker of capacity? 1 L, containing about 500 mL of bidistilled water, thermostated at 37?C, through a magnetic heating plate equipped with a thermometer for temperature control. 40 ?L of mucin dispersion were placed on a sheet of filter paper positioned on a Plexiglas support, immersed in the beaker, below the sample holder probe.

The probe? was then lowered to contact the mucin dispersion with the hydrated lyophil sample. The probe? was raised at speed? constant (2.5 mm/s) until causing the complete detachment of the sample from the mucin dispersion.

Eight measurements were made for each of the two lyophiles, compared with the control measurements obtained using 40 ?L of artificial saliva instead of the mucin dispersion.

? was therefore measured the force necessary to detach the sample holder probe from the mucin dispersion (mucin) or from the artificial saliva (control) and ? the normalized differential parameter of mucoadhesion (?Fmax/Fmax) was calculated according to the following equation:

?Fmax / Fmax = (Fmaxmucin- Fmaxcontrol) / Fmaxcontrol

where: Fmaxmucin ? the maximum detachment force measured in the presence of mucin and Fmaxcontrol ? the maximal detachment force measured in the absence of mucin (control). The values of maximum detachment force in the presence and absence of mucin (control) and of the normalized differential parameter of mucoadhesion (?Fmax/Fmax) are shown in Table 22.

Table 22? Lyophilized B13Lio and B15Lio containing probiotics.

The increase of the alginate concentration from 0.5 to 1.0% w/w results in a significant increase in the properties? mucoadhesive properties of lyophilic B13Lio, compared to lyophilic B15Lio, both containing probiotics (L. reuteri KP-E1-100B, Danisco), as indicated by the increase in the ?Fmax/Fmax value obtained for B13Lio.

Example 12 ? Capacity determination? of L. reuteri KP-1E1 release from lyophiles B13Lio and B15Lio containing probiotics

The quantity? of probiotics released by the lyophilic formulations B13Lio and B15Lio in physiological solution? was evaluated at time intervals of 0.5, 1, 3, 5 and 8 hours. A quantity? note of each lyophile ? placed in a known volume of saline and left at room temperature. After 0.5, 1, 3, 5 and 8 hours of incubation, at 37 ?C, ? The number of bacteria (colony forming units, CFU) contained in the lyophilic formulation and those released in 1 mL of physiological solution was determined.

Table 23 shows the CFU contained in the lyophile and the CFU released over time.

Table 23 ? bacteria release from the two lyophiles, in physiological solution, up to 8 hours, at 37?C.

The results show that the quantity of probiotics in lyophilized B13Lio decreases as the incubation time in physiological solution increases, in accordance with the concomitant increase of bacteria released over time. The lyophilic formulation B13Lio ? characterized by the ability to release probiotics in a controlled manner for up to 8 hours. The lyophilic formulation B15 tends to flake already? after 30 minutes of incubation, making it difficult to determine the bacterial count and in fact preventing a correct evaluation of the bacteria remaining in the sample and the number of those released into the solution.

Example 13 ? Determination of stability? of the lyophilic formulation (B13lio) containing L. reuteri KP-1E1

The stability? of the lyophilized B13Lio containing L. reuteri (AL0008), left at 4?C, ? was evaluated by bacterial count, after nine months (T9) at 4?C. The technique used for the bacterial count? that BioTimer, effective why? able to count live bacteria, even if present in aggregate form. [Pantanella F. et al. Journal of Microbiological Meth., 2008, 75: 478-484]. To the freeze-dried formulation? been added 1 ml of the Lacto-Counter reagent (at 37?C) and yes ? proceeded to count the bacteria, with the use of a calibration curve. The data is shown in Table 24.

Table 24 ? bacterial load in the lyophile, at time zero (T0) and after nine months (T9), at 4?C.

Example 14 ? Determination of the release of L. reuteri KP-1E1 from lyophilic B13Lio The same lyophilic formulation containing B13Lio at T9, ? been placed in 1 ml of physiological solution (9 g/l of NaCl) at 37? C and evaluated the quantity? of bacteria released after 1 and 5 hours in the solution.

Also in this case yes? used the BioTimer technique. To the solution ? 1ml of the Lacto-Counter reagent was added and the residual bacterial count was carried out (in the lyophile) and released into solution. Evaluated through a colorimetric reading, compared to a reference calibration curve.

Table 25 ? release of the bacteria from the lyophilized (at T9), in physiological solution

TABLE 4A

TABLE 4B

TABLE 5A

TABLE 6A

Claims (11)

1. A mucoadhesive formulation obtained by a process of drying a solution comprising a natural thickener/viscosifier at a concentration of 0.1 to 5.0% (w/w), a natural mucoadhesive agent at a concentration of 0.1 at 2% (w/w) and a calcium salt at a concentration of 0.02 to 0.1% (w/w) and probiotics in an amount? from 1x10<8>-1x10<13>CFU/100g. 2. The mucoadhesive formulation according to claim 1, wherein the thickening agent (viscosifier) is selected from the group comprising guar flour, gum tragacanth, corn starch, wheat starch, potato starch, rice starch, gelatin, isinglass, guar gum, xanthan gum, Kuzu root, agar-agar, sodium alginates , potassium alginates, ammonium alginate, propylene glycol alginate, acacia gum or gum arabic, karaya gum, tara gum, sorbitol, mannitol, sucrose acetate isobutyrate, cellulose derivatives and mixtures thereof; the bioadhesive agent (mucoadhesive) ? selected from the group comprising propolis, pectins, zeins, casein, gelatin, albumin, collagen, chitosan, oligosaccharides and polysaccharides such as cellulose, dextran, polysaccharides from tamarind seeds, xanthan gum, gum arabic, hyaluronic acid and mixtures thereof. 3. The mucoadhesive formulation according to claims 1 and 2 obtained by a drying process of a solution comprising alginate at a concentration of from 0.5 to 1% (w/w), xyloglucan at a concentration of from 0.2 to 0, 5% (w/w), calcium chloride at a concentration of 0.02 to 0.1% (w/w) and glycerol at a concentration of 1 to 2% (w/w), relative to the finished solution and probiotics in a quantity? from 1x10<8>-1x10<13>CFU/100g. 4. The formulation according to claim 3 obtained by a drying process of a solution comprising alginate 1.0% (w/w), xyloglucan 0.25% (w/w), calcium chloride 0.05% (w /p) and glycerol 2.0% (w/w), compared to the finished solution and probiotics in an amount? from 1x10<8>-1x10<13>CFU/100g . 5. The formulation according to claim 1 characterized by having a deformation force from 4500 to 16000 mN and a deformation work from 4000 to 10000 mN?mm. 6. The formulation according to claim 1 characterized by the? absorb an aqueous solution in a percentage by weight from 250 to 550% of the weight before hydration, in a time of three to eight hours. 7. The formulation in accordance with claims 1 including probiotics chosen in the group including Lactobacillus salivarus, Lactobacillus Brevis, Lactobacillus Helveticus, Lactobacillus paraacasei, lattoBacillus rhamnosus, lactobacillus animalis, lactobacillius reuteri, and bifidobacteri chosen in the component bifidobacterium group, Bifidobacterium infantis, and they mixtures in a quantity? from 1x10<8>-1x10<13 >(CFU/100 g). 8. The formulation according to claim 7 wherein the probiotics are released in an aqueous solution in a time ranging from three to eight hours, in a percentage higher than 50%. 9. A process for preparing a mucoadhesive formulation comprising the steps of: a) adding to an aqueous solution a natural thickening agent at a concentration of 0.5 to 1.0% (w/w), a bioadhesive agent (mucoadhesive) at a concentration of 0.1 to 1.0% (w/w p), and glycerol from 1.0 to 2.0% with respect to the final solution; b) add a probiotic chosen from lactobacilli or bifidobacteria or mixtures thereof in a quantity? from 1x10<8>-1x10<13 >(CFU/100g); c) drying the partitioned aliquots of the solution or the whole solution and then partitioning the solid. 10. The process according to claim 9 comprising the steps of: a) add an alginate at a concentration of 0.5 to 1.0% (w/w), xyloglucan at a concentration of 0.2 to 1.0% (w/w), and calcium chloride at a concentration of 0.02 to 0.1%, to an aqueous solution of maltodextrin at a concentration of 6.0% to 10.0% (w/w); b) add to the homogeneous solution a probiotic selected from among Lactobacillus salivarus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus animalis, Lactobacillus reuteri, and bifidobacteria, selected from the group comprising Bifidobacterium longum, Bifidobacterium infantis, and mixtures thereof in a quantity ? 1x10<8>-1x10<13 >(CFU/100g); c) drying by freeze-drying or spray-drying or wet granulation the divided aliquots of the solution or the solution as a whole and then dividing the solid. 11. A formulation according to claim 1 for use in the treatment and/or prevention of pathologies and/or disorders of the cavity? oral, associated with alteration of the local bacterial flora (oral dysbiosis) responsible for dental caries, periodontal problems, peri-implantitis, mucositis and bad breath.