BUCCAL MUCOADHESIVE GEL CONTAINING PROBIOTICS FOR USE IN THE PREVENTION AND TREATMENT OF PERIODONTAL DISEASES

OBJECT OF THE INVENTION

The present invention relates to mucoadhesive gels comprising probiotics, to the process for their preparation and to their use for the prevention and treatment of pathologies of the oral cavity.

STATE OF THE ART

Dental plaque consists of a microbial biofilm and is the main component of the bacterial ecosystem (microbiota) present in the oral cavity. There are many bacterial species that make up the microbiota of the oral cavity, in the order of several thousand, and it is estimated that about a thousand of these make up the biofilm colonizing the surface of the teeth.

The oral microbiota constitutes a defense system of the oral cavity, preventing the colonization of the mouth by pathogenic bacteria. An imbalance in the composition of the dental biofilm can give rise to the appearance of dental caries and gingival and periodontal infections/inflammations [Rinkee Mohanty, et al. J Family Med Prim Care. 2019, 8: 3480- 3486], For example, endogenous bacteria, mainly Streptococci mutans present in the biofilm, produce weak organic acids as a by-product of the metabolism of fermentable carbohydrates, with a decrease in pH and consequent teeth tissues demineralization [Selwiz R.H. et al., Lancet 2007; 369: 51 -59], Periodontal diseases (gingivitis and periodontitis) are largely due to specific infections caused by anaerobic Gram-negative bacteria which, if neglected, lead to damage of the soft connective tissue of the gum and, subsequently, to the loss of the underlying alveolar bone and ligament supporting the teeth. Among the bacterial species mainly involved in the development and progression of periodontitis there are Porphyromonas gingivalis, Treponema denticola, Prevotella denticola, Tannerella forsythia, Aggregatibacter actynomycetecomitans and Fusobacterium nucleatum, which together constitute the red complex, a group of bacteria classified together based on their association with severe forms of periodontal disease.

According to the definition of the FAO and the WHO, the term probiotic means "alive microorganisms that, administered in adequate amounts, provide benefit to the health of the host'. The FAO/WHO document giving this definition refers to non-pathogenic microorganisms, lactobacilli and bifidobacteria, present or added to food, which are able to exert beneficial effects to the body.

In recent years, the effect of pathogenic bacterial species on dental implants also has been studied [Tamrakar A.K. et al. J. Oral Biology and Craniofacial Research 2020, 10: 180-183], In fact, an oral dysbiosis can lead to implant loss. Chemotherapy treatments and radiotherapy can also cause oral mucositis (stomatitis), resulting in very painful inflammations and ulcerations which become an easy target for bacterial and viral infections.

According to WHO data, there is a high incidence of periodontal disease among adults, at least in the early stages. For example, in Europe, about 20% of the population in the middle age group (35-44 years old) and up to 40% in the older age group (65-74 years old) suffer from this disease, which is multifactorial. Furthermore, the health of the oral cavity has impacts on the overall well-being of the organism, starting from the prevention of cardiovascular problems [Aguilera, E.M. et al., Cardiovascular Res. 2019, 116: 28-39],

It is possible to promote actions against pathogenic bacteria of the oral microbiota, with preventive and curative effects of periodontal disorders. A possible action is introducing probiotic lactobacilli to partially replace pathogenic microorganisms, with promising results in controlling oral infections [Hoare et al. Microbiol Spectr. 2017, 5: doi: 10.1128/microbiolspec. BAD-0006-2016.]

Lactobacilli are Gram-positive bacteria, they obtain the energy necessary for their survival thanks to a fermentation process of hexose sugars (for example, glucose, mannose, fructose, etc.) by producing lactic acid and ethanol. Other bacteria producing lactic acid from sugars, albeit via a different metabolic pathway, are bifidobacteria. Lactobacilli and bifidobacteria belong to the group of bacteria defined as "probiotics", which carry out a series of beneficial actions, especially, but not only, in the intestine of Mammals and in particular in Humans. These probiotics secrete several antimicrobial substances, such as enzymes, organic acids, hydrogen peroxide and bacteriocins, and compete with buccal pathogens for mucosal adhesion sites [Meurman J.H. et al. Eur J Oral Sci 2005; 113: 188-1962],

Therefore, to overcome the disorders caused by the pathological effects of microorganisms of the oral cavity, the use of lactic acid bacteria and bifidobacteria represents an alternative and promising therapeutic opportunity, as well as being an effective method for maintaining correct dental and oral cavity health. Probiotic lactobacilli and bifidobacteria can compromise the viability of pathogenic microorganisms through the modulation of the pH and/or the redox potential of the environment in which they live, by stimulating non-specific immune mechanisms and modulating the humoral and cellular immune response [Marco M.L. et al., Curr. Opin. Biotech. 2006, 17: 204-210],

In recent years, to enrich the oral microbiota with beneficial bacteria (oral eubiosis) several probiotics-containing formulations in the form of granules for oral suspension have been developed. There are many probiotic strains with recognized therapeutic properties against periodontal infections, however, the analysis of clinical studies has shown that further research and studies focused on dosages, routes of administration and strains to be used are needed [Vives-Soler A. et al. Med Oral Patol Oral Cir Bucal. 2020; 1 : e161 -7].

Recently it has been shown that Lactobacillus brevis ATCC 4006 may have beneficial effects in the treatment of some oral diseases. This probiotic is currently marketed in an orodispersible tablet formulation, under the name Inersan®. In a group of patients suffering from chronic periodontitis treated with this formulation, an anti-inflammatory effect determined by the composition was found.

Piyush Shah et al. J. Clin. Diagn. Res., 2013, 7: 595-600, describes a clinical study wherein the effect of the probiotic Inersan® administered to patients with periodontitis is compared with the combination of the probiotic with the antibiotic doxycycline, and with the effect determined by the administration of the antibiotic alone. The study has shown that the probiotic composition based on lactobacilli was able to perform a favorable action on gingival inflammation and in preventing the appearance of caries, without the need to take doxycycline and therefore eliminating the problems associated with drug resistance towards antibiotics. Toiviainen, A. et al. Clin Oral Investig., 2015; 19: 77-83 discloses the use of a tablet formulation comprising Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12, with a 4-week treatment in healthy patients. The study has shown that shortterm consumption of L. rhamnosus LGG and B. animalis BB-12 is sufficient to determine a decrease in the amount of bacterial plaque associated with gingival inflammation.

The strain Lactobacillus rhamnosus SP1 (DSM 21690) was used in a study aimed to evaluate the effect of administering this probiotic on improving dental health in a group of 205 preschool children in Chile. The L. rhamnosus SP1 strain is given in freeze-dried form (1 .5 billion of alive lactic ferments) as a food supplement in 150 ml of milk in the afternoon snack consumed at school. Dental plaque analysis at the beginning and at the end of treatment lasting ten months, has shown a slight decrease in caries in the group that received the probiotic (54.4%) compared to the control group (65.8 %), but after two more months without further administration of the probiotic (12 months from the beginning of the test) the carious lesions in the children who had drunk the enriched milk, were significantly lower (9.7%) than in the control group (24.3%) showing that the likelihood of developing caries after the administration of the probiotic was significantly decreased. The study has demonstrated the efficacy of this treatment with probiotics in pediatric patients, in preventing the development of caries, but not of periodontal infections, such as gingivitis and pyorrhea [L’lntegratore Nutrizionale 2017 , 20: 56-57],

The efficacy of a mixture comprising several bacterial strains including Lactobacillus helveticus SP27 (DSM29575) used as an adjuvant in the treatment of some forms of pneumonia resistant to a wide range of antibiotics has been demonstrated [Piatek J. et al. Int. J. Med. Res. Health Sci., 2019, 8: 12-20],

The same probiotic composition, containing more bacterial strains, has also been used in the treatment of persistent forms of enteric infection caused by Salmonella typhimurium. In this case it has been observed that the inhibitory effect of the individual strains varies significantly, Streptococcus thermophilus ST-21 and Lactobacillus helveticus SP27 strains present in the mixture show the strongest inhibitory effect [Piatek J. et al. Beneficial Microbes. 2018; 10: 211 -217], No specific effect against periodontal infections is mentioned in the two Piatek's publications.

The nutraceutical product Kijimea® K53 comprising probiotic strains Lactobacillus rhamnosus SP1 (DSM 21690) and Lactobacillus helveticus SP27 (DSM 29575), together with other 51 strains of probiotic bacteria, is indicated for use in states of tiredness and fatigue. The composition also includes vitamin B12 and biotin.

Vuotto et al. (Oral Diseases, 2013; 20: 668-674) describes the use of Lactobacillus brevis to prevent the formation of the buccal biofilm caused by Prevotella melaninogenica, one of the causative agents of periodontitis.

WO 2018/169296 discloses mucoadhesive buccal films comprising probiotic bacteria (e.g. Lactobacillus brevis). The films contain gelling agents and are made by drying of highly viscous solutions. Mucoadhesive films for the release of alive lactobacilli represent a suitable dosage form for the treatment of oral cavity disorders but require long production times with consequent loss of vitality during the preparation process and during the storage.

Patent application CN 107865891 describes probiotic compositions, in form of gel, useful for inhibiting the growth of periodontal pathogens and for preventing and treating periodontitis. These probiotics include Lactobacillus acidophilus (10-30%), Streptococcus salivarius M18 (10-50%) and Streptococcus salivarius K12 (20-80%) bacteria, overall in amount from 10 ⁷ to 10 ¹⁰ CFU/ml, and pharmaceutically acceptable vehicles or excipients, wherein the bacterial suspension is mixed with a matrix comprising 1 -10% modified cellulose-based polymeric gel, in oily carrier, to which hydroxypropyl methylcellulose as bioadhesive material is added, and a silicon-based rheological modifier. The document shows composition stability data for up to six months of storage.

Given the beneficial activity of the use of probiotics in all the inflammations and disorders of the oral cavity, it is useful to have alternative pharmaceutical/nutraceutical compositions that are able to keep probiotics alive and to release them in the mouth in a controlled manner. It is also important that the composition remains adhered to the buccal mucosa and is also accepted by the subjects.

Being able to ensure the presence of probiotics for prolonged periods compared to current formulations that disperse probiotics in few minutes, can ensure a significant therapeutic advantage for the principle that probiotics act against pathogenic bacteria through a competition mechanism. For this reason, it is important to develop formulations including probiotics able of being released alive and active at the site of action for a prolonged time. Semi-solid dosage forms such as mucoadhesive oral gels have the advantage of ensuring a more intimate contact and an easier and faster dispersion of the active ingredients, the probiotic, its enzymes and pharmacologically active substances, through the oral mucosa.

A composition in the form of a mucoadhesive gel, comprising probiotics in amounts higher than 1x10 ⁷ CFU/g which are released in a time longer than six hours, has surprisingly been found, and it is the object of the present invention.

The gel is stable at room temperature and is characterized by an extreme ease of administration.

SUMMARY OF THE INVENTION

The object of the invention is a mucoadhesive gel composition comprising: a probiotic in an amount from 1x10 ⁷ to 1x10 ¹⁵ CFU/g; from 50 to 85% (w/w), with respect to the weight of the final composition, of a dispersing agent selected from the group consisting of: esters of fatty acid (C4-C30) with glycols, lecithins, non-ionic waxes, phospholipids, polaxamers, polyoxy alkyl esters, castor oil derivatives, polyoxyethylene esters of sorbitan and fatty acids, polyglycerides, povidone, stearic acid, esters of stearic acid and glycerin, sorbitan esters, starch, hydroxy stearate, and mixture thereof; from 5 to 20% (w/w), with respect to the weight of the final composition, of a bioadhesive agent selected from the group consisting of: glyceryl monooleate, hydroxypropylmethyl cellulose with viscosity from 750 to 1400 mPa sec in a 2% (w/w) aqueous solution, polycarbophil, polyethylene oxide, carbomers, dextrins, glucans, and mixture thereof; from 10 to 30% (w/w), with respect to the weight of the final composition, of a viscosifying mixture comprising: from 60.0 to 90.0% (w/w), with respect to the weight of said mixture of a viscosifying agent selected from the group consisting of: glyceryl behenate, agar, bentonite, xanthan gum, xyloglucans, hyaluronic acid esters with molecular weight higher than 300KDa, linseed, acacia gum, arabic gum, alginic acid and its esters, corn starch, colloidal bentonite, lanolin, waxes, beeswax, palmitic acid esters cellulose, ethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, cellulose, myristilic acid, polyvinyl acetate, povidone, alginate, carrageenan, cetostearyl alcohol, chitosan, gelatin, glycerin, type 1 vegetable oil, silica, sucrose, hydroxypropyl methylcellulose with a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution and mixture thereof; from 10.0 to 40.0% (w/w), with respect to the weight of said mixture, of a substituted silica with linear or branched alkyl groups, optionally including one or more heteroatoms.

The probiotics according to the present invention are preferably selected from the group consisting of: lactobacilli selected from the group consisting of: Lactobacilli salivarus, Lactobacilli brevis, Lactobacilli helveticus, Lactobacilli paracasei, Lactobacilli rhamnosus, Lactobacilli animalis, Lactobacilli reuteri, Lactobacilli plantarum, Lactobacilli acidophilus, Lactobacilli belbrueckir, Bifidobacteria selected from the group consisting of: Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium brevis, Streptococcus thermophilus, and mixtures thereof.

A second aspect of the present invention is to provide a process for the preparation of a mucoadhesive gel composition according to claim 1 comprising the following steps: a. providing said dispersing agent in an amount of 50-85 % (w/w) and adding said viscosifying mixture in an amount of 10-30% (w/w), at a temperature between 50 and 80 °C; b. adding said bioadhesive agent in an amount of 5 to 20% (w/w); c. cooling down to room temperature; d. adding under stirring said probiotics in an amount from 1x10 ⁷ to 1x10 ¹⁵ CFU/g; and e. aliquoting in a chosen dosage.

A third object of the invention is the use of the mucoadhesive composition for the prevention and treatment of disorders, pathologies and inflammations of the oral cavity, gingivitis, peri- implantitis, periodontitis, stomatitis and caries. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Release of probiotics included in the GEL 6 formulation (AL0005)

• GEL 6/I loaded with B. animalis BCL01 DSM 17741 (1A);

• GEL 6/II loaded with L. reuteri LR92 DSM 26866 (1 B);

• GEL 6/III loaded with L. brevis SP48 DSM 16806 (1 C);

• GEL 6/IV loaded with L. helveticus SP27 DSM 29575 (1 D);

• GEL 6/V loaded with L. rhamnosus SP1 DSM 21690 (1 E);

• GEL 6/VI loaded with L. paracasei LGM S-26420 (1 F).

Figure 2: Release of probiotics (L. rhamnosus SP1 DSM 21690 and L. helveticus SP27 DSM 29575) included in GEL 6A formulation (AL0006).

Figure 3: Release of probiotics (L rhamnosus SP1 DSM 21690, L. helveticus SP27 DSM 29575 and L. paracasei LGM S-26420) included in the GEL 6D formulation (AL0007).

Figure 4: Graph showing the variation over time in the body weight of mice over the 28-day study period.

Figure 5: Graph showing the variation over time in mm of the maxillary alveolar bone in animals over the 28-day treatment period.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a mucoadhesive gel comprising at least one probiotic for the treatment and/or prevention of disorders, pathologies of the oral cavity. The mucoadhesive gel is characterized by releasing the probiotics included in the formulation in a time longer than six hours.

The mucoadhesive gel comprises a dispersing agent in an amount from 50 to 85% (w/w), a viscosifying mixture in an amount from 10 to 30% (w/w), and a bioadhesive agent in an amount from 5 to 20% (w/w), with respect to the total weight of the composition. The gel may further comprise pharmaceutical excipients such as sweetening, preserving and flavoring agents.

The dispersing agent is selected from the group consisting of: esters of fatty acid (C4-C30) with glycols, lecithins, non-ionic waxes, phospholipids, polyxamers, polyoxy alkyl esters, castor oil derivatives, polyoxyethylene esters of sorbitan and fatty acids, polyglycerides, povidone, stearic acid, esters of stearic acid and glycerin, sorbitan esters, starch, hydroxy stearate, and mixture thereof.

The bioadhesive agent is selected from the group consisting of: glyceryl monooleate, hydroxypropylmethyl cellulose with viscosity from 750 to 1400 mPa sec in a 2% (w/w) aqueous solution, polycarbophil, polyethylene oxide, carbomers, dextrins, glucans, and mixture thereof;

The viscosifying mixture according to the invention comprises: from 60.0 to 90.0% (w/w), with respect to the weight of said mixture of a viscosifying agent selected from the group consisting of: glyceryl behenate, agar, bentonite, xanthan gum, xyloglucans, hyaluronic acid esters with molecular weight higher than 300KDa, linseed, acacia gum, arabic gum, alginic acid and its esters, corn starch, colloidal bentonite, lanolin, waxes, beeswax, palmitic acid esters cellulose, ethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, cellulose, myristilic acid, polyvinyl acetate, povidone, alginate, carrageenan, cetostearyl alcohol, chitosan, gelatin, glycerin, type 1 vegetable oil, silica, sucrose, hydroxypropyl methylcellulose with a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution and mixture thereof; from 10.0 to 40.0% (w/w), with respect to the weight of said mixture, of a substituted silica with linear or branched alkyl groups, optionally including one or more heteroatoms.

The preferred viscosifying agent is a mixture of viscosifying agents consisting of ethyl cellulose, glyceryl behenate, hydroxypropyl methylcellulose with a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution.

According to the present invention, the term dispersing agent refers to a compound or a compounds mixture that, added to a composition improving the separation of particles and prevents settling or clumping. According to the present invention, the term bioadhesive or mucoadhesive agent refers to natural materials that act as adhesives, in particular refers to a material designed to adhere to biological tissue.

According to the present invention, the term viscosifying agent, or thickening agent, refers to a substance that can increase the viscosity of a liquid without substantially changing its other properties.

According to the present invention the preferred heteroatoms are nitrogen, oxygen, sulfur, phosphorus, chlorine, bromine, and iodine, as well as the metals lithium and magnesium.

Preferably the substituted silica is dimethyl silylated silica.

Preferably the viscosifying agent comprises a said substituted silica in an amount from 2.5 % to 5.0 % (w/w), with respect to the weight of the final composition.

Preferably in the mucoadhesive gel composition according to the invention said dispersing agent is in an amount from 60 to 75% (w/w), with respect to the weight of the final composition; said bioadhesive agent is in amount from 5 to 10% (w/w), with respect to the weight of the final composition; said viscosifying mixture is in an amount from 20 to 30% (w/w), with respect to the weight of the final composition.

In a preferred aspect, the mucoadhesive gel composition comprises probiotics in an amount from 1x10 ⁷ to 1x10 ¹⁵ CFU/g, a dispersing agent consisting of a mixture of esters of fatty acids (C4-C30) with glycols, in an amount from 60 to 75% (w/w) with respect to the total weight of the composition, a viscosifying mixture consisting of ethyl cellulose, glyceryl behenate, substituted silica and hydroxypropyl methylcellulose with a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution respectively in a weight ratio 1 :0.5: 1 to 1 :1 :5, a bioadhesive agent consisting of hydroxypropyl methylcellulose with viscosity from 750 to 1400 mPa sec in a 2% (w/w) aqueous solution in an amount from 5 to 10% (w/w), relative to the total weight of the composition.

The mucoadhesive gel composition comprises the viscosifying mixture consisting of a mixture of ethyl cellulose, glyceryl behenate, substituted silica and hydroxypropyl methylcellulose with a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution (Benecell K4M), in a weight ratio from 1 : 0.5: 1 : 1 to 1 : 1 : 5: 10. The mucoadhesive gel composition is characterized by comprising a mixture of high viscosity hydroxypropyl methylcellulose, in which a hydroxypropyl methylcellulose characterized by producing a viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution and a hydroxypropyl methylcellulose characterized by producing a viscosity from 750 to 1400 mPa sec in a 2% (w/w) aqueous solution, which are respectively in a weight ratio from 0.5: 1 .5 to 1 :5.

In a more preferred embodiment, the mucoadhesive gel composition comprising esters of caprylic and/or capric acid with propylene glycol in an amount from 50.0 to 80.5% (w/w), ethyl cellulose 5.0-10.0% (w/w), glyceryl behenate 2.5-10.0% (w/w), dimethyl silylated silica 2.5-5.0% (w/w), hydroxypropylmethyl cellulose with a viscosity from 750 to 1400 mPa sec in a 2% (w/w) aqueous solution, in an amount from 5 to 20% (w/w), and hydroxypropylmethyl cellulose with viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution, in an amount from 5.0 to 10.0% (w/w), with respect to the final composition.

The mucoadhesive gel composition according to the present invention can also comprise pharmaceutically acceptable excipients such as preserving agents, buffering agents, flavoring agents.

Flavoring agents are selected from the group comprising natural plant, citrus and fruit extracts.

The mucoadhesive gel composition is characterized by being lipophilic and is homogeneous without separation of the oily phase from the gel phase.

The mucoadhesive gel composition according to the invention is characterized by a viscosity measurement between W and 40 mPa sec when a shear stress from 1 to 10 s ^-1 is applied.

In a particular aspect the viscosity of the formulation is between 14 and 38 mPa sec when a shear stress from 1 to 10 s ^_1 is applied.

The composition of the mucoadhesive gel is characterized by an adhesion to a mucin layer by more than 70% compared to an aqueous solution.

The mucoadhesive composition offers the advantage of releasing probiotics into the oral cavity in a controlled manner, increasing the therapeutic efficacy with less frequency of administration. The composition in gel form has the advantage to be easily administered one or more time in a day and well accepted by the subject.

The mucoadhesive gel composition according to the present invention does not contain water or is substantially free of water.

The gel composition includes one or more probiotics, belonging to the lactobacilli and/or bifidobacteria species selected from the group comprising: Lactobacilli salivarus, Lactobacilli brevis, Lactobacilli helveticus, Lactobacilli paracasei, Lactobacilli rhamnosus, Lactobacilli animalis, Lactobacilli reuteri, and bifidobacteria, as for example, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium brevis and/or their mixtures.

In one embodiment, the mucoadhesive gel composition preferably comprises the probiotics selected from the group comprising: Lactobacillus rhamnosus (DSM 21690), Lactobacillus helveticus (DSM 29575), Lactobacillus paracasei (LGM S-26420), and/or their mixtures, in amounts of 1x10 ⁹ -1x10 ¹⁷ (CFU/100g).

The probiotics Lactobacillus rhamnosus (DSM 21690), Lactobacillus helveticus (DSM 29575) and Lactobacillus paracasei (LGM S-26420) in amounts of 1x10 ⁹ -1x10 ¹⁷ (CFU/100g) and/or their mixtures are released in a time from 10 minutes to 6 hours.

In a preferred aspect the unitary composition is reported in Table 1 :

Table 1

In a preferred aspect the unitary composition is reported in Table 2: Table 2

Preferred unitary mucoadhesive gel compositions comprising probiotics are reported in Tables 3 and 4. Table 3

Table 4 The mucoadhesive gel composition according to the invention is characterized by the ability to release probiotic microorganisms in a controlled manner, in a percentage greater than 70%, in a time from ten minutes to six hours, in which the probiotics maintain their vitality.

It has been shown that the gel has a good adhesion to several types of materials, both of non-specific nature, such as filter paper, and polycrystalline bioceramic, a dentin mimicking material.

By in vitro studies it has been shown that the gel allows the release of probiotics from the gel into the mucous membrane of the oral cavity and into the dental biofilm.

It has been shown that the composition is characterized by releasing the probiotics in a solution (artificial saliva), in a time from ten minutes to two hours, in which a percentage greater than 60% of the probiotics are released after 2 hours, and a percentage of the 75% after 5 hours.

In one particular aspect, it has been shown that the mucoadhesive gel composition releases a percentage of L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575) equal to 62%, and in a percentage from 60% to 70% after two hours and adheres to the surface of microcrystalline bioceramic, the release percentage remains practically constant from 50% to 70% from ten minutes to six hours.

The percentage of probiotics released from the mucoadhesive gel containing L. rhamnosus SP1 (DSM 21690), L. helveticus SP27 (DSM 29575) and L. paracasei CBA-L87 (LGM S- 26420) and adhered to the surface ranges from 50 to 70% after two hours, and it rises up to 80% after 8 hours. Therefore, the bacteria released from the gel preparation maintain their ability to adhere to the surface and to maintain their vitality for a very long time and such as to allow them to exert their beneficial effect at the application site, up to eight hours after administration, thus favoring a greater distribution and release of probiotics even in the gingival pockets.

The mucoadhesive gel composition includes probiotics selected in the group comprising L. rhamnosus SP1 (DSM 21690), L. helveticus SP27 (DSM 29575) and L. paracasei CBA-L87 (LGM S-26420) and their mixtures, which inhibit the growth of pathogenic bacteria responsible for periodontal infections in the oral microbiome. This effect has been demonstrated in vitro on pathogenic strains responsible for mouth infections/inflammations such as Porphyromonas gingivalis (ATCC 33277), Terponema denticola (ATCC 35045), Tannarella forsythia (ATCC 43037), Prevotella melaninogenica (ATCC 25845) and Aggregatibacter actinomycetemcomans (ATCC 700685).

In a second aspect of the present invention, it is described the process for the preparation of the mucoadhesive composition as described before, comprising the steps of: a. providing a dispersing agent in an amount of 50-85 % (w/w) and adding a viscosifying mixture in an amount of 10-30% (w/w), at a temperature between 50 and 80 °C; b. adding a bioadhesive agent in an amount of 5 to 20% (w/w); c. cooling down to room temperature; d. adding under slight stirring probiotics in an amount from 1x10 ⁷ to 1x10 ¹⁵ CFU/g; and e. aliquoting in a chosen dosage form.

Preferably, in the process according to the invention, the dispersing agent is an ester of capric and/or caprylic acid with propylene glycol, in an amount of 50.0 to 80.5% (w/w); the viscosifying mixture is ethyl cellulose, in an amount from 5.0 to 10.0% (w/w), glyceryl behenate in an amount from 2.5 to 10.0% (w/w) hydroxypropyl methylcellulose with viscosity from 75000 to 140000 mPa sec in a 2% (w/w) aqueous solution from 5.0 to 20.0% (w/w) and dimethyl silylated silica in an amount from 2.0 to 5.0% (w/w); the bioadhesive agent is hydroxypropyl methylcellulose with viscosity from 750 to 1400 mPa- sec in a 2% (w/w) aqueous solution in an amount from 5.0 to 20.0% (w/w); and the probiotics are selected from the group consisting of: L. rhamnosus, L. paracasei, L. reuteri, L. helveticus, L. brevis, L. salivarius, L. plantarum, Streptococcus thermophilus, L. acidophilus, L. belbrueckii, B. longum, B. infantiand B. brevis.

The mucoadhesive gel composition according to the invention is dispensed in tubes of 5, 10, 15, 50 grams and stored at room temperature.

Another object of the present invention is a medical device comprising the mucoadhesive gel comprising probiotics.

The mucoadhesive gel composition according to the invention is stable for at least 6 months, at room temperature, period wherein the viability of the probiotic strains remains unaltered. The mucoadhesive gel composition according to the invention is useful for the treatment and/or prevention of oral diseases, in the prevention of periodontitis, gingivitis, stomatitis and caries.

In a study on an animal model of periodontitis it has been shown that the composition is effective in preventing damage to the teeth and their loss.

The effectiveness of the formulation according to the invention has been confirmed in a mouse model in which inflammation was induced by ligating the teeth. The mucoadhesive gel comprising probiotics L. rhamnosus SP1 (DSM 21690), L. helveticus SP27 (DSM 29575) and L. paracasei CBA-L87 (LGM S-26420) was found to be significantly effective compared to the gel without probiotics. The composition comprising L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575) is effective in preventing tooth loss in a percentage from 80 to 90% compared to the gel without probiotics in which the percentage ranges from 20 to 40 %.

The mucoadhesive gel composition according to the invention is useful for recovering alveolar bone loss which was particularly sensitive in the group treated with the mucoadhesive gel comprising L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575), as shown in Figure 5.

The mucoadhesive gel composition according to the invention useful for preventing problems in the oral cavity, such as periodontitis (pyorrhea), halitosis, gingival recession, gum bleeding, abscess and swelling of the gums, pain and difficulty while chewing. The composition is also useful for preventing/treating peri-implantitis, or the bacterial infection that develops around dental implants.

The mucoadhesive gel composition according to the invention can be used in combination with antibiotics, chemotherapy, radiotherapy, immunosuppressants.

The mucoadhesive gel composition according to the invention can be applied to the gums one to five times a day; it is well tolerated and has no toxic or adverse effects either on the mucosa or at a systemic level. EXAMPLES

Example 1 : PREPARATION OF BUCCAL GEL

Preparations of 100 grams of six gels were obtained by placing in a mixer in the following order: ethyl cellulose and ester of capric and caprylic acid with propylene glycol at a temperature of 85°C. The mixture was kept stirring until homogeneous, then brought to room temperature.

Glyceryl beetate, dimethyl silylated silica (Aerosil® R972), hydroxy-propylmethylcellulose (Benecell K4M) and (Benecell K100M) were added to the six homogeneous mixtures and the final mixtures were kept stirred until homogeneous mixtures were obtained. The unitary compositions are reported in Table 5.

Table 5

Gel compositions 1 -6 were placed in glass tubes and after 24 hours centrifuged at 3000 rpm for 15 min and their appearance was evaluated: Gel 1 : the oily phase is separated from the gel phase by approximately 50%

Gel 2: the oily phase is separated from the gel phase by about 20%

Gel 3: the oily phase is separated from the gel phase by about 10%

Gel 4: the oily phase is separated from the gel by about 5%

Gel 5: the oily phase is separated from the gel by about 1 % Gel 6: the gel is homogeneous without separation of the oily phase from the gel phase.

Gel 6 Formulation (AL0005) was chosen for preparations of gel containing probiotics.

Example 2: PREPARATION OF THE GEL COMPOSITION CONTAINING A PROBIOTIC STRAIN

The lyophilized bacteria of one of the probiotic strains were added to an amount of gel equal to 10 grams of GEL 6 obtained as shown in example 1 : B. animalis subsp. lactis BCL01 (DSM 17741 ), Lactobacillus rhamnosus SP 1 (DSM 21690) Lactobacillus reuteri LR92 (DSM 26866), Lactobacillus brevis SP48 (DSM 16806), Lactobacillus helveticus SP27 (DSM 29575), all registered with the Deutsche Organism Braunschweig, Germany, or the lyophilized bacteria of the Lactobacillus paracasei strain (LGM S-26420), deposited at the Belgian Coordinated Collection of Microorganism - BCCM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University.

Six probiotic gels as reported in Table 6 were obtained.

Table 6

Example 3: DETERMINATION OF THE RELEASE CAPACITY OF THE SINGLE BACTERIAL STRAINS FROM THE GEL

The amount of bacteria released from the formulation of GEL 6 loaded with bacteria of several strains in single form was evaluated both in physiological solution and in an artificial saliva solution at time intervals of 30 minutes, 2 hours, 5 hours and 8 hours. Through the microbiological analysis performed by the BioTimer, the initial and residual bacterial load in the gels at various time intervals, and in parallel the amount of bacteria released in the two types of solution were determined [Pantanella F. et al. Journal of Microbiological Meth., 2008, 75: 478-484], In particular, 400 mg of each probiotic gel preparation were divided into 10 tubes, each containing 40 mg of gel, and divided into two series of 5 tubes each. To each of the 5 tubes of a serie, 0.2 ml of artificial saliva solution (8.0 g/L sodium chloride, 2.38 g/L dibasic disodium phosphate, 0.19 g/L monobasic potassium phosphate, pH 6.8), and to each of the 5 tubes of the second series 0.2 ml of physiological solution were added. In Table 7 the release data in artificial saliva up to 5 hours, and in Table 8 the release data in physiological solution up to 8 hours, expressed as CFU/g for the gel and CFU/ml for the solution in which the release took place, are shown.

Table 7 Table 8

The amounts of probiotics released by Gels 6/l-VI have shown that in artificial saliva an important amount of L. paracasei (LGM S-26420) is released already after 30 minutes, while in physiological solution the release occurs more slowly. No bacteria are released from gel formulations containing L. brevis SP48 (DSM 16806) and B. animalis BCL01 (DSM 17741) in artificial saliva solution. In physiological solution the formulation containing L. brevis SP48 (DSM 16806) begins to release bacteria from the gel only after 8 hours, the formulation containing B. animalis BCL01 (DSM 17741) begins to release in minimum amounts after two hours and the maximum release is also in this case after 8 hours. Bacteria of the L. reuteri strain were not very stable in the formulation of GEL 6.

Example 4: PREPARATION OF THE GEL COMPOSITION CONTAINING MIXTURES OF PROBIOTIC STRAINS Based on the results obtained with the probiotic gel preparations containing only one probiotic strain, the gel formulations containing the probiotic strains L. paracasei (LGM S- 26420), L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575) in association with each other as indicated in Table 9 were prepared. The preparations were carried out on a 10 grams scale by adding the bacteria in lyophilized form to the preparation 6 of Example 1 . In Table 9 the starting bacterial load of the single strains in lyophilized form and of the probiotic mixtures before being added to GEL 6 is indicated by CFU count and the BioTimer method.

Table 9 Example 5: DETERMINATION OF THE GEL RELEASE CAPACITY OF BACTERIAL

STRAIN ASSOCIATIONS

The amount of bacteria released from GEL 6/l-VI compositions (Example 2) was evaluated in physiological solution at time intervals of 30 min, 2 hours, 5 hours and 8 hours as described in Example 4. The results obtained for the four compositions are reported in Table 10.

Table 10 The results obtained from the analyses have shown that the probiotics are released in physiological solution from Gel 6/C after 8 hours. The release profile of Gel 6/B is comparable to that of Gel 6/A.

Gel 6/A (AL0006) and Gel 6/D (AL0007) were selected for the preparations on a 100 grams scale. Example 6: DETERMINATION OF THE VISCOSITY OF THE GEL FORMULATIONS

The viscosity of the GEL 1 -6 compositions, prepared according to Example 1 , and of the GEL 6A and GEL 6D compositions, comprising the probiotic consortia and prepared according to Example 4, was measured by the rotational rheometer Kinexus Pro + (Malvern Analytical ), at a temperature of 37 °C depending on the shear stress. Table 11 shows the viscosity values with shear stress equal to 10 s’ ¹ .

Table 11

From the analysis of the rheological properties of the compositions it has been found that the addition of probiotic bacteria to the gel composition does not substantially affect the viscosity of the formulation.

Example 7: DETERMINATION OF MUCOADHESIVENESS

The in vitro determination of the mucoadhesiveness of the GEL 6 formulation, prepared according to example 1 , was carried out using the inert inclined plane method in the presence of a mucin biofilm, compared to an aqueous solution. The test was carried out by sliding the sample on an inert support (steel) covered with a layer of 8% porcine gastric mucin. The adhesion on the mucin layer was evaluated in relation to the adhesion of the same sample on the inclined inert support not covered with mucin. The actual mucoadhesion is determined by the delay in the flow of the sample and in the detachment from the plane caused by the presence of mucin. In order to be considered mucoadhesive, a sample have to demonstrate an adhesion compared to the control without mucin significantly higher than that one demonstrated by water, which interacts with the protein for hygroscopic absorption only.

Two ml of each sample: Gel 6, Gel 6/mucin, water, and water/mucin were placed on the table and let to adhere for 2 minutes. The plane was inclined at 45° and at the end of the run the sample portion was collected and weighed.

The mucoadhesive values obtained are reported in Table 12.

Table 12

The results of the test carried out on GEL 6 (without probiotics) confirm the mucoadhesive properties of the gel, with a capacity of adhesion to mucin greater than 70%. This mucoadhesion value is reached after 10 minutes and remains constant for the entire time of the test (60 minutes).

Example 8: PREPARATION OF THE GEL COMPOSITION CONTAINING PROBIOTIC ON AN INDUSTRIAL SCALE

Caprylic/capric triglyceride and ethyl cellulose were placed in a mixer, the temperature was brought to 85-90° C until a homogeneous mixture was obtained. Glyceryl behenate, dimethyl silylated silica and the mixture of hydroxypropyl methylcellulose, (Benecell™ K4M and Benecell™ K100M) were added, and the homogeneous mass was brought to room temperature. Subsequently, blueberry and aloe extracts were added. The mixture was kept under stirring until a homogeneous and finely dispersed gel was obtained. Finally, lemon essential oil and freeze-dried probiotics are added.

The unitary composition of the two gels obtained is reported in Table 13.

Table 13 Example 9: DETERMINATION OF THE BACTERIA RELEASE FROM GELS

The amount of bacteria released from the gel compositions Gel 6A and 6D was evaluated in physiological solution at time intervals of 30 min, 2 hours, 5 hours and 8 hours as described in example 3. The results obtained for the Gel 6A formulation, including the probiotic strains L. rhamnosus and L. helveticus are shown in Table 14 and the results obtained for the GEL 6D formulation, including the probiotic strains L. rhamnosus and L. helveticus and L. paracasei are shown in Table 15.

Table 14 Table 15

Example 10 - DETERMINATION OF THE VIABILITY OF THE BACTERIA RELEASED FROM THE GEL IN SIMULATED SALIVARY

The ability of the probiotic strains released by the gel to remain viable for a prolonged time at the administration site, for example, the oral cavity, was evaluated by means of an in vitro test and by comparing the recovery of probiotic strains in MRD solution (Maximum Recovery Diluent) and in SS (artificial saliva). MRD (enzymatic digest of casein or peptone 1 g/L, sodium chloride 8.5 g/L) is a protective and isotonic diluent for microbiological examination indicated by the ISO 6887 standard, its composition promotes maximum recovery of the microorganisms present in an initial suspension and in decimal dilutions of test samples [Charteris W P. et al. J. Applied Microbiology, 1998, 759-768],

An amount equal to 1 gram of each of the two Gels 6A or Gel 6D was added to a volume of 99 ml of MRD, Biolife. The sample was shaken manually, or mechanically, to ensure a uniform distribution of microorganisms. One ml of the obtained suspension was transferred to a tube containing 9 ml of diluent for serial decimal dilutions. An aliquot of known volume of the initial suspension and its serial dilutions was plated on MRS agar plates (Man, Rogosa and Sharpe), selective medium used for the isolation and cultivation of Lactobacillus sp. from clinical samples, food and dairy products in accordance with ISO/FDIS 15214. The plates were incubated at 37 °C for 2 and 5 hours and then the colonies grown on the plates corresponding to the alive colonies released from the gel were counted. At the same time, the same procedure was carried out by adding an amount equal to 1 gram of each of the two 6A or 6D gels to a volume of 99 ml of artificial saliva (8.0 g/L sodium chloride, 2.38 g/L dibasic sodium phosphate, 0.19 g/L monobasic potassium phosphate, pH 6.8). The results of this test are shown in Table 16.

Table 16

The obtained data demonstrate that the bacteria of both associations of probiotics released from the gel maintain their viability up to 5 hours under the conditions that simulate the environment of the oral cavity suspended in the saliva solution at a level slightly lower than those considered to be the optimal conditions for the recovery of the growth of a microorganism after stressful conditions.

Example 11 - DETERMINATION OF THE VIABILITY OF THE BACTERIA RELEASED BY THE GEL ADHERING TO A SURFACE OF A NON-SPECIFIC NATURE

The viability of the probiotic bacteria released from the gel and adhered to a surface of a non-specific nature, such as filter paper, was evaluated using an amount equal to 1 gram of each of the two gels: Gel 6A and Gel 6D diluted respectively in a volume of 99 ml of MRD (Maximum Recovery Diluent) Biolife, (enzymatic digest of casein or peptone 1 g/L, sodium chloride 8.5 g/L) or in SS (artificial saliva) (8.0 g/L sodium chloride, 2.38 g/L dibasic sodium phosphate, 0.19 g/L monobasic potassium phosphate, pH 6.8). For each suspension, decimal serial dilutions were made as described in Example 8. An aliquot of known volume of each initial suspension and its serial dilutions was placed in contact with a sterile filter paper disk and coated with MRD or SS, depending on the dilution medium initially used, for 2 and 5 hours. At the end of the time the filter paper discs were removed with sterile tweezers and placed on an agar plate with growth medium where they were left and incubated at 37 °C for 72 hours. After incubation, the number of colonies grown on the plates deriving from the bacteria adhered to the filter paper and corresponding to the bacteria remained viable after non-specific adhesion was counted.

Table 17 shows the obtained values expressed as Log CFU.

Table 17

The data show that the viability of probiotic strains released from the gel into a liquid vehicle, such as saliva, or released and adhered to a surface, albeit non-specific, remains substantially the same for a period of time up to 5 hours.

Example 12 - DETERMINATION OF GEL ADHESION TO POLYCRYSTALLINE BIOCERAMIC

The adhesive property of the bacteria released from the gels was determined using a [3- tricalcium phosphate ([3-TCP) disc model; 3D, Biotek, according to what is described in Honglue T et al. Biomaterials 2012, 33: 365-77.

An amount equal to 1 gram of each preparation of Gel 6A or Gel 6D was diluted in MRD solution (Maximum Recovery Diluent, Biolife) and 1 gram was diluted in artificial saliva solution (8.0 g/L sodium chloride, 2.38 g/L dibasic sodium phosphate, 0.19 g/L monobasic potassium phosphate, pH 6.8), until a bacterial concentration of approximately 1x10 ⁶ CFU/ml is obtained for each diluted gel suspension. [3-TCP Discs were immersed for 2 and 5 hours in 1 ml of each diluted gel suspension, either in MRD or in SS, at 37 °C. After 2 and 5 hours the discs were recovered and placed in a tube containing 9 ml of diluent (MRD or SS) depending on the one initially used. The bacteria adhered to each disk were removed by immersing the tubes in an ultrasonic bath followed by a short vortexing to evenly distribute the bacteria. Starting from the obtained suspensions, serial decimal dilutions in same dilution medium (MRD or SS) were made and then they were plated on agar plates and incubated at 37 °C for 24 hours. In order to get a measure of the bacteria adhering to the surface of the [3-tricalcium phosphate disk the number of colonies grown on the plates was determined.

The percentage of adhesion of probiotics on the surface of the tricalcium [3-phosphate disk is calculated using the formula: P = ([1/ M) * 100 wherein:

P represents the percentage of adhesion of the mixture of bacteria to the [3-TCP disc 3D Biotek;

[i represents the number of alive bacteria of the analyzed gel bound to the [3-TCP disc 3D Biotek expressed as a logarithmic value;

M represents the number of alive bacteria of the analyzed gel expressed as a logarithmic value of the total suspension load at T = 0.

The results obtained are reported in Table 18.

Table 18

Example 13: DETERMINATION OF EFFICACY OF THE MUCOADHESIVE GEL COMPOSITION

The efficacy of the gel composition comprising the probiotic strains L. paracasei (LGM S- 26420), L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575) was determined in a mouse model of periodontitis induced by tooth ligation, as described by Abe T. and Hajishengallis G. in Optimization of the ligature-induced periodontitis model in mice, Journal of Immunological Methods 2013, 394, 49-54.

Sixty C57BI/6 male mice, 9-10 weeks of age, were fed ad libitum with sterile food and water. On Day "0" the second maxillary molar tooth of the mice, mildly anesthetized, with a combination of Tiletamine and Xylazine (respectively, 25 mg/kg and 4 mg/kg), was tied with a 5-0 non-absorbable silk surgical suture, to induce inflammation of the periodontium. The mice were divided into four groups of 15 animals per group:

Group 1 . Ligature of the molar. No-treatment

Group 2. Molar ligation + 6A Gel treatment [L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575)]

Group 3. Molar ligation + 6D Gel treatment [L. paracasei (LGM S-26420), L. rhamnosus SP1 (DSM 21690) and L. helveticus SP27 (DSM 29575)]

Group 4. Molar ligation + treatment with Gel 6 (No-probiotic bacteria)

Starting from Day “1” the mice of Groups 2-4, under anesthesia, were treated daily, 5 days a week, for three weeks, with the corresponding Gels. Gels were applied in the late afternoon. Subsequently, the animals were left without water for an hour, and then overnight fasted but with free access to water. Three animals per group were sacrificed on day "8", day "15" and day "22". The remaining six animals were sacrificed on day “29”, after one week without Gel treatment.

In the various samples obtained, the loss of alveolar bone was evaluated with respect to the height of the bone in the unbound counterpart, furthermore, the amount of the gingival myeloperoxidase (MPO) enzyme was determined. Traditionally, myeloperoxidase acts as a bactericide by catalyzing the reaction of superoxide ions O2 produced by polymorph-nucleated leukocytes during phagocytosis, creating cytotoxic compounds which, in addition to favor the killing of bacteria and of other pathogens, can perform a significant pro-inflammatory action, and directly contribute to tissue damage in the body. The increase in its presence in saliva is an indicator of the presence of inflammatory phenomena of the mucous membranes or gingival tissue. On all animals a general clinical evaluation was also performed, and the body weight was checked and the loss of the bonded tooth was observed. The latter data is shown in Table 19. Table 19

The results of the analysis of the variation in mm of the maxillary alveolar bone in animals in the treatment period of 28 days are shown in Figure 5.

Example 14: DETERMINATION OF THE STABILITY OF THE GEL COMPOSITION

The stability of the two gels was evaluated by determining the bacterial load detected in the two formulations stored at room temperature for three and six months. The data are reported in Table 20.

Table 20

The number of bacterial counts of the two formulations does not undergo alteration and remains substantially unchanged after six months.