WO2020141201A1

WO2020141201A1 - Antibacterial oral compositions

Info

Publication number: WO2020141201A1
Application number: PCT/EP2020/050044
Authority: WO
Inventors: Cleo Fabbro GUAETTA; Andrea Zanardi; Elena GELFI
Original assignee: Rottapharm Spa
Priority date: 2019-01-02
Filing date: 2020-01-02
Publication date: 2020-07-09
Also published as: EP3906011A1; GB201900031D0

Abstract

A liquid oral care composition comprising: • a galactooligosaccharide; and • xylitol; for use in the treatment or prophylaxis of an oral bacterial infection.

Description

ANTIBACTERIAL ORAL COMPOSITIONS

This invention relates to a liquid oral care composition comprising a galactooligosaccharide and xylitol which is effective in preventing or reducing symptoms associated with halitosis and cavities.

BACKGROUND OF THE INVENTION

Oral hygiene is an important consideration in the daily life of children and adults around the globe. The maintenance of good oral hygiene is vital to keep the mouth, teeth and gums free from disease, and to prevent the growth of bacteria which can lead to the development of halitosis (bad breath) and also to dental caries (tooth decay).

Bacteria, in particular Fusobactehum nucleatum and Porphyromonas gingivalis, located in periodontal pockets and at the back of the tongue break down sulfur-containing amino acids, in particular cysteine and methionine, to produce the volatile sulfur compounds, including hydrogen sulfide and methyl mercaptan.

These volatile sulfur compounds have an extremely unpleasant odor, even at low concentrations, and also penetrate the epithelium and cause damage to cells of the underlying tissue. It has also been suggested that volatile sulfur compounds produced by bacteria located within periodontal pockets may be an important factor in the development of periodontal disease.

Bacteria, in particular Streptococcus mutans, are also responsible for dental caries (tooth decay or cavities). Streptococcus mutans metabolises sucrose to lactic acid creating an acidic environment in the mouth that can cause tooth enamel to be vulnerable to decay.

Therefore, antibacterial agents are often used in preventive dentistry to reduce the levels of these bacteria and thereby prevent or reduce symptoms associated with halitosis and cavities, and it is an object of the present invention to provide a liquid oral care composition which is effective in preventing or reducing these symptoms.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a liquid oral care composition comprising:

• a galactooligosaccharide; and

• xylitol; for use in the treatment or prophylaxis of an oral bacterial infection.

In a second aspect, the invention provides an oral care composition comprising:

• a galactooligosaccharide; and

• xylitol; for use in the treatment or prophylaxis of halitosis or dental caries.

In a third aspect, the invention provides an oral care composition which is a mouthwash, mouthrinse or spray comprising:

• a galactooligosaccharide; and

In a fourth aspect, the invention provides an oral care composition which is a mouthwash, mouthrinse or spray comprising:

• a galactooligosaccharide; and

Surprisingly, the applicant has found that a formulation comprising galactooligosaccharide and xylitol has a beneficial effect on deleterious bacteria in the mouth and therefore is useful for the treatment or prophylaxis of oral bacterial infections, in particular halitosis and dental caries.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an oral care composition comprising:

• a galactooligosaccharide; and

• xylitol; for use in the treatment or prophylaxis of (i) an oral bacterial infection or (ii) halitosis or dental caries.

Galactooligosaccharide

The topical formulation includes a galactooligosaccharide. For the avoidance of doubt, the term galactooligosaccharide includes mixtures (i.e. one or more) galactooligosaccharides, and formulations comprising mixtures of galactooligosaccharides are covered by the invention.

Galactooligosaccharides belong to the group of prebiotics, which are non-digestible food ingredients that beneficially affect the host by stimulating the growth and/or the activity of beneficial bacteria. Galactooligosaccharide is present in commercially available products such as food for both infants and adults. Galactooligosaccharides are produced through the enzymatic conversion of lactose or from a botanical source, and its composition of the galactooligosaccharide fraction varies in chain length and type of linkage between the monomer units. Galactooligosaccharides are oligomers or polymers of galactose molecules ending mainly with a glucose or sometimes ending with a galactose molecule and have varying degrees of polymerization and types of linkages. In one embodiment, galactooligosaccharide comprises galactose and glucose molecules. In another embodiment, galactooligosaccharide comprises only galactose molecules. In a further embodiment, galactooligosaccharide are galactose-containing oligosaccharides of the form of [p-D-Gal-(1 -6)]n-p-D-Gal-(1 -4)-D-Glc wherein n is 2-20. In another embodiment, the galactooligosaccharide are galactose-containing oligosaccharides of the form Glc-a-1 -4-[p-Gal1 -6]_n where n=2-20. In another embodiment, the galactooligosaccharides are in the form of a-D-Glc(1 -4)-[p- D-Gal-(1 -6)]n, where n=2-20. Gal is a galactopyranose unit and Glc (or Glu) is a glucopyranose unit.

Galactooligosaccharides are found in human and bovine maternal milk. Galactooligosaccharides can be produced from lactose syrup using the transgalactosylase activity of the enzyme b-galactosidase (Crittenden, (1999) Probiotics: A Critical Review. Tannock, G. (ed) Horizon Scientific Press, Wymondham, pp. 141 -156). b-D-galactosidase is known to catalyze not only the hydrolysis of the b-D- galactoside linkage of lactose to give D-glucose and D-galactose but also to carry out transgalactosylation reactions where the D-galactosyl group of a b-D-galactoside is transferred onto a hydroxylated acceptor. For example, when a b-D-galactoside such as lactose or another carbohydrate is present, it is possible to obtain new glycoside linkages between the D-galactose unit and the acceptor. The starting galactoside such as lactose can also be present in a galactooligosaccharide mixture following the transgalactosylation reactions.

As used herein, galactooligosaccharide comprises one or more saccharides that have been produced from a glycoside and the transgalactosylation reaction of a b-galactosidase. Thus, galactooligosaccharides include saccharides such as transgalactosylated oligosaccharides (i.e. transgalacto-oligosaccharides) or transgalactosylate disaccharides. The degrees of polymerization (DP) of the formed oligosaccharide can vary, typically from 2-20, depending on the enzyme source. In one embodiment, a galactooligosaccharides composition is a blend of one more saccharides with a DP range of 2-6 (i.e. di- through hexasaccharides).

In another embodiment, a galactooligosaccharides composition is a blend of one or more saccharides with a DP range of 2-8 (i.e. di- through octa-saccharides). In another embodiment, a galactooligosaccharide composition is a blend of one or more saccharides with a DP range of greater than 8. In yet another embodiment, a galactooligosaccharides composition is a blend of one or more saccharides with a DP range of 9-15. In another embodiment, a galactooligosaccharides composition is a blend of one or more saccharides with a DP of 1 , a DP range of 2-6, a DP range of 6-8, and DP range of greater than 8.

In one embodiment, the oral care composition comprises 0.01 % to 10% w/w galactooligosaccharide, for example 0.01 % to 5% w/w galactooligosaccharide. Alternatively, the composition comprises 0.1 % to 5% w/w galactooligosaccharide or 0.5% to 5% w/w galactooligosaccharide. In a preferred embodiment, the oral care composition comprises 0.01 % to 5% w/w galactooligosaccharide.

Xylitol

The oral care composition includes xylitol. Xylitol is a naturally occurring sugar alcohol used as a sweetener. It is found in low concentrations in the fibers of many fruits and vegetables and can be extracted from various berries, oats, and mushrooms, as well as fibrous material such as cornhusks and sugar cane bagasse.

In one embodiment, the oral care composition comprises 0.01 % to 10% w/w, in particular 0.01 % to 5% w/w xylitol. Alternatively, the oral care composition comprises 0.1 % to 5% w/w xylitol or 0.5% to 5% w/w xylitol.

In a preferred embodiment, the oral care composition comprises 0.01 % to 5% w/w xylitol.

In one embodiment, the oral care composition comprises a weight ratio of galactooligosaccharide:xylitol of from 10:1 to 1 :10, from 8:1 to 1 :8, from 5:1 to 1 :5, or from 3:1 to 1 :3.

In a preferred embodiment, the oral care composition comprises a weight ratio of galactooligosaccharide:xylitol of from 2:1 to 1 :2.

Uses of the invention

The applicant has surprisingly discovered that the oral care compositions of the invention have antibacterial effect against and reduce biofilm formation by Fusobactehum nucleatum, Porphyromonas gingivalis and Streptococcus mutans.

Fusobacterium nucleatum and Porphyromonas gingivalis, located in periodontal pockets and at the back of the tongue break down sulfur-containing amino acids, in particular cysteine and methionine, to produce volatile sulfur compounds, including hydrogen sulfide and methyl mercaptan. These volatile sulfur compounds have an extremely unpleasant odor, even at low concentrations, and also penetrate the epithelium and cause damage to cells of the underlying tissue. It has also been suggested that volatile sulfur compounds produced by bacteria located within periodontal pockets may be an important factor in the development of periodontal disease.

Other bacteria, in particular Streptococcus mutans, are responsible for dental caries (tooth decay or cavities). Streptococcus mutans metabolises sucrose to give lactic acid, which creates an acidic environment in the mouth that can cause tooth enamel to be vulnerable to decay.

Accordingly, the oral care compositions are for use in therapy for the treatment or prophylaxis of an oral bacterial infection, halitosis or dental caries, in particular by topical application to the oral cavity i.e. by oral topical application. In one embodiment, the invention provides a liquid oral care composition comprising:

• a galactooligosaccharide; and

In particular, the oral bacterial infection is associated with or caused by Fusobactehum nucleatum or Porphyromonas gingivalis.

In one embodiment, the invention provides a liquid oral care composition comprising:

• a galactooligosaccharide; and

In particular, the halitosis or dental caries is associated with or caused by Streptococcus mutans.

• a galactooligosaccharide; and

• xylitol; for use in therapy by topical application to the oral cavity, for example for the treatment or prophylaxis of an oral bacterial infection, halitosis or dental caries.

In a further embodiment, the invention provides a liquid oral care composition which is a mouthwash, mouthrinse or a spray, comprising:

• a galactooligosaccharide; and

• xylitol.

In a further aspect, the invention provides a liquid oral care composition comprising a galactooligosaccharide, for example 0.01 % to 10% w/w galactooligosaccharide, 0.01 % to 5% w/w galactooligosaccharide, 0.1 % to 5% w/w galactooligosaccharide or 0.5% to 5% w/w galactooligosaccharide, for use in the treatment or prophylaxis of an oral bacterial infection halitosis or dental caries.

In a further embodiment, the invention provides an oral care composition which is a mouthwash, mouthrinse or a spray, comprising a galactooligosaccharide, for example 0.01 % to 10% w/w galactooligosaccharide, 0.01 % to 5% w/w galactooligosaccharide, 0.1 % to 5% w/w galactooligosaccharide or 0.5% to 5% w/w galactooligosaccharide. In one embodiment, the invention provides a method for the treatment or prophylaxis of an oral bacterial infection, halitosis or dental caries, comprising the topical application of an effective amount of a liquid oral care composition comprising a galactooligosaccharide and xylitol to the oral cavity of a patient in need thereof.

In another embodiment, the invention provides the use of a galactooligosaccharide and xylitol for the manufacture of a medicament for use in the treatment or prophylaxis an oral bacterial infection, halitosis (i.e. neutralising bad breath) or dental caries.

The invention provides the use of a topical formulation of the invention for preventing the growth of Fusobactehum nucleatum, Porphyromonas gingivalis, and Streptococcus mutans, in particular in the oral cavity of a human or animal.

Again without being bound by theory, it is thought that the topical formulations of the invention prevent the formation of a biofilm of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans or remove such a biofilm once formed, in the oral cavity of a human or animal.

Accordingly, the invention provides the use of an oral composition of the invention for preventing the formation of a biofilm of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans or removing such a biofilm once formed, in the oral cavity of a human or animal.

Other uses of the compositions of the invention described herein, in addition to the use forthe treatment of an oral bacterial infection, halitosis or dental caries, are as follows.

The liquid oral care composition comprising a galactooligosaccharide and xylitol as defined herein, including any sub-embodiments thereof, for use in a therapeutic or cosmetic method for:

• cleaning the teeth;

• reducing plaque;

• reducing gingivitis,

• reducing dentinal hypersensitivity;

• removing or preventing stains;

• reducing, inhibiting or repairing dental enamel erosion;

• promoting remineralization of dental enamel; and

• improving the whiteness of the teeth.

The compositions

In one embodiment, the oral care composition is a liquid. In one embodiment, the oral care composition is a mouthwash, mouthrinse or a spray. In particular, the liquid oral care composition is a mouthwash, for example an aqueous mouthwash. Other components

The oral care composition generally includes water. In addition, the oral care composition includes other additives and adjuvants commonly found in oral care compositions, for example water, fluoride, a masking agent, a humectant, a flavouring agent, an emulsifying agent, and a buffering agent.

Water

Typically, water (i.e. demineralised water) is present as the balance of the composition. In other words, the composition is an aqueous oral care composition.

Fluoride

In one embodiment, the oral care composition includes one or more fluoride salts.

In one embodiment, the fluoride salt is selected from stannous fluoride, sodium fluoride, potassium fluoride, potassium monofluorophosphate, sodium monoflourophosphate, ammonium monoflourophosphate, sodium flourosilicate, ammonium flourosilicate, amine fluorides such ammonium fluoride, and combinations thereof.

In one embodiment, the oral care composition includes an amount of a fluoride salt such that the composition includes from 50 to 5000 ppm, 100 to 1000 ppm fluoride ions, 200 to 500 ppm fluoride ions e.g. about 250 ppm fluoride ions.

In one embodiment, the oral care composition comprises 0.01 to 1 % w/w, 0.01 to 0.1 % w/w, or 0.02 to 0.07% w/w e.g. 0.01 % w/w fluoride salt.

Masking agent

In one embodiment, the oral care composition includes a masking agent, such as tannic acid, glycyrrhizin derivatives, or acesulfame e.g. acesulfame potassium. The masking agent is present in an amount sufficient to reduce the aftertaste of any minerals present in the oral care preparation. In particular, when present, the composition includes 0.01 to 0.1 % w/w masking agent, for example 0.01 to 0.05% w/w masking agent.

Humectant

In one embodiment, the oral care composition includes one or more humectants, such as hydrogenated starch hydrolysate, glycerin, and sorbitol. The humectant is present in an amount sufficient to increase the viscosity and provide a certain mouth feel to the oral care composition. In one embodiment, the composition includes 1 to 10% w/w humectant, for example 5 to 10% w/w e.g. 7.50% w/w of humectant. Flavouring agent

In one embodiment, the oral care composition includes one or more flavouring agents, such as peppermint, spearmint, cinnamon, wintergreen oils, menthol, or methyl salicylate derivatives. Other flavouring agents may be used individually or in combination. In one embodiment, the composition includes 0.01 to 1 % w/w e.g. 0.2 to 0.5% w/w of a flavouring agent.

Emulsifying agent

In one embodiment, the oral care composition includes one or more emulsifying agents, such as PEG- 40 hydrogenated castor oil, or polysorbate-20. In one embodiment, the composition includes 0.1 to 10% w/w e.g. 1 % w/w of a flavouring agent.

Buffering agent

In another embodiment, the oral care composition includes a buffering agent, such as citric acid, and benzoic acid. In one embodiment, the oral care composition includes a buffering agent in an amount such that the pH of the oral care preparation is from 5 to 8, for example from 5 to 7, and in particular from 5 to 6.

Other components

A variety of additives and adjuvants can be included to make the oral care composition more amenable for use in a particular end-use application without negatively affecting its efficacy or the stability of chlorhexidine in a substantial manner. Examples include, but are not limited to, emollients, fragrances, pigments, dyes, flavors, abrasives, bleaching agents, preservatives, antioxidants, and the like.

Other components

EXAMPLES

The invention will now be illustrated by reference to the following examples.

Example 1 : Exemplary formulation

An example of a mouthwash according to the invention is as follows (Formulation 1)

It will be appreciated that the above composition is only an example, and the invention is not limited to only such compositions.

The formulations according to the invention are stable, and exhibit good physical and chemical stability over time, even at a temperature above ambient temperature. Example 2: Activity against periodontal and cariogenic pathogens

The aim of the present study is to get information on the in vitro effect of a new mouthwash formulation based on galactooligosaccharide and xylitol against periodontal pathogens (Fusobacterium nucleatum, Porphyromonas gingivalis) and cariogenic pathogens (Streptococcus mutans).

The following formulations were tested: Mouthwash 1

Antibacterial agents: Chlorhexidine diacetate (0.025% w/w)

Other ingredients: Water (77.565 w/w%), zinc acetate (0.3 w/w%), potassium acesulfame (0.03 w/w%), sodium fluoride (0.05 w/w%), hydrogen starch hydrolysate (7.5 w/w%), alcohol (1.2 w/w%), aroma (0.8 w/w%), PEG-40 hydrogenated castor oil (2.4 w/w%), glycerin (10.1 w/w%) and citric acid (0.03 w/w%). Mouthwash 3

Antibacterial agents: Galactooligosaccharide (2% w/w) and xylitol 2% (w/w)

Other ingredients: Water (74.150 w/w%), zinc acetate (0.3 w/w%), potassium acesulfame (0.03 w/w%), sodium fluoride (0.05 w/w%), hydrogen starch hydrolysate (7.5 w/w%), aroma (0.6 w/w%), PEG-40 hydrogenated castor oil (2.4 w/w%), glycerin (10.1 w/w%), sodium metabisulfite (0.15 w/w%), benzyl alcohol (0.5 w/w%) and citric acid (0.02 w/w%).

Bacterial strains

Fusobactehum nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277) and Streptococcus mutans (CH 02) reference clinical strain were used. Fusobacterium nucleatum and Porphyromonas gingivalis were cultured overnight in trypticase soy broth (TSB) with hemin and vitamin K at 37°C for 48-72h and Streptococcus mutans was cultured overnight in TSB with 1 % sucrose at 37°C for 24-48h, under anaerobic atmospheres. After incubation, all broth cultures were adjusted to 0.5 McFarland. The strains were stored at -80°C in microbanks.

The effect on planktonic phase: Colony-forming unit (CFU) determination

The CFU determination was performed to evaluate the effect of the compositions on the bacterial planktonic phase (i.e. the single-cells in the liquid medium).

Afterthe contact of the broth cultures with either the formulations (1 :1 v/v) orthe saline solution (control) for a particular time period under anaerobic conditions at 37 °C, serial ten-fold dilutions were performed and were spread on Anaerobe Basal Agar (ABA) with 5% sheep blood for Fusobacterium nucleatum and Porphyromonas gingivalis, and on Tripticase Soy Agar (TSA) with 5% sheep blood for Streptococcus mutans.

After incubation under anaerobic condition at 37°C for 48-72h and for 24-48h respectively, the number of CFU/ml were recorded to detect the bacterial count. Each measurement was be performed in triplicate.

The efficacy of the compositions was measured by determining the % log reduction in CFU/ml and the % reduction in CFU/ml:

%log reduction (CFU/ml) = 100— \ og[CFU /ml]/\og[CFU (control) /ml] ]x 100 % reduction (CFU/ml) = 100— [[ CFU/ml]/[CFU(control)/ml ] ] x 100

The effect on microbial biofilm formation

The efficacy of the compositions on biofilm formation was evaluated by determining biomass production. Standardized bacterial suspensions grown in TSB with hemin and vitamin K (for Fusobacterium nucleatum and Porphyromonas gingivalis) and with 1 % sucrose (for Streptococcus mutans) were incubated on 96-wells flat-bottomed microtiter plates at 37°C for 48-72h under anaerobic conditions.

After incubation, each well was washed twice with sterile PBS (pH 7.4), dried, stained for 1 min with 0.1 % crystal violet, and eluted with ethanol. The biomass was measured by spectrophotometer using an enzyme-linked-immune adsorbent assay (ELISA) reader OD570. Each measurement was performed in triplicate.

Statistical analysis

The significance of the results relative to the controls was evaluated using Student’s t-test.

Periodontal pathogens The amount of bacteria was measured in terms of Colony-Forming Unit i.e. CFU/ml after treatment with formulations 1 and 3, and the amount of biofilm reduction was determined relative to the control.

All data show statistical significance i.e p<0.01 reductions.

Porphyromonas gingivalis

Fusobacterium nucleatum

Cariogenic pathogens The amount of bacteria was measured in terms of Colony-Forming Unit i.e. CFU/ml after treatment with formulations 1 and 3, and the amount of biofilm reduction was determined relative to the control.

All data show statistical significance i.e p<0.01 reductions.

Streptococcus mutans

The amount of bacteria was measured in terms of Colony-Forming Unit i.e. CFU/ml after treatment with formulations 1 and 3, and the amount of biofilm reduction was determined relative to the control.

Discussion

The mouthwash comprising galactooligosaccharide and xylitol provides a comparable action against to that obtained with the leading composition comprising chlorhexidine diacetate and zinc zcetate on periodontal and cariogenic pathogens and against planktonic and sessile phases.

Claims

1 . A liquid oral care composition comprising:

• a galactooligosaccharide; and

2. A liquid oral care composition comprising:

• a galactooligosaccharide; and

3. The liquid oral care composition for use as claimed in claim 1 or 2, wherein the formulation comprises 0.01 % to 10% w/w xylitol.

4. The liquid oral care composition for use as claimed in any one of claims 1 to 3, wherein the formulation comprises 0.01 % to 5% w/w xylitol.

5. The liquid oral care composition for use as claimed in any one of claims 1 to 4, wherein the formulation comprises 0.01 % to 10% w/w galactooligosaccharide.

6. The liquid oral care composition for use as claimed in any one of claims 1 to 5, wherein the formulation comprises 0.01 % to 5% w/w galactooligosaccharide.

7. The liquid oral care composition for use as claimed in any one of claims 1 to 6, wherein the formulation comprises a weight ratio of galactooligosaccharide:xylitol of from 10:1 to 1 :10.

8. The liquid oral care composition for use as claimed in any one of claims 1 to 7 wherein the formulation comprises a weight ratio of galactooligosaccharide:xylitol of from 2:1 to 1 :2.

9. The liquid oral care composition for use as claimed in any one of claims 1 to 8, wherein the composition is mouthwash, mouthrinse or a spray.

10. The oral care composition for use as claimed in any one of claims 1 to 9, wherein the formulation is an aqueous mouthwash.

1 1 . A liquid oral care composition comprising:

• a galactooligosaccharide; and

• xylitol; for use in therapy by topical application to the oral cavity.

12. The liquid oral care composition for use as claimed in any one of claims 9 to 1 1 , wherein the formulation is for the treatment or prophylaxis of an oral bacterial infection, halitosis or dental caries.

13. The liquid oral care composition for use as claimed in claim 12, wherein the oral bacterial infection, halitosis or dental caries is associated with Fusobactehum nucleatum, Porphyromonas gingiva!is, or Streptococcus mutans.

14. The use of a liquid oral care composition as defined in any one of claims 1 to 10 as a cosmetic.