ION SOURCE

TECHNOLOGICAL FIELD

The present disclosure concerns products for oral care.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

- US5,683,678

- US6,303, 125

- US6,843,993

- US2013/0089506

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Adhesion of bacterial species to each other (intraspecies) and to other bacterial species (intergeneric co-aggregation) as well as to host tissues and cells all of which lead to biofilm formation and hence contribute significantly to disease progression and pathology as, for example, in dental caries and plaque as well as in the persistence of Helicobacter pylori infection. It would therefore be useful to have compounds which can interrupt microbial adhesion and/or aggregation and or biofilm formation.

U.S. Patent No. 5,683,678 describes oral compositions containing cranberries or parts of cranberries such as cranberry extract for treating plaque/gingivitis and other periodontal diseases as well as dental calculus and carries. US Patent Nos. 6,303, 125 and 6,843,993 describe cranberry extract non- dialyzable material (NDM) having a molecular weight greater than or equal to 14,000Da, for use in oral hygiene compositions. The NDM is describes as exhibiting adhesion inhibitory activity against bacteria.

US patent application publication No. US2013/0089506 describes oral care compositions and the use of such oral care compositions for inhibiting co-aggregation of oral bacteria and inhibiting bacterial growth. The oral care compositions include cranberry extract non-dialyzable material (NDM) present in an amount effective to inhibit co-aggregation of oral bacteria and/or inhibit bacterial growth.

GENERAL DESCRIPTION

The present disclosure provides an oral care composition comprising, in an orally acceptable vehicle, (i) a fluoride ion source; and (ii) cranberry extract non- dialyzable material (NDM) or a phenolic functional analog of NDM, wherein said composition has an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone.

Further, the present disclosure provides A method of inhibiting bacterial biofilm formation in an oral cavity comprising applying to the oral cavity an oral care composition comprising (i) a fluoride ion source; and (ii) cranberry extract non- dialyzable material (NDM) or a phenolic functional analog of NDM, said composition exhibiting an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone.

Further provided by the present disclosure is a method of treating a subject's oral cavity, the method comprises applying to the oral cavity an oral care composition comprising (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, said composition exhibiting an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone.

Yet further, provided by the present disclosure is the use of (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM for the preparation of a composition, the composition being effective to inhibit bacterial biofilm formation in an oral cavity and/or to treat a subject's oral cavity.

Finally, provided by the present disclosure is a kit comprising (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, and instructions for use of same for the preparation of a composition for oral care.

The kit can comprise a single composition comprising said fluoride ion source; and said cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, or two compositions each comprising said fluoride ion source; or said cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM and instructions for use of the two compositions in combination.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a graph showing the effect of the tested compositions comprising fluoride, NDM or fluoride + NDM on % biofilm formation and on % bacterial growth according to one example of the present disclosure.

Fig. 2 is a graph showing the effect of the tested compositions comprising fluoride, NDM or fluoride + NDM on % biofilm formation and % bacterial growth according to another example of the present disclosure.

Fig. 3 is a graph showing the effect of the tested compositions comprising fluoride, PAC or fluoride + PAC on % biofilm formation and % bacterial growth.

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure provides oral compositions and methods of using the same for dental care. Also provided herein are kits for use in oral care.

In its broadest aspect, the present disclosure provides an oral care composition comprising, in an orally acceptable vehicle, (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM. In the following description reference is made in particular to the oral care composition. However, the following equally supports other aspects of the present disclosure including methods of using the components of the composition, and kits comprising the same. Specifically, embodiments encompassed by the present disclosure also include:

A method of inhibiting bacterial biofilm formation in an oral cavity comprising applying to the oral cavity an oral care composition comprising (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material ( DM) or a phenolic functional analog of NDM;

A method of treating a subject's oral cavity, the method comprises applying to the oral cavity an oral care composition comprising (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, wherein said combination has an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone;

Use of (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM for the preparation of an oral care composition, wherein said composition has an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone;

Use of (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, for the preparation of a composition for treating a subject's oral cavity, wherein said composition has an inhibitory effect on bacterial biofilm formation that is greater than the effect obtained by the sum of effects of each component when given alone;

A kit comprising in a single or separate compositions, (i) a fluoride ion source; and (ii) cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM, and (iii) instructions for use of said (i) and (ii) for the preparation of a composition for oral care and applying same onto a subject's oral cavity. When said fluoride ion source (i) and said cranberry extract non-dialyzable material (NDM) or a phenolic functional analog of NDM (ii) are provided in two separate compositions the kit's instructions comprise also use of same in combination. It has been surprisingly found by the inventors, as also exemplified herein below, that the combination of a low amount of fluoride ion source (e.g. lower than 0.05%w/w) and a low amount of DM (e.g. less than 0.001%w/w) or its examined functional analog PAC, provided an inhibitory effect of bacterial biofilm (co- aggregation) that is super-additive (synergistic) according to conventional determination methods for synergism. It has thus been concluded by the inventors that the combination disclosed herein has a synergistic effect, i.e. it is a synergistic combination.

For determining a synergistic effect one may use any quantitative method of evaluation or calculation known in the art. For example, to determine synergy in an in vitro assay any one of checkerboard, time-kill, and E test quantitative methods can be used [Roger LW et al. "Comparison of three different in vitro methods of detecting synergy: Time Kill, Checkerboard, and E tesf Antimicrobial, Agents and Chemotherapy 40(8) : 1914- 1918 ( 1996)] .

In some embodiments, synergy is determined by defining the Fractional Inhibitory Concentration Index (FICI, also known as the synergy factor, SF). FICI is calculated based on in vitro determination of Minimal Inhibitory Concentration (MIC) of a tested drug A or B in combination and alone, according to the following equation [See, for example, Roger LW et al. "Comparison of three different in vitro methods of detecting synergy: Time Kill, Checkerboard, and E tesf Antimicrobial, Agents and Chemotherapy 40(8) : 1914- 1918 ( 1996)] :

_ MIC (A + B) MIC (A + B)

F ^{ICI ~} MIC (A) ⁺ MIC(B)

A denotes fluoride ion;

B denotes NDM or its polyphenolic analogue

MIC (A+B) denotes the MIC οΐΑ+Β;

MIC(A) denotes the MIC A

MIC(B) denotes the MIC B

MIC can be determined by various ways. In some embodiments, MIC is determined by biofilm ELISA (reflecting the amount of bacteria in the biofilm), as described, for example, in the Experimental section hereinbelow. According to this equation and as commonly acceptable, 'synergy' is determined for FICI < 0.5; antagonism is determined for FICI > 4.0; and no interaction is determined for FICI > 0.5-4.0.

As provided in the following non-limiting Examples, the FICI for the combination of fluoride and NDM or its polyp henolic analogue, PAC, was below 0.5, even below 0.4. In some embodiments, the FICI for the combination of NMD and fluoride was below 0.25; and in yet some other embodiments, the FICI for the combination of NMD polyphenol analogue and fluoride was below 0.35.

Without being bound by theory, the results presented herein with respect to Fluoride+ PAC suggest that the synergistic effect is dependent on the phenol(s) present in cranberry juice.

In some embodiments, the amount of the fluoride is less than 0.01%w/w (about 3mM), at times, less than 0.0005% (about 0.125mM), less than 0.025%, or even less than 0.01%). In one embodiment, the amount of the fluoride is less than 0.05%>w/w.

In some embodiments, the amount of fluoride is between 0.0005%> to 0.1%> out of the total weight of the composition, at times, between 0.0005%> to 0.05%> out of the total weight of the composition.

The source of the fluoride ion can be of any kind known to be used for oral care, in particular as anti-carries agent. In some embodiments, the fluoride ion source is an orally acceptable particulated fluoride ion. In some embodiments, the fluoride ion source comprises monofluorophosphates.

Examples of monofluorophosphates include, without being limited thereto, potassium, sodium and ammonium fluorides, each representing a separate and independent embodiment. In some embodiments, the fluoride ion source comprises stannous fluoride. In some embodiments, the fluoride ion source comprises indium fluoride. In yet some other embodiments, the fluoride ion source comprises amine fluorides such as olaflur (N'-octadecyltrimethylendiamine-N,N,N'-tris(2-ethanol)- dihydrofluoride). The fluoride ion source can comprise any combination of the above.

Also NDM is known in the art. It was first described by Zafriri et al. [Zafriri, D., Ofek L, Adar R., Pocino M., Sharon, N. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eukaryotic cells. Anti Microb. Agents Chemothe. 33 :92-98, 1989] and by Weiss et al. [Weiss EI, Lev-Dor R, Kashman Y, Goldhar J, Sharon N, and Ofek I. Inhibiting interspecies co-aggregation of plaque bacteria with cranberry juice constituent (1998). JADA 129: 1719-1723] the content of which with respect to the cranberry juice extract or DM is incorporated herein by reference.

NDM is a high molecular weight material isolated from cranberry juice in order to avoid any effect of the low pH of cranberry juice on bacterial growth.

NDM can be obtained by dialysis of cranberry against distilled water in dialysis bags or tubes having a MW cut-off of >12,000 and the non-dialysable material (NDM) is collected and typically lyophilized and chilled until use.

NDM can be extracted from various cranberry varieties, all belonging to the species Vaccinium macrocarpon {Vaccinium oxycoccos).

In some embodiments, the NDM is enriched with total phenol. In this context, when referring to total phenol it is to be understood as denoting from a single type of phenol molecule, to a combination/mixture of several, chemically distinct, phenol molecules. In some embodiments, when referring to total phenol content it is to be understood as referring to a combination of two or more phenol molecules.

Further, in the context of the present description, when referring to total phenol it is to be understood as encompassing the total phenol content in NDM as well as the total phenol content in the phenolic functional analogs of NDM.

The term phenol is to be understood as any one or more compounds comprising a phenol in its chemical backbone, be it a compound containing a single phenol ring or containing several (chemically/covalently) chemically linked phenol rings. The phenol can be in a form of a monomer, oligomer or polymer.

In some embodiments, the phenol encompasses any one or combination of tannins. In some embodiments, the phenol encompasses tannins. In some embodiments, the tannins encompass any one or combination of anthocyanins and proanthocyanins.

In some embodiment, the NDM, or the total phenol comprises at least one phenolic oligomer or polymer and mixtures of same.

In some embodiments, the phenol comprises at least a phenol dimmer. In some embodiments, the phenol dimer is a proanthocyanidin (PAC). In some embodiments, the PAC is cranberry PAC.

Cranberry PACs have been found to be primarily composed of epicatechin units with a combination of 4β- 8 (B-type) direct carbon-carbon bond linkages (also found in apples, grape seed, and cacao), and linkage featuring both 4β- 8 and 2β- 0- 7 interflavanoid bonds (A-type) that has been associated with the anti-bacterial adhesion properties of cranberry [Foo, LY, Lu, Y, Howell, AB, Vorsa, N. A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P- fimbriated Escherichia coli. J Nat Prod, 2000. 63(9): 1225-28.].

In some embodiments, the PAC is proanthocyanidin A-2 (i.e. A-type) having the chemical structure:

In some embodiments, the phenolic analog is or comprises naturally occurring phenols. In yet some other embodiments, the phenolic analog is or comprises synthetic phenols.

The phenolic analog is functionally equivalent to NDM, i.e. having at least the bacterial co-aggregation inhibition activity exhibited for NDM. This can be determined, for example, in a test assay where an amount of NDM or of the tested analog are each incubated with a bacterial sample and the growth or biofilm formation of the bacteria is inhibited thereby under statistically acceptable criteria.

In some embodiments, the amount of the NDM or the phenolic analog is in the range of 50 -100 μg/ml. At times, the amount thereof is not more than 0.001 % out of the total composition.

The oral care composition disclosed herein was found to be effective in inhibiting bacterial biofilm. When referring to bacterial biofilm or co-aggregation it is to be understood as any phenomena associated with bacterial plaque formation or dental plaque deposition. The composition may act by inhibiting bacterial growth, inhibiting bacterial adhesion, reversing the interspecies binding of bacteria etc.

The oral care composition disclosed herein can comprise one or more further agents. Such additional agents may include one or more of: flavorants (flavouring agent), sweetening agent, mouth feel agent, colorant, whitening agent, abrasive, cleaning agent, adhesion agents, surfactant, foam modulator, pH modifying agent, humectant, , tartar control (anti-calculus) agent, saliva stimulating agent, nutrients and combinations thereof.

In some embodiments, the composition comprises any one or combination of such additional agents, as described, for instance, in PCT application publication No. WOl 1/162758, as transcribed below:

Flavorants- any material or mixture of materials operable to enhance the taste of the composition. Any orally acceptable natural or synthetic flavorant can be used, such as flavoring oils, flavoring aldehydes, esters, alcohols, similar materials, and combinations thereof. Without being limited thereto, the flavorants can be any one or combination of vanillin, sage, marjoram, parsley oil, spearmint oil, cinnamon oil, oil of wintergreen (methylsalicylate), peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, citrus oils, fruit oils and essences including those derived from lemon, orange, lime, grapefruit, apricot, banana, grape, apple, strawberry, cherry, pineapple, etc., bean- and nut-derived flavors such as coffee, cocoa, cola, peanut, almond, etc., adsorbed and encapsulated flavorants, and mixtures thereof.

At times, the flavorants can include materials that provide fragrance and/or other sensory effect in the mouth, including cooling or warming effects. Such ingredients include menthol, menthyl acetate, menthyl lactate, camphor, eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone, [a]-irisone, propenyl guaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine, N,2,3~trimethyl-2- isopropylbutanamide, 3-l-menthoxypropane-l,2-diol, cinnamaldehyde glycerol acetal (CGA), methone glycerol acetal (MGA), and mixtures thereof.

Sweetening agents- such as, without being limited thereto, dextrose, polydextrose, sucrose, maltose, dextrin, dried invert sugar, mannose, xylose, ribose, fructose, levulose, galactose, com syrup, partially hydrolyzed starch, hydrogenated starch hydrolysate, sorbitol, mannitol, xylitol, maltitol, isomalt, aspartame, neotame, saccharin and salts thereof, sucralose, dipeptide-based intense sweeteners, cyclamates, dihydrochalcones, and mixtures thereof.

Mouth-feel agents - materials imparting a desirable texture or other feeling during use of the composition. These may include, without being limited thereto, agglomerated silica particles that are designed to break down with agitation, such as SORBOSIL(R) BFG series, (e.g., BFG 10, BFG 50, BFG 100, etc.), CBT60S, CBT70, or AC33/43 silicates, commercially available from PQ Corporation, Valley Forge, Pennsylvania.

Colorants - any material imparting a particular luster or reflectivity such as pearling agents. In some embodiments, colorants are operable to provide a white or light-colored coating on a dental surface, to act as an indicator of locations on a dental surface that have been effectively contacted by the composition, and/or to modify appearance. Any orally acceptable colorant can be used, including FD and C dyes and pigments, talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silica, titanium dioxide, zinc oxide, red, yellow, brown and black iron oxides, ferric ammonium ferrocyanide, manganese violet, ultramarine, titaniated mica, bismuth oxychloride, and mixtures thereof.

Abrasives - also known as polishing agents known to be used for oral use, i.e. that do not excessively abraded tooth enamel during normal use of the composition. Non-limiting examples of abrasives include silica, for example in the form of precipitated silica or as admixed with alumina, insoluble phosphates, calcium carbonate, and mixtures thereof. Among insoluble phosphates useful as abrasives are orthophosphates, polymetaphosphates and pyrophosphates. Illustrative examples are dicalcium orthophosphate dihydrate, calcium pyrophosphate, calcium pyrophosphate, tricalcium phosphate, calcium polymetaphosphate and insoluble sodium polymetaphosphate.

Tartar control (anticalculus) agents - agents including their salts, for example, the alkali metal and ammonium salts of phosphates and polyphosphates (for example pyrophosphates), polyaminopropanesulfonic acid (AMPS), polyolefin sulfonates, polyolefin phosphates, diphosphonates such as azacycloalkane-2,2- diphosphonates (e.g., azacycloheptane-2,2-diphosphonic acid), N-methyl azacyclopentane- 2,3- diphosphonic acid, ethane- 1 -hydroxy- 1, 1-diphosphonic acid (EUDP) and ethane- 1 - amino- 1, 1 -diphosphonate, phosphonoalkane carboxylic acids and. Useful inorganic phosphate and polyphosphate salts include monobasic, dibasic and tribasic sodium phosphates, sodium tripolyphosphate, tetrapolyphosphate, mono-, di-, tri- and tetrasodium pyrophosphates, sodium trimetaphosphate, sodium hexametaphosphate and mixtures thereof.

Saliva stimulating agent - agent for causing wetting of dry mouth, including, for example, food acids such as citric, lactic, malic, succinic, ascorbic, adipic, fumaric and tartaric acids, and mixtures thereof. One or more saliva stimulating agents are optionally present in saliva stimulating effective total amount.

Nutrients - including vitamins, minerals, amino acids, and mixtures thereof. Vitamins include Vitamins C and D, thiamine, riboflavin, calcium pantothenate, niacin, folic acid, nicotinamide, pyridoxine, cyanocobalamin, para-aminobenzoic acid, bioflavonoids, and mixtures thereof. Nutritional supplements include amino acids (such as L-tryptophane, L- lysine, methionine, threonine, levocaraitine and L-carnitine), lipotropics (such as choline, inositol, betaine, and linoleic acid), and mixtures thereof.

The oral care composition disclosed herein is for oral treatment. As such, it is in a form suitable for being retained in the oral cavity for a time duration sufficient to achieve the desired effect and then be removed from the cavity.

In some embodiments, the oral care composition is in a form suitable for application onto the teeth. This includes, without being limited thereto, liquid, gel and paste.

In some embodiments, the oral care composition was found effective for inhibiting biofilm adhesion and thereby plaque formation or in other words, for inhibiting of bacterial co-aggregation. In this respect, there is also disclosed herein a method for oral care, namely, for treating a subjects oral cavity. To this end, the oral composition is applied to the oral cavity.

The oral composition can be applied once, twice, thrice or more times a day, for a single day, for a period of several days, or every day (consecutive days or with intervals) to achieve the desired oral effect.

As noted above, the oral composition can be in a form of a gel, paste, mouthwash etc. Accordingly, the manner of application will depend on its form, e.g. brushing when in a form of a paste, washing when in liquid form, etc. The method of application should not be a limiting factor in performing the invention as numerous products for mouth care are known and any end user will easily understand the possibilities of using the composition disclosed herein.

NON-LIMITING EXAMPLES

Preparation of cranberry materials

Non-dialyzable material ( DM) was prepared from concentrated cranberry juice (50Brix, Ocean Spray Cranberries Inc., Lackeville-Middleboro Mass. 02349) as described previously [Zafriri, D., Ofek L, Adar R., Pocino M., Sharon, N. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Anti Microb. Agents Chemothe. 33 :92-98, 1989; Weiss EI, Lev-Dor R, Kashman Y, Goldhar J, Sharon N, and Ofek I. Inhibiting interspecies coaggregation of plaque bacteria with cranberry juice constituent (1998). JADA 129: 1719-1723]. Briefly, concentrated cranberry juice (CCJ) was dialyzed extensively against distilled water (>10x the volume of CCJ) and six changes for six days using dialysis tubes of 12- 14 KDa MW cut-off. The non-dialyzable material was collected, lyophilized and chilled until use (about 6g NDM from one liter CCJ).

Preparation of NDM Fluoride composition

Non-dialyzable material (NDM) was obtained from the cranberry juice concentrate (50Brix, Ocean Spray) as described previously [Zafriri, D., Ofek L, Adar R., Pocino M., Sharon, N. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Anti Microb. Agents Chemothe. 33 :92-98, 1989).

Briefly, The cranberry concentrated juice was dialyzed extensively against distilled water (>5xthe volume of concentrated juice, and five changes for six days) in dialysis tubes of MW cut-off of >12,000 and the nondialysable material was collected, lyophylized and chilled until use (about 6g NDM from one liter of concentrated juice). For the experiments NDM was dilluted in Trypticase Soy Broth (TSB; Difco Laboratories, Detroit, MI) to a concentration of 0.5 mg/ml from which 0.02 ml was added to 0.2 ml of the reaction mixture containing the TSB broth and NDM and into 96 microtiter wells to obtain final concentration of 0.05 mg/ml of NDM (see below). For testing effect of Protoanthocyanin (PAC), PAC powder was dissolved in TSB to a concentration of 3mg/ml from which 0.02 ml was added to 0.2 ml reaction mixture containing the TSB broth and PAC and into 96 microtiter wells to obtain final concentration of 0.3 mg/ml of PAC (see below).

Estimation of biofilm formation by bacteria in presence of inhibitors

The bacterial strain used for determining inhibition of coagulation is the Streptococcus mutans UA159 (S. mutans UA159), was employed as described by Nassar et al. [Nassar HM, Li M, Gregory RL (2012) Effect of honey on Streptococcus mutans growth and biofilm formation. Appl Environ Microbiol 78:536-540].

Briefly, as described, the bacteria, Streptococcus mutans UA159, [Ajdic ^' D, et al. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. U. S. A. 99: 14434-14439] were grown in TSB at 37°C for 18 h under anaerobic conditions (GasPack Anaerobic System; BBL). The bacterial cultures were then diluted in fresh broth medium (TSB + sucrose 2%) to obtain an optical density at 655 nm (OD ₆ 55) of 0.01.

Equal volumes (100 μΐ) of the diluted bacteria and concentrations of NDM, Sodium Fluoride (NaF, Sigma) or Protoanthocyanin (PAC, Synthetase, France), in TSB medium at the concentrations indicated in Tables 1A-1C were mixed into (triplicate) wells of 96-well plates (Thermo Scientific, Waltham, MA, USA). As negative control, wells with bacteria and no composition were used. Control wells with no bacteria were also prepared and considered as the 100% plaque formation.

Table 1A: Composition comprising NDN (50μg/ml) and NaF (0 125mM)

Composition NDM NaF

1 - -

2 - 0.125mM

3 50μg/ml -

4 50μg/ml 0.125mM (0.0005%v/v) Table IB: Composition comprising NDN (lOC^g/ml) and NaF (3mM)

Following incubation for 24h at 37°C, 5% C0 ₂ , bacterial growth was recorded by measuring the OD ₆ 55 using a micro-plate reader.

Biofilm formation was determined by first aspirating the supernatants from the wells followed by three washes with PBS to remove planktonic and loosely adherent bacteria. The bacterial mass in the biofilm adherent to the bottom of the weils was stained with 200 μΐ of Gram's crystal violet for 45 min. The wells were then rinsed with water and dried. The amount, of biofilm biomass was quantified by de-staining the wells with 200 μ! of 33% acetic acid and then measuring the absorhance of the crystal violet solution in a microplate spectrophotometer set at 595 ran.

Results are presented in Figures 1 to 3 as % of control and in Table 2 as % inhibition values where in the Control group no inhibitors were added, i.e. 0% (the % representing the amount out of no inhibition/no treatment group). The results show that the biofilm formation was significantly inhibited by the combination disclosed. The synergism was determined as FICI which was calculated from the ELISA readings representing amount of biofilm bacteria remaining on the bottom of microtiter plate in the presence of the text composition and is presented in the following Table 2.

As described hereinabove, the FICI is calculated from (the amount of biofilm formation for the combined composition divided by the amount of biofilm formation with fluoride only) + (the amount of biofilm formation for the combined composition divided by the amount of biofilm formation with DM/PAC only) this being in line with the equation.

The values of MIC(A), MIC(B) and MIC(A+B) were determined by ELISA (See also description hereinabove regarding calculation of FICI (SF)).

Table 2 shows that with the combined composition the FICI was below 0.5, meaning that these combinations are synergistic.

Table 2 - Inhibition of biofilm formation

Composition NDM NaF ELISA % of Control FICI (SF)

No. (MIC) (% inhibition)

1 (Control) - - 0.7015 100% (0%)

2 - 0.125mM 0.4192 60% (40%)

3 5(^g/ml - 1.124 100% (0%)

4 5(^g/ml 0.125mM 0.054 8% (92%) 0.17

5 - 3.0mM 0.2802 40% (60%)

6 10(^g/ml - 0.9623 100% (0%)

7 10(^g/ml 3.0mM 0.0477 9% (91%) 0.22

8 30(^g/ml - 0.573 80% (20%)

9 30(^g/ml 3.0mM 0.062 9% (91%) 0.33