INHIBITION OF MATURATION OF DENTAL BIOFILM AND CARIOGENIC PROPERTIES

Abstract

The present invention relates to a small molecule inhibitor for use in the prevention of maturation of dental plaque biofilms. A further application may be the reduction of the risk for diseases associated with functions that derive from matured dental plaque biofilms. The invention also relates to the use of an effective amount of a small molecule inhibitor in a cosmetic product for the prevention of maturation of dental biofilms. The present invention also relates to oral compositions comprising an effective amount of a small molecule inhibitor suitable for the prevention of maturation of dental plaque biofilms. The present invention further relates to a device for application of oral compositions further comprising an oral composition comprising an effective amount of a small molecule inhibitor suitable for the prevention of maturation of dental plaque biofilms.

Claims

1. A small molecule inhibitor for use in the prevention of maturation of dental plaque biofilms and/or the reduction of the risk for diseases associated with functions that derive from matured dental plaque biofilms selected from the group consisting of caries, gingival inflammation and infections such as gingivitis and periodontitis, wherein the small molecule inhibitor is characterized by the general Formula 1 ##STR00015## wherein R1 is an unbranched hydrocarbon with three to nine carbon atoms, and wherein R2 is ##STR00016##

2.-4. (canceled)

5. A small molecule inhibitor according to claim 1, wherein the small molecule inhibitor is presented to the dental plaque biofilms at a concentration, which is sub-inhibitory.

6. A small molecule inhibitor according to claim 1 wherein the small molecule inhibitor is presented to the dental plaque biofilms at a concentration of between 1 and 1000 μM.

7. A small molecule inhibitor according to claim 1, wherein the small molecule inhibitor affects a reduction of lactate production in dental plaque biofilms.

8. Oral composition for use in the prevention of maturation of dental plaque biofilms and/or the reduction of the risk for diseases associated with functions that derive from matured dental plaque biofilms, selected from the group consisting of caries, gingival inflammation and infections such as gingivitis and periodontitis, comprising an effective amount of a compound of general Formula 1 ##STR00017## wherein R1 is an unbranched hydrocarbon with three to nine carbon atoms, and wherein R2 is ##STR00018##

9.-11. (canceled)

12. Oral composition according to claim 8, which comprises the compound of general Formula 1 at a concentration of 1-10,000 μM.

13. Oral composition according to claim 8 wherein the oral composition is a liquid or a paste.

14. Oral composition according to claim 8, wherein the oral composition is a chewing gum.

15. An electronic device, such as a powered toothbrush, an interdental cleaner, an oral irrigator, a tongue scraper, or any other appropriate intra-oral delivery system, for application of an oral composition according to claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 shows the in vitro oral plaque bio film formation expressed as colony forming units (CFU). The concentrations of the compounds are shown on the x-axis. Statistically significant differences in comparison with the control are marked with an asterisk (*).

[0074] FIG. 2 shows total lactic acid production for biofilms grown in the presence of homoserinelactone molecules with different C-chain lengths. Also 3-Oxo-N(2-oxocyclohexyl)dodecanamide was tested, and DMSO is used as control. 100 μM of each compound is used. Lactic acid production is given per biofilm after 3 h incubation in buffered peptone water (BPW) containing 0.2% sucrose. Significance compared to the control is shown: *** P<0.01.

[0075] FIG. 3 shows total lactic acid production for biofilms grown in the presence of different concentrations 3-Oxo-N(2-oxocyclohexyl)dodecanamide and HSL, in mM per biofilm after 3 h incubation in BPW containing 0.2% sucrose. All concentrations of 3-Oxo-N(2-oxocyclohexyl)dodecanamine result in a statistical significant difference compared to the control.

[0076] FIG. 4A shows lactate production in mM lactate; and FIG. 4B shows lactate production corrected for CFU, expressed in μM lactate/1×10.sup.6 CFU. Concentrations of the compounds are shown on the x-axis. Statistically significant differences in comparison with the control samples are marked with an asterisk (*).

[0077] FIG. 5 shows the compositional shift of in vitro oral plaque biofilms (A %) with respect to the most abundant species (Streptococcus (A) and Veillonella (B)) grown after 48 and 96 hours upon addition of 100 μM 3-oxo-N(2-oxocyclohexyl)dodecanamide (a), 10 μM Furanone C30 (b) or 10 μM 3,4-Dibromo-2(5H)-furanone (c), compared to the control biofilms.

EXAMPLES

Materials & Methods

Inoculum Collection

[0078] Stimulated saliva, used as inoculum, was collected on ice from ten donors. Saliva was donated 24 h after last brushing. The saliva was diluted 2-fold with 60% sterile glycerol, aliquoted and stored at −80° C. Before use, a pooled sample was prepared by mixing 200 μl of thawed saliva of each donor and vortexing for 30 seconds. In vitro oral plaque biofilms were inoculated 1:50 with pooled saliva.

Test Compounds

[0079] All compounds tested of Table 1 were obtained from Sigma Aldrich. All compounds of Table 2, except 3-oxo-N(2oxocyclohexyl)dodecanamide, were kindly provided by M. Meijler, University of the Negev, Israel. The compounds were dissolved in DMSO and stored at −20 C. The compounds were continuously present at the indicated concentrations during biofilm growth, but not during further phenotypic analysis, i.e. lactate production.

TABLE-US-00001 TABLE 1 Compounds tested in in vitro oral plaque biofilm model (Example 1) Concentration Compound used (in μM) Reference 3-Oxo-N 3-oxo-N-(2- 0.01-100 Smith et al. oxocyclohexyl)dodecanamide (Jan. 2003) 3,4- 3,4-Dibromo-2(5H)-furanone 10 dibromo Furanone (Z-)-4-Bromo-5- 10 Wu et al. C30 (bromomethylene)- (2004) 2(5H)-furanone

TABLE-US-00002 TABLE 2 Compounds tested for effect on lactic acid production (Example 2) Compound HSL-C6 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-hexanamide HSL-C7 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-heptanamide HSL-C8 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-octanamide HSL-C9 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-nonanamide HSL-C10 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-decanamide HSL-C11 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-undecanamide HSL-C12S N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide HSL-C12R N-[(3R)-tetrahydro-2-oxo-3-furanyl]-dodecanamide HSL-C13 N-[(3S)-tetrahydro-2-oxo-3-furanyl]-tridecanamide 3-Oxo-N 3-oxo-N-(2-oxocyclohexyl)dodecanamide

In Vitro Oral Plaque Biofilm Formation

[0080] In vitro oral plaque biofilms were grown in the Amsterdam Active Attachment Model (AAA-model, Exterkate et al. (2010)), assembled with glass coverslips (diameter 12 mm; Menzel, Braunschweig, Germany). The model was inoculated for 8 h with pooled saliva in buffered semi-defined McBain medium (2.5 g/l mucin (Sigma, St Louis, Mo.), 2.0 g/l Bacto peptone (Difco, Detroit, Mich.), 2.0 g/l Trypticase peptone (BBL, Cockeysville, Md.), 1.0 g/l yeast extract (BD Diagnostic Systems, Sparks, Md.), 0.35 g/l NaCl, 0.2 g/l KCl, 0.2 g/l CaCl.sub.2, 1 mg/l hemin (Sigma, St. Louis, Mo., USA), and 2 mg/l vitamin K1 (McBain et al. (2005)), with 50 mmol/1 PIPES at pH 7.0), with 0.2% sucrose.

[0081] In vitro oral plaque biofilms were grown in the AAA-model for 48-96 h in the presence of the compounds tested. The model was inoculated with saliva and incubated anaerobically at 37° C. for 8 hours to allow microbes to attach to the glass coverslips. After 8 hours of attachment, the inoculation medium was refreshed and in vitro oral plaque biofilms were grown for 16 hours. This refreshment routine was repeated daily until the day of harvesting.

[0082] In vitro oral plaque biofilms were harvested by transferring the glass coverslips into 2 ml phosphate buffered saline (PBS). The biofilms were dispersed using a Vibracell VCX130 sonicator with a maximum of 130 Watts and 20 kHz (Sonics & Materials, Newtown, USA). Bio films were sonicated on ice for 1 minute with a pulse rate of 50% and pulses of 1 second. Vibration amplitude was set to 40%.

CFU Determination

[0083] To estimate the amount of in vitro oral plaque biofilm formation, total anaerobic colony forming units were determined. Briefly, serial dilutions of the dispersed bio films were made and plated on tryptic soy agar blood plates. Plates were subsequently incubated anaerobically for 96 h at 37° C. and colony forming units were determined by counting the number of colonies for each dilution.

Lactic Acid Production Assay

[0084] To estimate the cariogenic phenotype, lactic acid production of the in vitro oral plaque biofilms was determined prior to harvesting (Exerkate et al 2010). In short, the biofilms on coverslips were placed in a 24-well plate containing 1.5 ml of BPW with 0.2% sucrose. Lactic acid formation was allowed for 3 hr at 37° C. under anaerobic conditions. The total amount of lactic acid produced in this period was analyzed using a colorimetric assay described previously (van Loveren et al. (2000)) and expressed as mM lactic acid, and as μM lactic acid per 1×10.sup.6 CFU.

DNA Isolation and PCR

[0085] DNA was isolated using phenol bead-beating followed by Agowa isolation, following the manufacturers instructions (LGC Genomics, Mag mini kit). Briefly, cells were mechanically disrupted four times at 1200 rpm for two minutes in addition of Tris-saturated Phenol, pH8, 0.1 mm zirconium beads and Mag lysis buffer. The DNA-containing phase was mixed with binding buffer and magnetic beads. DNA concentration was estimated by determining the absorbance at 260 nm using the nanodrop (Isogen, ND-1000).

[0086] The primers used for 16S rDNA PCR amplification are listed in Table 2. PCR was performed in a final volume of 25 μl containing 1 μl of each primer (10 mM), 1 μl dNTP (10 mM), 2.5 μl 10× DreamTaq Buffer including MgCl.sub.2 (Thermo Scientific), 0.2 μl DreamTaq DNA Polymerase (5 u/μ1, Thermo Scientific) and 50 ng of DNA. Initial denaturation was performed at 94° C. for 4 min, followed by 35 cycles of denaturation at 94° C. for 30 s, 54° C. annealing for 1 min, 72° C. for 1 min primer extension and a final extension at 72° C. for 5 min. Product formation was confirmed by electrophoresis of 5 μl on a 1% (w/v) agarose gel (Sphearo Q, Leiden, The Netherlands) stained with ethidium bromide.

[0087] These samples were analyzed by 16S rDNA Illumina sequencing at TNO Zeist (Netherlands).

Example 1

Testing of Compounds of Table 1 in Amsterdam Active Attachment Model

[0088] In vitro oral plaque biofilm formation was slightly affected by 3-Oxo-N-(2-oxocyclohexyl)dodecanamide, but only for the highest concentration tested (100 μM). With 100 μM of this compound present, in vitro oral plaque biofilms established up to 6.6×10.sup.8 Colony Forming Units (CFU) versus 1.5×10.sup.9 CFU for the control and all the other concentrations (FIG. 1). Furanone C30 and 3,4-dibromo-2(5H)-furanone also slightly effect in vitro oral plaque biofilm formation but since the difference is less than one log, this is not considered biologically relevant.

[0089] Caries is related to high lactate production by dental plaque biofilms. Therefore, lactate production was measured for all in vitro oral plaque biofilms. FIG. 4A shows that lactate production is almost completely blocked by the addition of 100 μM of 3-oxo-N(2-oxocyclohexyl)dodecanamide. Addition of 10 μM of this compound also significantly reduces lactate production of the in vitro oral plaque biofilm. Although lactate production of the control in vitro oral plaque biofilm was lower after 96 hours of growth than after 48 hours of growth, the reducing effect was still present. The highest concentration of 3-Oxo-N(2-oxocyclohexyl)dodecanamide also slightly reduced total biomass, but this cannot explain the lower lactate production. FIG. 4B shows that when corrected for CFU, lactate production is still lower upon addition of 100 μM or 10 μM of this compound.

[0090] The reduction of lactate production is not observed for furanone C30 and for 3,4-dibromo-2(5H)-furanone. After 96 h of growth both furanones even seem to up-regulate the lactic acid production of the in vitro oral plaque biofilms.

Example 2

Lactic Acid Production in Presence of C12-HSL and Structural Variants Thereof

[0091] Lactate production of biofilms grown in the presence of homoserine lactone (HSL) molecules with different C-chains (compounds of Table 2) was measured. 3-Oxo-N-(2-oxocyclohexyl)dodecanamide has a C12 chain, so the HSL-C12 QS molecule is the most similar structurally. HSL-C12(S) is the active form of the QS molecule, HSL-C12(R) is the inactive enantiomer. For the C12-chain, both enantiomers were tested.

[0092] Biofilms were grown for 48 h in buffered McBain medium supplemented with 0.2% sucrose in the presence of 100 μM of one of the compounds. Medium was refreshed twice every day. After 48 h of growth, biofilms were transferred to BPW with 0.2% sucrose, and biofilms were allowed to produce lactate for 3 h.

[0093] FIG. 2 shows the lactate production of the biofilms. Only HSL-C14 and 3-Oxo-N show a reduced lactic acid production. For HSL-C14, this can be explained by a lack of biofilm production as the compound is toxic to the formation of biofilm. This was not the case for 3-oxo-N, were normal amounts of biofilm were visible.

[0094] The data obtained show no effect of the natural homoserine lactone (C12-HSL) or its structural variants on lactic acid production. This indicates that it is not likely that 3-Oxo-N-(2-oxocyclohexyl)dodecanamide acts as a inhibitor of QS. The observed effect therefore has to be related to a currently unknown effect of 3-Oxo-N-(2-oxocyclohexyl)dodecanamide on the composition and behavior of in vitro oral dental plaque.

Example 3

[0095] No Interference of the Quorum Sensing Natural Homoserine Lactone with 3-Oxo-N-(2-Oxocyclohexyl)Dodecanamide

[0096] In this experiment the effect of the quorum sensing molecule C12-HSL on the lactate reducing activity of 3-Oxo-N-(2-oxocyclohexyl)dodecanamide was tested. Biofilms were grown for 48 h in buffered McBain medium supplemented with 0.2% sucrose, in the presence of 0, or 25 or 100 μM 3-oxo-N(2-oxocyclohexyl)dodecanamide and 0, or 25 or 100 μM of C12-HSL. Medium was refreshed twice every day. After 48 h of growth, biofilms were transferred to BPW with 0.2% sucrose, and biofilms were allowed to produce lactate for 3 h.

[0097] FIG. 3 shows the total lactic acid production per bio film. It can concluded that the QS molecule does not reduce the effect of our compound. This points towards mechanism for the reduction of lactate production by the compounds of the present invention different from quorum sensing.

Example 4

In Vivo Oral Plaque Biofilm Composition in the Presence of Quorum Sensing Inhibitors

[0098] The composition of the biofilms is studied using 16S rDNA sequencing (Tables 4A and 4B). For all bio films, Veillonella and Streptococcus are the two dominating genera, but their ratio is different. After 48 h, a shift in composition is observed for the biofilms grown with addition of 100 μM 3-Oxo-N(2-oxocyclohexyl)dodecanamide in comparison with the control. Veillonella, known to consume lactate, is 10% more abundant in biofilms grown with addition of this compound and Streptococcus is more than 30% reduced in these biofilms (Table 2). The decrease in Streptococcus spp in the 3-Oxo-N(2-oxocyclohexyl)dodecanamide grown biofilms is accompanied with an increase in Actinobacillus species. For both furanone C30 and 3,4-dibromo-2(5H)-furanone this effect is absent or even inversed.

[0099] All 96 h biofilms grown with addition of a quorum sensing inhibitor show a decrease in Streptococcus and an increase in Veillonella and Actinobacillus (Table 2).

[0100] The compositional shift of biofilms (A %) with respect to the most abundant species (Streptococcus (A) and Veillonella (B)) grown after 48 and 96 hours is shown in FIG. 5.

[0101] Table 3. Microbial composition of biofilms grown for 48 (Table 3A) or 96 h (Table 3B) in the presence of different QS inhibitors. Composition was determined by 16S rDNA sequencing. The composition of the original inoculum used for all biofilms was also determined. “Unclass.” means: unclassified.

TABLE-US-00003 TABLE 3A 48 h taxon Control 3-Oxo-N Furanone C30 3,4-Di-bromo Inoculum Streptococcus 60.8% 34.3% 66.2% 60.8% 19.5% Veillonella 32.5% 36.0% 28.6% 34.8% 23.4% Actinobacillus 5.3% 27.6% 4.9% 3.7% 15.0% Prevotella 0.3% 0.0% 0.0% 0.0% 16.8% Campylobacter 0.3% 1.1% 0.0% 0.2% 1.1% Neisseria 0.0% 0.0% 0.0% 0.0% 6.4% Megasphaera 0.0% 0.0% 0.0% 0.0% 1.2% Porphyromonas 0.0% 0.0% 0.0% 0.0% 2.7% Solobacterium 0.1% 0.0% 0.1% 0.1% 0.1% Unclass. Pasteurellaceae 0.4% 0.6% 0.0% 0.2% 0.5% Fusobacterium 0.0% 0.0% 0.0% 0.0% 2.4% Granulicatella 0.1% 0.0% 0.0% 0.1% 0.8% Haemophilus 0.1% 0.2% 0.0% 0.0% 0.9% Actinomyces 0.0% 0.0% 0.1% 0.0% 0.8% Rothia 0.0% 0.0% 0.0% 0.0% 1.3% Other 0.1% 0.1% 0.0% 0.1% 7.2%

TABLE-US-00004 TABLE 3B 96 h taxon Control 3-Oxo-N Furanone C30 3,4-Di-bromo Inoculum Streptococcus 40.7% 28.7% 34.4% 38.0% 19.5% Veillonella 43.8% 51.7% 59.6% 52.9% 23.4% Actinobacillus 2.1% 12.0% 4.5% 3.4% 15.0% Prevotella 3.8% 0.0% 0.0% 2.3% 16.8% Campylobacter 3.7% 7.2% 0.0% 1.6% 1.1% Neisseria 0.0% 0.0% 0.0% 0.0% 6.4% Megasphaera 2.8% 0.0% 0.0% 0.0% 1.2% Porphyromonas 0.0% 0.0% 0.0% 0.0% 2.7% Solobacterium 0.6% 0.0% 0.8% 0.7% 0.1% Unclass. Pasteurellaceae 0.3% 0.0% 0.0% 0.0% 0.5% Fusobacterium 0.2% 0.0% 0.0% 0.0% 2.4% Granulicatella 0.1% 0.0% 0.3% 0.2% 0.8% Haemophilus 0.0% 0.1% 0.0% 0.0% 0.9% Actinomyces 0.1% 0.0% 0.3% 0.1% 0.8% Rothia 0.0% 0.0% 0.0% 0.0% 1.3% Other 1.7% 0.3% 0.1% 0.7% 7.2%

Example 5

Toothpaste

[0102] Toothpaste according to the present invention may contain the following ingredients (in weight/weight %): [0103] 20-50% abbrassive; [0104] 20-30% humectant such as sorbitol; [0105] 1-5% surface-active compound; 1-5% thickener; 1-2% flavor compound; 0.5-2% white dye compound; 0.05-0.5% preservative; 0.5-5% 3-oxo-N-(2-oxocyclohexyl)dodecanamide [0106] 20-30% water

REFERENCES CITED

[0107] 1) Smith et al. (January 2003): Smith, K. M., Bu, Y. and Suga, H. “Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs” Chem. Biol. 2003 January; 10(6): 81-9 [0108] 2) Smith et al. (June 2003): Smith, K. M., Bu, Y. and Suga, H. “Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer” Chem. Biol. 2003 June; 10(6): 563-71. [0109] 3) Wu et al. (2004): Wu, H., Song, Z., Hentzer, M., Andersen, J. B., Molin, s., Givskov, M. and Hoiby, N. “Synthetic furanones inhibit quorum-sensing and enhanced bacterial clearance in Pseudomonas aeruginosa lung infection in mice” J. Antimicrob. Chemother. 2004, 53, 1054-61. [0110] 4) Exterkate et al. (2010): R. A. M. Exterkate, W. Crielaard, J. M. Ten Cate “Different response to amine fluoride by Streptococcus mutans and polymicrobial bio films in a novel hight-throughput active attachment model”; Caries Res. 2010; 44:372-379 [0111] 5) McBain et al. (2005): A. J. McBain, C. Sissons, R. G. Ledder, P. K. Sreenivasan, W. De Vizio, P. Gilbert “Development and characterization of a simple perfused oral microcosm” J. Appl. Microbiol. 2005, 98, 624-634 [0112] 6) Van Loveren et al. (2000): van Loveren C., Buijs J. F., ten Cate J. M. “The effect of triclosan toothpaste on enamel demineralization in a bacterial demineralization model” J. Antimicrob. Chemother. 2000; 45:153-158.