METHOD AND PRODUCT FOR CARIES TREATMENT

Abstract

The present invention provides new dental care products comprising self-assembling peptides (SAP) that are capable of undergoing self-assembly at a certain pH for use in dental care, e.g. preventing and/or treating a tooth lesion such as a caries lesion, remineralising a tooth surface, increasing smoothness or shine or increasing hardness of a tooth surface. The dental care products may be administered following a simple protocol involving multiple steps, e.g., comprising tooth brushing with a sonic tooth-brush such as an ultrasonic toothbrush and/or use of an oral irrigator, thereby enabling non-targeted treatment of a plurality of teeth in a subject, independent of the diagnosis of active caries, i,e., not previously successfully treated caries. The invention also relates to said method as well as kits and devices suitable therefor.

Claims

1. A method of treating or preventing a tooth lesion and/or remineralising a tooth surface in a subject, wherein the method comprises (i) removing dental biofilm from a plurality of teeth by a method selected from the group comprising tooth brushing, flossing of teeth, use of an oral irrigator, and rinsing with a salt and detergent solution; (ii) removing dental pellicle from the plurality of teeth by a method selected from the group comprising tooth brushing, flossing of teeth, use of an oral irrigator, and rinsing with a salt and detergent solution; (iii) optionally, removing inorganic deposit from the plurality of teeth by a method selected from the group comprising tooth brushing, rinsing with an acidic solution or rinsing with a salt and detergent solution, and; (iv) optionally, rinsing of teeth with a solution having a pH of 7 or more before step (v); and (v) applying a dental care product to the plurality of teeth; wherein the dental care product comprises self-assembling peptides and wherein the self-assembling peptides maintain monomeric form after application in step (v) for at least 1 minute, wherein the self-assembling peptides are capable of undergoing self-assembly at a pH below 7.5, and wherein steps (i), (ii) and (iii) may be carried out sequentially in any order or simultaneously, and wherein step (v) is carried out after the other steps.

2. The method of claim 1, wherein steps (i), (ii), (iii), (iv), (v), or any combination thereof, are carried out using an oral irrigator.

3. The method of claim 1, wherein the method comprises tooth brushing followed by using an oral irrigator, wherein the oral irrigator is at least used for step (v).

4. The method of claim 2, wherein use of an oral irrigator in any one or more of steps (i), (ii) and (iii) involves oral irrigation with a solution comprising water, salt and detergent.

5. The method of claim 1 wherein the subject has not been diagnosed with active caries.

6. The method of claim 1, wherein the dental care product is used without prior etching or NaOCl treatment.

7. The method of claim 1 a wherein the tooth lesion is a caries lesion.

8. The method of claim 1, wherein the dental care product remineralizes the tooth surface.

9. The method of claim 1, wherein the pH of the dental care product is 7.5-9.0

10. The method of claim 1, wherein the pH of the solution is more than 0.5 pH units above the pH at which the peptides start to undergo self-assembly.

11. The method of claim 1, wherein the concentration of the self-assembling peptides is 1-50000 mg/kg.

12. The method of claim 1, wherein said self-assembling peptides comprise the sequence of SEQ ID NO: 3.

13. The method of claim 1, wherein said self-assembling peptides comprise a sequence having at least 80% sequence identity to one of the sequences of SEQ ID NOs: 1 or 2.

14. The method of claim 1, wherein the product is applied at least every 6.

15. (canceled)

16. A kit for use in the method of claim 1, comprising (a) the dental care product comprising self-assembling peptides, wherein the self-assembling peptides maintain monomeric form after application in step (v) for at least 1 minute, wherein the self-assembling peptides are capable of undergoing self-assembly at a pH below 7.5 (b) a solution suitable for use in step (i) and (ii) according to any of the preceding claims, (c) optionally, a solution suitable for use in step (iii); and (d) optionally, a solution suitable for use in step (iv).

17. (canceled)

18. The method of claim 1, wherein the pH of the dental care product is 7.8-8.5.

19. The method of claim 1, wherein the pH of the dental care product is 8.0-8.2.

20. The method of claim 1, wherein said self-assembling peptides comprise the sequence of any one of SEQ ID NOs: 4 or 5.

21. The method of claim 1, wherein said self-assembling peptides comprise the sequence of SEQ ID NO: 1.

22. The The method of claim 1, wherein the product is applied at least every 2 months or at least every month.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0135] FIG. 1 shows a flow scheme of an exemplary treatment of the invention

[0136] FIG. 2 shows exemplary containers for use in the invention. A, B show a set of single use cartridges (containers) for oral irrigator. C shows a single cartridge comprising two compartments. (1) container; (2) in 2A, acidic pellicle removing solution; in 2B, solution of the invention comprising self-assembling peptide; (3) adaptor to oral irrigation unit; (4) direction of flow; (5) compartment comprising acidic pellicle removing solution; (6) membrane; (7) compartment comprising solution of the invention comprising self-assembling peptide; (8) thorns.

[0137] FIG. 3 shows self-assembling kinetic of P11-4, assessed by measurements of elastic modulus G. P11-4 was prepared at 15 mg/ml with different buffer compositions (see below) measured at 0.1% strain, 37 C. and a frequency of 1 rad/second. Self-assembly of peptide monomers to polymers takes more than 5 min independent of the used ionic strength at neutral pH.

[0138] Squares (top line of symbols) NaCl: Tris (0.055 M)+NaCl, final ionic strength 142 mM;

[0139] Artifical saliva (second line of symbols from top): Tris, Ca(NO.sub.3).sub.2, KH.sub.2PO.sub.4 (ratio 0.77:0.14: 0.08), ionic strength 142 mM;

[0140] Dulbecco's Modified Eagle Medium (DMEM): NaCl, NaHCO.sub.3,KCl,CaCl.sub.2, MgSO.sub.4 (ratio 0.66:0.4:0.05: 0.02:0.007), ionic strength 165 mM; MgSO.sub.4: 0.055 M Tris+0.192 M MgSO.sub.4 0.14 M Ionic strength

[0141] FIG. 4A shows generation of artificial lesions in extracted human molars. FIG. 4B, C, D and E show penetration of fluorescence-marked peptide P11-4 into artificial lesions after incubation with the peptide at pH 8.4-8.5. FIG. 4F and G show fluorescence-marked peptide P11-4 after incubation with the peptide at pH 6.3-6.8. Confocal pictures show detection of labelled peptide at 4 magnification before incubation (a), after 1 min incubation (b), 2 min incubation (c), 5 min incubation (d) and after 4 days' washing in remineralization buffer (e). FIG. 4C, D, E and G shows fluorescence-marked peptide P11-4, wherein incubation with peptide was after pre-incubation of the tooth with human saliva. In FIG. 4D and E, the pellicle formed by saliva was removed before incubation with the P11-4 by NaClO (FIG. 4D) and sonication (FIG. 4E). The experiments are described in detail in Example 3.

EXAMPLES

Example 1

Exemplary Solutions for Use in the Invention

[0142] Base Fomulation Solution I

TABLE-US-00001 Ingredient % (w/w) Glycerol 1-30% Flavour 0.1-2 Citric acid 0.05-1 Potassium sorbate 0.05-0.2 Poloxamer 407 0.1-1 Natrium fluoride 0.1-1 Sodium Chloride 0.5-1.0 Di-Sodium hydrogen phosphate 0.1-1 Potassium chloride 0.001-0.1 Potassium dihydrogen phosphate 0.01-0.1 Surfactant 0.1-2 Water ad 100

[0143] Formulation for Solution II

TABLE-US-00002 Ingredient % (w/w) PVP 1-10 Glycerol 1-30 Sucarose 1-10% TRIS 0.01-1 Sodium Hydroxyde 0.01-1 P11-4 0.001-1 Water ad 100

Example 2

Exemplary Treatment Methods of the Invention

[0144] Mouthwash

[0145] Step A: Removal of Biofilm [0146] Subject cleans teeth with toothpaste, e.g., abrasive toothpaste [0147] spit out remnants of tooth paste

[0148] Step B: Removal of Pellicle [0149] Subject takes 5 ml of mouthwash IA comprising Water, Glycerol, Flavour, Citric acid, Potassium sorbate, Poloxamer 407, sodium fluoride, Sodium chloride, Di-Sodium hydrogen phsophate, Potassium chloride, Potassium dihydrogen phosphate, EDTA (concentrations cf. Example I); [0150] or, alternatively, Subject takes 5 ml of mouthwash IB comprising Water, Glycerol, Flavour,

[0151] Citric Acid, Potassium sorbate, Poloxamer 407, sodium fluoride, Sodium chloride, Di-Sodium hydrogen phsophate, Potassium chloride, Potassium dihydrogen phosphate, Sodium Lauryl Sulfate SDS (concentrations cf. Example I); [0152] or, alternatively, Subject takes 5 ml of mouthwash IC comprising Water, Glycerol, Flavour, Citric Acid, Potassium sorbate, Poloxamer 407, sodium fluoride, Sodium Chloride, Di-Sodium hydrogen phsophate, Potassium Chloride, Potassium dihydrogen phosphate, Cocoamidopropylbetaine (concentrations cf. Example I) [0153] and swirls the Mouthwash for about 5 min in the oral cavity [0154] Subject spits out the mouthwash

[0155] Step C: Regeneration [0156] Subject takes 5 ml of mouthwash II comprising PVP/Glycerol, water, ethanol, sucrose, P11-4, Sodium hydroxide, TRIS (concentrations cf. Example I) and swirls the solution for about 5 min in the oral cavity [0157] Subject spits out the mouthwash after 5 min.

[0158] Oral Irrigator

[0159] Step A: Temoval of Biofilm [0160] Subject cleans teeth with toothpaste, e.g., abrasive toothpaste [0161] spit out remnants of tooth paste

[0162] Step B: removal of pellicle [0163] Subject places cartridge I on oral irrigator. Cartridge I contains 300 m1-600 ml acidic solution (pH 4-7) comprising: [0164] Solution IA: Water, Glycerol, Flavour, Citric acid, Potassium sorbate, Poloxamer 407, sodium fluoride, Sodium chloride, Di-Sodium hydrogen phosphate, Potassium chloride, Potassium dihydrogen phosphate, EDTA (concentrations cf. Example I); [0165] or, alternatively Solution IB comprising Water, Glycerol, Flavour, Citric Acid, Potassium sorbate, Poloxamer 407, sodium fluoride, Sodium chloride, Di-Sodium hydrogen phosphate, Potassium chloride, Potassium dihydrogen phosphate, Sodium Lauryl Sulfate SDS (concentrations cf. Example I); [0166] or, alternatively, Solution IC comprising Water, Glycerol, Flavour, Citric Acid, Potassium um sorbate, Poloxamer 407, sodium fluoride, Sodium Chloride, Di-Sodium hydrogen phosphate, Potassium Chloride, Potassium dihydrogen phosphate, Cocoamidopropylbetaine (concentrations cf. Example I) [0167] Subject starts cleaning the teeth with the oral irrigator until cartridge I is finished. During the procedure, subject spits out the surplus fluid.

[0168] Step C: Regeneration [0169] Subject removes cartridge I and places cartridge II on oral irrigator. Cartridge II contains 100 ml of a slightly basic (pH>7.5) solution comprising PVP/Glycerol, water, ethanol, sucrose, P11-4, Sodium Hydroxide, TRIS solution (concentrations cf. Example I). [0170] Subject starts cleaning the teeth with the oral irrigator until cartridge II is finished. During the procedure, subject spits out the surplus fluid.

Example 3

Penetration of Monomeric Self-Assembling Peptide into Subsurface Lesions

[0171] a) Creation of Artificial Carious Lesions

[0172] Creation of artificial lesions on extracted human molar teeth was performed as described by Lo et al. 2010 (J Dent. 38(4):352-359). To define the position of the demineralized subsurface area (i.e., artificial carious lesion or white spot), the tooth was covered with colorless nail varnish, leaving a window of approximately 44 mm. The tooth was placed in demineralization buffer (2.2 mM CaCl.sub.2, 2.2 mM NaH.sub.2PO.sub.4, 50 mM acetic acid; pH adjusted with 1 M KOH to 4.4) for 3 d at 37 C. To ensure that all pores are open, as in a typical active caries lesion, the resulting subsurface lesions were treated with 10 L of 2% NaCIO (incubation for 1 min), rinsed, and air-dried at room temperature.

[0173] Placebo-treated samples underwent identical pre-treatment.

[0174] Typical artificial lesions are shown in FIG. 4A.

[0175] b) Treatment [0176] A 200 ppm (i.e., 200 g/ml) solution of P11-4 (SEQ ID NO: 2), spiked with 10 ppm ATT0647-P11-4 (fluorescence labelled P11-4 (SEQ ID NO: 6): Ac-QQRFEWEFEQQSGSGC-(ATT0647)-NH.sub.2) , i.e., 1:20, in 25 mM TRIS buffer was prepared at different pH, in particular, at 8.4 or 8.5 or 6.3, 6.5 or 6.8). [0177] As specified in the table below, tooth were optionally pretreated, e.g., by incubation in human saliva for one day (about 24 hours), optionally followed by 5 min sonication (258 hz with a sonic toothbrush, Sonicare Diamond Clean (Philips)) or by immersion in 2% NaClO for 1 min. [0178] Tooth were immersed in the P11-4 solution for a defined period of time, 1, 2 or 5 min, and then removed and washed in water for 10 min or in remineralization buffer (2 mM Ca(NO3), 1.2 mM KHPO4, and 60 mM Tris/HC1, pH adjusted to 7.4 with 1 M KOH) for 4 days and/or 6 days. [0179] Confocal assessment after 1, 2 or 5 min incubation was carried out while the teeth were in P11-4 solution, and optionally, after washing in water or in remineralization buffer after 10 min or 4 or 6 days.

[0180] c) Confocal Microscopy

[0181] Samples were placed in a -slide I (ibidi, Martinsried) in dest. water and analyzed by a confocal laser microscope (Olympus IX81). The recorded stack of four 2-dimensional images each projected the lesion volume of 51.4 gm thickness, giving a total assessment depth of 205 gm (objective: UPLSAPO 20x/NA 0.75; helium-neon gas laser; excitation: 633 nm and emission: 668 nm). Images were analyzed by Olympus software (FluoView FV1000).

[0182] d) Conclusion

[0183] While the visual analysis does not allow for exact quantitative conclusions, tendencies detected in repeated experiments allow for comparisons between different conditions of incubation and pretreatment.

TABLE-US-00003 TABLE 1 Conditions of treatment and assessment of the presence of fluorescent P11-4 in exemplary lesions is shown (1 = no or low presence to 4 = high presence of fluorescent P11-4) Average sonifi- Remin. presence tooth pH saliva NaClO cation 1 mm 2 mm 5 mm H.sub.2O buffer 4 d 6 d of P11-4 4 6.3 1 1 2 x 1 1.3 12 6.5 2 3.5 2 x 1 2.1 3 6.3 x 1 1 1 x 1 1.0 13 6.8 x 1 2 2 x 1 1.5 10 8.5 1 4 4 x 2 2.8 1 8.4 4 2 2 2.7 2 8.4 1 3 3 x 1 2.0 ZR1 8.5 x 4 4 4 4.0 11 8.5 x 1 1 1 x 1 1.0 5 8.5 x 2 1 1 1.3 ZR4 8.5 x 1 1 2 1.3 ZR3 8.5 x x 1 3 3 2.3 ZR2 8.5 x x 3 4 4 3.7

[0184] While, after incubation of the lesions with P11-4 at pH8.5 (teeth 10 and 1, tooth 10 in FIG. 4B), peptide was already detectable after 1 min incubation (FIG. 4Bb), most peptide was detected in the lesions after 2 min incubation (FIG. 4Bc) or 5 min incubation (FIG. 4Bd). Lower but significant amounts of peptide could reproducibly still be detected in the lesions after 4 days' incubation in remineralization buffer (FIG. 4Be).

[0185] A comparative experiment which differed only insofar as washing was carried out in water (tooth 2, pictures not shown) shows a tendency that remineralization buffer instead of water stabilizes presence of the self-assembling peptide in the lesion.

[0186] These experiments show that monomeric fluorescently labelled P11-4 at a concentration of 200 ppm in a slightly basic buffer can diffuse into the artificial carious lesions and form a stable network there. Previous art demonstrates that this leads to remineralization of lesions. No significant amounts of peptide are detected outside the lesions. It is believed that through assembly of the peptides in the lesion, the gradient is shifted, and further diffusion of peptide from the solution into the lesion is enabled, which leads to concentration of the self-assembling peptide in the lesion. Incubation in remineralization buffer, which mirrors human saliva, after contact with P11-4 containing solution improves maintenance of the self-assembling peptide in the lesion, probably by formation of complexes and remineralization.

[0187] The conclusion can be drawn that non-labelled P11-4 behaves the same way as the labelled detected peptide, and it can thus, at this or similar low concentrations be used for non-targeted treatment of early caries lesions such as subsurface caries lesions per the invention.

[0188] Sonification as a pretreatment before incubation with P11-4 led to an increased presence of peptide in the lesions (tooth ZR1, pictures not shown).

[0189] After pre-incubation of a tooth with artificial lesions with human saliva, which is known to lead to formation of a pellicle on the tooth (teeth 11, 5 and ZR4, tooth 10 shown in FIG. 4C), there was a trend that incubation with fluorescence-marked peptide P11-4 at pH 8.5 for 1, 2, or 5 min led to significantly lower presence of the fluorescent peptide on the tooth or in the lesions than without such pre-incubation (FIG. 4B).

[0190] It could be shown that this negative effect of pellicle could be compensated for by cleaning the tooth by either sonication (tooth ZR3, shown in FIG. 4D) or treatment with NaCIO (tooth ZR2, shown in FIG. 4E) after incubation with human saliva.

[0191] After formation of pellicle and cleaning by sonification, the self-assembling peptide could thus surprisingly be detected in even higher amounts than without pre-incubation with saliva.

[0192] After incubation at pH 6.3-6.8 (teeth 4, 12 (both without saliva), 3, 13 (both with saliva), teeth 12 and 13 shown in FIG. 4F and 4G, respectively), i.e., when the peptide was in polymerized form, lower amounts of peptide were generally detectable in the lesions than after incubation at pH 8.4 or 8.5. Pellicle formation by incubation with saliva (Teeth 3, 13) further decreased infiltration by peptide.