PERSONAL DENTAL CARE PRODUCT FOR CARIES TREATMENT

Abstract

The present invention provides new dental care products comprising self-assembling peptides 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 or increasing smoothness or shine, or for increasing hardness of a tooth surface. The present invention provides dental care products such as chewing gum, toffees or toothpaste, in which the peptides are present in monomeric form for a prolonged period after application into the oral cavity, thereby enabling non-targeted treatment of a plurality of teeth, independent of the diagnosis of an active lesion. Products of the invention are useful for animals and humans.

Claims

1. A method for treating a tooth, comprising applying a dental care product to a plurality of teeth of a subject, wherein the dental care product comprises (i) self-assembling peptides that are capable of undergoing self-assembly at a pH below 7.5, wherein the self-assembling peptides are present in the dental care product in monomeric form, and (ii) a pharmaceutically acceptable basis, wherein the dental care product is applied to a plurality of teeth of the subject independent of diagnosis of caries, and wherein, after application, self-assembling peptides are present in monomeric state for at least 1 minute.

2. The method of claim 1, wherein the subject has caries lesion.

3. The method of claim 1, wherein the method remineralizes a tooth surface.

4. The method of claim 1, wherein at least 70% of the self-assembling peptide are present in a monomeric state in the dental care product before application.

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

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

7. The method of claim 1, wherein the concentration of self-assembling peptides is 0.1-1000 mg/kg.

8. The method of claim 1, wherein said peptide comprises the sequence of SEQ ID NO: 3.

9. 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.

10. The method of claim 1, wherein the dental care product is selected from the group comprising candy, lozenge, gelatin-gum, toffee, chewing gum, chew toy, biscuit, capsule, toothpaste, toothgel, prophylactic paste, toothpowder, mouthwash, mouthspray, solution, coated dental floss, coated interdental brush or coated toothbrush.

11. The method of claim 1, wherein the self-assembling peptide is encapsulated.

12. The method of claim 1, wherein the product is applied for at least 1 minute.

13. The method of claim 1, wherein the product is for application at least once a week.

14-16. (canceled)

17. The method of claim 1, wherein, preferably, at least 50% of the self-assembling peptides are present in a monomeric state for at least 1 minute after application in the oral cavity.

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 8-8.2.

20. The method of claim 1, wherein said peptide comprises the sequence of any one of SEQ ID NOs: 4 or 5.

21. The method of claim 1, wherein said peptide comprises the sequence of SEQ ID NO: 1.

22. The method of claim 1, wherein the dental care product is a toffee.

23. The method of claim 1, wherein the dental care product is a chewing gum.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0113] FIG. 1 shows exemplary lozenges of the invention. FIG. 1A shows a lozenge consisting of an outer shell (A) comprising abrasive agents, e.g., dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is dissolved in a buffered, basic environment, e.g., at pH 8 (B). FIG. 1B shows a lozenge consisting of an outer shell (A) comprising abrasive agents, e.g., dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is present in encapsulated form in a buffered, basic environment, e.g. at pH 8 (B).

[0114] FIG. 2 illustrates exemplary toffees of the invention. FIG. 2A shows a toffee comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g., sodium carbonate and SDS, whereas monomeric self-assembling peptide is incorporated in the basic buffered toffee. FIG. 2B shows a toffee comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents e.g sodium carbonate and SDS, whereas monomeric self-assembling peptide is incorporated in encapsulated form in the basic buffered toffee. FIG. 2C shows a toffee consisting of an outer shell (A) comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is dissolved in a buffered, basic environment, e.g. at pH 8, (B). FIG. 2D shows a toffee consisting of an outer shell (A) comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is present in encapsulated form in a buffered, basic environment, e.g. at pH 8 (B).

[0115] FIG. 3 shows chewing gums of the invention. FIG. 3A illustrates a chewing gum comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS and/or phosphate buffer, wherein the chewing gum contains monomeric self-assembling peptide in a basic environment, e.g. at pH 8. FIG. 3B shows a chewing gum consisting of an outer shell (A) comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is dissolved in a buffered, basic environment, e.g. at pH 8, (B). FIG. 3C shows a chewing gum consisting of an outer shell (A) comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS, and/or phosphate buffer, wherein the monomeric self-assembling peptide is present in encapsulated form in a buffered, basic environment, e.g. at pH 8 (B).

[0116] FIG. 4 shows gelatin gums of the invention. FIG. 4A illustrates a gelatin gum comprising abrasive agents e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS and/or phosphate buffer, wherein the gelatin gum contains monomeric self-assembling peptide in a basic environment, e.g. at pH 8. FIG. 4B illustrates a gelatin gum comprising abrasive agents, e.g. dicalcium phosphate and biofilm removing agents, e.g. sodium carbonate, SDS and/or phosphate buffer, wherein the gelatin gum contains monomeric self-assembling peptide in encapsulated form in basic environment, e.g. at pH 8.

[0117] FIG. 5 A shows soft capsules comprising self-assembling peptides. The capsules were produced as indicated in Example 2. FIG. 5 B shows exemplary toffees of the invention prepared according to Example 1 containing 230 mg/kg P11-4, which were used in Example 5.

[0118] FIG. 6 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.

[0119] Squares (top line of symbols)NaCl: Tris (0.055 M)+NaCl, final ionic strength 142 mM;

[0120] Artificial 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;

[0121] 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;

[0122] MgSO.sub.4: 0.055 M Tris+0.192 M MgSO.sub.4 0.14 M Ionic strength

[0123] FIG. 7 shows self-assembling kinetic studies with quartz crystal microbalance with dissipation monitoring (QCM-D) over 4 hours. P11-4 peptide was layered on the surface of the amine-coupling sensor at 1 mg/ml in PBS at pH8.5. After 1 hour and 1 hour 25 minutes, the sensor was washed with PBS pH 8.5. After 1 hour and 38 minutes, 10 mg/ml P11-4 peptide in PBS pH 8.5 were injected. After 2 hours, 0.1 M HCl were injected to induce self-assembly.

[0124] It can be seen that essentially complete self-assembly has occurred about 30 min after injection of 0.1 M HCl. Resonance frequency F (left scale, Hz) and energy dissipation factor D (right scale), triangle=frequency, square=dissipation are shown.

[0125] FIG. 8 shows transfer of peptide from chewing gum to tooth. A chewing gum of the invention comprising P11-4 was incubated with a tooth as described in detail in Example 6. A shows a MALDI-TOV analysis of peptide P11-4 in water as a standard, B shows the presence of said peptide in drilling dust of the tooth after 2 h of incubation and C shows the presence of said peptide in the supernatant after 2 h of incubation.

[0126] FIG. 9A shows generation of artificial lesions in extracted human molars. FIGS. 9B, 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. FIGS. 9F 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). FIGS. 9C, 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 FIGS. 9D and E, the pellicle formed by saliva was removed before incubation with the P11-4 by NaClO (FIG. 9D) and sonication (FIG. 9E). The experiments are described in detail in Example 7.

EXAMPLES

Example 1: Preparation of a Peptide Toffee

[0127] Toffees of the invention were compounded as follows:

TABLE-US-00001 Ingredient % (w/w) Maltitol syrup 49.00 Isomalt 31.00 Coconut oil 5.50 Gelatine 3.00 Vitamin mixture 1.25 Citric acid 0.90 Flavour, e.g., raspberry flavour 0.85 Peptide P11-4 e.g., 0.03 to 0.25 Colorant (e.g., anthocyanes) 0.02 Acesulfame K 0.01 Sucralose 0.004 Water ad 100

[0128] All ingredients except for the peptide P11-4 were warmed. Monomeric peptide in powder form was added and all ingredients mixed. Toffees were formed. Toffees were employed for the chewing test in Example 5.

[0129] Due to the low water content, the peptide stays in monomeric form. This applies even though the dry peptide is compounded with citric acid. Assembly can only occur after contact with saliva. Toffees were employed for Example 5.

Example 2: Encapsulation of Peptides

[0130] Self-assembling peptides were encapsulated in soft-gelatin capsules as follows.

[0131] Filling material comprising the self-assembling peptides, MCT oil, soybean lecithin, beeswax and silicon dioxide in amounts indicated below was prepared and encapsulated. The solution for the capsule shells comprised, e.g. gelatine, glycerine, sorbitol and, optionally, colorants. Capsules were dried subsequently, and may be packaged. Resulting capsules are shown in FIG. 5 A.

TABLE-US-00002 Ingredient mg/capsule Peptide P11-4 1 MCT oil 829 Soybean lecithin 35 Beeswax 120 Silicon dioxide 15

Example 3: Preparation of Peptide Chewing Gum

[0132] Exemplary chewing gums of the invention may be prepared, e.g., according to Zumb et al., 2001, British Journal of Nutrition 85, Suppl. 1, S31-S45, FIG. 8. Peptide in monomeric form is added after all other ingredients are mixed.

TABLE-US-00003 Ingredient % w/w Peptide 0.01 Milled xylitol 20 Sorbitol syrup 15.2 Glycerine 2 Flavour 1.8 Baking Soda 1 Aspartame 0.05 Milled sorbitol 35.0 Gum base ad 100

Example 4: Preparation of a Toothpaste

[0133] A toothpaste may be prepared according to WO2004/069170.

TABLE-US-00004 Ingredient % (w/w) Sorbitol (70%) 43.00 CaCO.sub.3 14.50 Glycerin 9.60 Sodium lauryl sulfate 0.34 Na monofluorophosphate 0.75 Xanthan Gum 0.36 Cassia Gum 0.04 Carbopol 980 NF Polymer 0.4 Na saccharin 0.2 Peptide 0.05 Buffer 0.1 Baking Soda 1 Deionized water ad 100 pH 8.2

Example 5: Chewing Test

[0134] Groups of four subjects were given either a toffee containing 30 mg/kg (group A) and 230 mg/kg (group B) monomeric self-assembling peptide P11-4 and group C with no peptide in it (Placebo). Each subject chewed the toffee for 5 min and reported regarding the sensoric experience without knowledge of the content of peptide.

[0135] The toffee had a nice texture and pleasant taste. All members of groups A and B reported a smooth, clean feeling on the tooth surface while and after chewing the toffee, whereas group C did not report such a finding. Within the two peptide containing groups A and B, group B showed a faster onset of the smooth feeling.

Example 6: Transfer of Peptide from Chewing Gum to Tooth

[0136] A chewing gum of the invention comprising P11-4, prepared according to Example 3, was cut into pieces of 4-5 mm particle size and then chewed with a denture either in water or remineralization buffer ((2 mM Ca(NO3), 1.2 mM KHPO4, and 60 mM Tris/HCl, pH adjusted to 7.4 with 1 M KOH) for 5 minutes. The resulting saliva with extracted peptide was then used to incubate a human tooth in it at 37 C. for about 2 h on a shaker.

[0137] After incubation, the tooth was air dried and the upper enamel layer mechanically extracted with a high speed drill. The resulting dust was suspended in 200 l of distilled water adapted to pH 8 with ammonia, followed by purification with Cleanup C18 Pipette Tips. The tips were rinsed twice with 10 l with 50 wt % acetonitrile in H.sub.2O, then 2 with 10 l 0.1M triethylammonium acetate in H.sub.2O+1% 1M NH.sub.4OH and then 30.1% TFA in H.sub.2O. The sample was eluted with 10 l 50% acetonitrile in H.sub.2O.

[0138] The sample holder MTP384 was prepared with 1.5 l matrix (20 mg/ml 3,5-Dimethoxy-4-hydroxycinnamonacid in H.sub.2O/Acetonitrile (1:1)+0.1% TFA), loaded with 1.5 l sample solution on the matrix. On a separate spot, 2 l of the calibration standard were placed. Then the Mass spectra over a mass/charge (m/z) ratio of 520-3200 was acquired with a N.sub.2)-laser, =337 nm, Laser power 22%, laser shots over 5 sides on each sample and calculated with FlexControl (Version 2.4) and FlexAnalysis (Version 2.4) software.

[0139] The result in FIG. 8 shows that both the tooth dust and the supernatant comprise the self-assembling peptide P11-4, i.e., the experiment proves a transfer of peptide to the tooth.

Example 7: Penetration of Monomeric Self-Assembling Peptide into Subsurface Lesions

[0140] a) Creation of Artificial Carious Lesions

[0141] 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 colourless nail varnish, leaving a window of approximately 44 mm. The tooth was placed in demineralization buffer (2.2 mM CaCl2, 2.2 mM NaH2PO4, 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% NaClO (incubation for 1 min), rinsed, and air-dried at room temperature.

[0142] Placebo-treated samples underwent identical pre-treatment.

[0143] Typical artificial lesions are shown in FIG. 9A.

[0144] b) Treatment [0145] A 200 ppm (i.e., 200 g/ml) solution of P11-4 (SEQ ID NO: 2), spiked with 10 ppm ATTO647-P11-4 (fluorescence labelled P11-4 (SEQ ID NO: 6): Ac-QQRFEWEFEQQSGSGC-(ATTO647)-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) [0146] 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. [0147] 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/HCl, pH adjusted to 7.4 with 1 M KOH) for 4 days and/or 6 days. [0148] 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.

[0149] c) Confocal Microscopy

[0150] 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 m thickness, giving a total assessment depth of 205 m (objective: UPLSAPO 20/NA 0.75; helium-neon gas laser; excitation: 633 nm and emission: 668 nm). Images were analyzed by Olympus software (FluoView FV1000).

[0151] d) Conclusion

[0152] 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-00005 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 Remin. presence tooth pH saliva NaClO sonification 1 min 2 min 5 min 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

[0153] While, after incubation of the lesions with P11-4 at pH8.5 (teeth 10 and 1, tooth 10 in FIG. 9B), peptide was already detectable after 1 min incubation (FIG. 9Bb), most peptide was detected in the lesions after 2 min incubation (FIG. 9Bc) or 5 min incubation (FIG. 9Bd). Lower but significant amounts of peptide could reproducibly still be detected in the lesions after 4 days' incubation in remineralization buffer (FIG. 9Be).

[0154] 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.

[0155] 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.

[0156] 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.

[0157] Sonification as a pretreatment before incubation with P11-4 led to an increased presence of peptide in the lesions (tooth ZR1, pictures not shown).

[0158] 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. 9C), 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. 9B).

[0159] 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. 9D) or treatment with NaClO (tooth ZR2, shown in FIG. 9E) after incubation with human saliva.

[0160] 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.

[0161] 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 FIGS. 9F and 9G, 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.