SELF-ASSEMBLING PEPTIDES IN THE PREVENTION AND TREATMENT OF CAVITATED CARIOUS LESIONS

Abstract

The invention is in the field of caries prevention and treatment, in particular minimally-invasive, or non-invasive dental decay treatment. It relates to a composition and a kit comprising a self-assembling peptide, such as the self-assembling peptide P11-4 or P11-8, and a dental agent, namely a dental sealant, e.g. a glass-ionomer cement-based sealant or a resin-based sealant, a fluoride varnish, a dental restorative material or a bonding agent. The invention further relates to a composition and kit comprising a self-assembling peptide and a dental agent for use in the treatment of a carious lesion, preferably a cavitated carious lesion. The self-assembling peptide prevents secondary caries after failure of the dental agent used in treatment of cavitated caries, especially in case of a failing interface between the restoration with the dental agent and the tooth. The composition and kit of the present invention may also be used for pulp capping.

Claims

1. A composition comprising: a) a self-assembling peptide, and b) a dental agent selected from the group consisting of a dental sealant, a fluoride varnish, and a dental bonding agent, wherein the composition preferably is a pharmaceutical composition.

2. The composition of claim 1, wherein the dental agent is a dental sealant selected from the group comprising (i) glass-ionomer cement-based sealants selected from the group comprising glass-ionomer cements and resin-modified glass-ionomer cements, and (ii) resin-based sealants selected from the group comprising polyacid-modified composite resins and resin-based composites, wherein, preferably, the dental sealant is a glass-ionomer cement-based sealant.

3. The composition of claim 1, wherein the dental agent is a fluoride varnish.

4. The composition of claim 1, wherein the dental agent is a dental bonding agent.

5. A kit comprising, in separate form the components: a) a self-assembling peptide, and b) a dental agent that is a glass-ionomer cement-based dental sealant selected from the group comprising glass-ionomer cements and resin-modified glass-ionomer cements, wherein the kit is a pharmaceutical kit.

6. The kit of claim 5, wherein the dental agent is capable of releasing fluoride ions, wherein, preferably, the dental agent is also capable of releasing calcium ions.

7. The kit of claim 5, wherein the dental agent is permeable for and to ions.

8. The kit of claim 5, wherein the self-assembling peptide undergoes self-assembly at a pH below 7.5.

9. The kit of any of claims 5-7 claim 5, wherein the self-assembling peptide undergoes self-assembly at a pH of at least 7.5.

10. The kit of claim 5, wherein the self-assembling peptide comprises a sequence of the formula X1-X2-X1-X2-X1, wherein X1 is an amino acid with an acidic side chain and X2 is an amino acid with a hydrophobic side chain.

11. The kit of claim 5, wherein the self-assembling peptide comprises the amino acid sequence SEQ ID NO: 1 or an amino acid sequence having at least 80% identity thereto.

12. The kit of claim 5, wherein the self-assembling peptide is in predominantly monomeric form.

13. The kit of claim 5, wherein the self-assembling peptide is in predominantly assembled form.

14. A method for preventing secondary caries after failure of the dental agent in treatment of a cavitated carious lesion comprising administering to a subject the components of the kit claim 5.

15. The method of claim 14, wherein the treatment of the carious lesion comprises steps of a) cleaning of the tooth surface, b) optionally, drying of the tooth surface, c) optionally, etching the tooth surface, d) administering the composition or the components of the kit to the carious lesion, and e) optionally, curing.

16. A method for pulp capping comprising administering to a carious lesion in contact with pulp components of a kit comprising a) a self-assembling peptide, and b) a dental material selected from the group comprising a dental sealant, a dental bonding agent and a filler.

17. (canceled)

18. (canceled)

19. The kit of claim 8, wherein the self-assembling peptide is P11-4.

20. The kit of any of claim 9, wherein the self-assembling peptide is P11-8.

21. A method for preventing secondary caries after failure of the dental agent in treatment of a cavitated carious lesion comprising administering to a subject the composition of claim 1.

22. A method for pulp capping comprising administering to a carious lesion in contact with pulp a composition comprising: a) a self-assembling peptide, and b) a dental material selected from the group comprising a dental sealant, a dental bonding agent and a filler.

Description

FIGURE LEGENDS

[0106] FIG. 1.1 Treatment of a partially cavitated carious lesion (ICDAS class 3).

[0107] A: Cross section of partially cavitated tooth; partial cavitation on the left.

[0108] B: After tooth cleaning, here, without excavation, application of predominantly monomeric self-assembling peptide (e.g., P11-4, strips) solution onto tooth surface. The self-assembling peptide starts to diffuse into the cavity.

[0109] C: The self-assembling peptide, e.g., P11-4, assembles in the cavity due to the low pH, generating a matrix.

[0110] D: The tooth surface is dried, leaving the assembled P11-4 ribbons & fibres within the lesion

[0111] E: After the solution has been absorbed into the cavity, a sealant such as a glass-ionomer cement (GIC) is applied, allowing the diffusion of ions (calcium and phosphate) from the saliva and from the glass-ionomer cement into the lesion body.

[0112] F: Mineralization (hexagons) within the lesion protects the failing sealant, resulting in a mineralised structure underneath the lesion (protected by the sealant, e.g., GIC).

[0113] After failure of the GIC, a fully regenerated tooth surface and structure remains.

[0114] FIG. 1.2

[0115] A: Cross section of partially cavitated tooth; partial cavitation on the left.

[0116] B: Excavated caries lesion

[0117] C: Application of monomeric P11-4 (strips), either at the same time as filler, or sequentially.

[0118] D: Excogitated tooth with Filler (grey) and fibrillar P11-4 (longer strips) at the interface between tooth and filler

[0119] E: Mineralisation (hexagons) within the microgaps between filler and tooth. Calcium Phospahte supplied either from filler (if glass-ionomer cement), saliva or from pulp (via dentinal fluid).

[0120] FIG. 2 Treatment of a cavitated carious lesion (ICDAS class 4) with a composition of the invention in a caries model, as described in Example 2. In the right and left column, two representative examples are shown.

[0121] A, B: Drilling of artificial cavity

[0122] C, D: Application of 1 drop of P11-4, 10 mg/mL, pH 8

[0123] E, F: Application of Glass-Ionomer Cement (Aqual Ionofill Plus, VOCO)

[0124] FIG. 3 Treatment of a cavitated carious lesion (ICDAS class 4) with a kit of the invention in a caries model, as described in Example 3. In the right and left column, two representative examples are shown.

[0125] A, B: Drilling of artificial cavity.

[0126] C, D: Application of Glass-Ionomer Cement (Aqual Ionofill Plus, VOCO) mixed with 1 drop of

[0127] P11-4, 10 mg/mL, pH 8.

[0128] FIG. 4 Release profile of the model substance Congo Red and P11-4 from varnish, glass-ionomer cement and Paro-Amin Fluor Gelée. The experiment was conducted as specified in Examples 6 and 7. The release was measured as described and is specified in Arbitrary units (AU). FIG. 4A shows release of Congo Red from the varnish Duraphat (sample A1) and glass-ionomer cement Aqua Ionofil Plus (sample B1), and FIG. 4B shows release of Congo Red from paro Amin Fluor Gelée (Sample 2). FIG. 4C shows release of monomeric P11-4 from the varnish Duraphat (sample AP) and glass-ionomer cement Aqua Ionofil Plus (sample BP).

EXAMPLES

Example 1

[0129] A partially cavitated lesion (ICDAS class 3) is cleaned. It is either excavated (FIG. 1.2) or not excavated (FIG. 1.1). After cleaning, optionally including excavation, self-assembling peptide (e.g., P11-4) is applied onto the cleaned surface of the tooth, in particular, to the cavity. The self-assembling peptide starts to diffuse into the cavity. There, the self-assembling peptide assembles due to the low pH, generating a matrix. After the solution has been absorbed in to dentine or enamel (10 sec-5 min, dependent e.g. on the surface, quicker absorption in dentine than enamel), a sealant such as a glass-ionomer cement is applied, allowing the diffusion of ions (Ca and phosphate) from the saliva or the glass ionomer into the lesion body. This allows for a start of mineralization in the matrix protected by the sealant. After the sealant has fallen off the tooth, typically, after several months or years, a fully regenerated tooth surface and structure remains, in the absence of secondary caries.

Example 2

[0130] An artificial ICDAS class 4 lesion, generated by drilling in an excised bovine incisor was treated with a kit of the invention, in particular by application of 1 drop of P11-4, 10 mg/mL, pH 8, followed by a glass-ionomer cement (Aqual Ionofill Plus, VOCO) according to the manufacturer's instructions. In brief, polyacrylic acid conditioner was applied for 10 seconds, rinsed, slightly dried, glass ionomer applied and the material allowed to mature prior to finishing procedures. The main steps are shown in FIG. 2. The glass-ionomer cement cured to yield a hard restoration undistinguishable from a restoration applied without combination with P11-4 (data not shown). No differences could be determined up to one week after application either.

Example 3

[0131] An artificial ICDAS class 4 lesion, generated by drilling in an excised bovine incisor was treated with a composition of the invention. Glass-ionomer cement powder (Aqual Ionofill Plus, VOCO) was prepared according to the manufacturer's instructions. 1 mg P11-4 per 100 mg was added (10 mg per 1 ml Powder) and mixed with water. The powder was dissolved per manufacturer's recommendations. The solution was mixed for 30 s and applied to the tooth surface as recommended by the manufacturer (as described above). The main steps are shown in FIG. 3.

[0132] The glass-ionomer cement cured to yield a hard restoration undistinguishable from a restoration applied without combination with P11-4 (data not shown). No differences could be determined up to one week after application.

Example 4—Prevention of Secondary Caries

[0133] Teeth with artificial ICDAS class 4 lesions, generated by drilling in an excised bovine incisor, are treated with dental sealant (e.g., glass-ionomer (for example, Aqual Ionofill Plus, VOCO)) in the absence or presence of P11-4 according to three different protocols:

[0134] 1) The dental sealant is applied to the cleaned surface of the cavity according to the manufacturer's instructions.

[0135] 2) A drop of P11-4, 10 mg/mL, pH 8, is applied to the cleaned surface of the cavity so that the surface of the cavity is covered. After the solution has been essentially absorbed by the tooth surface, the dental sealant is applied to the cleaned surface of the cavity according to the manufacturer's instructions.

[0136] 3) Glass-ionomer cement powder (Aqual Ionofill Plus, VOCO) is prepared according to the manufacturer's instructions. 1 mg P11-4 per 100 mg is added (10 mg per 1 ml Powder) and mixed with water. The powder is dissolved per manufacturer's recommendations. The solution was mixed for 30 s and applied to the tooth surface as recommended by the manufacturer (as described above).

[0137] Teeth were stored in distilled water for 24 h. Then, the teeth were thermocycled (500 cycles, 5-55° C.), immersed in basic fuchsine, sectioned, and analysed for dye penetration and remineralisation using a stereomicroscope (e.g., as described by Hepdeniz et al., 2016. Eur J Dent. 10(2): 163-169).

[0138] The formation of mikrogaps or microleakage and shrinking of the dental sealant is monitored over time, and the remineralisation in the location of microgaps analyzed. The different groups are compared.

Example 5—Pulp Capping—Remineralization Due to Pulp and Calcium Phosphate from Blood Supply

[0139] A cavity exposing the pulp is excavated due to dental caries. The pulp is covered with P11-4 solution (e.g., 100 μl, 10 mg/mL, pH 8) before placing of a dental material, e.g., Resin Composite according to the manufacturer's instructions (e.g., application in layers and light curing, as appropriate).

[0140] The pulp remains symptoms free (i.e. no pulpitis), and in a follow-up x-ray after 1 year, retraction of the pulp is assessed.

Example 6— Release Profile

[0141] As well-known to the person skilled in the art, a release profile of an agent, i.e. fluoride or self-assembling peptide, depends on the matrix of the material (Mousavinasab et al., 2009, Dent Res J (Isfahan) 6(2):75-81). In the following experiment, the release profile of a model substance mimicking self-assembling peptide, Congo Red, was assessed after the incorporation into the dental material.

[0142] The material was prepared and applied as per instructions for use (IFU), usually describing the use of 0.5-0.75 mL of the respective products. To mimic the saliva, Phosphate Buffered Saline (PBS) was used. PBS matches the osmolarity and ion concentrations of the human body—it is isotonic. To mimic the constant saliva flux, the applied amount was placed in a solution equal to 1 h saliva flux, which is about 20 mL. The diffusion of the material was assessed at 500 nm with a photometer.

TABLE-US-00003 Material Article Sample Name Supplier ID Lot ID Duraphat Colgate Na   227955 A (Duraphat ® dental suspension, varnish) Aqua Ionofil Plus Voco 1513   1827129 B (glass ionomer restorative material) Paro-Amin Esro — 1919-1 2 Fluor Gelée PBS-Taps Sigma P4417 Water Credentis — — Oligopeptide-104 Bachem E104 1000020803 — Congo Red Sigma C6767 BCBR1055 Ethanol Credentis — — Syringe 1 mL Codan — — Balance Mettler AE200 — Toledo UV VIs Pharmacia LKB Ultraspec II — UV cuvette Brand — — Spatula Microscopic VWR — — Slides Eppendorf VWR — — tube 2 mL 50 mL VWR — — centrifugation tube

[0143] Release Profile [0144] a) Preparation of varnish (Duraphat) and paro Amin Fluor Gelée [0145] Per suppliers IFU, it is recommend to add 0.5 -1 mL of the varnish to a carious site. The concentration of Congo Red was chosen to lead a distinct signal in the UV Vis. [0146] Place 12 mg Congo Red in 2 mL Eppendorf tube [0147] Add 2 mL of the varnish directly into the tube with a syringe [0148] Mix Congo Red and varnish vigorously until content uniformity is reached [0149] Pick up product with syringe [0150] Place 0.5-0.75 ml of the product on a microscopic slide as a thin film—as recommended per IFU [0151] Let it air dry—as per IFU [0152] b) Preparation of Ionofil (glass ionomer restorative material) [0153] Place 12 mg Congo Red directly in a dental cup [0154] Add 2 spoons of Ionofil Plus as per IFU [0155] Add 2 drops of water and mix as per IFU [0156] Place amount of product on microscopic slide [0157] c) Incubation [0158] Prepare the PBS Solution with 1% EtOH for preservation [0159] Place slide in 50 ml centrifugation tube [0160] Add 20 mL of PBS [0161] Place on rotating wheel [0162] d) Measurement and calculation [0163] Directly decant 1 mL of incubated fluid into UV-Cell [0164] Measure at 500 nm, control/blank=PBS with 1% EthOH [0165] subtract control w/o Congo Red from measured value

[0166] Results and Discussion

[0167] Independent on the formulation of the material, the model substance incorporated easily into the products. This is shown by the visual assessment of content uniformity, since a homogenous red/brown dispersion is visible.

[0168] The model substance is also able to diffuse from all three fluoride containing preparations into the artificial saliva. Depending on the formulation, the release profile of the individual formulations is different.

[0169] The model substance Congo red was released from the varnish Duraphat and the glass ionomer cement (FIG. 4A), as well as from paro Amin Fluor Gelée used for comparison (FIG. 4B). Release from the varnish was quicker than from the glass ionomer cement. It can be seen that release from the glass ionomer cement was constant and increases with time.

Example 7— Release Profile of P11-4

[0170] Example 7 was in essence carried out as Example 6, but with the self-assembling peptide P11-4, if not otherwise described. 12 mg of P11-4 (credentis AG), in monomeric form, were added to either the varnish Duraphat (Sample ID: AP) or the glass ionomer cement Aqua Ionofil Plus (Sample ID BP) in the amounts and under the conditions described above. The incubation was carried out in a static manner at room temperature. Measurement was at 280 nm, and the control value for buffer without peptide was subtracted from the measured value.

[0171] The results are shown in FIG. 4C. Confirming the data before, the experiment shows a surprisingly good diffusion of the peptide out from the matrix tested, i.e. Duraphat (Sample AP) or Aqua Ionofil Plus (BP), confirming the data gained with the model substance.

[0172] P11-4 is slowly and substantially constantly released from both the varnish and the glass ionomer cement. The amount of released peptide increases with time, which was measured for several days. The release from varnish is quicker than the release from the gloss ionomer cement. Thus, depending on the formulation, a slow release can be obtained by using a sealant such as a glass ionomer cement, or a “fast” release with a varnish, e.g., a collophonium containing formulation.

[0173] A fast release as seen with the varnish e.g., Duraphat, may have beneficial impact on the performance of the peptide shortly after application, which may be particularly helpful, e.g., for remineralisation of lesions covered by varnish.

[0174] On the other hand, a longer release may further be particularly advantageous for prevention or treatment of secondary lesions—that may slowly develop over time-under, or at the edges of sealant on treated (filled) lesions. It is reasonable to conclude that the agent will be released from the sealant, e.g., the glass ionomer cement over a longer period of time than from varnish. This is appropriate, as the average time of the sealant, e.g., glass-ionomer cement, on the tooth is also longer than for varnish. Further, self-assembling peptide released from a filling may remain in place for a long time.