ACCELERATING TOOTH REMINERALISATION AND BONE REGENERATION WITH SELF-ASSEMBLING PEPTIDES AND AMORPHOUS CALCIUM PHOSHATE

Abstract

The present invention relates to the field of medicinal tissue mineralisation, in particular, i.e. tooth remineralisation and bone regeneration with self-assembling peptides. Use of self-assembling peptides, such as P11-4, also designated Oligopeptide-104, in these processes leads to generation of hydroxyapatite, which is also present in natural enamel, dentin and bone. The inventors have discovered that both tooth remineralisation and bone regeneration can be significantly accelerated by adding amorphous calcium phosphate or calcium and phosphate ions that, when mixed in solution, can lead to immediate precipitation of calcium phosphate, preferably, amorphous, i.e., non-crystalline calcium phosphate. The presence of self-assembling peptide however changes the structure of the precipitated calcium phosphate, and advantageously induces crystallisation and a synergistic accelerated formation of crystalline calcium phosphate, in particular, hydroxyapatite (HA). The invention thus provides a kit comprising, a self-assembling peptide and either calcium and phosphate ions in separate compositions suitable for immediately forming calcium phosphate precipitates, e.g., amorphous calcium phosphate (ACP), if the compositions are mixed in the presence of water, or calcium phosphate particles, preferably, in the form of a suspension of calcium phosphate particles comprising at least 50% ACP. The invention also provides medical use of said kit, in particular, for in the tooth for remineralisation of lesions, mineralisation of pits and fissures, pulp capping, and for bone regeneration.

Claims

1. A kit comprising, a) a self-assembling peptide comprising the amino acid sequence SEQ ID NO: 1 or an amino acid sequence having at least 70% identity thereto, and b) (i) calcium and phosphate ions in separate compositions suitable for immediately forming calcium phosphate precipitates if the compositions are mixed in the presence of water, wherein the compositions optionally are solutions, or (ii) calcium phosphate particles, optionally, in the form of a suspension of calcium phosphate particles comprising at least 50% ACP.

2. The kit of claim 1 for use in inducing tooth remineralization or bone regeneration in at least a region of a subject in need thereof.

3. The kit of claim 1, comprising b) calcium and phosphate ions in separate compositions suitable for immediately forming calcium phosphate precipitates, ACP if the compositions are mixed in the presence of water, wherein the compositions are solutions.

4. The kit of claim 3, wherein the solution comprising calcium ions comprises Ca.sup.2+ in a concentration of 0.01-0.5 M, e.g., 0.1-0.15 M.

5. The kit of claim 3, wherein the solution comprising phosphate ions comprises phosphate in a concentration of 0.01-0.5 M, e.g., 0.1-0.15 M.

6. The kit of claim 3, wherein the self-assembling peptide is a part of the composition comprising calcium ions.

7. The kit of claim 3, wherein the self-assembling peptide is a part of the composition comprising phosphate ions.

8. The kit of claim 3, further comprising fluoride ions.

9. The kit of claim 1, comprising b) calcium phosphate particles.

10. The kit of claim 1, wherein the self-assembling peptide comprises the amino acid sequence SEQ ID NO: 1, wherein the self-assembling peptide optionally consists of said amino acid sequence.

11. The kit of claim 1, wherein the self-assembling peptide is provided in a composition selected from the group consisting of a dry composition, a lyophilized composition, a water-based solution, a toothpaste, a tooth gel, a mouthwash, a mouth spray, an oral care foam, a chewing gum, a toffee, a lozenge, or a candy.

12. The kit of claim 1, wherein the composition comprising the calcium phosphate particles is provided as a composition selected from the group consisting of a dry powder, a water-based suspension, a water-free product, a toothpaste, a tooth gel, a mouthwash, a mouth spray, an oral care foam, a chewing gum, a toffee, a lozenge, a tablet or a candy.

13. The kit of claim 1 that is a solid or semi-solid composition comprising, a) in a first part, the self-assembling peptide, and b) in a second part, which is optionally surrounded by the first part, the phosphate ions or the calcium phosphate particles.

14-16. (canceled)

17. The method of claim 1, wherein the calcium phosphate precipitates are amorphous calcium phosphate (ACP).

18. The method of claim 4, wherein the solution comprises CaCl.sub.2.

19. The method of claim 5, wherein the solution comprises Na.sub.2HPO.sub.4.

20. The method of claim 9, comprising an aqueous suspension of at least 50% ACP.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0240] FIG. 1 is a table summarizing the conditions and results of Example 1. n.a.: not applicable

[0241] FIG. 2 shows SEM pictures of crystals, or, if no crystals were formed, of dried solutions formed in the experiments of Example 1. A: HA (control) B: ACP (control), C: CaNa, D: CaPaNa, E: CaPaNaF, F: alpha, G: beta, H: gamma-1, I: gamma-2, J: delta. [0242] a/b) different scales as specified [0243] c) photo of solution formed by mixing [0244] d) microscopic picture of crystals formed, as seen by a digital magnifying glass, if applicable.

EXAMPLES

Example 1

[0245] Individual solutions of CaCl.sub.2) (calcium chloride dihydrate, pH 6) and Na.sub.2HPO.sub.4 (di-Sodium Phosphate Dihydrate, pH 8) were prepared at different concentrations and in the presence or absence of self-assembling peptide P.sub.11-4 (Bachem, E104) and/or NaF. pH was corrected to 8, if required, with NaOH. Remineralisation solution (also known as artificial saliva) was prepared according to Kirkham et al. 2007, consisting of 1.5 mM Ca(NO 3) 2, 0.9 mM KH.sub.2PO.sub.4, 130 mM KCl, 60 mM Tris (pH 7.4). All salts were obtained from Sigma Aldrich.

[0246] The individual compositions, as shown in FIG. 1, were transferred to a chamber of a dual chamber syringe (Sulzer Mixpac), the mixing unit, a static mixer, e.g. 1:1, DN2, brown (Sulzer Mixpac) added, and the compositions extruded. The concentrations relate to the concentration in the syringe if not specified otherwise. After extrusion, the respective concentrations are half of those specified, as each solution is diluted 1:1.

[0247] Further, with all compositions comprising sufficiently high concentrations of calcium and phosphate of at least 0.01 M, i.e., higher than the remineralisation buffer, calcium phosphate crystals precipitated, basically, immediately. The crystals, and for comparison, HA and ACP obtained from Sigma Aldrich, were analysed by SEM (FIG. 2) and by analysing the Ca/P ratio (FIG. 1), which allows for assessment of the calcium phosphate form formed. A Ca/P ratio of 1.6 or more is regarded as HA (e.g., Jeffre et al., 1993).

[0248] Further details of preparation and results for each experiment are provided below.

Example 1A—SCaNa/CaPaNa/CaPaNaF

[0249] Fresh solutions of 0.36M CaCl.sub.2) (Calcium Chloride Dihydrate, Sigma Aldrich, pH 6) and 0.4M Na.sub.2HPO.sub.4 (di-Sodium Phosphate Dihydrate, Sigma Aldrich, pH 8) were prepared.

[0250] CaNa: The CaCl.sub.2) solution and the Na.sub.2HPO.sub.4 solutions were each transferred into one chamber of the double chamber syringe system equipped with a static mixer (1:1). The two solutions were transferred through the mixer resulting in mixed liquids (pH 6-7), wherein a precipitate formed immediately. SEM showed spherical precipitate, wherein the particles were typical of ACP (cf. ACP control). The SEM further shows crystals typical of NaCl that were generated by the drying process.

[0251] CaPaNa: The self-assembling peptide P.sub.11-4 was added to the Na.sub.2HPO.sub.4 solutions. 150 mg self-assembling peptide P.sub.11-4 was weighed into a 5 mL Eppendorf tube and added to 3 mL of Na.sub.2HPO.sub.4 solution (0.4M). pH was adjusted with NaOH (1N) to pH=8. The solution was sonicated (30 s), yielding a clear and transparent solution having a P.sub.11-4 concentration of 30 mg/mL. This solution comprising the self-assembling peptide P.sub.11-4 and Na.sub.2HPO.sub.4 and the CaCl.sub.2) solution were each transferred to a chamber of the double chamber syringe system. The two solutions were transferred through the mixer 1:1, resulting in mixed liquids (pH 6-7), wherein a precipitate formed immediately. SEM showed spherical precipitate particles typical of ACP.

[0252] The addition of self-assembling peptide P.sub.11-4 to the Na.sub.2HPO.sub.4 solution let to formation of calcium phosphate having a low Ca/P-ratio of 0.65, indicating still less crystalline character than without self-assembling peptide. The solution appears to be so highly supersaturated in Ca.sup.2+ and PO.sub.4.sup.3−-ions that precipitation occurs immediately, and addition of self-assembling peptide fibres cannot control/catalyse formation of more ordered crystals. Recrystallization on the self-assembling peptides may further lead to HA formation.

[0253] CaPaNaF: Na.sub.2HPO.sub.4 solution (0.108 M Ca.sup.2+ and 0.12 M phosphate) containing 30 mg*mL.sup.−1 self-assembling peptide P.sub.11-4 was enriched with NaF (1450 ppm Sodium Fluoride; i.e. the fluoride concentration of typical commercial toothpaste) to assess the influence of fluoride ions on the crystal growth. The resulting Na.sub.2HPO.sub.4/self-assembling peptide P.sub.11-4/Fluoride and the CaCl.sub.2) solution (0.108 M) were transferred to a double chamber syringe system and mixed 1:1. The liquid, having a pH or 6-7 and a final P11-4 concentration of 15 mg/mL, immediately showed 3D effects. Immediately, crystal-like structures formed that were similar to HA. The Ca/P ratio was >2. Thus, addition of fluoride at (1450 ppm) favoured HA formation, even though the self-assembling peptide was favoured despite the self-assembling peptide P.sub.11-4 being incorporated into the CaCl.sub.2) solution.

Example 1B—“Greek” Series

Investigating the Influence of Self-Assembling Peptide P.SUB.11.-4 and Ion Concentrations.

[0254] Fresh solutions of 0.14M CaCl.sub.2) (pH=6-7; Calcium Chloride Dihydrate) and 0.14M Na.sub.2HPO.sub.4 (pH=8; di-Sodium Phosphate Dihydrate) and remineralisation buffer were prepared.

[0255] alpha: In the absence of self-assembling peptide, 0.14 M CaCl.sub.2) solution and 0.14 M Na.sub.2HPO.sub.4 solution were transferred separately into the dual chamber syringe system equipped with a static mixer (1:1). The two solutions were transferred trough the mixer, resulting in immediately mixed, pH neutral liquids. Flat square crystals formed immediately. The crystals visible on the SEM appear to be NaCl cubic crystals. In between the cubic NaCl crystals, spheres of calcium phosphate precipitate can be seen, similar to in appearance to ACP. The Ca/P ratio was 1.26, also similar to ACP.

[0256] beta: Double concentrated remineralisation buffer/artificial saliva-solution was freshly prepared and immediately transferred one chamber of the dual chamber syringe, whereas in the other barrel, plain water was transferred. The two solutions were transferred trough the mixer, resulting in immediately mixed, pH neutral liquids. No precipitation or crystal formation occurred within the next 12 hours.

[0257] delta: Double concentrated remineralisation buffer/artificial saliva-solution was prepared as for experiment beta. However, self-assembling peptide P.sub.11-4 dissolved in water at 20 mg/mL (pH adjusted to pH=8 with 1N NaOH), and sonicated for 30 s, resulting in a homogenous, clear solution, was placed in the other chamber of the dual chamber syringe instead of water. The two solutions were transferred through the mixer resulting in immediately mixed, pH neutral liquids. No precipitation or crystal formation occurred within the next 12 hours. The crystals seen on the SEM picture only formed upon drying of the solution in the recording process.

[0258] gamma-1: The self-assembling peptide P.sub.11-4 (20 mg/mL) was added to 0.14M sodium phosphate solution (pH adjusted with 1 N NaOH to pH=8), sonicated for 30 s, resulting in a clear solution. 0.14 M CaCl.sub.2) solution and 0.14 M Na.sub.2HPO.sub.4 solution were transferred separately into the double chamber syringe and mixed 1:1. This resulted in pH neutral mixed liquids, wherein, immediately, a precipitate having an interwoven or network-like morphology precipitated, with crystals visible. The Ca/P-Ratio remained similar at Ca/P-Ratio=1.1. On the SEM pictures, it appears that self-assembling peptide P.sub.11-4 fibres had formed, and that calcium phosphate precipitated around it.

[0259] gamma-2: The experiment was prepared as for gamma-1, however, the self-assembling peptide P.sub.11-4 peptide was added into the 0.14M CaCl.sub.2) solution at 20 mg/mL (pH adjusted with 1 N NaOH to pH=8), resulting in a whitish/opaque solution even after 30 s sonication. The solutions were transferred separately into the dual chamber syringe and mixed 1:1. This resulted in pH neutral mixed liquids, wherein, immediately, a precipitate having an interwoven or network-like morphology precipitated, with crystals visible.

[0260] Surprisingly, when self-assembling peptide P.sub.11-4 was incorporated into the CaCl.sub.2) solution, the Ca/P-Ratio of the formed precipitate increased to Ca/P-Ratio=1.7, clearly indicating a formed apatite structure. The SEM pictures showed thin needle-type crystals around self-assembling peptide P.sub.11-4 fibres.

[0261] In conclusion, addition of self-assembling peptide P.sub.11-4 led to precipitation of calcium phosphate around the self-assembling peptide P.sub.11-4 fibres when the concentration of Ca.sup.2+ and PO.sub.4.sup.3− ions was in the range of physiological salt concentration, e.g., about 0.05-0.15 M. The crystalline form of the precipitated calcium phosphates depended on the presence of self-assembling peptide P.sub.11-4, the concentration of Ca.sup.2+ and PO.sub.4.sup.3− ions, the presence of fluoride, and the solution in which self-assembling peptide P.sub.11-4 was included prior to mixing.

[0262] If the concentration of calcium and phosphate ions was high, e.g., 0.36M, the precipitate tended to be amorphous both in absence and presence of self-assembling peptide P.sub.11-4, indicating that the Ca.sup.2+ and PO.sub.4.sup.3− supersaturation was so high that the precipitation reaction was faster than crystal nucleation (in other words, templating of the calcium phosphate crystals on the fibrilliar surface of self-assembling peptide was too slow to compete).

[0263] In the range of physiological salt concentration of Ca.sup.2+ and PO.sub.4.sup.3− ions (0.14M, or, in the resulting solution, 0.07 M), the precipitation reaction and the nucleation and secondary crystal growth seem to compete with regard to the formation of calcium phosphates. If the self-assembling peptide is included in the phosphate containing solution, ACP was formed, indicating that no templating nor prepositioning of Ca.sup.2+ ions occurs prior to precipitation. However, when the self-assembling peptide is included in the Ca.sup.2+ containing solution, hydroxyapatite was formed. Without intending to be bound by the theory, this may indicate that calcium ions were prepositioned, probably by being bound to low order aggregates of the self-assembling peptide, leading to fast nucleation of HA and secondary crystal growth, dominating over precipitation of ACP.

[0264] Interestingly, the addition of fluoride ions to the system resulted in formation of HA, even if the self-assembling peptide was included in the Na.sub.2HPO.sub.4 solution. It seems that presence of fluoride-ions favours nucleation of HA crystals.

Sample Preparation for Microscopic Evaluation

[0265] The samples were dried (12 h; 40° C.), rinsed—if possible—and dried again. The resulting precipitates were collected for scanning electron microscope (SEM) and Energy-dispersive X-ray spectroscopy (EDX) investigation.

[0266] Samples were individually placed on sample holders equipped with carbon tape and sputtered with gold (30 s). The coating had an average thickness of 3-4 nm. Following sputtering, samples were transferred to an SEM (SEM Supra 40 VP; 10 kV; WD=˜8 mm, Zeiss) and visually assessed.

[0267] The atomic composition was investigated using the EDX beam installed on the SEM. The standard magnification was defined with 1000×.

Calculation of Ca/P

[0268] EDX: The % element ratio was determined by critically assessing the Ca and P-peak using software (Thermoscientific NSS, Version 3.3). For calculation purposes, the Au (from the gold coating) was deducted in the calculations to avoid overestimation of the P-ratio. The calculation was as follows:

(Element wt (Ca)/Element wt (P))=Ca/P ratio.