METHOD FOR PRODUCING A COMPOSITION FOR TREATING A TOOTH LESION

Abstract

The invention relates to a method for preparing a composition for treating a tooth lesion, said composition comprising peptides that are capable of undergoing self-assembly at a certain pH. The compositions of the invention are highly suitable for being used in the medical field, in particular for remineralising a tooth lesion such as a subsurface caries lesion.

Claims

1-8. (canceled)

9. A composition in lyophilized undissolved form comprising a peptide that undergoes self-assembly at a pH value below 7.5, wherein the peptide is predominantly in a monomeric form; a non-reducing sugar; and a non-volatile buffer; wherein upon reconstitution of the composition in water, a solution with a pH that is 0.1 to 0.5 pH units above the pH at which the peptide starts to undergo self-assembly is obtained in which the peptide is predominantly present in a monomeric form.

10. The composition of claim 9, wherein said peptide comprises the sequence of SEQ ID NO:3.

11. The composition of claim 9, wherein said peptide comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

12. The composition of claim 9, wherein said peptide comprises the sequence depicted in SEQ ID NO:1 or SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto.

13. The composition of claim 9, wherein at least 70%, preferably at least 80%, more preferably at least 90%, of the peptide are present in monomeric form.

14. (canceled)

15. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0073] FIG. 1 depicts the sequences of the self-assembling peptide P11-4 (SEQ ID NO:1) and a sequence of P11-4 that has been N- and C-terminally modified (SEQ ID NO:2).

[0074] FIGS. 2A to 2D show example compositions in bulk solution prior to lyopilization (FIG. 2A), after filling prior to lyophilization (FIG. 2B) and after lyophilisation (FIGS. 2C and 2D). The compositions in FIG. 2B have been stained solely for the purpose of visualization.

[0075] FIG. 3 shows the phase behaviour of P11-4 (c=6.3 mM) as a function of pH (DCl/NaOD): I=nematic gel, II=flocculate, III=nematic fluid, IV=isotropic fluid. o=zero shear viscosity, and .circle-solid.=% p-sheet determined using FTIR spectroscopy: the continuous line denotes the proportion of peptide in fibrils. The broken vertical lines separating regions I, II, and III denote approximate boundaries between different macroscopic fibrillar states, while that separating regions III and IV denotes a first order nematic-to-isotropic transition. (Figure replicated with permission from Aggeli et al. (2003, J am ChemSoc, 125, 9619-9628).)

[0076] The method of the invention is exemplified in more detail in the following examples.

EXAMPLES

Example 1

Preparation of Peptide Solutions

[0077] Modified P11-4 peptide with the sequence depicted in SEQ ID NO:2 was thawed 24 hours before use at 5+/3 C. 4 mM Tris are added to water of the WFI (water for injection) grade under stirring for at least 10 minutes at room temperature. Trehalose-Dihydrate is added under continued stirring to a final concentration of 10 mg/g. Subsequently, the peptide P11-4 is added to a final concentration of 2 mg/ml and dissolved under stirring to clarity. This peptide solution initially has a pH of about 8.2.

[0078] The pH of the peptide solution is adjusted to 8.5 +/0.4 with 1% NH.sub.4OH solution.

[0079] The peptide solution is subsequently filtered sterile through Sartopore filters. The sterile solution is filled into vials which are then submitted to lyophilisation in a multi shelve freeze-drier. After freezing for 1 to 3 h at 45 C., the vials are submitted to a main drying step at 30 C. for 10 to 13 h and a after drying step at 30 C. for 7 to 10 h. Airing is supplied with nitrogen at 200 to 800 mbar. The vials are closed at 30 C. and 200 mbar and capped for transport.

[0080] The vials are subsequently inspected visually for integrity of the cake (i.e. the lyophilisate).

[0081] Results: The peptide solution was in the form of a nematic viscoelastic fluid after dissolution. After adjusting the pH to 8.5, the peptide solution appeared clear and non-viscous as an isotropic fluid (FIG. 2 A). After filling of the solution into vials, the solution still appeared clear and non-viscous (FIG. 2B: example aliquots which were stained blue solely for visualization of the solution). Lyophilization lead to an intact cake, i.e. lyophilisate, (FIG. 2C) that showed good storage stability.

Example 2

Preparation of Peptide Solutions with Different Concentrations

[0082] As described above, peptide solutions with different buffer (TRIS), peptide (P11-4) and bulking agent (Trehalose-Dihydrate) concentrations were prepared. The pH was adjusted as described above.

TABLE-US-00001 Peptide Bulking conc Agent Buffer Example [mg * ml1] [mg * ml1] [mM] 1 1 50 4 2 2 25 5 3 4 10 6

[0083] Results: After filling of the solution into vials, all three solutions appeared clear and non-viscous. Lyophilization lead to an intact cake, i.e. lyophilisate, that showed good storage stability.

Example 3

Viscosity of P11-4 Peptide Solution

[0084] To estimate the assembly state in a peptide solution, passage of the P11-4 peptide solution through different filling needles was tested.

[0085] Composition of P11-4 Bulk Solution

[0086] A P11-4 bulk solution was compounded as follows:

TABLE-US-00002 TABLE 1 Table 1 shows the composition of the solution. Concentration in final solution Substances Weight [g] [mg/g] P11-4 0.500 2 Trehalose dihydrate 2.5 10 TRIS 0.121 0.484 add. to with dist. 250 / Water

[0087] P11-4, trehalose dihydrate (Hayashibara Co. Ltd., Lot Nr. 9D081) and TRIS (Carl Roth, A411.1) were dissolved in approximately 200 g purified water. The pH was adjusted to 8.5 using about 0.5 mL 1% NH3-solution. Final weight of the bulk solution was adjusted with purified water to 250 g.

[0088] The solution was then filtered using an Acrodisc 25 mm syringe filter (0.22 m Fluorodyne II membrane) connected to a peristaltic pump (520Di, Watson-Marlow Pumps Group, Falmouth, United Kingdom) delivering the bulk solution at a pump speed of 10 rpm.

[0089] Density of the Peptide Solution

[0090] P11-4 bulk solution was compounded and the density of the solution was determined using a DMA300 portable density meter (Anton Paar Germany GmbH, Ostfildern, Germany). Bulk solution was filled into the density meter using a syringe. Temperature compensation to 25 C. was automatically performed by the density meter.

[0091] Density measurements of the peptide solution were performed. The solution reached a density of 1.001 mg/mL at 25 C., n=2.

[0092] Viscosity of the Peptide Solution

[0093] The EDM 3295 dosing machine (Bausch+Strobel Maschinenfabrik GmbH+Co. KG, Ilshofen, Germany) was adjusted to a filling weight of 250 mg. Test inlay vials were weighted empty, filled with bulk solution and weighted again after filling.

[0094] Table 2 gives the results for the test filling using filling needle 443009L (0 1.6 mm, length 96 mm; material code: AI-SI316L).

TABLE-US-00003 TABLE 2 Statistical evaluation test filling using filling needle 443009L ( 1.6 mm) Average [mg] 248.4 SD [mg] 0.3 Rel. SD [%] 0.1 2 SD [mg] 0.7 2 rel. SD [%] 0.3 Minima [mg] 248.0 Maxima [mg] 249.3 n 20

[0095] Table 3 gives the results of the test filling using filling needle 443008L (0 1.1 mm, length 96 mm; material code: AI-SI316L)

TABLE-US-00004 TABLE 3 Statistical evaluation test filling using filling needle 443008L ( 1.1 mm) Average [mg] 248.7 SD [mg] 0.3 Rel. SD [%] 0.1 2 SD [mg] 0.7 2 rel. SD [%] 0.3 Minima [mg] 248.2 Maxima [mg] 249.4 n 20

[0096] Both filling needles showed a comparable filling accuracy of less than 1.0 mg, i.e. a minimal loss of substance, at a filling weight of 250 mg. Consequently, the peptide solution was able to pass efficiently through both needles. Essentially no clogging of the needle occurred. The viscosity of the solution was low enough to allow needle passage. This indicates that the peptide solution is present as an isotropic fluid (compare Aggeli et al., 2003, J Am Chem Soc, 125: 9619-9628 for the phase behaviour of peptide P11-4 as a function of the pH).