PHARMACEUTICAL COMPOSITION FOR JOINT DISEASE TREATMENT AND METHOD FOR PRODUCING SAME

Abstract

A pharmaceutical composition for treating joint diseases has a peptide hydrogel constituted of a self-assembled peptide. The peptide contains a β-hairpin structure constituted of β-sheet structures composed of alternating sequences of hydrophilic amino acids and hydrophobic amino acids, and a β-turn structure having biological activity. A method for producing the pharmaceutical composition involves preparing an aqueous solution containing said peptide, and adding concentrated phosphate buffered saline to the aqueous solution to generate a peptide hydrogel, and further comprising adjusting a concentration of the phosphate buffered saline in order to adjust an optimum elastic modulus of the peptide hydrogel according to the site to which the pharmaceutical composition is applied.

Claims

1. A pharmaceutical composition for treating joint diseases the pharmaceutical composition comprising a peptide hydrogel constituted of a self-assembled peptide, wherein the peptide contains a β-hairpin structure constituted of β-sheet structures composed of alternating sequences of hydrophilic amino acids and hydrophobic amino acids, and a β-turn structure having biological activity.

2. The pharmaceutical composition according to claim 1, wherein the peptide hydrogel has a difference between a storage elastic modulus G′ and a loss elastic modulus G″ of 3900 Pa or more.

3. The pharmaceutical composition according to claim 1, wherein the peptide is composed of an amino acid sequence of SEQ ID NO: 1.

4. The pharmaceutical composition according to claim 1, wherein the joint disease is meniscus injury.

5. A method for producing the pharmaceutical composition as described in claim 1, the method comprising: a step of preparing an aqueous solution containing said peptide, and a step of adding concentrated phosphate buffered saline (PBS) to the aqueous solution to generate a peptide hydrogel.

6. The method according to claim 5, wherein the concentrated PBS is added to the aqueous solution such that the final concentration of PBS is 3×PBS or more.

7. The method according to claim 5, further comprising a step of adjusting a concentration of the phosphate buffered saline in order to adjust an optimum elastic modulus of the peptide hydrogel according to the site to which the pharmaceutical composition comprising a peptide hydrogel constituted of a self-assembled peptide, wherein the peptide contains a β-hairpin structure constituted of β-sheet structures composed of alternating sequences of hydrophilic amino acids and hydrophobic amino acids, and a β-turn structure having biological activity is applied.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] FIG. 1 is a graph showing the storage elastic modulus G′ (left bar) and loss elastic modulus G″ (right bar) of peptide hydrogels obtained using 5 wt % NaCl aqueous solution, 1×PBS, 3×PBS and 5×PBS, respectively.

[0027] FIG. 2 A is a photograph showing a defect in the meniscus of a rabbit prepared in the present example, and FIG. 2 B is a photograph showing a peptide hydrogel used for transplantation in the present example.

[0028] FIG. 3 provides photographs for explaining Quantitative Analysis of the Occupation Ratio of Tissue Regeneration (R/D ratio) performed in the present example.

[0029] FIG. 4 provides photographs showing the meniscus after 2, 4, 8 and 12 weeks in the case where a peptide hydrogel was transplanted into the rabbit meniscus defect (KI24RGDS group) and where it was not transplanted (Control group), respectively.

[0030] FIG. 5 provides photographs showing safranin O-stained meniscus tissue at 2, 4, 8 and 12 weeks after a peptide hydrogel was transplanted into the meniscus defect of a rabbit (KI24RGDS group).

[0031] FIG. 6 provides photographs showing safranin O-stained meniscus tissue at 2, 4, 8 and 12 weeks after the preparation of the defect in the meniscus defect of a rabbit (control group).

[0032] FIG. 7 is a graph showing the results of Ishida Score of the KI24RGDS group and the control group in the present example.

[0033] FIG. 8 is a graph showing the results of Quantitative Analysis of the Occupation Ratio of Tissue Regeneration (RID ratio) of the KI24RGDS group and the control group in the present example.

[0034] FIG. 9 provides photographs and a model diagram showing the meniscus tissue 2 weeks after a peptide hydrogel was transplanted into the meniscus defect of a rabbit, and FIG. 9A is a HE-stained photograph, FIG. 9B is a safranin O-stained photograph, and FIG. 9C is a model diagram showing the state of tissue regeneration.

DESCRIPTION OF EMBODIMENTS

[0035] Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The following description of preferred embodiments is merely exemplary and is not intended to limit the invention, its application methods or its uses.

[0036] The pharmaceutical composition according to the present invention contains a peptide hydrogel constituted of self-assembled peptides, and is particularly a pharmaceutical composition used for the treatment of joint diseases. Further, in the pharmaceutical composition according to the present invention, the peptide is characterized by containing a β-hairpin structure constituted of β-sheet structures composed of alternating sequences of hydrophilic amino acids and hydrophobic amino acids and a β-turn structure having biological activity.

[0037] In the pharmaceutical composition according to one embodiment of the present invention, the peptide hydrogel is a hydrogel obtained by encapsulating water when a peptide having a structure having both a hydrophilic surface and a hydrophobic surface is self-assembled under predetermined conditions. Further, the peptide constituting the peptide hydrogel is a peptide having a β hairpin structure formed of β sheets and a β turn located between the β sheets and connecting them as described above. The β sheet is formed by alternating sequences of hydrophilic amino acids and hydrophobic amino acids as described above, and has both a hydrophilic surface and a hydrophobic surface. In the present embodiment, the β sheet is preferably formed by repeating sequences of lysine (K) and isoleucine (I).

[0038] The β-turn usually refers to a turn structure stabled due to hydrogen bond formation between the carbonyl oxygen atom of the n-th amino acid residue and the amide proton of the n+3-th amino acid residue. There are various amino acid sequences that form β-turns, which are well known to those of skill in the art. In the present embodiment, the amino acid sequence forming a β-turn has biological activity, and the biological activity is preferably cell adhesion activity. In the present embodiment, the β-turn preferably consists of an arginine-glycine-aspartic acid-serine (RGDS) sequence. The RGDS sequence is known as a sequence forming a β-turn, and further, the RGDS sequence is a sequence derived from fibronectin and is known to bind to integrin αVβI and exhibit cell adhesion activity.

[0039] In the present embodiment, the β hairpin structure is formed by shielding the electrostatic repulsion of the hydrophilic surface (lysine residue) of the β sheet by the action of a salt on the peptide having the above sequence. The β-hairpin structure is stably formed by intramolecular hydrophobic interaction between side chains and an intramolecular hydrogen bond between the carbonyl and amide moieties of the peptide backbone, which facilitates self-assembly.

[0040] In particular, in the present embodiment, the β hairpin structure is preferably a β hairpin structure in which the RGDS sequence is arranged between the repeating sequences of K and I. The number of repetitions of K and I in the β sheet is not particularly limited, but for example, the number of repetitions is preferably 5, and therefore, the amino acid sequence forming the β hairpin structure in the present embodiment is preferably IKIKIKIKIKRGDSKIKIKIKIKI (KI24RGDS: SEQ ID NO: 1).

[0041] In the present embodiment, the peptide hydrogel preferably has high mechanical strength. Specifically, it is preferable that the peptide hydrogel has a mechanical strength that makes it difficult for the peptide hydrogel to flow to the surroundings at a desired transplanted portion in a living body, and in particular, the difference between the storage elastic modulus G′ and the loss elastic modulus G″ is preferably 3900 Pa or more, and more preferably 10000 Pa or more.

[0042] In the pharmaceutical composition according to the present embodiment, the joint disease refers to a disease in which an abnormality such as deformation, injury or inflammation occurs in the joint. In the present embodiment, the joint refers to, but not limited to, a portion where bones are connected to each other in parts such as shoulders, elbows, fingers, crotch, knees and ankles. The joint disease in the present embodiment is specifically a disease of a part of the joint other than the bone, and includes, for example, diseases of cartilage, ligament, and meniscus.

[0043] The pharmaceutical compositions according to the present embodiment is used transplanted into the affected area of a joint disease, and more specifically is implanted into the defect of the site of a joint disease. The defect may be a portion that is defective due to the disease itself, or may be a portion that is created by active excision by surgery.

[0044] The pharmaceutical composition according to the present embodiment may contain various pharmaceutically acceptable additives in addition to the peptide hydrogel. However, it is preferable that the additive does not reduce the mechanical strength of the peptide hydrogel and does not reduce the mechanical strength of the pharmaceutical composition.

[0045] The method for producing a pharmaceutical composition according to the present invention is a method including the preparation of a peptide hydrogel as described above, and specifically, comprises a step of preparing an aqueous solution containing a peptide and a step of adding phosphate buffered saline (PBS) to the aqueous solution to generate a peptide hydrogel.

[0046] In the method for producing a pharmaceutical composition according to an embodiment of the present invention, the peptide and the peptide hydrogel are a peptide and a peptide hydrogel having the above-mentioned characteristics, respectively. The peptide having a desired sequence can be synthesized by a method well known to those skilled in the art, for example, by using a well-known Fmoc solid-phase synthesis method. The synthesized peptide may be purified by a well-known method such as high performance liquid chromatography (HPLC) or the like. Then, the obtained peptide can be dissolved in water to prepare an aqueous peptide solution. The peptide concentration of the aqueous solution is not particularly limited, but can be adjusted to, for example, about 1 to 10%.

[0047] In the present embodiment, a peptide hydrogel is obtained by adding PBS to the aqueous solution prepared as described above. Specifically, the salt in PBS shields the electrostatic repulsion of the hydrophilic surface of the peptide to form a β-hairpin structure, which becomes stable by the intramolecular hydrophobic interaction between side chains of the peptide and an intramolecular hydrogen bond between the carbonyl and amide moieties of the peptide backbone, facilitating self-assembly. At this time, water in the aqueous solution is encapsulated, and as a result, a peptide hydrogel is generated. When PBS is added to an aqueous solution containing a peptide, it is preferable to add an equal amount of PBS to the aqueous solution. In this case, PBS at a concentration twice the desired final concentration of PBS will be added.

[0048] In the present embodiment, the concentration of PBS is not particularly limited, but the mechanical strength of the resultant peptide hydrogel can be increased by increasing the concentration of PBS. However, if the concentration of PBS is increased excessively, salt precipitation may occur, so it is not preferable to increase the concentration excessively. In the present embodiment, for example, it is preferable to mix 10×PBS with the peptide-containing aqueous solution in an equal amount to obtain a final concentration of 5×PBS. In addition, 1×PBS can be prepared by a well-known method, for example, it can be prepared by adding 1 L of water to 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 1.44 g of disodium hydrogen phosphate and 0.24 g of potassium dihydrogen phosphate, and adjusting pH to 7.4 with hydrochloric acid or the like. Therefore, 2×PBS can be prepared by doubling the amount of each of the above salts, and 10×PBS can be prepared by multiplying the amount of each of the above salts by 10 times. As described above, since the mechanical strength of the resultant peptide hydrogel can be increased by increasing the concentration of PBS, the mechanical strength of the peptide hydrogel can be adjusted by adjusting the concentration of PBS used.

Examples

[0049] Hereinafter, examples for explaining in detail the pharmaceutical composition according to the present invention and the method for producing the same are shown.

[0050] [Example 1: Preparation of peptide hydrogel] First, in order to prepare the peptide hydrogel according to the present example, a peptide consisting of an amino acid sequence of IKIKIKIKIKRGDSKIKIKIKIKI (KI24RGDS: SEQ ID NO: 1) was synthesized by using the Fmoc solid-phase synthesis method, which is a conventional method for peptide synthesis. The synthesized peptide was isolated and purified using well-known HPLC.

[0051] Six milligrams (6 mg) of the KI24RGDS peptide obtained as described above was dissolved in 100 μL of ultrapure water to prepare a 6% aqueous peptide solution.

[0052] Next, an equal amount (100 μL) of 2×PBS, 4×PBS, 6×PBS or 10×PBS was added to the aqueous peptide solution and mixed. Therefore, the final concentration of PBS in the mixture was 1×PBS, 2×PBS, 3×PBS, and 5×PBS, respectively. Thereafter, it was allowed to stand still in a cool and dark place for one day. As a result, the peptide in the aqueous peptide solution self-assembled by the action of the salt in PBS, and a peptide hydrogel was obtained. In addition, as a comparative example, Eagle's minimum essential medium (EMEM), which contains salts necessary for gel formation but is considered to be toxic in vivo, or 10 wt % NaCl aqueous solution (final concentration is 5 wt %) was added in an equal amount to the peptide aqueous solution and mixed, and the mixture was allowed to stand still in a cool and dark place for 1 day. A peptide hydrogel was also obtained in this comparative example.

[0053] [Example 2: Measurement of mechanical strength of peptide hydrogel] In order to evaluate the mechanical strength of each peptide hydrogel obtained as described above, a rheometer (HAAKE viscosity/viscoelasticity measuring device (HAAKE MARS40) manufactured by Thermo Scientific) was used for each peptide hydrogel and the rheology thereof was measured. FIG. 1 shows the storage elastic modulus G′ and the loss elastic modulus G′ of each peptide hydrogel obtained by the rheology measurement. Specifically, FIG. 1 shows a graph comparing the measurement results of peptide hydrogels produced by 5 wt % NaCl, 1×PBS, 2×PBS, 3×PBS, 5×PBS using a peptide of KI24RGDS.

[0054] As shown in FIG. 1, the difference between the storage elastic modulus G′ and the loss elastic modulus G″ was larger and a peptide hydrogel having higher mechanical strength with higher rigidity could be obtained when 5×PBS was used than when EMEM or 5 wt % NaCl aqueous solution was used as the salt, and specifically, the storage elastic modulus G of the peptide hydrogel was about 15,000, and the loss elastic modulus G″ was about 800, and thus it became clear that a peptide hydrogel having higher mechanical strength with higher rigidity could be obtained. Though it is not shown, the result when EMEM was used was similar to that when 5 wt % NaCl was used.

[0055] Further, as shown in FIG. 1, as the concentration of PBS used for the production of the peptide hydrogel was increased, the difference between the storage elastic modulus G′ and the loss elastic modulus G″ was increased, that is, a peptide hydrogel having higher mechanical strength with higher rigidity was obtained. If compared with the case of using a 5 wt % NaCl aqueous solution, the difference between the storage elastic modulus G and the loss elastic modulus G″ was larger (approximately 3950 for 3×PBS and approximately 3750 for 5 wt % NaCl aqueous solution) and a peptide hydrogel having higher mechanical strength was obtained when PBS having a concentration of 3×PBS or higher was used than when a 5 wt % NaCl aqueous solution was used. From the above results, it became clear that the rigidity of the peptide hydrogel obtained could be improved depending on the concentration of PBS used.

[0056] [Example 3: Application of peptide hydrogel to rabbit meniscus] Next, in order to examine the meniscus regeneration effect by transplanting the peptide hydrogel into the meniscus defect, the effect was evaluated using a rabbit meniscus defect model. The method and results will be described below.

[0057] (Method)

[0058] First, 20 male Japanese white rabbits weighing 3.0 to 3.4 kg at 6 months of age were prepared, and a cylindrical defect with a diameter of 2.0 mm was prepared in the medial anterior segment of the meniscus of both knees (FIG. 2A). Further, a peptide hydrogel produced by the above method using a peptide of KI24RGDS and 5×PBS was prepared (FIG. 2B), and the peptide hydrogel was filled in the defect. The group in which the defect was filled with peptide hydrogel was designated as the KI24RGDS group, and the group in which no treatment was applied to the defect was designated as the control group. After transplantation, tissue evaluation was performed on the knees of each group using hematoxylin/eosin staining (HE staining) or safranin O staining. Tissue evaluation was performed at 2, 4, 8 and 12 weeks after transplantation, and 5 animals (10 knees) were evaluated at each evaluation period. In addition, the tissue evaluation used Ishida score and Quantitative Analysis of the Occupation Ratio of Tissue Regeneration (R/D ratio). In the evaluation based on Ishida score, scores were given from the three viewpoints shown in Table 1 below, and the degree of tissue repair was evaluated by the total. On the other hand, in the evaluation based on the R/D ratio, the degree of tissue repair was evaluated by analyzing the area of the entire defect (D) and the area of the repaired tissue (R) and calculating the ratio of R/D using ImageJ software as shown in FIG. 3.

TABLE-US-00001 TABLE 1 1. Connection of repaired tissue 2 point Connected to surrounding meniscus on both sides 1 point Partially connected to surrounding meniscus 0 point No connection to surrounding meniscus 2. Presence of fibrocartilage cell 2 point Widespread presence of fibrocartilage cell in repaired tissue 1 point Local presence of fibrocartilage cell in repaired tissue 0 point No fibrocartilage cell present in repaired tissue 3. Safranin O-staining 2 point Deeply stained with safranin O 1 point Slightly stained with safranin O 0 point Not stained with safranin O

[0059] (Result)

[0060] FIG. 4 shows photographs of the meniscus at 2, 4, 8 and 12 weeks after transplantation of the KI24RGDS group and the control group, FIG. 5 shows photographs of the safranin O-stained meniscus defect at 2, 4, 8 and 12 weeks after transplantation of the KI24RGDS group, and FIG. 6 shows photographs of the safranin O-stained meniscus defect at 2, 4, 8 and 12 weeks after the preparation of the defect in the control group. In addition, FIG. 7 shows the results of Ishida scores in each group, and FIG. 8 shows the results of the R/D ratio. Furthermore, FIG. 9 shows photographs of the meniscus defect stained with HE and safranin O at 2 weeks after transplantation of the KI24RGDS group, and a model diagram of the process of tissue regeneration by the peptide hydrogel.

[0061] As shown in FIG. 4, it is clear in the macroscopic findings of the meniscus that in the KI24RGDS group, the defect (part indicated by the arrow in the figure) became smaller and repaired as 2, 4, 8 and 12 weeks after transplantation. On the other hand, in the control group, although the defect became smaller as 2, 4, 8 and 12 weeks after the preparation of the defect, the degree of repair was clearly lower than that in the KI24RGDS group, and even after 12 weeks, the presence of holes penetrating the meniscus was observed. Also, in the photograph of the meniscus tissue stained with safranin O, it is clear as shown in FIG. 5 that in the KI24 RGDS group, cells gathered in the defect and the tissue was repaired as 2, 4, 8 and 12 weeks after the transplantation. On the other hand, as shown in FIG. 6, no clear state of repair was observed in the control group as compared with the KI24RGDS group. This is clear also from the results of Ishida score shown in FIG. 7 and the R/D ratio shown in FIG. 8, and the Ishida score and R/D ratio were higher in the KI24RGDS group than in the control group at any stage of 2, 4, 8 and 12 weeks after transplantation, suggesting that tissue repair is progressing.

[0062] Next, the process of tissue regeneration by transplantation of the peptide hydrogel into the meniscus defect will be described with reference to FIG. 9. FIG. 9A is a HE-stained photograph of the meniscus tissue at 2 weeks after transplantation of the KI24RGDS group, and FIG. 9B is a safranin O-stained photograph. A model diagram for facilitating understanding is shown in FIG. 9C. As shown in FIGS. 9A to 9C, when the defect is filled with the peptide hydrogel, the peptide hydrogel serves as a scaffold, and cells migrate from the tissue adjacent to the defect along the periphery of the peptide hydrogel. It is considered that, as a result, cells that can differentiate into meniscus tissue gather in the defect and regenerate the tissue.

[0063] From the above results, the peptide hydrogel according to the present example has high mechanical strength, can be maintained in the defect when transplanted into the tissue defect of the joint tissue, and can exhibit an excellent effect of promoting tissue regeneration at the detect, thus, is extremely useful for regenerative therapy for joint diseases.

[0064] [Sequence List]