JP2011012041A - Sustained release carrier for nucleic acid complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sustained release carrier for a nucleic acid complex having high safety and high expression of effect in a target tissue or cell, and enabling to continue the expression of the effect of the nucleic acid complex in an affected part for a long period.SOLUTION: There is provided the transfer carrier for a nucleic acid complex containing a self-assembling peptide, wherein the self-assembling peptide is an amphiphilic peptide containing hydrophilic amino acids and hydrophobic amino acids alternately bonded with each other, containing 8-200 amino acid residues, and exhibiting a β sheet structure in an aqueous solution at physiological pH and/or in the presence of a univalent ion.

Description

  The present invention relates to a transfer carrier for a nucleic acid complex containing a self-assembling peptide, and a sustained-release nucleic acid pharmaceutical in which the nucleic acid complex is supported on a self-assembling peptide carrier.

  A variety of transfer carriers have been conventionally developed as means for transporting nucleic acid complexes to target cells or tissues. In the current technology, a transfer carrier when using a nucleic acid complex as a nucleic acid medicine has low expression efficiency in the affected part of the nucleic acid complex, and its toxicity has been pointed out. New transfer carriers have been developed to solve these problems, but it is known that the duration of the effect of the introduced nucleic acid complex in vivo is short.

Carriers for sustained release of genes using biocompatible materials have already been developed (Patent Document 1). However, mainly used materials are animal-derived materials such as collagen and gelatin, and there are problems such as infectious diseases. It has been pointed out.
JP-A-9-71542

  Considering the clinical application in nucleic acid medicine including plasmid DNA complex, the carrier of nucleic acid medicine to the affected area is extremely low in toxicity and at the same time has high expression efficiency in target tissues / cells. Development of a sustained-release nucleic acid-introduced preparation that continues to exhibit its effects is awaited.

    The problem to be solved by the present invention is to provide a nucleic acid complex sustained-release carrier that is highly safe and highly effective in target tissues and cells. Further, the effect of the nucleic acid complex in the affected area can be expressed for a long time. It is to provide a sustained-release nucleic acid complex transfer carrier that can be sustained.

    As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that the self-assembled peptide is useful as a transport carrier for the nucleic acid complex, and further the in vivo of the transferred nucleic acid complex. As a result, the present invention was completed.

Therefore, the gist of the present invention is as follows.
[1] A carrier for transferring a nucleic acid complex containing a self-assembling peptide.
[2] The self-assembling peptide is an amphiphilic peptide having amino acid residues 8 to 200 in which hydrophilic amino acids and hydrophobic amino acids are alternately bonded, and in an aqueous solution in the presence of monovalent ions. The nucleic acid complex transfer carrier according to [1], which exhibits a stable β sheet structure.
[3] The transport carrier of the nucleic acid complex is a self-assembling peptide having a repeating sequence of arginine, alanine, aspartic acid and alanine, preferably a self-assembling peptide having 16 amino acid residues.
[4] A medicine for local administration or intraperitoneal or intrathoracic administration, comprising the nucleic acid complex transport carrier according to any one of [1] to [3] above and a nucleic acid complex.
[5] A sustained-release nucleic acid pharmaceutical, which is a transfer carrier for the nucleic acid complex according to any one of [1] to [3] above.
[6] A sustained-release nucleic acid drug that can be injected with the drug according to [4].

    The nucleic acid complex sustained-release carrier of the present invention has high safety, high effect expression in target tissues / cells of nucleic acid pharmaceuticals, and further improves retention of the nucleic acid complex in the affected area, thereby prolonging the expression of the effect. Has the effect of lasting for a period.

    In the present specification, the “nucleic acid complex” is a complex comprising “a nucleic acid, an oligonucleic acid, or a derivative thereof” and “a cationic polymer or a cationic lipid or an assembly containing the same”, It is a composite that also includes an “aggregate including an anionic polymer”.

    The nucleic acid complex transfer carrier of the present invention is an amphiphilic peptide having amino acid residues of 8 to 200 in which hydrophilic amino acids and hydrophobic amino acids are alternately associated, and in an aqueous solution in the presence of monovalent ions. The main component is a self-assembling peptide that exhibits a stable β-sheet structure.

The self-assembling peptide used in the present invention can be represented by, for example, the following four general formulas.
((XY) _1- (ZY) _m ) _n (I)
((YX) _1- (YZ) _m ) _n (II)
((ZY) _1- (XY) _m ) _n (III)
((YZ) _1- (YX) _m ) _n (IV)
In the formulas (I) to (IV), X represents an acidic amino acid, Y represents a hydrophobic amino acid, Z represents a basic amino acid, and 1, m and n are both integers (n × (1 + m) <200). . )
Moreover, the N terminal may be acetylated and the C terminal may be amidated.

    Here, as the hydrophilic amino acid, an acidic amino acid selected from aspartic acid and glutamic acid and a basic amino acid selected from arginine, lysine, histidine and ornithine can be used. As the hydrophobic amino acid, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine or glycine can be used.

Among these self-assembling peptides, self-assembling peptides having repetitive sequences of arginine, alanine, aspartic acid and alanine (RADA) can be preferably used, and the sequence of such a peptide is Ac- (RADA). represented by _{p -NH 2 (p = 2~50)} .

Preferred examples of the self-assembled peptide in the present invention, (Ac- (RADA) 4- NH 2) include peptide RAD16-I having the sequence, its aqueous solution as PuraMatrix (TM) are commercially available.

PuraMatrix (registered trademark) is an oligopeptide having 16 amino acids (Ac- (RADA) ₄ -CONH ₂ ) and a length of about 5 nm, and the solution shows a liquid state when the pH is 5.0 or less. By changing to 0 or more, self-assembly of the peptide occurs, and nanofibers having a diameter of about 10 nm are formed. As a result, the peptide solution gels.

    The method for producing a self-assembled peptide is synthesized by a solid phase synthesis method. Since the peptide can be artificially synthesized, it does not contain biological substances and there is no risk of infection.

    In the present invention, the nucleic acid complex sustained-release carrier is used for transferring a nucleic acid complex containing a nucleic acid, oligonucleic acid, or a derivative thereof to an intended tissue of a living body, and introducing it into a cell. It refers to a composition that can be enhanced and exhibits a sustained release effect of the nucleic acid complex.

Examination of the sustained release effect of plasmid DNA complex in a mouse model for local administration A plasmid DNA complex containing a luciferase gene mixed with a self-assembling peptide was directly injected into a tumor-bearing mouse transplanted with tumor cells subcutaneously. The gene transfer effect was analyzed by measuring the amount of luciferase luminescence, and it was confirmed that the gene expression of the plasmid DNA complex persisted in the self-assembled peptide / plasmid DNA complex mixture.

<Material>
・ PuraMatrix (registered trademark)
Sequence: Ac- (RADA) ₄ -NH ₂ , manufactured by CPC, PuraMatrix (registered trademark) concentration 10 mg / mL (1%)
・ Luciferase DNA plasmid (commercially available)
Plasmid DNA complex (prepared in Patent Document 2 claim 1 at a mixing ratio of 1:12:12)
Cell lysate (0.1% Triton X-100; 2 mM EDTA; mixed solution of 0.1 M Tris HCl)
・ Tumor cells B16 cells

<Method>
Creation of tumor model mice B16 cells were treated with Eagle's minimum essential medium (EMEM) (invitrogen) + 10% bovine serum (FBS) (Gibco) + penicillin streptomycin (PS) (Gibco) at 37 ° C under 5% CO ₂ . Cultured. B16 cells were collected by trypsin treatment, suspended in MEM medium, and implanted with 8 × 10 ⁶ cells / 200 μl of the lower abdomen subcutaneously in a 5-week-old male ddY mouse with an injection needle to prepare a tumor mouse.
-Preparation of Dosing Solution A lyophilized (method described in Patent Document 2) plasmid complex was prepared by the method described in Example 1 of Patent Document 2. In the test group, the plasmid DNA complex solution prepared as described above was sucked up with a syringe, and then the same volume of PuraMatrix (registered trademark) was sucked up into the same syringe to prepare a two-layer administration solution. In the control group, the plasmid DNA complex solution was sucked up with a syringe, and then the same volume of PBS was sucked up into the same syringe to prepare an administration solution.
-Administration In the test group, a prepared solution containing PuraMatrix (registered trademark) was administered in a total amount of 200 μl per tumor site. On the other hand, a total amount of 200 μl of the prepared solution containing no PuraMatrix (registered trademark) was administered to the control group.
・ Measurement of luciferase gene expression level After 2 days, 4 days and 7 days after administration, all tumor sites were excised, added with cell lysate, homogenized with a homogenizer (As One Co., Ltd.), and then a luminometer (Lumat). LB 9507; Berto Japan Co., Ltd.), and the amount of luciferase luminescence was measured. The measured value was evaluated in terms of the amount of luminescence per gram of tumor.

<Result>
The results are shown in FIG. In the control group, the highest expression was observed 2 days after the administration, and the expression was almost lost after 4 days and 7 days after the administration. In the test group, the luminescence level after 2 days after the administration was 4 days and 7 days after the administration. It was measured continuously after the day. From this, it was considered that gene expression was sustained for a long time by encapsulating the plasmid DNA complex in the self-assembling peptide.
Japanese Patent Application No. 2006-138201

Examination of sustained release effect of plasmid DNA complex in mouse intraperitoneal administration model Intraperitoneal injection of plasmid DNA complex expressing luciferase gene mixed with self-assembling peptide into the peritoneal cavity of mice transplanted with tumor cells intraperitoneally, When the gene expression efficiency in the tumor site was analyzed by measuring the amount of luciferase luminescence, it was confirmed that the gene expression of the plasmid DNA complex persisted in the self-assembled peptide / plasmid DNA complex mixture.

<Material>
・ PuraMatrix (registered trademark)
Sequence: Ac- (RADA) ₄ -NH ₂ , manufactured by CPC, PuraMatrix (registered trademark) concentration 10 mg / mL (1%)
・ Luciferase DNA plasmid (commercially available)
Plasmid DNA complex (prepared in Patent Document 2 claim 1 at a mixing ratio of 1:12:12)
Cell lysate (0.1% Triton X-100; 2 mM EDTA; mixed solution of 0.1 M Tris HCl)
・ Tumor cells B16 cells

<Method>
-Creation of tumor model mice B16 cells were treated with Eagle's minimum essential medium (EMEM) (Invitrogen) + 10% bovine serum (FBS) (Gibco) + penicillin streptomycin (PS) (Gibco) at 37 ° C under 5% CO ₂ . Cultured. B16 cells were collected by trypsin treatment, suspended in MEM medium, and 4 × 10 ⁶ cells / 1 ml were administered into the abdominal cavity of a 5-week-old male ddY mouse with an injection needle to prepare tumor mice.
-Preparation of Dosing Solution A lyophilized (method described in Patent Document 2) plasmid complex was prepared by the method described in Example 1 of Patent Document 2. In the test group, the plasmid DNA complex solution prepared as described above was sucked up with a syringe, and then the same volume of PuraMatrix (registered trademark) was sucked up into the same syringe to prepare a two-layer administration solution. In the control group, the plasmid DNA complex solution was sucked up with a syringe, and then the same volume of PBS was sucked up into the same syringe to prepare an administration solution.
-Administration In the test group, 1 mL of the solution containing the prepared PuraMatrix (registered trademark) was administered into the abdominal cavity from the lower abdomen of the mouse. On the other hand, in the control group, a total amount of 1 mL of the prepared solution containing no PuraMatrix (registered trademark) was administered.
・ Measurement of luciferase gene expression level 2 days, 4 days, and 7 days after administration, tumor tissues in the peritoneal cavity that can be visually observed were collected, added with cell lysate and homogenized with a homogenizer (As One Co., Ltd.). The amount of luciferase luminescence was measured with a meter (Lumat LB 9507; Bertoold Japan KK). The measured value was evaluated in terms of the amount of luminescence per gram of tumor.

<Result>
The results are shown in FIG. In the control group, slight expression was observed 2 days after administration, but almost no expression was observed 4 days and 7 days after administration. In the test group, expression after 4 days of administration increased compared to the expression level after 2 days of administration, and high expression persisted until 7 days after administration. From this, it was considered that inclusion of the plasmid DNA complex in PuraMatrix (registered trademark) would sustain the gene expression of the plasmid DNA in the tumor cells for a long period of time and increase the effect.

<General consideration>
Combined with the results of Examples 1 and 2, the self-assembling peptide is useful for increasing the in vivo retention of the encapsulated plasmid DNA complex in both the local administration and intraperitoneal administration and obtaining a high healing effect. It was thought that.

  It is confirmation of the persistence of gene expression in the local administration of the plasmid DNA complex by the self-assembling peptide (PuraMatrix®). “Plasmid DNA complex + PBS” means a control group, and “plasmid DNA complex + PM” means a test group in which a plasmid DNA complex is mixed with PuraMatrix (registered trademark).   It is confirmation of the persistence of gene expression in the intraperitoneal administration of the plasmid DNA complex by the self-assembling peptide (PuraMatrix (registered trademark)). “Plasmid DNA complex + PBS” means a control group, and “plasmid DNA complex + PM” means a test group in which a plasmid DNA complex is mixed with PuraMatrix (registered trademark).

Claims (9)

  A transport carrier comprising a self-assembling peptide of a nucleic acid complex or a complex of oligonucleic acid or a derivative thereof (hereinafter referred to as a nucleic acid complex).   The self-assembling peptide is an amphiphilic peptide having amino acid residues 8 to 200 in which hydrophilic amino acids and hydrophobic amino acids are alternately bonded, and is stable in an aqueous solution in the presence of monovalent ions. The carrier for transporting a nucleic acid complex according to claim 1, which is a self-assembling peptide exhibiting a sheet structure. The transport carrier for a nucleic acid complex according to claim 1, wherein the peptide is a self-assembling peptide having a repeating sequence of arginine, alanine, aspartic acid and alanine.   A medicinal product that can be administered locally, intraperitoneally, and intrathoracically, having a high expression of the effect of the nucleic acid complex in the target tissue / cell by including the carrier for transporting the nucleic acid complex of claim 1.   3. A pharmaceutical product comprising the nucleic acid complex transfer carrier according to claim 2, wherein the nucleic acid complex is highly effective in target tissues and cells, and can be administered locally, intraperitoneally, and intrathoracically.   A medicinal product capable of local administration and intraperitoneal or intrathoracic administration, comprising the carrier for transporting the nucleic acid complex of claim 3, wherein the effect expression of the nucleic acid complex in the target tissue / cell is high.   A sustained-release nucleic acid pharmaceutical that can improve the retention of the nucleic acid complex in the target tissue / cell and maintain the effect in the affected area, using the carrier for the nucleic acid complex of claim 1.   A sustained-release nucleic acid pharmaceutical that improves the retention of the nucleic acid complex in the target tissue / cell and can maintain the expression of the effect in the affected area, using the carrier for the nucleic acid complex of claim 2.   A sustained-release nucleic acid pharmaceutical that can improve the retention of the nucleic acid complex in the target tissue / cell and maintain the effect in the affected area, using the carrier for the nucleic acid complex of claim 3.

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