JPWO2009063998A1 - Hydrophobic substance-added nucleic acid and use thereof - Google Patents

Abstract

To provide a hydrophobic substance-added nucleic acid and its nanoparticles that bind to midkine and inhibit its activity. Hydrophobic substance-added nucleic acid that binds to midkine and inhibits its activity and its nanoparticles; nucleic acid characterized by having binding activity to pleiotrophin; said nucleic acid and functional substance (for example, affinity substance, A complex containing a labeling substance, an enzyme, a drug delivery vehicle, or a drug; a pharmaceutical or a reagent containing the nucleic acid or the complex; a method for detecting midkine using the nucleic acid or the complex; Particle manufacturing method etc.

Description

  The present invention relates to a hydrophobic substance-added nucleic acid, a method for using the same, a detection method, and the like.

  Midkine (hereinafter referred to as “MK” where necessary) is a proliferation discovered as a product of a gene that is transiently expressed in the process of induction of differentiation of pulmonary tumor cells (EC) by retinoic acid. It is a differentiation factor, a polypeptide having a molecular weight of 13 kDa rich in basic amino acids and cysteine (see, for example, Non-Patent Document 1 and Non-Patent Document 2).

  The steric structure of MK is determined by NMR (for example, see Non-Patent Document 3). MK is mainly composed of two fragments due to structural features. Specifically, for example, in the case of human MK, an N-terminal fragment consisting of amino acids 1 to 52 (hereinafter referred to as “N-fragment”), a C-terminal fragment consisting of amino acids 62 to 121 (hereinafter referred to as “N-fragment”) , Referred to as “C-fragment”) and a loop region (the 53rd to 61st amino acids) connecting them. Each fragment consists of a domain and a tail rich in basic amino acids. The domain has three reverse β-sheet structures (hereinafter, the domain in the N-fragment consisting of amino acids 15 to 52 is referred to as “N-domain”, and the domain in the C-fragment consisting of amino acids 62 to 104 is referred to as "C-domain"). Further, the tail moves freely and does not take a specific structure (hereinafter, the tail in the N-fragment consisting of the 1st to 14th amino acids is referred to as “N-tail”, the C-fragment consisting of the 105th to 121st amino acids). The tail is called “C-tail”).

  Receptor-type protein tyrosine phosphatase ζ (PTPζ), LRP (low density lipoprotein receptor-related protein), ALK (anaplastic leukemia kinase), integrin, syndecan and the like are known as MK receptors. MK is a positively and strongly charged protein that is rich in lysine (K) and arginine (R), which are basic amino acids. It is known to have a heparin binding site in the C domain and to bind strongly to negatively charged molecules such as heparin and chondroitin sulfate E. As a result of mutation introduction analysis and NMR analysis, it is considered that cluster I composed of K79, R81 and K102 and cluster II composed of K86, K87 and R89 are important for binding to heparin. On the other hand, there is a report that only cluster I is important for binding to chondroitin sulfate E. When R81 of cluster I is changed to A, the ability to bind heparin decreases. As a result, the ability to bind to PTPζ is reduced, and the elongation and migration of neurons by MK is inhibited. For example, when a petri dish is coated with MK and a mouse embryo nerve cell is spread on this, a neurite is elongated. At this time, when nerve cells are digested with heparitinase, neurite outgrowth is inhibited. On the other hand, when vascular endothelial cells are cultured and MK is added, the plasminogen activating enzyme activity of the cells increases. In this case as well, when the cells are digested with heparitinase, the increase in plasminogen activity is inhibited.

  Thus, MK often binds to glycosaminoglycan and exhibits activity. However, anything that is negatively charged does not bind to MK. It is known that chondroitin sulfate E and heparin bind strongly to MK, but chondroitin sulfate A, B, C and D do not bind to MK. In addition, it binds to sulfatide, cholesterol-3-sulfate, and phosphatidylserine and phosphatidic acid at high concentrations, but does not bind to lecithin, phosphatidylinositol, sphingomyelin, cholesterol, and the like (see, for example, Non-Patent Document 4).

  MK is known to have various biological activities. For example, it is known that the expression of MK is increased in human cancer cells. This increased expression is caused by esophageal cancer, thyroid cancer, bladder cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, neuroblastoma, glioblastoma, uterine cancer, ovary It has been confirmed in various cancers such as cancer and Wilms tumor (see, for example, Patent Document 1 and Non-Patent Document 5). MK is also believed to promote cancer cell survival and migration, promote angiogenesis, and help cancer progression.

  In addition, MK is known to be one of the molecules that occupy the core in the process of inflammatory image formation. For example, it is known that the formation of neointima when a blood vessel is injured and the development of nephritis at the time of ischemic injury are reduced in knockout mice lacking the MK gene. It is also known that rheumatic models and post-surgical adhesions are greatly reduced by knockout mice (see, for example, Patent Document 2, Patent Document 3, and Patent Document 4). Thus, MK is arthritis, autoimmune disease, rheumatoid arthritis (chronic rheumatoid arthritis (RA), osteoarthritis (OA)), multiple sclerosis, postoperative adhesion, inflammatory bowel disease, psoriasis, It is known to be involved in inflammatory diseases such as lupus, asthma and neutrophil dysfunction. Furthermore, MK is known to promote migration (migration) of inflammatory cells such as macrophages and neutrophils. Since this movement is necessary for the establishment of an inflammation image, it is considered that if the midkine is deleted, a disease based on inflammation is unlikely to occur (see, for example, Patent Document 5).

  In addition, in the peritoneal fluid of women with advanced endometriosis, MK levels are increased and MK stimulates the proliferation of cultured endometrial stromal cells, so that MK develops endometriosis. It is known to be involved in progress (see, for example, Patent Document 6).

  In addition, since MK has an intimal thickening action, restenosis after vascular reconstruction, cardiac coronary vascular occlusive disease, cerebrovascular occlusive disease, renal vascular occlusive disease, peripheral vascular occlusive disease, arteriosclerosis, It is known to be involved in vascular occlusive diseases such as cerebral infarction (for example, see Patent Document 2).

  Furthermore, MK is known to suppress proliferation of regulatory T cells. Therefore, MK inhibitors can be used as therapeutic agents for diseases related to regulatory T cells such as multiple sclerosis, rheumatoid arthritis, type I diabetes, Crohn's disease, systemic lupus erythematosus, myasthenia gravis ( Patent Document 7, Non-Patent Document 6).

  Cell migration is known to be important for mechanisms such as invasion / metastasis of cancer cells, intimal thickening in arteriosclerotic lesions, and angiogenesis. It is also known that inflammatory cell migration is deeply involved in cardiovascular diseases such as angina pectoris, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hypertension.

  Pleiotrophin (PTN or HB-GAM; hereinafter referred to as “PTN” where appropriate) is the only family protein of MK and has about 50% homology with MK. Both MK and PTN are proteins containing a large amount of cysteine and basic amino acids. All 10 cysteines present in MK and PTN are conserved, and PTN can be structurally divided into N-domain and C-domain, similar to MK. NMR analysis shows that the three-dimensional structures of these two molecules are very similar. Each domain consists of three β-sheets connected by a flexible linker region. K79, R81, and K102, which are thought to be important for binding to chondroitin sulfate and heparin, are conserved between both proteins. K79 and R81 exist on the same β sheet, but K102 exists on another β sheet. When MK and PTN form a three-dimensional structure, these basic amino acids appear near the protein surface.

  Substances that inhibit the activity of MK include low molecular weight compounds, antibodies, siRNA, antisense and the like, which are considered useful for the treatment or prevention of the above-mentioned diseases. There are no drug candidates under study. In recent years, high-performance RNA aptamers for MK have been produced and are being developed as drug candidates (see Patent Documents 7 and 8). In addition, low molecular weight compounds that bind to MK have also been reported (see Non-Patent Document 4).

Attempts have already been made to commercialize nucleic acids to which cholesterol is bound. Many of them are siRNAs bound with cholesterol, which improves delivery efficiency into cells (for example, Non-Patent Documents 7 and 8). In addition, pharmacokinetics in the body is improved by encapsulating cholesterol-bound aptamers in liposomes (Patent Document 9, Non-Patent Documents 9 and 10). On the other hand, a cholesterol-binding aptamer that has not been formulated into a liposome has been reported to have a significantly different in vivo pharmacokinetics from an aptamer to which cholesterol is not bound (Non-patent Document 11).
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  It is an object of the present invention to provide a hydrophobic substance-added nucleic acid. In particular, an object of the present invention is to provide a hydrophobic substance-added nucleic acid having the ability to inhibit midkine activity and nanoparticles thereof. It is another object of the present invention to provide a usage method, a detection method, and the like.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have succeeded in producing a hydrophobic substance-added nucleic acid having the ability to inhibit midkine activity. It was also found that the hydrophobic substance-added nucleic acid can exist as nanoparticles. The inventors of the present invention have further studied earnestly and have completed the present invention.

That is, the present invention provides the following inventions and the like:
[1] A hydrophobic substance-added nucleic acid characterized by binding to midkine and inhibiting its activity;
[2] The nucleic acid according to [1], further having an inhibitory activity against pleiotrophin;
[3] The nucleic acid according to [1] or [2], wherein the hydrophobic substance is cholesterol;
[4] A nucleic acid represented by any one of SEQ ID NOs: 1 to 10, or a nucleic acid having 1 to 3 bases substituted, deleted, added or inserted into the nucleic acid represented by any of SEQ ID NOs: 1 to 10 The nucleic acid according to any one of [1] to [3];
[5] A complex comprising the nucleic acid according to any one of [1] to [4] and a functional substance;
[6] The complex according to [5], wherein the functional substance is an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle, or a drug;
[7] A medicine or reagent comprising the nucleic acid according to any one of [1] to [4] or the complex according to [5] or [6];
[8] A method for detecting midkine, comprising using the nucleic acid according to any one of [1] to [4] or the complex according to [5] or [6];
[9] A method for forming a nucleic acid into nanoparticles by binding a hydrophobic substance to the nucleic acid;
[10] (a) binding a hydrophobic substance to a nucleic acid; (b) dissolving the hydrophobic substance-added nucleic acid obtained in (a) in a neutral salt solution; A method of granulating;
[11] The method according to [10], wherein the hydrophobic substance-added nucleic acid is dissolved in a neutral salt solution at a concentration of 0.1 mg / mL to 10 mg / mL;
[12] A method for improving in vivo pharmacokinetics of a nucleic acid, which comprises binding a hydrophobic substance;
[13] The method according to [12], wherein the nucleic acid is a nucleic acid having physiological activity;
[14] The method according to any one of [9] to [13], wherein the hydrophobic substance is cholesterol;
[15] Nucleic acid nanoparticles formed by binding a hydrophobic substance;
[16] A medicine or reagent comprising the nucleic acid nanoparticle according to [15];
[17] The medicament or reagent according to [7] or [16], which is a cell migration inhibitor;
[18] The pharmaceutical or reagent according to [7] or [16], which is a proliferation promoter for regulatory T cells;
[19] The medicament according to [7] or [16], which is a prophylactic / therapeutic agent for autoimmune disease or cancer;
[20] A method for preventing / treating an autoimmune disease or cancer, comprising administering an effective amount of the nucleic acid according to [1] to a mammal;
[21] Use of the nucleic acid according to [1] for producing a prophylactic / therapeutic agent for autoimmune disease or cancer.

  The hydrophobic substance-added nucleic acid and complex of the present invention can be useful as a reagent for various diseases such as autoimmune disease, cancer, postoperative adhesion, endometriosis, or a diagnostic agent. Nucleic acids and complexes to which the hydrophobic substances of the invention have been added may also be useful for MK purification and concentration, and MK detection and quantification.

  The nucleic acid nanoparticles obtained by the present invention not only retain the physiological activity possessed by the nucleic acid molecule, but also have improved physiological activity and in vivo pharmacokinetics. Useful for the production of nucleic acid reagents.

It is a figure which shows the result of the binding activity measurement with respect to MK of the nucleic acid 1 using the surface plasmon resonance method. It is a figure which shows the observation result of the clinical symptom change in an EAE model mouse | mouth by administration of the cholesterol addition nucleic acid represented by sequence number 10. It is a figure which shows the analysis result of the nucleic acid nanoparticle by gel filtration HPLC. It is a figure which shows the result of non-denaturing polyacrylamide gel electrophoresis. It is a figure which shows that the in-vivo pharmacokinetics of a nucleic acid nanoparticle is improving.

(1) Hydrophobic substance-added nucleic acid The present invention provides a hydrophobic substance-added nucleic acid having a binding activity with midkine (MK). The present invention also provides a hydrophobic substance-added nucleic acid that inhibits the activity of MK.

  Examples of the activity of the MK include migration activity of cells (eg, macrophages, neutrophils, eosinophils, vascular smooth muscle cells, tumor cells, osteoblasts, nerve cells and their progenitor cells) (Takada et al , 1997, J. Biochem. 122, 453-458; Horiba et al., 2000, J. Clin. Invest. 105, 489-495; Maeda et al., 1999, J. Biol. 12479; Qi et al., 2001, J. Biol. Chem. 276, 15868-15875, etc.), cells (eg, tumor cells, fibroblasts, keratinocytes, neurons, chondrocytes and their progenitor cells) And differentiation promoting activity (Muramatsu and Muramatsu, 991, Biochem.Biophys.Res.Commun.177,652-658; Muramatsu et al., 1993, Dev.Biol.159,392-402; Takei et al., 2001, Cancer Res.61, 8486-8491, etc. ), Regulatory T cell proliferation inhibitory activity, regulatory T cell function inhibitory activity, neuronal neurite outgrowth promoting activity, cell (eg, tumor cell, neural cell) apoptosis inhibitory activity, cell (eg, tumor) Cell) angiogenesis inducing activity, myoblast synapse formation inducing activity, vascular endothelial cell fibrinolytic system promoting activity, vascular smooth muscle cell IL-8 production promoting activity and the like.

  The hydrophobic substance-added nucleic acid of the present invention can bind to any part of MK derived from any mammal and inhibit its activity.

  Examples of mammals include primates (eg, humans, monkeys), rodents (eg, mice, rats, guinea pigs), and pets, livestock and working animals (eg, dogs, cats, horses, cows, goats). , Sheep, pigs), with primates and rodents being preferred and humans being most preferred.

Among the MKs derived from various mammals, for example, the amino acid sequence of human MK is shown in GenBank accession number BC011704. Among these, the secreted protein that functions as MK is composed of 121 amino acids from the 23rd lysine to the 143rd aspartic acid. Therefore, in the present specification, when the amino acid number is indicated in the amino acid sequence of MK, the 23rd lysine is generally indicated as the first amino acid.
That is, “human MK” in the present specification comprises an N-fragment consisting of amino acids 1 to 52, a C-fragment consisting of amino acids 62 to 121, and a loop region connecting them. Similarly, MKs derived from other mammals also refer to mature proteins excluding secretion signals.

  The position on MK to which the hydrophobic substance-added nucleic acid of the present invention binds is not particularly limited, and examples thereof include N-fragment and C-fragment of MK. One hydrophobic substance-added nucleic acid may have binding activity to a plurality of positions of MK. The hydrophobic substance-added nucleic acid of the present invention can inhibit the activity of MK by binding to any position (for example, N-fragment, C-fragment, etc.) of MK.

  In the present specification, the hydrophobic substance-added nucleic acid refers to a nucleic acid such as RNA, DNA, modified nucleic acid, or a mixture thereof bound to the hydrophobic substance. That is, the hydrophobic substance-added nucleic acid of the present invention refers to a substance in which a “nucleic acid” part and a “hydrophobic substance” part are combined. The “nucleic acid” portion and the “hydrophobic substance” portion may be linked by a “linker” portion.

  The type of nucleotide constituting the “nucleic acid” portion of the hydrophobic substance-added nucleic acid of the present invention is not particularly limited as long as the hydrophobic substance-added nucleic acid binds to MK and inhibits the activity of MK, such as DNA and RNA. Any of publicly known nucleotides, modified nucleic acids or mixtures thereof may be used, and they may be double-stranded or single-stranded. Further, the nucleotide sequence itself is not particularly limited. The “modified nucleic acid” refers to a “nucleotide substituted (modified) at a substitutable position” shown below, unless otherwise specified.

  Each nucleotide constituting the “nucleic acid” portion may be any nucleotide that is substituted (modified) at a substitutable position as long as the hydrophobic substance-added nucleic acid binds to MK and inhibits the activity of MK. It may be a nucleotide that is not substituted (modified) at all. In the case of substitution (modification), those skilled in the art will know the “substitutable position”, and it is also possible to select a substituent known per se for the substituent.

Among nucleotides constituting the “nucleic acid” part, nucleotides substituted (modified) at substitutable positions (sometimes referred to as modified nucleic acids in this specification) are preferably such that each nucleotide is ribose. (Eg, a ribose at the 2 ′ position of a pyrimidine nucleotide) (ie, an unsubstituted nucleotide), or at the 2 ′ position of the ribose, the hydroxyl group is the same or different, and any atom or substitution A nucleotide that is substituted with a group.
Examples of the atom or substituent include a hydrogen atom, a halogen atom (eg, fluorine atom), an —O-alkyl group (eg, —O—Me group), an —O-acyl group (eg, —O—CHO group). ), An amino group (eg, —NH ₂ group) and the like.

  The “nucleic acid” portion of the hydrophobic substance-added nucleic acid of the present invention is one in which sugar residues (eg, ribose) of each nucleotide are further modified in order to enhance the binding property, stability, drug delivery property, etc. to MK. May be. Examples of the substitutable site in the sugar residue include, in addition to the 2 'position, for example, the 3' position and / or the 4 'position of the sugar residue. Further, the portion corresponding to the sugar residue is not necessarily pentose, but may be hexose or other substances.

As the substitution (modification), each hydroxyl group is, for example, a fluoro group, an O-alkyl group (eg, O-methyl group, O-ethyl group), an O-allyl group, an S-alkyl group (eg, S-alkyl group). A methyl group, an S-ethyl group), an S-allyl group, and an amino group (eg, —NH ₂ ). In addition, 4′-SRNA in which oxygen at the 4′-position is substituted with sulfur, LNA (Locked Nucleic Acid) in which the 2′-position and the 4′-position are cross-linked via methylene, and the hydroxyl group at the 3′-position as an amino group Substituted 3′-N-phosphoramidate nucleic acids and the like can be mentioned as examples. Such substitution (modification) of a sugar residue can be performed by a method known per se (for example, Sproat et al., (1991) Nucle. Acid. Res. 19, 733-738; Cotton et al., (1991) Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973) Biochemistry 12, 5138-5145, etc.).

  The “nucleic acid” part of the nucleic acid-added nucleic acid of the present invention is one in which a nucleobase (eg, purine or pyrimidine) is further substituted (modified) (eg, chemically substituted) in order to enhance binding to MK, etc. It may be. Examples of such substitution (modification) include 5-position pyrimidine modification, 6 or / and 8-position purine modification, modification with exocyclic amine, substitution with 4-thiouridine, 5-bromo or 5-iodo-uracil. Substitution.

In the “nucleic acid” part of the nucleic acid-added nucleic acid of the present invention, even if the nucleic acid has one or more bases substituted, deleted, added or inserted, as long as it retains the function of the nucleic acid, Included in the nucleic acids of the present invention. The function of the nucleic acid is not particularly limited, and examples thereof include MK binding activity and MK inhibitory activity.
The number of bases that may be substituted, deleted, added or inserted is not particularly limited, but is usually 1 to several, preferably 1 to 5, more preferably 1 to 3.

  Furthermore, the nucleic acid also has a DNA that can hybridize under stringent conditions with a DNA having a base sequence complementary to the nucleic acid comprising part or all of the “nucleic acid” portion of the nucleic acid-added nucleic acid of the present invention. As long as it retains its function, it is included in the DNA of the present invention. Here, the stringent conditions are, for example, “Molecular Cloning: A Laboratory Manual 2nd Edition” (edited by T. Maniatis et al., Cold Spring Harbor The conditions described in Laboratory Issue, 1989) can be mentioned.

Further, the phosphate group contained in the hydrophobic substance-added nucleic acid of the present invention may be further substituted (modified) so as to be resistant to nuclease and hydrolysis. For example, one or more of the P (O) O groups (phosphodiester bond) may be P (O) H (H-phosphonate bond), P (O) S (phosphorothioate bond), P (S) S (phosphorodithiol bond). Ate linkage), P (O) NR ₂ (phosphoramidate linkage), P (O) R, R (O) OR ′, CO or CH ₂ (form acetal) or 3′-amine (—NH—CH ₂ Optionally substituted with —CH ₂ —), wherein R, R ′ and R ₂ are each independently H, or substituted or unsubstituted alkyl (eg, methyl Ethyl). And may be substituted with a boranophosphate bond, a phosphoroselenate bond, or the like.
The substitutions (modifications) may also include 3 ′ and 5 ′ end modifications such as capping.

The above substitution (modification) further includes polyethylene glycol, amino acid, peptide, inverted dT, nucleic acid, nucleoside, Myristoyl, Lithocholic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleoyl, other lipids, steroids, cholesterol, caffeine, Vitamins, dyes, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin, and the like can be added to the 3 ′ end or 5 ′ end of the “nucleic acid” moiety.
For such modifications, reference can be made, for example, to US Pat. Nos. 5,660,985 and 5,756,703.

The length of the “nucleic acid” portion of the hydrophobic substance-added nucleic acid of the present invention is not particularly limited as long as the hydrophobic substance-added nucleic acid binds to MK and inhibits the activity of MK, and is usually 1 to 200 nucleotides. However, it is preferably 5 to 100 nucleotides, more preferably 5 to 60 nucleotides, and further preferably 10 to 40 nucleotides.
If the total number of nucleotides in the “nucleic acid” portion is small, chemical synthesis and mass production of the hydrophobic substance-added nucleic acid of the present invention will be easier, and this is advantageous in terms of cost. In addition, a hydrophobic substance-added nucleic acid can be obtained that facilitates chemical modification, has high in vivo stability, and low toxicity. On the other hand, if the total number of nucleotides in the “nucleic acid” portion is too small, the activity of MK may not be inhibited. An appropriate number of nucleotides can be appropriately determined by those skilled in the art according to the purpose.

  The substance constituting the “hydrophobic substance” portion of the hydrophobic substance-added nucleic acid of the present invention can be substituted at any substitutable position as long as the hydrophobic substance-added nucleic acid binds to MK and inhibits the activity of MK ( It may be a modified substance. A person skilled in the art knows the “substitutable position” and can select a substituent known per se for the substituent.

  Examples of substances constituting the “hydrophobic substance” part include strophanthidine, cholestanol, steroid hormones (testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycorticosterone, etc.), sterols Steroids such as cholesterol (eg, cholesterol, cholesterol ester, stigmasterol, lanosterol, ergosterol, etc.), sitosterol, ergosterol, etc., preferably sterols, more preferably cholesterols It is. Cholesterols may be derivatized, and examples of such cholesterol derivatives include hydrogenated dihydrocholesterol, esters with lower or higher fatty acids, such as cholesteryl hydroxystearate, cholesteryl oleate, isostearin. Cholesteryl acid, lanolin fatty acid cholesteryl, macadamia nut oil fatty acid cholesteryl, cholesteryl nonanoate, cholesteryl stearate, cholesteryl butyrate and the like are commercially available. Further, vitamin A, vitamin D, vitamin E, vitamin K and the like may be used.

  The hydrophobic substance-added nucleic acid of the present invention may be one in which the “nucleic acid” part and the “hydrophobic substance” part are directly bonded, or the “nucleic acid” part and the “hydrophobic substance” part are designated as “ It may be formed by bonding at a “linker” portion. The “nucleic acid” portion and the “hydrophobic substance” portion can be bound by methods known to those skilled in the art.

In the hydrophobic substance-added nucleic acid of the present invention, the “linker” part that can bind the “nucleic acid” part and the “hydrophobic substance” part binds the hydrophobic substance-added nucleic acid to MK and inhibits MK activity. It does not specifically limit as long as it is a person skilled in the art and can determine suitably. Examples of such a linker include a saturated hydrocarbon chain (eg, a saturated hydrocarbon chain having 12 carbon atoms), a nucleotide chain (eg, 1 to about 20 nucleotides), a non-nucleotide chain (eg, — (CH ₂ ) _n. - linker, a linker comprising a peptide (e.g., -Gly-Cys -), - linker containing S-S- bonds _{(e.g., - (CH 2) m -S} -S- (CH 2) n -), - CONH - linker comprising a binding _{(e.g., -CONH -, - (CH 2} ) m -CONH- (CH 2) n -, - O-CO-NH- (CH 2) n -), - OPO 3 - including binding linker _{(e.g., - (CH 2) m -O} -PO 2 -O- (CH 2) n -), polyethylene glycol linker (e.g., pentaethylene glycol linker, hexaethylene glycol linker), amidite (e.g., S acer9, Spacer18)) m and n in the mentioned are (each linker such denotes an any integer).
The linker portion may be one obtained by branching the linker exemplified above and adding a functional molecule such as a dimethoxytrityl group (DMT) or a fluorescent material (when these functional molecules are finally removed). Is). Further, it may be a peptide that can be recognized and cleaved by an enzyme such as thrombin, matrix metalloprotease (MMP), Factor X, or a polynucleotide that can be cleaved by a nuclease or a restriction enzyme.

  Examples of the “saturated hydrocarbon chain” include those having 3, 6, 12, 18, 24 carbon atoms. Further, it may be branched.

  The “nucleotide chain” is a nucleotide containing a hydroxyl group at the 2 ′ position of ribose (eg, ribose of a pyrimidine nucleotide, ribose of a purine nucleotide) or a hydroxyl group at the 2 ′ position of ribose Nucleotides substituted (modified) with a group (eg, hydrogen atom, fluorine atom or —O-methyl group), LNA, phosphate moiety modified, nucleobase moiety modified, etc. Or a combination thereof. The modification of phosphoric acid and nucleobase is not particularly limited, and examples thereof include modifications known per se known to those skilled in the art.

The bonding between the “linker” portion and the “hydrophobic substance” portion is not particularly limited, and both portions can be bonded by methods known to those skilled in the art.
Further, the binding between the “linker” portion and the “nucleic acid” portion is not particularly limited, and both portions can be bound by a method known to those skilled in the art.

  The “linker” part and the “hydrophobic substance” part of the hydrophobic substance-added nucleic acid of the present invention may be bound to the 5 ′ end side of the “nucleic acid” part, or may be bound to the 3 ′ end side. Good. It may also be bound to both the 5 'end and the 3' end of the "nucleic acid" part. Alternatively, it may be bound to a nucleobase, ribose or phosphate part of the sequence in the middle of the “nucleic acid” part.

The hydrophobic substance-added nucleic acid of the present invention can be chemically synthesized by a method known per se in the art. For example, cholesterol can be added to the 5 ′ end of a nucleic acid synthesized using a nucleic acid synthesizer using commercially available cholesterol TEG phosphoramidite (manufactured by Glen Research). In this case, any linker can be added between the nucleic acid and cholesterol by simultaneously using Spacer18 (manufactured by Glen Research), which is a commercially available amidite body.
Also, cholesterol can be added to the 3 ′ end of the nucleic acid by using commercially available 3′-cholesterol TEG-CPG (manufactured by Glen Research). Similarly to the above, an optional linker can be added between the nucleic acid and cholesterol by simultaneously using a commercially available amidite body, such as Spacer 18 (manufactured by Glen Research).
By using the same method, cholesterol can be added to the nucleobase, ribose, and phosphate part of the intermediate sequence.

Furthermore, by adding an amino group to the terminal or in the middle of the nucleic acid, cholesterol can be added by a coupling reaction after nucleic acid synthesis using a synthesizer.
In order to add an amino group to the 5 ′ end of the nucleic acid, a commercially available 5′-amino-modifier C6-TFA (manufactured by Glen Research) or the like can be used.
In addition, in order to add an amino group to the 3 ′ end of the nucleic acid, commercially available 3′-amino-modifier C7-CPG (manufactured by Glen Research) or the like can be used.
Cholesterol can be added in the middle of a nucleic acid by using an amino group in a nucleobase, or by introducing an amino group at the 5-position of pyrimidine, the 6-position of purine, or the like. Moreover, you may introduce | transduce an amino group into 2 'position of ribose or a phosphoric acid part.
The coupling reaction between cholesterol and an amino group can be easily performed by adding an active group such as NHS (N-hydroxysuccinimide) to cholesterol. Thereby, the hydrophobic substance-added nucleic acid of the present invention can be produced.

In the present specification, each symbol in the nucleic acid described below has the following meaning.
chol: Cholesterol PEG 40k: Two 20 kDa polyethylene glycols are linked, and 40 kDa polyethylene glycol branched at a position where polyethylene glycol is possible N: Ribonucleotide N (M): The 2 ′ position of ribose is modified with an O-methyl group Nucleotide N (F): nucleotide 2 ′ modified with ribose at the 2′-position C6: C6 saturated hydrocarbon chain linker C12: C12 saturated hydrocarbon chain linker S18: Spacer18 linker idT: inverted dT modification

  As a preferred embodiment of the hydrophobic substance-added nucleic acid of the present invention, 1 to 3 bases are substituted for the nucleic acid represented by SEQ ID NO: 1 to 10, or the nucleic acid represented by any one of SEQ ID NO: 1 to 10, Examples include nucleic acids that are deleted, added or inserted. The nucleic acid may be further substituted (modified). Preferred examples of the substitution (modification) include the above-described substitution (modification).

As a particularly preferred embodiment of the hydrophobic substance-added nucleic acid of the present invention, the following nucleic acids and derivatives thereof:
chol-A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) (SEQ ID NO: 1),
chol-C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) (SEQ ID NO: 2),
chol-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M),
chol-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT,
chol-C12-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G ( M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT,
chol-G (H) C (H) A (H) C (H) A (H) G (H) G (H) G (H) G (H) U (H) U (H) G (H) G (H) U (H) G (H) U (H) C (H) G (H) G (H) G (H) U (H) G (H) C (H) and G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M ) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -chol (hereinafter, SEQ ID NO: 3),
chol-C12-U (M) U (M) G (M) G (M) U (M) G (M) U (M) -idT (SEQ ID NO: 4),
chol-G (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) G (M) G (M) A (M) A (M) G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G ( M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT, and chol-C12- G (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) G (M) G (M) A (M) A (M) G ( M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (above, SEQ ID NO: 5),
chol-G (F) G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) AGGG (M) G (M) U (F) U (F) G (M) G (M) U ( F) G (M) U (F) C (F) GGGU (F) G (M) C (F) A (M) C (F) -idT and GGGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F ) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -Chol (above, SEQ ID NO: 6),
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -idT,
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -S18-PEG40k,
chol-G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) G (F) G (M) G (M) G (M) U (F) U (F) G ( M) G (M) U (F) G (M) U (F) C (F) G (F) G (F) G (M) U (F) G (M) C (F) A (M) C (F) -idT,
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -PEG40k,
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -C12-PEG40k, and chol-G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) G ( F) G (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) G (F) G (F) G (M) U (F) G (M) C (F) A (M) C (F) -C12-PEG40k (above, SEQ ID NO: 7),
chol-C12-G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U ( F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -IdT (SEQ ID NO: 8),
chol-C12-GGGA (M) GA (M) GGA (M) GA (M) A (M) GA (M) GGA (M) A (M) GU (F) G (M) C (F) A ( M) C (F) AGGGGG (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGGU (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 9), and chol-G (M) G (M) A (M) A (M) G (M) G (M) A (M ) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) A (M) A (M) A (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M ) U (M) U (M) C (M) C (M) -idT (SEQ ID NO: 10)
Is mentioned.

  The present invention also provides a hydrophobic substance-added nucleic acid characterized by having an inhibitory activity against pleiotrophin in addition to inhibiting the activity of MK (eg, cell migration activity of MK).

  Although MK knockout mice and PTN knockout mice grow without major defects, MK and PTN double knockout mice are embryonic lethal, and thus MK and PTN are thought to complement each other in their functions. It is also known that both MK and PTN expression are elevated in experimental autoimmune encephalomyelitis (EAE) mice, which are models of various cancers, Alzheimer's disease, osteoporosis, and multiple sclerosis. Therefore, it is considered that nucleic acids having inhibitory activity against not only MK but also PTN are useful for these diseases.

  The cholesterol-added nucleic acid of the present invention inhibits both MK and PTN. However, when cholesterol is removed, the inhibitory activity against both MK and PTN may be lost (SEQ ID NO: 5 and nucleic acid 5 in Table 1 described later). 1 and nucleic acid 5-2). In this case, by inserting a peptide sequence or the like that is cleaved by a certain enzyme between cholesterol and nucleic acid, cholesterol can be cleaved and detoxified quickly when target-dependent side effects occur. Cholesterol-added nucleic acids inhibit both MK and PTN. However, nucleic acids to which cholesterol is not added may inhibit MK but not PTN. In this case, by including a cleavage site in the cholesterol linker, it is possible to control to inhibit both MK and PTN, or to inhibit only MK. Such adjustment can be easily performed by those skilled in the art.

  The inhibitory activity of MK and PTN in the nucleic acid-added nucleic acid of the present invention and the nucleic acid from which the hydrophobic substance has been removed can be measured, for example, according to the methods described in the examples below.

(2) Complex containing hydrophobic substance-added nucleic acid and functional substance The present invention also provides a complex containing the hydrophobic substance-added nucleic acid and functional substance of the present invention. The bond between the “hydrophobic substance-added nucleic acid” and the “functional substance” in the complex of the present invention can be, for example, a covalent bond or a non-covalent bond. The complex of the present invention may be a conjugate of the hydrophobic substance-added nucleic acid of the present invention and one or more (eg, 2 or 3) of the same or different functional substances.

  As the above-mentioned “functional substance”, a substance that newly adds some function to the hydrophobic substance-added nucleic acid of the present invention or a property that can be retained by the hydrophobic substance-added nucleic acid of the present invention (for example, binding activity to MK) Having an inhibitory activity on MK, having an inhibitory activity on PTN, drug pharmacokinetics, nuclease resistance, etc.) is not particularly limited.

Examples of the functional substance include proteins, peptides, amino acids, lipids, carbohydrates, monosaccharides, polynucleotides, and nucleotides.
Other functional substances include, for example, affinity substances (eg, biotin, streptavidin, polynucleotides having affinity for target complementary sequences, antibodies, glutathione sepharose, histidine), labeling substances (eg, fluorescent substances) , Luminescent substances, radioisotopes), enzymes (eg, horseradish peroxidase, alkaline phosphatase), drug delivery vehicles (eg, liposomes, microspheres, peptides, polyethylene glycols, cholesterols), drugs (eg, calicheamicin and Those used in missile therapy such as duocarmycin, nitrogen mustard analogues such as cyclophosphamide, melphalan, ifosfamide or trophosphamide, ethyleneimines such as thiotepa, nitrosourea such as carmustine, temozolomide Is a folate-like antimetabolite such as dacarbazine, methotrexate or raltitrexed, a purine analogue such as thioguanine, cladribine or fludarabine, a pyrimidine analogue such as fluorouracil, tegafur or gemcitabine, a vinca alkaloid such as vinblastine, vincristine or vinorelbine and its analogues, Podophyllotoxin derivatives such as etoposide, taxane, docetaxel or paclitaxel, anthracyclines and analogs such as doxorubicin, epirubicin, idarubicin and mitoxantrone, other cytotoxic antibiotics such as bleomycin, mitomycin, minomycin, cisplatin, carboplatin And platinum compounds such as oxaliplatin, pentostatin, miltefosine, est Musuchin, topotecan, irinotecan and bicalutamide), toxins (e.g., ricin toxin, rear toxins and Vero toxins), and the like.

“Covalent bond” exemplified as a bond between “hydrophobic substance-added nucleic acid” and “functional substance” includes a saturated carbon chain linker (eg, — (CH ₂ ) _n —), a linker containing a peptide (eg, , -Gly-Cys -), - the linker _(e.g., - (CH2) containing the S-S- bond _{m -S-S- (CH 2)} n -), - CONH- linker comprising a binding (e.g., - ( CH ₂ ) _m —CONH— (CH ₂ ) _n —, —O—CO—NH— (CH ₂ ) _n —), a linker containing an —OPO ₃ — linkage (eg, — (CH ₂ ) _m —O—PO _2- O— (CH ₂ ) _n —), polyethylene glycol linker (eg, hexaethylene glycol linker)) and the like (the symbols in each linker are the same as above). These linkers may be branched at possible positions in the molecule. Furthermore, it may be a peptide that can be recognized and cleaved by an enzyme such as thrombin, matrix metalloprotease (MMP), Factor X, or a polynucleotide that can be cleaved by a nuclease or a restriction enzyme.

  Similarly, examples of non-covalent bonds include hydrophobic bonds, ionic bonds, hydrogen bonds, bonds using chelating agents, bonds using biotin and avidin bonds, bonds using antibodies, and aptamers. And bonds using base pairs.

(3) Method for Nanoparticulating Nucleic Acid The present invention provides a method for nanoparticulating nucleic acid, characterized by binding a hydrophobic substance.
In the present specification, the “nanoparticle” does not specify the particle diameter, but as a temporary measure, it means a particle having a nano unit, specifically, a particle diameter of about 10 to about 1000 nm. As used herein, “nanoparticulation” refers to growing a nucleic acid molecule into an aggregate of a plurality of nucleic acid molecules having a particle diameter of about 10 to about 1000 nm. The nanoparticle of the present invention is produced by binding a hydrophobic substance to a nucleic acid. Specifically, the nanoparticles of the present invention are formed by assembling nucleic acid molecules to which a hydrophobic substance is bound.

  In the method for nanoparticulating the nucleic acid of the present invention, the method for binding the hydrophobic substance to the nucleic acid includes the same method as the production of the “hydrophobic substance-added nucleic acid” described in (1) above. Examples of the “hydrophobic substance” to which the nucleic acid binds include those similar to the hydrophobic substance described in the above (1), preferably cholesterol.

In the method for nanoparticulating the nucleic acid of the present invention, the “nucleic acid” to which the hydrophobic substance binds is not particularly limited, and itself known nucleic acids such as aptamers, siRNAs, antisense nucleic acids, decoy nucleic acids and the like are useful per se. Various nucleic acid molecules having physiological activity can be applied, and preferably the same nucleic acid as described in (1) above can be used.
The number of bases of the nucleic acid is preferably a nucleic acid having 5 to 100 bases, more preferably a nucleic acid having 10 to 50 bases, and most preferably a nucleic acid having 15 to 45 bases.

  The method for forming the nucleic acid of the present invention into nanoparticles is characterized in that a hydrophobic substance is bound to the nucleic acid, but other methods are not particularly limited. Therefore, a method known per se can be applied to other methods.

As a method for converting the nucleic acid of the present invention into nanoparticles, preferably,
(A) binding a hydrophobic substance to the nucleic acid;
(B) a step of dissolving the hydrophobic substance-added nucleic acid obtained in (a) in a neutral salt solution;
A method comprising Hereinafter, each step will be described.

(A) Step of binding a hydrophobic substance to a nucleic acid This step is a step of binding a hydrophobic substance to a nucleic acid molecule. Examples of the method for binding a hydrophobic substance to a nucleic acid include the same method as in the production of the “hydrophobic substance-added nucleic acid” described in (1) above. Moreover, as a hydrophobic substance, the hydrophobic substance as described in said (1) is mentioned, Preferably it is cholesterol. In addition, the “nucleic acid” to which the hydrophobic substance binds is not particularly limited, and various nucleic acid molecules having useful physiological activities such as aptamers, siRNAs, antisense nucleic acids, decoy nucleic acids, etc. are applied. Preferably, the nucleic acid described in (1) above and the same aptamer described in International Publication WO2008 / 059877 can be used.

(B) Step of dissolving hydrophobic substance-added nucleic acid This step is a step of dissolving the hydrophobic substance-added nucleic acid obtained in (a) above in a neutral salt solution.
As the “neutral salt” of the “neutral salt solution” in this step, for example, sodium chloride, magnesium chloride, potassium chloride, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium sulfate, potassium sulfate, calcium sulfate, Examples thereof include magnesium sulfate, sodium phosphate, and potassium phosphate. Of these, sodium chloride, potassium chloride, and magnesium chloride are preferable, and sodium chloride is particularly preferable.

In addition, the solution for dissolving the “neutral salt” is not particularly limited as long as the reaction for forming the nucleic acid nanoparticles proceeds by dissolving the hydrophobic substance-added nucleic acid, and water, physiological saline, alcohol (eg, methanol) And ethanol). The concentration of the hydrophobic substance-added nucleic acid in the solution is not particularly limited as long as the reaction for forming the nucleic acid nanoparticles proceeds by dissolving the hydrophobic substance-added nucleic acid, and is usually 0.1 mg / ml to 100 mg / ml. Yes, preferably 0.1 mg / ml to 10 mg / ml. If the concentration of the hydrophobic substance-added nucleic acid is less than 0.1 mg / ml, the nucleic acid molecules are difficult to assemble and nanoparticles are not properly produced. On the other hand, if the concentration of the hydrophobic substance-added nucleic acid is higher than 100 mg / ml, the nucleic acid Since the concentration is too high and gelation occurs, nanoparticles having an appropriate size cannot be produced.
The neutral salt concentration in the solution is not particularly limited as long as the reaction for forming the nucleic acid nanoparticles proceeds by dissolving the hydrophobic substance-added nucleic acid, and is usually 1 mM to 1 M, preferably 10 mM to 300 mM. is there.

For dissolution in this step, it is sufficient to add the hydrophobic substance-added nucleic acid to the neutral salt solution and dissolve it. However, as long as the reaction for dissolving the hydrophobic substance-added nucleic acid to form nucleic acid nanoparticles proceeds, stirring, etc. Additional operations may be added. The solution is allowed to stand to produce nucleic acid nanoparticles, but the standing time is not particularly limited, and is usually 1 hour to 96 hours, preferably 1 hour to 48 hours, more preferably 1 hour. ~ 24 hours.
In this step, the nucleic acid nanoparticles are promoted by dissolving the hydrophobic substance-added nucleic acid in a neutral salt solution, preferably at a concentration of 0.1 mg / ml to 10 mg / ml, and then preferably heating. Can do. Although it does not specifically limit as temperature at the time of melt | dissolving, Usually, it is 0 to 100 degreeC, Preferably it is room temperature of 10 to 40 degreeC. Moreover, the temperature at the time of heating is not particularly limited, but is usually 50 ° C to 100 ° C, preferably 60 ° C to 90 ° C. Moreover, you may melt | dissolve and heat simultaneously at the temperature of 50 to 100 degreeC, Preferably 60 to 90 degreeC.

(4) Method for Improving the Pharmacokinetics of Nucleic Acids Nucleic acid nanoparticles obtained by the method of the present invention not only maintain the physiological activity of non-nanoparticulate nucleic acids, but also significantly improve their in vivo pharmacokinetics. It is useful in that That is, the present invention provides a method for improving the in vivo pharmacokinetics of a nucleic acid, characterized by binding a hydrophobic substance. In the present specification, “improving pharmacokinetics in the body” means improving the blood stability of a substance administered into the body as a drug or the like. That is, according to the present invention, blood stability of a nucleic acid administered into the body can be increased. The target for improving the in vivo pharmacokinetics is not particularly limited.
The binding of the hydrophobic substance to the nucleic acid is as described in (3) above. In addition, the method for making nucleic acid molecules into nanoparticles is as described in (3) above.
Examples of the “nucleic acid” in the method of the present invention include the nucleic acid described in (1) above. Maintaining the inhibitory effect on MK because the nucleic acid nanoparticles are formed by binding a hydrophobic substance to the nucleic acid, so that the stability of the nucleic acid in the blood is improved and the residence time in the blood is significantly increased. Is possible.

As described above, the present invention improves the pharmacokinetics in the body by converting the cholesterol-added nucleic acid itself having physiological activity into nanoparticles. The nanoparticle of the present invention itself has physiological activity. This is different from those using other delivery agents such as the liposomes described above. When using other delivery agents such as liposomes, it is necessary not only to confirm the stability and effectiveness of the drug substance itself, but also to check the safety and efficacy of these delivery agents separately, clinical trials It takes a lot of money and time.
Since the nucleic acid nanoparticles obtained from the cholesterol-added nucleic acid of the present invention are the drug substance itself, this leads to a reduction in time and cost in drug development. In addition, the drug encapsulated in the liposome exhibits a medicinal effect only when it is gradually released from the liposome, but the nucleic acid nanoparticle of the present invention itself exhibits a medicinal effect, and is also useful in terms of high immediate efficacy.

(5) A method for improving the pharmacokinetics in the body by converting the nucleic acid having physiological activity into nanoparticles The liposome has a spherical shell shape, and the drug is encapsulated and administered into the body, whereby the drug is targeted to the target disease site. It is a delivery agent that reaches On the other hand, the present invention does not require such a delivery agent, and improves the stability of the nucleic acid in blood by forming nanoparticles of the nucleic acid itself having physiological activity (drug activity). Since the hydrophobic substance-added nucleic acid maintains its physiological activity even when it is made into nanoparticles, the nanoparticles themselves can be used as a drug.
The binding of the hydrophobic substance to the nucleic acid is as described in (3) above. In addition, the method for making nucleic acid molecules into nanoparticles is as described in (3) above.
Examples of the “nucleic acid” in the method of the present invention include the nucleic acid described in (1) above. By binding a hydrophobic substance to the nucleic acid to form nucleic acid nanoparticles, the pharmacokinetics in the body can be improved, and as a result, a highly effective medicine can be provided. Here, the “physiological activity” of the “nucleic acid having physiological activity” is not particularly limited, and includes all the activities reported as the activity of the nucleic acid and the physiological activity, drug activity, side effects and the like that can be caused thereby. For example, in the case of the nucleic acid described in (1) above, preferred examples include MK inhibitory activity and regulatory T cell proliferation activity.

(6) Pharmaceutical containing hydrophobic substance-added nucleic acid, etc. The hydrophobic substance-added nucleic acid, complex, and nucleic acid nanoparticle of the present invention can be used as a medicine. In particular, the nucleic acid nanoparticles of the present invention are extremely useful as an active ingredient of the medicine because the nanoparticles themselves act as drugs as described above. That is, the present invention provides a pharmaceutical comprising the hydrophobic substance-added nucleic acid, complex, or nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention).
Hydrophobic substance-added nucleic acid is characterized in that its specific use as a pharmaceutical differs depending on the physiological activity of the hydrophobic substance-added nucleic acid. For example, the drug is bound to the midkine of the present invention and inhibits its activity. Examples of the nucleic acid include cell migration inhibitors, regulatory T cell proliferation promoters, regulatory T cell inhibitory function promoters, apoptosis inhibition inhibitors, cell proliferation inhibitors, cell differentiation inhibitors and the like.

  Specifically, autoimmune diseases (eg, multiple sclerosis, systemic lupus erythematosus (SLE), Sjogren's syndrome, polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (rheumatoid arthritis (RA), Osteoarthritis (OA), inflammatory bowel disease (Crohn's disease, etc.), progressive systemic sclerosis (PSS), nodular periarteritis (PN), thyroid disease (eg Graves' disease), Guillain-Barre syndrome , Primary biliary cirrhosis (PBC), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), type I diabetes, psoriasis, asthma , Neutrophil dysfunction, etc.), cancer (eg, esophageal cancer, thyroid cancer, bladder cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, neuroblastoma, glio Blastoma, uterine cancer Ovarian cancer, Wilms tumor, prostate cancer, etc.), postoperative adhesion, endometriosis, transplant rejection, allergy, restenosis after vascular reconstruction, cardiovascular occlusive disease, cerebrovascular occlusive disease, renal vascular It can be used for the prevention or treatment of various diseases such as occlusive disease, peripheral vascular occlusive disease, arteriosclerosis, cerebral infarction and the like.

  In particular, since it inhibits the cell migration activity of MK, it is useful for the prevention or treatment of multiple sclerosis, postoperative adhesion, endometriosis, rheumatoid arthritis, vascular stenosis, and nephritis. Moreover, it is particularly useful for the prevention or treatment of multiple sclerosis because of the proliferation activity of regulatory T cells.

  The medicament of the present invention may be formulated with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Lubricant such as sodium lauryl sulfate, Citric acid, Menthol, Glycyrrhizin / Ammonium salt, Glycine, Orange powder, etc. Fragrance, Nato benzoate Preservatives such as sodium, sodium bisulfite, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  The medicament of the present invention may be a preparation suitable for oral administration. Preparations suitable for oral administration include solutions in which an effective amount of ligand is dissolved in a diluent such as water, physiological saline, orange juice, capsules and sachets containing an effective amount of the ligand as solids or granules. Or a tablet, a suspension in which an effective amount of an active ingredient is suspended in an appropriate dispersion medium, an emulsion in which a solution in which an effective amount of an active ingredient is dissolved is dispersed in an appropriate dispersion medium, and the like are included. .

Further, the medicament of the present invention can be coated by a method known per se for the purpose of taste masking, enteric property or sustainability, if necessary. Examples of the coating agent used for coating include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm, Germany, methacrylic acid / acrylic acid copolymer) and pigments (eg, Bengala, titanium dioxide, etc.) are used. The medicament of the present invention can be used together with a sustained release agent, particularly for the purpose of sustaining. Examples of the sustained-release base include liposomes, atelocollagen, gelatin, hydroxyapatite, PLGA, and polysaccharide schizophyllan.
The medicament of the present invention may be either an immediate release preparation or a sustained release preparation.

  Suitable formulations for parenteral administration (eg, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary, etc.) are aqueous and non-aqueous isotonic. Examples include sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like. Aqueous and non-aqueous sterile suspensions can also be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile solvent immediately before use. In addition to injectable solutions, inhalants and ointments are also possible. In the case of an inhalant, the active ingredient in a lyophilized state is refined and administered by inhalation using an appropriate inhalation device. In the inhalant, conventionally used surfactants, oils, seasonings, cyclodextrins or derivatives thereof can be appropriately blended as necessary.

  Examples of the surfactant include oleic acid, lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl trioleate, glyceryl monolaurate, glyceryl monooleate, glyceryl monostearate, Glyceryl monolysinoate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetylpyridinium chloride, sorbitan trioleate (trade name span 80), sorbitan monooleate (trade name span 80), sorbitan monolaurate (trade name span 20), Polyoxyethylene hydrogenated castor oil (trade name HCO-60), polyoxyethylene (20) sorbitan monolaurate (trade name Tween 20), polyoxy Ethylene (20) sorbitan monooleate (trade name Tween 80), lecithin derived from natural resources (trade name Epicron), oleyl polyoxyethylene (2) ether (trade name Brigi 92), stearyl polyoxyethylene (2) ether ( Brand name Brizi 72), lauryl polyoxyethylene (4) ether (trade name Brigi 30), oleyl polyoxyethylene (2) ether (trade name Genapor 0-020), block copolymer of oxyethylene and oxypropylene ( Product name simperonic). Examples of the oil include corn oil, olive oil, cottonseed oil and sunflower oil. In the case of an ointment, an appropriate pharmaceutically acceptable base (yellow petrolatum, white petrolatum, paraffin, plastibase, silicone, white ointment, beeswax, pig oil, vegetable oil, hydrophilic ointment, hydrophilic petrolatum, purified lanolin, hydrolyzed lanolin , Water-absorbing ointment, hydrophilic plastibase, macrogol ointment, etc.) and mixing with active ingredients to formulate and use.

  Inhalants can be manufactured according to conventional methods. That is, the aptamer and the complex of the present invention can be produced by making a powder or liquid, blending in an inhalation propellant and / or carrier, and filling an appropriate inhalation container. In addition, when the aptamer and complex of the present invention are powder, a normal mechanical powder inhaler can be used, and when it is liquid, an inhaler such as a nebulizer can be used. Here, conventionally known propellants can be widely used, such as Freon-11, Freon-12, Freon-21, Freon-22, Freon-113, Freon-114, Freon-123, Freon-142c, Freon-134a. , Freon compounds such as Freon-227, Freon-C318, 1,1,1,2-tetrafluoroethane, hydrocarbons such as propane, isobutane and n-butane, ethers such as diethyl ether, nitrogen gas, carbonic acid Examples thereof include compressed gas such as gas.

  The concentration of the hydrophobic substance-added nucleic acid of the present invention in the medicament of the present invention is not particularly limited, but a multimer of the nucleic acid can be formed by increasing the concentration. Forming multimers is advantageous in that nuclease resistance can be improved and excretion can be suppressed. In addition, since the multimer can be easily made into a monomer by heating at a reduced concentration, after being formulated as a high-concentration multimer, it is diluted and heated at the time of use according to the purpose. It may be used as a monomer. Further, the concentration of the nucleic acid nanoparticles of the present invention in the medicament of the present invention is not particularly limited. Both the multimer and the monomer of the hydrophobic substance-added nucleic acid or nucleic acid nanoparticle (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention) have cell migration inhibitory activity. ing.

  The dosage of the medicament of the present invention is the type and activity of the active ingredient, the characteristics of the preparation itself (enteric, sustained, sustained release, etc.), the severity of the disease, the animal species to be administered, the drug to be administered Usually, the amount of active ingredient per adult is about 0.0001 to about 100 mg / kg, such as about 0.0001 to about 10 mg / kg, preferably about 0.005 to about It can be 1 mg / kg.

  The hydrophobic substance-added nucleic acid, complex and nucleic acid nanoparticles of the present invention (preferably, nucleic acid nanoparticles produced using the hydrophobic substance-added nucleic acid of the present invention) are also reagents (eg, diagnostic agents, test agents, etc.). Including laboratory reagents). That is, the present invention provides a reagent comprising the hydrophobic substance-added nucleic acid, complex or nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention).

  The reagent utilizes the function of the above-described hydrophobic substance-added nucleic acid, complex or nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention). As long as it is, it is not particularly limited. The use related to the reagent is a use related to the nucleic acid, complex or nucleic acid nanoparticle, and uses known to those skilled in the art are widely applied.

When the reagent is characterized by binding to MK and inhibiting its activity, the reagent is preferably used as an experimental reagent (eg, cell migration inhibitor, regulatory T cell proliferation). Promoter, Regulatory T Cell Suppression Function Promoter, Apoptosis Inhibitor, Cell Growth Inhibitor, Cell Differentiation Inhibitor, In Vivo Imaging Probe, MK Blood Concentration Probe, Tissue Staining Probe, ELISA Probe , Separation agents for MK and PTN purification, etc.), diagnostic agents (eg, in vivo diagnostic agents, MK blood concentration measuring agents, tissue staining agents, ELISA diagnostic agents, etc.), and labeling agents (eg, in vivo labeling) Agent, tissue staining labeling agent, ELISA labeling agent, etc.).
For use of the above-described hydrophobic substance-added nucleic acid, complex and nucleic acid nanoparticle (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention), more preferably, regulatory T cell Examples include use as a growth promoter, cell migration inhibitor, anticancer agent, diagnostic agent, or labeling agent. That is, the present invention provides a regulatory T cell proliferation promoter comprising the hydrophobic substance-added nucleic acid, complex or nucleic acid nanoparticle (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention). Cell migration inhibitors, anticancer agents, diagnostic agents or labeling agents.

  A person skilled in the art can appropriately determine the amount of the reagent of the present invention.

The present invention also provides a solid phase carrier on which the hydrophobic substance-added nucleic acid, complex, and nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention) is immobilized. provide. Examples of the solid phase carrier include a substrate, a resin, a plate (eg, multiwell plate), a filter, a cartridge, a column, and a porous material.
Examples of the substrate include those used for DNA chips and protein chips, and examples thereof include nickel-PTFE (polytetrafluoroethylene) substrates, glass substrates, apatite substrates, silicone substrates, and alumina substrates. Further, those obtained by coating these substrates with a polymer or the like are also included.
Examples of the resin include agarose particles, silica particles, copolymers of acrylamide and N, N′-methylenebisacrylamide, polystyrene-crosslinked divinylbenzene particles, particles obtained by crosslinking dextran with epichlorohydrin, cellulose fibers, and allyldextran. And N, N'-methylenebisacrylamide cross-linked polymers, monodisperse synthetic polymers, monodisperse hydrophilic polymers, Sepharose, Toyopearl, etc., and resins with various functional groups attached to these resins It is. The solid phase carrier of the present invention can be useful for, for example, purification of MK and PTN, and detection and quantification of MK and PTN.

  The hydrophobic substance-added nucleic acid, complex and nucleic acid nanoparticles of the present invention can be immobilized on a solid phase carrier by a method known per se. For example, an affinity substance (eg, those described above) or a predetermined functional group is introduced into the hydrophobic substance-added nucleic acid, complex, or nucleic acid nanoparticle of the present invention, and then the affinity substance or the predetermined functional group is used. For example, a method of immobilizing on a solid support. The present invention also provides such a method. The predetermined functional group may be a functional group that can be subjected to a coupling reaction, and examples thereof include an amino group, a thiol group, a hydroxyl group, and a carboxyl group. Also, biotin-streptavidin binding, GST tag, Flag tag, histidine tag, binding using an antibody, nucleic acid hybridization, and the like can be used. The present invention also provides a hydrophobic substance-added nucleic acid and a nucleic acid nanoparticle into which such a functional group has been introduced.

The present invention also provides methods for purification and concentration of MK and PTN. The purification and concentration method of the present invention may include a step of adsorbing MK to the solid phase carrier of the present invention and eluting the adsorbed MK with an eluent. Adsorption of MK to the solid phase carrier of the present invention can be carried out by a method known per se. For example, a sample containing MK (eg, bacterial or cell culture or culture supernatant, blood) is introduced into the solid phase carrier of the present invention or its contents. MK can be eluted using an eluent such as a neutral solution. The neutral solution is not particularly limited, but the pH of the solution is, for example, about pH 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 7 to about 8. Neutral solutions also include, for example, potassium salts (eg, NaCl, KCl), magnesium salts (eg, MgCl ₂ ), surfactants (eg, Tween 20, Triton, NP40, etc.), glycerin, chelating agents (eg, EDTA) And one or more substances selected from urea, organic solvents, and the like.
The purification and concentration method of the present invention may further include a step of washing the solid support using a washing solution after the adsorption of MK. Examples of the cleaning liquid include urea, a chelating agent (eg, EDTA), Tris, an acid, and an alkali. The purification and concentration method of the present invention may further include a step of heat-treating the solid phase carrier. By this process, the solid phase carrier can be regenerated and sterilized.

  The present invention also provides a method for detecting or quantifying MK and PTN. The detection or quantification method of the present invention uses the hydrophobic substance-added nucleic acid, complex or nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle produced using the hydrophobic substance-added nucleic acid of the present invention). And a method for detecting MK or a method for quantifying MK. The method for detecting and quantifying MK can be performed by a method similar to a known immunological method except that the hydrophobic substance-added nucleic acid, complex or nucleic acid nanoparticle of the present invention is used instead of the antibody. That is, by using the hydrophobic substance-added nucleic acid, complex, or nucleic acid nanoparticle of the present invention instead of an antibody, enzyme immunoassay (EIA) (eg, direct competitive ELISA, indirect competitive ELISA, sandwich ELISA), radioimmunity Similar to methods such as measurement method (RIA), fluorescence immunoassay method (FIA), Western blot method (eg, use as a substitute for secondary antibody in Western blot method), immunohistochemical staining method, cell sorting method, etc. The method can detect and quantify MK. Such a method may be useful, for example, for measuring the amount of MK in a biological or biological sample, or diagnosing a disease associated with MK.

  The hydrophobic substance-added nucleic acid or nucleic acid nanoparticle of the present invention can be used as a drug delivery substance by adding another drug or toxin. By using the hydrophobic substance-added nucleic acid or nucleic acid nanoparticle of the present invention (preferably, a nucleic acid nanoparticle manufactured using the hydrophobic substance-added nucleic acid of the present invention), an inflammatory site where MK and PTN are overexpressed It is possible to efficiently deliver a drug to a cancer site. Therefore, the present invention provides a drug delivery system that efficiently delivers a drug to an inflammatory site or a cancer site where MK and PTN are overexpressed.

  The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples.

[Example 1] Cholesterol-added nucleic acid that inhibits cell migration activity (1)
Midkine is known to have a cell invasion effect on osteoblast precursor cells (Qi et al., J. Biol. Chem. 276 (19), 15868-15875, 2001). Therefore, whether or not the cholesterol-added nucleic acid shown below inhibits the cell migration activity of midkine was examined using UMR106 cells (ATCC No. CRL1661) which are rat osteoblast precursor cells. 30 μL of 1.5 μM midkine was applied to the outer surface of chemotaxel (membrane pore diameter: 8 μm, manufactured by Kurabo Industries), and midkine was immobilized on the outer surface of the membrane. Chemotaxel with immobilized midkine was placed in a 24-well culture plate containing 500 μL of a medium containing cholesterol-added nucleic acid 500 nM (0.3% bovine serum albumin added, Dulbecco's Modified Eagle's medium). In the inner layer of the chemotaxel chamber, 200 μL of UMR106 cells at a concentration of 1 × 10 ⁶ cells / mL was placed and cultured at 37 ° C. for 4 hours. Cells remaining in the inner layer of the chemotaxel chamber were removed, and the cells that had infiltrated and adhered to the midkine-coated surface were fixed with methanol. The chemotaxel chamber was immersed in a 1% crystal violet aqueous solution for 30 minutes to stain the cells. After the chemotaxel chamber was washed with distilled water and dried, the dye was extracted with a mixture of 200 μL of 1% SDS and 1% triton X100. 150 μL of the extract was transferred to a 96-well microplate and the absorbance at 590 nm was measured. The pleiotrophin cell migration inhibition experiment was performed in the same manner.

  The cholesterol-added nucleic acid used in the experiment is shown below.

Nucleic acid 1
chol-A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) (SEQ ID NO: 1)

  This cholesterol-added nucleic acid is a 23-nucleotide nucleic acid consisting of a repeating sequence of A and G, with cholesterol added to the 5 'end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked to five polyethylene glycol chains by amide bonds.

Nucleic acid 2
chol-C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) (SEQ ID NO: 2)

  This cholesterol-added nucleic acid is a 23-nucleotide nucleic acid consisting of a C and U repeat sequence, with cholesterol added to the 5 'end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked to five polyethylene glycol chains by amide bonds.

Nucleic acid 3
chol-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) (SEQ ID NO: 3)

  This cholesterol-added nucleic acid is a 23-nucleotide nucleic acid consisting of A, G, C, and U, with cholesterol added to the 5 'end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked to five polyethylene glycol chains by amide bonds.

Nucleic acid 3-1.
chol-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (SEQ ID NO: 3 )

  This cholesterol-added nucleic acid is a modified nucleic acid represented by nucleic acid 3, and the 3 'end is idT-modified.

Nucleic acid 3-2
chol-C12-G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G ( M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (sequence) Number 3)

  This cholesterol-added nucleic acid is a modification of the nucleic acid represented by nucleic acid 3, and is formed by linking cholesterol and a nucleic acid by a saturated hydrocarbon chain having 12 carbon atoms, five polyethylene glycol chains, and an amide bond. The 3 'end is idT-modified.

Nucleic acid 3-3
chol-G (H) C (H) A (H) C (H) A (H) G (H) G (H) G (H) G (H) U (H) U (H) G (H) G (H) U (H) G (H) U (H) C (H) G (H) G (H) G (H) U (H) G (H) C (H) (SEQ ID NO: 3)

  This cholesterol-added nucleic acid is a modification of nucleic acid 3, and is a nucleic acid whose nucleotide is deoxyribonucleotide.

Nucleic acid 3-4
G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G ( M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -chol (SEQ ID NO: 3)

  This cholesterol-added nucleic acid is a modified version of nucleic acid 3 and has cholesterol added to the 3 'end.

Nucleic acid 4
chol-C12-U (M) U (M) G (M) G (M) U (M) G (M) U (M) -idT (SEQ ID NO: 4)

  This cholesterol-added nucleic acid is a 7-nucleotide nucleic acid consisting of G and U, with cholesterol added to the 5 'end and idT added to the 3' end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked by a saturated hydrocarbon chain having 12 carbon atoms, five polyethylene glycol chains, and an amide bond.

Nucleic acid 5
chol-G (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) G (M) G (M) A (M) A (M) G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G ( M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (SEQ ID NO: 5)

  This cholesterol-added nucleic acid is a 35 nucleotide nucleic acid consisting of A, G, C, and U, with cholesterol added to the 5 'end and idT added to the 3' end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked to five polyethylene glycol chains by amide bonds.

Nucleic acid 5-1
chol-C12-G (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) G (M) G (M) A (M) A ( M) G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G (M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (SEQ ID NO: 5 )

  This cholesterol-added nucleic acid is a modified form of nucleic acid 5 and is formed by linking cholesterol and nucleic acid via an amide bond via a saturated hydrocarbon chain having 12 carbon atoms and five polyethylene glycol chains.

Nucleic acid 5-2
idT-G (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) G (M) G (M) A (M) A (M) G (M) C (M) A (M) C (M) A (M) G (M) G (M) G (M) G (M) U (M) U (M) G (M) G ( M) U (M) G (M) U (M) C (M) G (M) G (M) G (M) U (M) G (M) C (M) -idT (SEQ ID NO: 5)

  This cholesterol-added nucleic acid is a modified version of nucleic acid 5 and is obtained by adding idT in place of cholesterol.

Nucleic acid 5-3
GGGAAGGAGGGAAGC (F) AC (F) AGGGGU (F) U (F) GGU (F) GU (F) C (F) GGGG (F) GC (F) (SEQ ID NO: 5)

  This cholesterol-added nucleic acid is a variant of nucleic acid 5 in which both end modifications are removed. G and A are ribonucleotides, and C and U are those in which the 2'-position of ribose is modified with F.

Nucleic acid 6
chol-G (F) G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) AGGG (M) G (M) U (F) U (F) G (M) G (M) U ( F) G (M) U (F) C (F) GGGU (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 6)

  This cholesterol-added nucleic acid is a 39-nucleotide nucleic acid consisting of A, G, C, and U, with cholesterol added to the 5 'end and idT added to the 3' end. Nucleotides include those in which the 2'-position of ribose is modified with a fluoro group and an O-methyl group, and unmodified ones.

Nucleic acid 6-1
GGGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG ( M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -Chol (SEQ ID NO: 6)

  This cholesterol-added nucleic acid is a variant of nucleic acid 6 in which cholesterol is added to the 3 'end and the modification at the 2' position of ribose is changed.

Nucleic acid 6-2
PEG40k-C6-GGGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A ( M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 6)

  This nucleic acid is a modified version of nucleic acid 6-1, in which cholesterol is removed, 40 kDa PEG is added to the 5 'end with a C6 linker sandwiched, and idT is added to the 3' end.

Nucleic acid 6-3
PEG40k-C12-GGGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A ( M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 6)

  This nucleic acid is a modification of nucleic acid 6-1, in which cholesterol is removed, 40 kDa PEG is added to the 5 'end with a C12 linker, and idT is added to the 3' end.

Nucleic acid 6-4
S18-GGGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 6)

  This nucleic acid is a modification of nucleic acid 6-1, in which cholesterol is removed and S18 is added to the 5 'end and idT is added to the 3' end.

Nucleic acid 7
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a variant of nucleic acid 6 in which G at the 5 'end is removed and the modification at the 2' position of ribose is changed.

Nucleic acid 7-1
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -S18-PEG40k (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a modification of nucleic acid 7, which is obtained by removing idT at the 3 'end and adding 40 KDa branched polyethylene glycol sandwiched between Spacer 18 instead.

Nucleic acid 7-2
chol-G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) G (F) G (M) G (M) G (M) U (F) U (F) G ( M) G (M) U (F) G (M) U (F) C (F) G (F) G (F) G (M) U (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a modified version of nucleic acid 7, which is obtained by changing the 2'-position modification of ribose.

Nucleic acid 7-3
G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U ( F) G (M) C (F) A (M) C (F) A (M) G (F) G (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) G (F) G (F) G (M) U (F) G (M) C (F) A (M) C ( F) (SEQ ID NO: 7)

  This nucleic acid is a modified version of nucleic acid 7-2, excluding terminal cholesterol and invertedT.

Nucleic acid 7-4
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -PEG40k (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a modification of nucleic acid 7-1 and is obtained by removing Spacer18.

Nucleic acid 7-5
chol-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -C12-PEG40k (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a modified version of nucleic acid 7-1 and uses a C12 linker instead of Spacer18.

Nucleic acid 7-6
chol-G (F) G (F) A (M) A (M) G (F) G (F) A (M) G (F) G (F) A (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) G (F) G (M) G (M) G (M) U (F) U (F) G ( M) G (M) U (F) G (M) U (F) C (F) G (F) G (F) G (M) U (F) G (M) C (F) A (M) C (F) -S18-PEG40k (SEQ ID NO: 7)

  This cholesterol-added nucleic acid is a modification of nucleic acid 7-2, which is obtained by removing idT at the 3 'end and adding 40 KDa branched polyethylene glycol across Spacer18 instead.

Nucleic acid 8
chol-C12-G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U ( F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) -IdT (SEQ ID NO: 8)
This cholesterol-added nucleic acid is a 27-nucleotide nucleic acid to which cholesterol is added at the 5 ′ end and idT is added at the 3 ′ end. Cholesterol and nucleic acid are linked by a saturated hydrocarbon chain having 12 carbon atoms. Nucleotides include those in which the 2 ′ position of ribose is modified with a fluoro group and an O-methyl group, and unmodified ones.
Nucleic acid 9
chol-C12-GGGA (M) GA (M) GGA (M) GA (M) A (M) GA (M) GGA (M) A (M) GU (F) G (M) C (F) A ( M) C (F) AGGGGG (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGGU (F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 9)

  This cholesterol-added nucleic acid is a 45-nucleotide nucleic acid with cholesterol added at the 5 'end and idT added at the 3' end. Cholesterol and nucleic acid are linked by a saturated hydrocarbon chain having 12 carbon atoms. Nucleotides include those in which the 2'-position of ribose is modified with a fluoro group and an O-methyl group, and unmodified ones.

  The results of the cell migration inhibition experiment are shown in Table 1. Inhibitory activity% is a value obtained by subtracting from 100 the number of cells that migrated when nucleic acid was added, with the number of cells that migrated when no nucleic acid was added being 100. Although the inhibitory activity of cholesterol alone was also measured, no inhibitory activity was observed even when 100 times the concentration of nucleic acid was added. On the other hand, as can be seen from the comparison of nucleic acids 5, 5-1, 5-2, and 5-3, nucleic acids without added cholesterol (nucleic acids 5-2 and 5-3) did not show inhibitory activity. Addition of cholesterol showed high inhibitory activity (nucleic acids 5, 5-1). From the above, it was shown that the cell migration activity of MK is inhibited by combining nucleic acid and cholesterol.

These results show the following.
(1) The position of cholesterol may be the 5 ′ end or the 3 ′ end.
(2) The type of linker that connects cholesterol and nucleic acid does not significantly affect the activity.
(3) When the length of the nucleic acid is short, the physiological activity decreases.
(4) A cholesterol-added nucleic acid that inhibits midkine inhibits pleiotrophin.
(5) Even if polyethylene glycol is added to the end of the cholesterol-added nucleic acid, the physiological activity does not decrease.

[Example 2] Evaluation of binding activity by surface plasmon resonance method Cholesterol addition represented by nucleic acids 1, 3, 3-2, 3-3, 4, 5, 5-1, 5-3, 7, 7-1 The binding activity of nucleic acid to midkine was examined by surface plasmon resonance. BIAcore2000 manufactured by BIAcore was used for the measurement. A CM4 chip having a carboxyl group immobilized thereon was used as the sensor chip, and midkine was immobilized by amino coupling. Amino coupling was carried out using N-hydroxysuccinimide (NHS, 11 mg / L) and N-ethyl-N ′-(3-dimethylaminopropyl) carbohydrate hydrate (EDC, 75 mg / L) according to the specifications of BIAcore. Moreover, the unreacted carboxyl group was blocked using ethanolamine. Use a mixed solution of 145 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM magnesium chloride, 1.8 mM calcium chloride, and 20 mM Tris (pH 7.6) as the running buffer (this solution is hereinafter referred to as “solution A”). It was. A 6M urea solution was used as the regeneration solution. 400 nM cholesterol-added nucleic acid was injected to confirm the interaction. As a result, all the cholesterol-added nucleic acids measured bound to midkine. Their binding strength varied depending on the nucleic acid. An example of a sensorgram when nucleic acid 1 is used is shown in FIG.

[Example 3] Onset suppression experiment of EAE model mouse using cholesterol-added nucleic acid 300 μg of Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG _35-55 ) (MEVGWYRSPFSRVVHLYRNG (sequence number 11)) ) And incomplete Feund's adjuvant, and subcutaneously administered to the lumbar region of wild type C57 / BL6 (female, 8 weeks old) for sensitization. Furthermore, immediately after sensitization and 48 hours later, 300 ng of pertussis toxin was dissolved in 200 μL of PBS and administered intraperitoneally to induce EAE. Thereafter, clinical symptoms were evaluated daily according to the following criteria.
A clinical score was assigned daily to the wild-type mouse group (n = 5) and the cholesterol-added nucleic acid-administered group (n = 5) represented by nucleic acid 10, and clinical symptoms were evaluated. Clinical score values are 0: no symptoms, 1: drooping tail, 2: unable to stand up on the back, 3: unstable walking, 4: mild hind limb paralysis, 5: severe hind limb paralysis, 6: death, The proportion of all animals up to 28 days after MOG _35-55 administration was recorded.
The cholesterol-added nucleic acid represented by the nucleic acid 10 is as follows.

Nucleic acid 10
chol-G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) G (M) A (M) A (M) G (M) A (M) A (M) A (M) U ( M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) U (M) U (M) C (M) C (M) -idT (sequence Number 10)

  This cholesterol-added nucleic acid is a 36-nucleotide nucleic acid consisting of A, G, C, and U, with cholesterol added to the 5 'end and idT added to the 3' end. The nucleotide is modified at the 2 'position of ribose with an O-methyl group. Cholesterol and nucleic acid are linked to five polyethylene glycol chains by amide bonds.

Cholesterol-added nucleic acid represented by nucleic acid 10 is dissolved in physiological saline containing 1 mM magnesium chloride, 8 times in total every 2 days until 14 days after administration of MOG _35-55 , then once a day A total of 13 administrations were performed at intervals of (Fig. 2, black squares). The dose was 1 mg / kg. As a control group, physiological saline containing 1 mM magnesium chloride was administered in the same manner (FIG. 2, black circles).
As a result of the experiment, clinical symptoms were reduced with a statistically significant difference of p <0.05 from day 17 to day 21 in the cholesterol-added nucleic acid administration group (FIG. 2). Statistical significance was analyzed using the Dunnett method. From the above, it was suggested that the cholesterol-added nucleic acid may be used as a therapeutic agent for multiple sclerosis.

[Example 4] Nanoparticle formation of cholesterol-added nucleic acid Cholesterol is a hydrophobic substance, while nucleic acid is a hydrophilic substance. In view of this, it was considered that the cholesterol-added nucleic acid formed by binding them was formed into nanoparticles depending on conditions. Therefore, whether or not cholesterol-added nucleic acid was dissolved and converted into nanoparticles under various conditions was examined using gel filtration HPLC. The conditions of gel filtration HPLC are as follows.
Column: Tosoh G3000SW _XL
Eluent: Solution A
Flow rate: 0.5 mL / min
Mode: Isocratic column temperature: 25 ° C
Detection wavelength: 260 nm

  When nucleic acid 7 was dissolved in physiological saline containing 1 mM magnesium chloride at a concentration of 1 mg / mL, formation of nanoparticles was confirmed (FIG. 3D). When this aqueous solution was heated at 90 ° C. for 10 minutes, nanoparticulation was promoted (FIG. 3E). On the other hand, when this aqueous solution was diluted with physiological saline containing 1 mM magnesium chloride to a concentration of 0.1 mg / mL or less and heated at 90 ° C. for 10 minutes, the gel filtration HPLC peak shifted to the low molecular weight side (this peak). Is hereinafter referred to as the monomer peak (FIG. 3F). Further, when nucleic acid 7 was dissolved in ultrapure water at a concentration of 1 mg / mL, a peak appeared at the position of the monomer peak, and formation of nanoparticles was not confirmed (FIG. 3C). When the same experiment was performed using nucleic acid 7-7 and nucleic acid 7-8 to which no cholesterol was added, formation of nanoparticles was not confirmed (FIG. 3B; FIG. 3B shows 1 mg of nucleic acid 7-8). This is the result when dissolved in physiological saline containing 1 mM magnesium chloride at a concentration of / mL.) Here, the nucleic acids 7-7 and 7-8, which are “nucleic acids to which cholesterol is not added”, are as follows.

Nucleic acid 7-7
GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG ( M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U (F) G (M) C (F) A (M) C (F) (SEQ ID NO: 7)
Nucleic acid 7-8
S18-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 7)

  When poly A having 40 continuous AMPs was injected as a standard sample, a sample in which nucleic acid 7 having a concentration of 0.1 mg / mL or less was heated (FIG. 3F), a sample in which nucleic acid 7 was dissolved in ultrapure water (FIG. 3C), and The retention time was similar to that of nucleic acid 7-8 (FIG. 3B) (near the monomer peak, FIG. 3A). These results suggest that the sample in which the nucleic acid 7 having a concentration of 0.1 mg / mL or less is heated and the sample in which the nucleic acid 7 is dissolved in ultrapure water are monomers.

Next, it was examined whether the formation of nanoparticles depends on the presence or absence of neutralized salts in the aqueous solution and the concentration of nucleic acid. When nucleic acid 7 was dissolved in ultrapure water at a concentration of 1 mg / mL, formation of nanoparticles was not confirmed. This solution was kept at room temperature for 23 hours, but no peak shift toward the high mass side was observed by gel filtration HPLC. On the other hand, when nucleic acid 7 was dissolved in ultrapure water at a concentration of 10 mg / mL, broad peaks were detected from high mass to low mass, confirming the formation of nanoparticles of various sizes. In addition, when nucleic acid 7 was dissolved in physiological saline containing 1 mM magnesium chloride at concentrations of 0.1, 0.5, 1.5, 2, 10 mg / mL and heated at 90 ° C. for 10 minutes, 0.1 mg Nanoparticle formation was confirmed at concentrations other than / mL. These nanoparticles were stable in neutral salt solution for at least 24 hours. On the other hand, the 0.1 mg / mL sample showed a peak at the position of the monomer peak after heating. In addition, when nucleic acid 7 was dissolved at room temperature in a physiological saline containing 1 mM magnesium chloride at a concentration of 0.1 mg / mL, it was confirmed that nanoparticles were formed.
From the above, it was found that the formation of nucleic acid nanoparticles requires a salt, and the formation is promoted by heating. Moreover, although the nanoparticle once formed is stable, it turned out that it dimerizes to the density | concentration of 0.1 mg / mL or less, and it becomes a monomer by heating.

  The nucleic acid 7 was then analyzed by 12% non-denaturing polyacrylamide gel electrophoresis (PAGE). Nucleic acid 7 was dissolved in physiological saline containing ultrapure and 1 mM magnesium chloride to make 0.1 mg / mL. Each of these solutions was electrophoresed at 5 μL. As a result, the nucleic acid 7 dissolved in ultrapure water showed a band at a place considered to be a monomer (FIG. 4). On the other hand, the nucleic acid 7 dissolved in physiological saline containing 1 mM magnesium chloride was collected in the well, and most of the nucleic acid 7 was not migrated. These results are consistent with the results of gel filtration HPLC, indicating that nucleic acid nanoparticles are likely to be formed in the presence of salt. In FIG. 4, M represents a marker, W represents nucleic acid 7 dissolved in ultrapure water, and S represents nucleic acid 7 dissolved in physiological saline containing 1 mM magnesium chloride.

  A similar experiment was performed using nucleic acid 3-2. As a result, nanoparticles were formed in the presence of salt in the same manner as nucleic acid 7. Heating this solution promoted nanoparticulation. In addition, nanoparticles were not formed in ultrapure water. Heating this solution promoted monomer formation.

In order to confirm that the hydrophobic substance-added nucleic acid having physiological activity did not lose its activity even when it was nanoparticulated, a cell migration inhibition experiment was performed by applying the same method as in Example 1. Nucleic acid 7 was dissolved in physiological saline containing 1 mM magnesium chloride so as to be 10 mg / mL, maintained for several hours, and then diluted with physiological saline to 0.069 mg / mL. This sample was divided into two (samples 1 and 2). Sample 1 was used for cell migration inhibition experiment as it was (nanoparticle state). Sample 2 was heated at 90 ° C. for 10 minutes and then used for cell migration inhibition experiments (non-nanoparticle state). As a result of the experiment, both samples 1 and 2 strongly inhibited cell migration.
From the above, it was shown that the nucleic acid was converted into nanoparticles by adding cholesterol. It was also found that nanoparticulation depends on the presence of salt, the concentration of nucleic acid, and the presence or absence of heating. Furthermore, it was shown that the physiological activity was retained even after nanoparticulation.

[Example 5] In vivo pharmacokinetics of nanoparticulate cholesterol-added nucleic acid Nucleic acid 7 and nucleic acid 7-8 not added with cholesterol were each dissolved in physiological saline at 1 mg / mL, and C57 / BL6 / J mice ( Female mice, 8 weeks old, N = 3) were intravenously administered at 10 mg / kg. Nucleic acid 7-8 has 5 ′ end modified with Spacer18, and nuclease resistance is considered to be the same as nucleic acid 7.

Blood was collected after 0.25, 1, 2, and 4 hours, and the residual nucleic acid concentration in the blood was measured using the ELOSA method (hybridization method). The ELOSA method is a method for quantifying nucleic acid in a biological sample using two types of nucleic acid probes having sequences complementary to the nucleic acid to be measured, and is a general method already used in clinical tests and the like. One probe was immobilized on a plate (96-well plate (round) kit for DNA immobilization, manufactured by Sumitomo Bakelite Co., Ltd.) and used to trap the target nucleic acid. The other probe was bound with FAM and used for detection. In order to increase the sensitivity, an anti-FAM antibody conjugated with HRP was used, and the analysis was performed based on the absorbance of the chromogenic substrate. Quantification was performed using a calibration curve prepared using samples of known concentration.
As a result of the measurement, it was shown that the nucleic acid 7 to which cholesterol was added had a significantly longer nucleic acid residence time in the blood and a higher blood concentration than the nucleic acid 7-8 to which cholesterol was not added (FIG. 5). 5).

By the way, Healy et al. Dissolved an aptamer for TGFβ-2 having a length of 32 nucleotides added with cholesterol in phosphate physiological saline at a concentration of 1 mg / mL, and administered intravenously to rats at 1 mg / kg. (See Pharmaceutical Research, Vol. 21, No. 12, 2004, 2234). In this experiment, the blood drug concentration was measured using the ELOSA method, and the pharmacokinetic parameters were determined. As a result, the characteristics of the aptamer to which cholesterol was not added and the aptamer to which cholesterol was not added were compared. Is reported to be good. This indicates that simply adding cholesterol does not improve the pharmacokinetics.
From the above, it can be seen that in vivo pharmacokinetics can be improved by adding a hydrophobic substance to a nucleic acid to form nanoparticles.

The hydrophobic substance-added nucleic acid and complex of the present invention can be useful as a reagent for various diseases such as autoimmune disease, cancer, postoperative adhesion, endometriosis, or a diagnostic agent. Nucleic acids and complexes to which the hydrophobic substances of the invention have been added may also be useful for MK purification and concentration, and MK detection and quantification.
The nucleic acid nanoparticles obtained by the present invention not only retain the physiological activity possessed by the nucleic acid molecule, but also have improved physiological activity and in vivo pharmacokinetics. Useful for the production of nucleic acid reagents.
This application is based on Japanese Patent Application No. 2007-295951 filed in Japan, the contents of which are incorporated in full herein.

Claims (21)

  A hydrophobic substance-added nucleic acid characterized by binding to midkine and inhibiting its activity.   Furthermore, it has inhibitory activity with respect to pleiotrophin, The nucleic acid of Claim 1 characterized by the above-mentioned.   The nucleic acid according to claim 1 or 2, wherein the hydrophobic substance is cholesterol.   A nucleic acid represented by any one of SEQ ID NOs: 1 to 10, or a nucleic acid in which 1 to 3 bases are substituted, deleted, added or inserted into the nucleic acid represented by any of SEQ ID NOs: 1 to 10. Item 4. The nucleic acid according to any one of Items 1 to 3.   A complex comprising the nucleic acid according to any one of claims 1 to 4 and a functional substance.   The complex according to claim 5, wherein the functional substance is an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle or a drug.   A medicine or reagent comprising the nucleic acid according to any one of claims 1 to 4 or the complex according to claim 5 or 6.   A method for detecting midkine, wherein the nucleic acid according to any one of claims 1 to 4 or the complex according to claim 5 or 6 is used.   A method of forming a nucleic acid into nanoparticles, comprising binding a hydrophobic substance to the nucleic acid. (A) binding a hydrophobic substance to the nucleic acid;
(B) a step of dissolving the hydrophobic substance-added nucleic acid obtained in (a) in a neutral salt solution;
A method for forming a nucleic acid into nanoparticles.   The method according to claim 10, wherein the hydrophobic substance-added nucleic acid is dissolved in a neutral salt solution at a concentration of 0.1 mg / mL to 10 mg / mL.   A method for improving the in vivo pharmacokinetics of a nucleic acid, comprising binding a hydrophobic substance.   The method according to claim 12, wherein the nucleic acid is a nucleic acid having physiological activity.   The method according to any one of claims 9 to 13, wherein the hydrophobic substance is cholesterol.   Nucleic acid nanoparticles formed by binding hydrophobic substances.   A medicine or reagent comprising the nucleic acid nanoparticles according to claim 15.   The medicament or reagent according to claim 7 or 16, which is a cell migration inhibitor.   The medicament or reagent according to claim 7 or 16, which is a growth promoter for regulatory T cells.   The medicament according to claim 7 or 16, which is a prophylactic / therapeutic agent for autoimmune disease or cancer.   A method for preventing / treating an autoimmune disease or cancer, comprising administering an effective amount of the nucleic acid according to claim 1 to a mammal. Use of the nucleic acid according to claim 1 for producing an agent for preventing / treating an autoimmune disease or cancer.