Aptamer preparation

Abstract

The present invention provides a preparation formula capable of stably maintaining the activity of an aptamer (especially an aptamer to FGF2) for a long time, and further provides a pharmaceutical preparation with the aptamer (especially an FGF2 aptamer) as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an aptamer preparation, in particular to an aptamer preparation containing an aptamer targeting basic fibroblast growth factor (FGF2) as an active ingredient. Background Art

[0002] In recent years, the application of RNA aptamers in therapeutic drugs, diagnostic reagents, and reagents has attracted much attention, and several RNA aptamers have entered the clinical stage or are in the practical stage. In December 2004, the world's first RNA aptamer drug Macugen (registered trademark) (generic name: pegaptanib sodium) was licensed in the United States as a therapeutic drug for age-related macular degeneration, and was also licensed in Japan in July 2008. Macugen (registered trademark) uses an aptamer targeting VEGF as the active subject. The aptamer is composed of a synthetic oligonucleotide containing 28 nucleic acid molecules, and a polyethylene glycol (PEG) derivative is bound to its 5' end.

[0003] Macugen (registered trademark) is in the form of a pre-filled syringe injection, which is injected into the lens. It is a colorless to slightly colored clear aqueous injection, which contains sodium hydrogen phosphate, sodium dihydrogen phosphate, an isotonic agent, and a pH adjuster. Using this formulation, it was shown to be stable in an accelerated test at 25±2°C for 6 months and in a long-term storage test at 5±3°C for 36 months (Non-Patent Document 1).

[0004] On the other hand, at the time of filing the present application, there were no drugs containing aptamers as active ingredients other than Macugen (registered trademark), and it was completely unknown what formulation was appropriate for aptamer preparations.

[0005] The applicant is developing a drug with an aptamer targeting FGF2 as an active ingredient and with age-related macular degeneration, cartilage hypoplasia, or cancer pain as indications (Patent Documents 1 and 2). In the process of developing an FGF2 aptamer preparation, the inventor used a preparation formulation that was believed to be roughly the same as Macugen (registered trademark) and used phosphate buffered saline (PBS) as a medium, but the result was that the activity of the FGF2 aptamer could not be maintained.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2011 / 099576

[0009] Patent Document 2: International Publication No. 2015 / 147017

[0010] Non-patent literature

[0011] Non-patent document 1: Drug interview form Age-related macular degeneration therapeutic agent Macugen (registered trademark) intracrystalline injection kit 0.3 mg March 2015 (revised 5th edition) Summary of the invention

[0012] Problem that the invention aims to solve

[0013] Therefore, the object of the present invention is to provide a preparation formula that can stably maintain the activity of an aptamer (especially an aptamer to FGF2) for a long time, and further provide a pharmaceutical preparation containing an aptamer (especially an FGF2 aptamer) as an active ingredient.

[0014] Methods used to solve problems

[0015] The present inventors have conducted intensive studies to achieve the above-mentioned object and, as a result, have successfully found the optimal formulation conditions for the FGF2 aptamer, thereby completing the present invention.

[0016] That is, the present invention provides the following contents.

[0017] [1] An aqueous liquid preparation comprising an aptamer or a salt thereof that binds to FGF2 and an osmotic pressure regulator that is a non-electrolyte, wherein the aptamer or the salt thereof is stable for a long period of time.

[0018] [2] The aqueous liquid preparation according to [1], which contains substantially no electrolyte other than the aptamer or a salt thereof.

[0019] [3] The aqueous liquid preparation according to [1] or [2], wherein the aptamer comprises a nucleotide sequence represented by the following formula (1):

[0020]

[0021] Among them, N 1 and N 6 Each independently represents 0 to 1 or more arbitrary bases, N 2 , N 3 , N 4 and N 5 independently represents an arbitrary base, wherein uracil may be thymine, and the aptamer is any one of the following (a) or (b):

[0022] (a) among the nucleotides contained in the aptamer,

[0023] (i) the 2′ position of the ribose sugar of each pyrimidine nucleotide is a fluorine atom,

[0024] (ii) the 2′ position of the ribose sugar of each purine nucleotide is a hydroxyl group;

[0025] (b) In the aptamer of (a),

[0026] (i) the fluorine atom at the 2′-position of the ribose of each pyrimidine nucleotide is independently unsubstituted or substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group,

[0027] (ii) The hydroxyl group at the 2′-position of the ribose of each purine nucleotide is independently unsubstituted or substituted with an atom or group selected from the group consisting of a hydrogen atom, a methoxy group and a fluorine atom.

[0028] [4] The aqueous liquid preparation according to [1] or [2], wherein the aptamer comprises a nucleotide sequence represented by the following formula (3):

[0029]

[0030] Among them, N 1 and N 62 Each independently represents 0 to 1 or more arbitrary bases.

[0031] [5] The aqueous liquid preparation according to [1] or [2], wherein the aptamer comprises any one of the nucleotide sequences shown in SEQ ID NO: 3, 8, 9, 10 or 12.

[0032] [6] The aqueous liquid preparation according to any one of [1] to [5], wherein the concentration of the aptamer is 1 to 60 mg / mL.

[0033] [7] The aqueous liquid preparation according to any one of [1] to [6], wherein the osmotic pressure regulator is contained in an amount of 2 to 7.5% (w / v) of the total amount of the aqueous liquid preparation.

[0034] [8] The aqueous liquid preparation according to any one of [1] to [7], wherein the osmotic pressure regulator is mannitol.

[0035] [9] The aqueous liquid preparation according to [8], wherein the content of mannitol is 1 to 50 mg per 1 mg of the aptamer.

[0036]

[10] The aqueous liquid preparation according to any one of [1] to [9], stored at 5°C or less.

[0037]

[11] The aqueous liquid preparation according to any one of [1] to

[10] , wherein the proportion of the monomeric aptamer after storage at 4°C for 3 months is 80% or more.

[0038]

[12] The aqueous liquid preparation according to any one of [1] to

[11] , which is an injection.

[0039]

[13] The aqueous liquid preparation according to any one of [1] to

[12] , which is used for preventing or treating a disease associated with angiogenesis, a bone / cartilage disease, or pain.

[0040]

[14] A method for preventing or treating a disease associated with angiogenesis, a bone / cartilage disease or pain, comprising administering the aqueous liquid preparation of any one of [1] to

[12] to a subject.

[0041]

[15] The aqueous liquid preparation according to any one of [1] to

[12] , which is used for preventing or treating a disease accompanied by angiogenesis, a bone / cartilage disease or pain.

[0042] Effects of the Invention

[0043] According to the present invention, an aptamer preparation can be provided which can stably store an aptamer or a salt thereof to FGF2 as an active ingredient in the form of an aqueous liquid for a long period of time and is therefore easy to handle. DETAILED DESCRIPTION

[0044] The present invention provides a pharmaceutical preparation (hereinafter also referred to as "aptamer preparation of the present invention") which contains an aptamer or a salt thereof for FGF2 as an active ingredient and which stably maintains the aptamer or the salt thereof for a long period of time. Here, "long-term stability" means that when the preparation is sealed in a glass bottle and stored at 4°C for 3 months, the proportion of monomeric aptamers is 70% or more. The proportion of monomeric aptamers is calculated by using size exclusion chromatography to separate and detect monomers and polymers under the following conditions, thereby calculating the value of (monomer peak area) / (sum of monomer and polymer peak areas)×100(%).

[0045] Equipment: ACQUITY UPLC H-Class Bio by Waters

[0046] Detector: TUV detector manufactured by Waters

[0047] Column: ACQUITY UPLC BEH200 SEC column manufactured by Waters

[0048] Sample concentration: 0.2 mg / mL

[0049] Injection volume: 5μL

[0050] Eluent: 10% acetonitrile / PBS

[0051] Flow rate: 0.3mL / min

[0052] Column temperature: 25°C

[0053] The aptamer preparation of the present invention contains an aptamer or a salt thereof for FGF2 as an active ingredient and an osmotic pressure regulator as a non-electrolyte.

[0054] An aptamer refers to a nucleic acid molecule having binding activity relative to a given target molecule. An aptamer can inhibit the activity of a given target molecule by binding to the target molecule. The aptamer as the active ingredient of the aptamer preparation of the present invention is an aptamer having binding activity for FGF2. In a preferred embodiment, the aptamer is an aptamer that can bind to FGF2 and inhibit the binding of FGF2 to an FGF receptor. That is, the aptamer has inhibitory activity against FGF2.

[0055] The aptamer used in the present invention is an aptamer that binds to FGF2, and more preferably an aptamer that can bind to FGF2 and inhibit the binding of FGF2 to FGF receptor. Whether the aptamer used in the present invention inhibits the binding of FGF2 to FGF receptor can be evaluated by, for example, a test using the surface plasmon resonance method such as Example 1.

[0056] There are no particular limitations on the aptamer for FGF2, and for example, the aptamer described in International Publication No. 2015 / 147017. Specifically, the aptamer comprises a nucleotide sequence represented by the following formula (1):

[0057]

[0058] Wherein, uracil may be thymine, and the aptamer may be any one of the following (a) or (b):

[0059] (a) among the nucleotides contained in the aptamer,

[0060] (i) the 2′ position of the ribose sugar of each pyrimidine nucleotide is a fluorine atom,

[0061] (ii) the 2′ position of the ribose sugar of each purine nucleotide is a hydroxyl group;

[0062] (b) In the aptamer of (a),

[0063] (i) the fluorine atom at the 2′-position of the ribose of each pyrimidine nucleotide is independently unsubstituted or substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group,

[0064] (ii) The hydroxyl group at the 2′-position of the ribose of each purine nucleotide is independently unsubstituted or substituted with an atom or group selected from the group consisting of a hydrogen atom, a methoxy group and a fluorine atom.

[0065] In the above formula (1), N 1 and N 6 Each independently represents 0 to 1 or more arbitrary bases, N 2 , N 3 , N 4 and N 5In the present specification, "base" refers to any one of adenine (A), guanine (G), cytosine (C), uracil (U) or thymine (T) constituting nucleic acid.

[0066] To N 1 There is no particular limitation on the number of bases, as long as the aptamer comprising the nucleotide sequence shown in formula (1) binds to FGF2. For example, it can be: 0 to about 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, etc., preferably 0 to 2.

[0067] To N 6 In terms of the number of bases, it is also related to N 1 There are no particular limitations on the number of moieties, and the number of moieties may be, for example, 0 to about 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, etc., and preferably 0 to 10, 3 to 9, or 5 to 8.

[0068] In a preferred embodiment, in the above formula (1),

[0069] N 1 is G, GG, AG, C or gap,

[0070] N 2 is A or U,

[0071] N 3 is G, C or A,

[0072] N 4 is G, C or U,

[0073] N 5 is G or U,

[0074] N 6 For UUCN 61 or AGUCN 62 (Where N 61 and N 62 Each independently represents 0 to 1 or more arbitrary bases). 1 "Vacant" means that N in formula (1) 1 Does not exist, that is, N 1 is 0 bases.

[0075] To N 61 The number of bases is not particularly limited, and may be, for example, 0 to about 10, 0 to 7, 0 to 6, 0 to 5, 0 to 4, etc., and preferably 0 to 5, 1 to 5, or 2 to 4.

[0076] To N 62The number of bases is not particularly limited, and may be, for example, 0 to about 10, 0 to 7, 0 to 5, 0 to 4, 0 to 3, etc., preferably 0 to 5, 0 to 4, or 0 to 3.

[0077] In another preferred embodiment, in the above formula (1),

[0078] N 1 is G, GG, AG or vacant,

[0079] N 2 is A or U,

[0080] N 3 is G or A,

[0081] N 4 is C or U,

[0082] N 5 is G or U,

[0083] N 6 For UUCN 61 or AGUCN 62 (Where N 61 and N 62 Synonymous with above).

[0084] In a preferred embodiment, the aptamer used in the present invention comprises a nucleotide sequence represented by the following formula (2) or (3):

[0085]

[0086] (Where N 1 , N 61 and N 62 The same meaning as above), more preferably comprising the nucleotide sequence represented by formula (3).

[0087] In a preferred embodiment, the aptamer used in the present invention comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 12. The nucleotide sequences shown in SEQ ID NOs: 1 to 12 are shown below (wherein uracil may be thymine), (hereinafter, A, G, C and U represent that the base of the nucleotide is adenine, guanine, cytosine and uracil, respectively).

[0088] SEQ ID NO: 1:

[0089]

[0090] SEQ ID NO: 2:

[0091]

[0092] SEQ ID NO: 3:

[0093]

[0094] SEQ ID NO: 4:

[0095]

[0096] SEQ ID NO: 5:

[0097]

[0098] SEQ ID NO: 6:

[0099]

[0100] SEQ ID NO: 7:

[0101]

[0102] SEQ ID NO: 8:

[0103] SEQ ID NO:9:

[0104]

[0105] SEQ ID NO: 10:

[0106]

[0107] SEQ ID NO: 11:

[0108]

[0109] SEQ ID NO: 12:

[0110]

[0111] In a preferred embodiment, the aptamer used in the present invention comprises SEQ ID NO: 1, 3, 4, 5, 6, 8, 9, 10 or 12, more preferably comprises the nucleotide sequence shown in SEQ ID NO: 3, 8, 9, 10 or 12.

[0112] In another preferred embodiment, the aptamer used in the present invention comprises the nucleotide sequence shown in SEQ ID NO: 2 or 7 (included in the above formula (2)).

[0113] In still another preferred embodiment, the aptamer used in the present invention comprises the nucleotide sequence represented by SEQ ID NO: 1, 3, 4, 5, 6 or 8 (included in the above formula (3)).

[0114] In one embodiment, the aptamer used in the present invention may include a nucleotide sequence in which one or more nucleotides in any of the above nucleotide sequences are replaced, deleted, inserted or added, as long as it still binds to FGF2. The aptamer may be

[0115] (a) among the nucleotides contained in the aptamer,

[0116] (i) the 2′ position of the ribose sugar of each pyrimidine nucleotide is a fluorine atom,

[0117] (ii) an aptamer in which the 2′ position of the ribose of each purine nucleotide is a hydroxyl group;

[0118] (b) In the aptamer of (a),

[0119] (i) the fluorine atom at the 2′-position of the ribose of each pyrimidine nucleotide is independently unsubstituted or substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group,

[0120] (ii) An aptamer in which the hydroxyl group at the 2′-position of the ribose of each purine nucleotide is independently not substituted or substituted with an atom or a group selected from the group consisting of a hydrogen atom, a methoxy group and a fluorine atom.

[0121] There is no particular limitation on the number of nucleotides substituted, deleted, inserted or added, as long as the FGF2 can be still bound after the substitution, deletion, insertion or addition. For example, it can be 1 to about 10, preferably 1 to 6, more preferably 1 to 5, further preferably 1 to 4, further preferably 1 to 3, and most preferably 1 or 2. There is no particular limitation on the site where the nucleotide is substituted, deleted, inserted or added, as long as the FGF2 can be still bound after the substitution, deletion, insertion or addition. In the above formulas (1), (2) and (3), in the site specifically designated by one nucleotide (i.e., A, G, C or U), nucleotide substitution, deletion, insertion or addition is performed at 1 to 3, preferably 1 or 2, and more preferably 1. On the other hand, in the formulas (1), (2) and (3), in the site that can take multiple nucleotides (i.e., N 1 , N 2 , N 3 , N 4 , N 5 , N 6 , N 61 or N 62), substitution, deletion, insertion or addition of more nucleotides (e.g., 1 to about 10, preferably 1 to 6, more preferably 1 to 5, and further preferably 1 to 4) may be allowed. For example, if the nucleotide sequence shown in SEQ ID NO: 3 is taken as the original sequence, SEQ ID NO: 1 is a sequence in which CC at the 3′ end of SEQ ID NO: 3 is replaced with UCGA, SEQ ID NO: 4 is a sequence in which one nucleotide is deleted at each end of SEQ ID NO: 3, SEQ ID NO: 5 is a sequence in which G is added to the 5′ end of SEQ ID NO: 3 and C is added to the 3′ end, SEQ ID NO: 6 is a sequence in which A is added to the 5′ end of SEQ ID NO: 3, SEQ ID NO: 8 is a sequence in which G at the 5′ end of SEQ ID NO: 3 is replaced with C and C at the 3′ end is replaced with G, SEQ ID NO: 9 is a sequence in which GG at the 5′ end of SEQ ID NO: 3 is replaced with CC and CC at the 3′ end is replaced with GG, SEQ ID NO: 10 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: 12 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: 13 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: 14 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: 15 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: 16 is a sequence in which A at position 14 of SEQ ID NO: 3 is replaced with G and U at position 19 is replaced with C, SEQ ID NO: A sequence in which the A at position 17 of NO:3 is substituted with U.

[0122] The length of the aptamer used in the present invention is not particularly limited, and can generally be about 10 to about 200 nucleotides, for example, about 20 nucleotides or more (for example, 25 nucleotides or more, 30 nucleotides or more, 31 nucleotides or more, 32 nucleotides or more, 33 nucleotides or more), preferably 25 nucleotides or more, more preferably 30 nucleotides or more, and further preferably 33 nucleotides or more. In addition, for example, it can be about 100 nucleotides or less, generally about 80 nucleotides or less, preferably about 70 nucleotides or less, more preferably about 60 nucleotides or less, further preferably about 50 nucleotides or less, and further preferably about 45 nucleotides or less (for example, 44 nucleotides or less, 43 nucleotides or less, 42 nucleotides or less, 41 nucleotides or less, 40 nucleotides or less). If the total number of nucleotides is small, chemical synthesis and mass production are easier, and the cost advantage is also large. In addition, it can be considered that chemical modification is also easy, the in vivo stability is also high, and the toxicity is also low.

[0123] Therefore, the length of the aptamer used in the present invention is generally about 10 to about 200 nucleotides, preferably 20 to 80 nucleotides, more preferably 25 to 60 nucleotides, further preferably 25 to 50 nucleotides, and most preferably 30 to 45 nucleotides.

[0124] The aptamer used in the present invention may also be a linker, which is selected from a group consisting of a plurality of linkers of an aptamer (aptamer (A)) comprising a nucleotide sequence represented by the above formula (1), a plurality of linkers of an aptamer (aptamer (B)) comprising a nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted or added in the nucleotide sequence represented by the above formula (1), and a linker of one or more aptamers (A) and one or more aptamers (B). These linkers may also bind to FGF2.

[0125] Here, the connection can be performed by tandem bonding. In addition, a linker can also be used when connecting. Examples of the linker include a nucleotide chain (e.g., 1 to about 20 nucleotides), a non-nucleotide chain (e.g., -(CH2) n -Linker, -(CH2CH2O) n -linker, hexaethylene glycol linker, TEG linker, peptide-containing linker, -SS-linker, -CONH-linker, -OPO3-linker). The number of the plurality of linkers is not particularly limited as long as it is 2 or more, and can be, for example, 2, 3 or 4.

[0126] The nucleotides contained in the aptamer used in the present invention can be the same or different, and can be a nucleotide containing a hydroxyl group at the 2' position of ribose (for example, ribose of pyrimidine nucleotides, ribose of purine nucleotides) (i.e., a natural ribonucleotide), or a nucleotide in which the hydroxyl group at the 2' position of ribose is replaced (modified) by an arbitrary atom or group (sometimes described as a "modified nucleotide" in this specification).

[0127] Examples include nucleotides substituted with any of the atoms or groups, such as hydrogen atoms, fluorine atoms, or -O-alkyl groups (e.g., -O-Me groups), -O-acyl groups (e.g., -O-CHO groups), and amino groups (e.g., -NH2 groups). In the aptamer used in the present invention, at least one (e.g., 1, 2, 3, or 4) nucleotide may be a modified nucleotide containing a hydroxyl group at the 2' position of the ribose, or any of the atoms or groups, such as at least two (e.g., 2, 3, or 4) groups selected from the group consisting of hydrogen atoms, fluorine atoms, hydroxyl groups, and -O-Me groups.

[0128] In the aptamer used in the present invention, all pyrimidine nucleotides may be nucleotides in which the 2′ position of the ribose is a fluorine atom, or the fluorine atom may be unsubstituted or substituted with any of the above atoms or groups, preferably, an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group. In particular, when the method for preparing the aptamer used in the present invention uses DuraScribe TMIn the case of the preparation method using T7 transcription kit (manufactured by Epicentre), aptamers in which the 2'-position of the ribose of all pyrimidine nucleotides is fluorinated can be obtained. Aptamers in which the fluorine atom is replaced by other atoms or groups described above can be prepared by the method described below.

[0129] The aptamer used in the present invention may be a nucleotide in which all purine nucleotides are ribose 2' positions with hydroxyl groups, or a nucleotide in which the hydroxyl groups are identically or differently not substituted, or substituted with any of the above atoms or groups, preferably, atoms or groups selected from the group consisting of hydrogen atoms, methoxy groups and fluorine atoms. The aptamer in which the hydroxyl groups are substituted with other of the above atoms or groups can be prepared by the method described below.

[0130] The aptamer used in the present invention may be a nucleotide in which the fluorine atom at the 2' position of the ribose of all pyrimidine nucleotides is replaced by any of the above atoms or groups, for example, the same atom or group selected from the group consisting of hydrogen atoms, hydroxyl groups and -O-Me groups.

[0131] In the aptamer used in the present invention, all purine nucleotides may have the hydroxyl group at the 2' position of ribose replaced by any of the above atoms or groups, for example, the same atom or group selected from the group consisting of hydrogen atoms, fluorine atoms and -O-Me groups.

[0132] In a preferred embodiment, each pyrimidine nucleotide contained in the aptamer used in the present invention is a nucleotide containing a fluorine atom at the 2' position of the ribose, and each purine nucleotide is a nucleotide containing a hydroxyl group at the 2' position of the ribose. In other embodiments, the fluorine atom at the 2' position of the ribose of each pyrimidine nucleotide can be independently replaced by an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group, and the hydroxyl group at the 2' position of the ribose of each purine nucleotide can be independently replaced by an atom or group selected from the group consisting of a hydrogen atom, a methoxy group and a fluorine atom.

[0133] In addition, in this specification, the nucleotide constituting the aptamer is assumed to be RNA (i.e., the sugar group is assumed to be ribose), and the embodiment of the modification of the sugar group in the nucleotide is described, but this does not mean that DNA is excluded from the nucleotide constituting the aptamer, and it can be appropriately interpreted as a modification of DNA. For example, when the nucleotide constituting the aptamer is DNA, the hydroxyl group at the 2' position of ribose is replaced by X, which can be interpreted as a method of replacing the hydrogen atom at the 2' position of deoxyribose with X.

[0134] In the aptamer used in the present invention, by replacing uracil with thymine, the binding property to FGF2, the inhibitory activity of binding of FGF2 to FGF receptor, the stability of the aptamer, the drug delivery property, the stability in blood, etc. can be improved.

[0135] In the aptamer used in the present invention, one or several, for example, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleotides in the phosphodiester bonds in the nucleotides may be modified or replaced with any substituent. For example, the phosphodiester bond may be replaced with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoroamidate bond, etc. Among them, for example, "a nucleotide is replaced with a phosphorothioate bond" means that the phosphate group at the binding site between adjacent nucleotides is sulfurized, that is, it means that the phosphodiester bond is changed to a phosphorothioate bond.

[0136] In the aptamer used in the present invention, one or several, for example, 1 to 2, 1 to 3, 1 to 4, 1 to 5 nucleotides may be replaced by a bridged nucleic acid (BNA) or a locked nucleic acid (LNA) for the purpose of stabilizing the aptamer and improving its activity. Here, "bridged nucleic acid" refers to a structure that limits the freedom of nucleic acid by intramolecular bridging, thereby increasing the binding affinity to the complementary sequence and obtaining nuclease resistance, and examples thereof include, but are not limited to, 2', 4'-BNA (LNA), 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA), etc.

[0137] The aptamers used in the present invention may be those in which the sugar residues (e.g., ribose) of each nucleotide are modified in order to improve the binding, stability, drug delivery, etc. to FGF2. As the modified site in the sugar residue, for example, those in which the oxygen atoms at the 2′, 3′, and / or 4′ positions of the sugar residue are replaced by other atoms can be cited. As the types of modification, for example, fluorination, O-alkylation (e.g., O-methylation, O-ethylation), O-allylation, S-alkylation (e.g., S-methylation, S-ethylation), S-allylation, amination (e.g., -NH2) can be cited. In addition, as examples, 4′SRNA in which the oxygen at the 4′ position is replaced by sulfur, LNA (Locked Nucleic Acid) in which the 2′ and 4′ positions are bridged by methylene, and 3′-N-phosphoramide nucleic acid in which the hydroxyl at the 3′ position is replaced by an amino group can be cited. The aptamer used in the present invention may be prepared in a manner such that the oxygen atom at the 2′ position of the ribose of the pyrimidine nucleotide has a certain modification, depending on the preparation method. For example, when DuraScribe is used TMIn the case of the preparation method of T7 transcription kit (manufactured by Epicentre), it is preferred to prepare an aptamer in which the 2′ position of the ribose of all pyrimidine nucleotides is fluorinated. Therefore, by applying such a subsequent change in the sugar residue to the obtained aptamer, it is possible to prepare various deformed aptamers with improved activity although the base sequence is the same. According to the above description, the aptamer used in the present invention can preferably be an aptamer in which the sugar residue of at least one nucleotide is modified. Such a change in the sugar residue can be carried out by a method known per se (see, for example: Sproat et al., (1991), Nucl. Acid. Res. 19, 733-738; Cotton et al., (1991), Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973), Biochemistry 12, 5138-5145). Specifically, based on an aptamer in which the hydroxyl group at the 2'-position of ribose of all pyrimidine nucleotides is replaced with a fluorine group, an aptamer in which the hydroxyl group at the 2'-position of ribose is replaced with an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group can be prepared.

[0138] The aptamers used in the present invention may be those in which the nucleic acid bases (e.g., purine, pyrimidine) are changed (e.g., chemically replaced) in order to improve the binding property to FGF2, prevent polymerization, stability, drug delivery, etc. Such changes include, for example, changes to the pyrimidine at position 5, changes to the purine at position 6 and / or 8, changes using an exocyclic amine, replacement using 4-thiouridine, and replacement using 5-bromo or 5-iodo-uracil. In addition, in order to make the aptamers used in the present invention resistant to nucleases and hydrolysis, the phosphate groups contained in the aptamers used in the present invention may also be changed. For example, the P(O)O group may be replaced by P(O)S (thio), P(S)S (dithio), P(O)N(R)R′ (amidation), P(O)R, P(O)OR, CO or CH 2 (methylal) or 3′-amine (-NH-CH 2 -CH 2 -) [wherein each R or R' is independently H, or a substituted or unsubstituted alkyl group (e.g., methyl, ethyl)].

[0139] Examples of the linking group include -O-, -N-, and -S-, and the adjacent nucleotide can be bonded via these linking groups.

[0140] Alterations may also include 3' and 5' alterations such as capping.

[0141] The modification can be further performed by adding polyethylene glycol (PEG), amino acids, peptides, inverted dT, nucleic acids, nucleosides, myristoyl, cholestyric acid-oleyl, behenyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl, lipids other than these, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin, etc. to the terminal. For such modification, for example, refer to: U.S. Patent Nos. 5,660,985 and 5,756,703.

[0142] In particular, when the modification is performed by adding PEG at the end, the molecular weight of PEG is not particularly limited, but is preferably 1000 to 100000, and more preferably 30000 to 90000. PEG may be a straight chain or may be branched into two or more chains (multi-arm PEG). The addition of PEG at the end is useful in preventing the multimerization of the aptamer described later.

[0143] There is no particular limitation on such PEG, and a person skilled in the art can appropriately select a commercially available or well-known PEG for use (for example, refer to: http: / / www.peg-drug.com / peg_product / branched.html). Preferred examples of PEG suitable for use in the aptamer used in the present invention include a 2-branched GS type PEG with a molecular weight of 40,000 (SUNBRIGHT GL2-400GS manufactured by NOFCO), a 2-branched TS type PEG with a molecular weight of 40,000 (SUNBRIGHT GL2-400TS manufactured by NOFCO), a 4-branched TS type PEG with a molecular weight of 40,000 (SUNBRIGHT GL4-400TS manufactured by NOFCO), a 2-branched TS type PEG with a molecular weight of 80,000 (SUNBRIGHT GL2-800TS manufactured by NOFCO), or a 4-branched TS type PEG with a molecular weight of 80,000 (SUNBRIGHT GL4-800TS manufactured by NOFCO).

[0144] In this case, PEG may be directly added to the terminal of the aptamer used in the present invention, or more preferably, PEG may be added to the aptamer used in the present invention by adding a linker having a group capable of binding to PEG to the terminal.

[0145] There is no particular limitation on the linker between PEG and the aptamer used in the present invention, and the number of carbon chains, functional groups, etc. can be appropriately selected according to the binding site, the type of PEG, etc. As such a linker, for example, a linker having an amino group can be cited, and specifically, when added to the 5' end, an ssH linker (SAFC) or DMS (O) MT-AMINO-MODIFIER (Glenresearch) can be exemplified, and when added to the 3' end, it is TFA Amino C-6lcaa CPG (ChemGenes) and the like. In the case of selecting this linker, an active group such as N-hydroxysuccinimide can be added to PEG, and it can react with the amino group on the linker side, thereby combining the aptamer used in the present invention with PEG through the linker.

[0146] It should be noted that commercially available PEG and linkers can be preferably used. In addition, the reaction conditions and the like involved in the binding of PEG, linkers and the aptamer used in the present invention can be appropriately set by those skilled in the art.

[0147] As a more preferred embodiment of the aptamer used in the present invention, the aptamer ID1 comprising the nucleotide sequence shown in SEQ ID NO: 3:

[0148] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A

[0149] (M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A(M)A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-idT;

[0150] Aptamer ID2 comprising the nucleotide sequence shown in SEQ ID NO: 8:

[0151] GL2-400TS-C6-C(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A( M)A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)G(M)-idT;

[0152] Aptamer ID3 comprising the nucleotide sequence shown in SEQ ID NO: 9:

[0153] GL2-400TS-C6-C(M)C(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A( M)A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)G(M)G(M)-idT;

[0154] Aptamer ID4 comprising the nucleotide sequence shown in SEQ ID NO: 10:

[0155] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)G(M)U(M)U(F)A( M)A(M)C(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-idT;

[0156] Aptamer ID5 comprising the nucleotide sequence shown in SEQ ID NO: 12:

[0157] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)U(M )A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-idT; and

[0158] Aptamer ID6 comprising the nucleotide sequence shown in SEQ ID NO: 3:

[0159] idT-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A(M)A(M)U(M )G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-C6-GL2-400TS

[0160] (In the above formulas, the brackets in each nucleotide indicate the modification of the 2′ position of the ribose, F represents a fluorine atom, and M represents a methoxy group.)

[0161] Since any of these aptamers can stably exist in the constitution of the aptamer preparation of the present invention when used, they are suitable as active ingredients of the aptamer preparation of the present invention.

[0162] The aptamer used in the present invention may be a free body or a pharmaceutically acceptable salt thereof. Examples of such salts include metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, and the like. Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts, and the like. Examples of ammonium salts include salts of ammonium and tetramethylammonium, and the like. Examples of organic amine addition salts include salts of trihydroxyaminomethane, and the like. Examples of amino acid addition salts include salts of lysine, arginine, histidine, tryptophan, ornithine, and the like.

[0163] The concentration of the aptamer for FGF2 used in the present invention is not particularly limited, and any amount of the aptamer preparation of the present invention can be contained as long as it exerts the intended effect. In this specification, "the concentration of the aptamer" refers to the ratio (mg / mL) of the weight of the nucleic acid portion connected by 5'→3' phosphate bonds constituting the aptamer molecule to the total volume of the preparation. Examples of the concentration of the aptamer include 1 to 60 mg / mL.

[0164] If the concentration of the aptamer for FGF2 exceeds 20 mg / mL, there is a tendency to easily produce multimers of the aptamer even at the normal storage temperature of 4 to 5°C. The aptamer for FGF2 binds to FGF2 when it exists in the form of a monomer, inhibits the function of FGF2 and exerts its pharmacological effect, but if it multimerizes, the binding to FGF2 is not observed in the measurement using BIAcore, and it is considered that it does not inhibit the function of FGF2 (reference example 4). When the concentration of the aptamer is low, the distance between molecules is far, and the tendency to become a multimer is low, but if the concentration of the aptamer exceeds 20 mg / mL, multimerization occurs at the normal storage temperature of 4 to 5°C, resulting in a tendency for the binding activity to FGF2 to decrease as a whole (reference example 5). Therefore, the upper limit of the FGF2 aptamer concentration in the aptamer preparation of the present invention is preferably a concentration not exceeding 20 mg / mL. In addition, when the aptamer concentration of the aptamer preparation of the present invention is low, there is a case where the effect as an injection cannot be obtained. Therefore, although the lower limit of the FGF2 aptamer concentration in the aptamer preparation of the present invention is not particularly limited, as long as the concentration can obtain the effect as an injection, for example, a concentration of 1 mg / mL or more is preferred.

[0165] The dosage form of the aptamer preparation of the present invention is not particularly limited as long as it is an aqueous liquid preparation, and is preferably an injection. Since the aptamer preparation of the present invention is an aqueous liquid preparation, the aptamer against FGF2 as an active ingredient is dissolved in a solvent and exists.

[0166] It is generally known that aptamers need inorganic salts as strong electrolytes to maintain their higher-order structures. Sodium hydrogen phosphate, sodium dihydrogen phosphate, isotonic agents and pH adjusters are also added to Macugen (registered trademark), the only aptamer drug currently on the market. In this state, Macugen showed stable existence in an accelerated test at 25±2°C for 6 months and in a long-term storage test at 5±3°C for 36 months. Therefore, it should be believed by those skilled in the art that inorganic salts (electrolytes) are necessary to keep the higher-order structure of the aptamer unchanged in the injection.

[0167] However, as shown in the examples described below, it is known that the above-mentioned aptamer for FGF2 as an active ingredient in the present invention loses stability under the influence of inorganic salts as strong electrolytes and tends to form multimers, resulting in loss of binding activity to FGF2, when PBS or saline is used as a solvent. For this reason, the present inventors found that when water containing no inorganic salts (electrolytes) was used as a solvent, the multimerization of the FGF2 aptamer was unexpectedly suppressed, and the proportion of the FGF2 aptamer existing in the form of monomers increased (reference Example 3).

[0168] Without intending to be limited by any theory, when the FGF2 aptamer is dissolved in water, the Tm value of the FGF2 aptamer cannot be measured as shown in Example 1, and the proton shift in NMR cannot be observed as shown in Example 2, so it is assumed that the FGF2 aptamer dissolved in water exists in a state of being extended as a monomer. That is, it is considered that by using water that does not contain inorganic salts as a solvent, the higher-order structure of the aptamer that is considered to be necessary for the construction of activity is destroyed and modified to exist in a single-chain state, thereby preventing inactivation (conversion into a multimer). In addition, as shown in Examples 4 and 5, when the FGF2 aptamer is dissolved in a solution containing an electrolyte and subjected to surface plasmon resonance (SPR) measurement, it is confirmed that if the FGF2 aptamer dissolved in water is administered to the body, the higher-order structure necessary for the activity of the aptamer is reconstructed due to the action of the electrolytes present in the body fluid, so that the activity can be exerted.

[0169] It was found that the higher-order structure of the FGF2 aptamer is intentionally collapsed and extended by dissolving in a solvent containing no electrolytes, thereby preventing multimerization even during long-term storage, and that the higher-order structure is reconstructed by exposure to a physiological salt concentration after administration to exert the drug effect, and as a result, the activity of the FGF2 aptamer can be maintained for a long time. The present invention was completed by carefully verifying the existence mode of the aptamer in such a solution, which is not easily conceivable even by those skilled in the art.

[0170] As described above, as a solvent that can be used in the aptamer preparation of the present invention, an aqueous solvent containing no inorganic salt (electrolyte) is preferred, and water is particularly preferred.

[0171] Since the aptamer preparation of the present invention uses a solvent that does not contain an inorganic salt (electrolyte) as described above, the osmotic pressure is low and cannot be used directly as an injection. Therefore, in the aptamer preparation of the present invention, an osmotic pressure regulator (osmotic agent) that is a non-electrolyte is included for the purpose of adjusting the osmotic pressure ratio relative to the plasma osmotic pressure to 1 or more, preferably 1 to 3.

[0172] As the osmotic pressure regulator used in the aptamer preparation of the present invention, there is no particular limitation, as long as it is a non-electrolyte, as long as it is an osmotic pressure regulator commonly used except for inorganic ions (potassium ions, chloride ions, etc.), it can be used. As such an osmotic pressure regulator, polyols (glycerol, mannitol, trehalose, glucose, sucrose, sorbitol, inositol, etc.), amino acids (alanine, glycine, glutamic acid, proline, GABA, taurine, ectoine, etc.), methylammonium (TMAO, choline, acetylcholine, glycine betaine, GPC, DMSP, etc.), ureas, etc., preferably polyols are used, and more preferably mannitol can be used.

[0173] The amount of the osmotic pressure regulator is not particularly limited, and those skilled in the art can make appropriate changes according to the amount of FGF2 aptamer contained in the preparation, the type (molecular weight) of the osmotic pressure regulator used, and the target osmotic pressure. For example, when mannitol is used as an osmotic pressure regulator, the osmotic pressure ratio relative to saline is about 1, and the preparation ratio of the osmotic pressure regulator in the total amount of the injection becomes 2 to 7.5% (w / v). More specifically, when the osmotic pressure ratio relative to saline is 1, if the concentration of the above-mentioned FGF2 aptamer is 2 mg / ml, the preparation ratio of mannitol becomes 4.9%, and if the concentration of the above-mentioned FGF2 aptamer is 20 mg / ml, the preparation ratio of mannitol becomes 3.6%.

[0174] The aptamer preparation of the present invention preferably contains substantially no electrolytes other than the aptamer or its salt for FGF2 as an active ingredient. Here, "substantially no electrolytes" means that a small amount of electrolytes may be contained within the range in which the FGF2 aptamer in the preparation can be stably stored for a long time. More preferably, in the aptamer preparation of the present invention, no electrolytes are contained other than the aptamer or its salt for FGF2 as an active ingredient.

[0175] The aptamer preparation of the present invention may further contain pharmaceutically acceptable additives as required. Such additives include, for example, stabilizers, preservatives, solubilizers, buffers, pH adjusters, soothing agents, etc., and preferably, non-electrolyte drug additives that are commonly used as additives for injections and are well known can be used.

[0176] The pH of the aptamer preparation of the present invention is not particularly limited. When used as an injection, the pH is preferably near neutral, and can be appropriately selected within the range of, for example, pH 5 to 9, preferably 6 to 8. When the aptamer or a salt thereof to FGF2 as an active ingredient is dissolved in water, the pH of the solution is within the above range. Therefore, it is not necessary to particularly add a pH adjuster or a buffer as an electrolyte to the aptamer preparation of the present invention.

[0177] In the aptamer preparation of the present invention, other active ingredients may be formulated as long as they have no adverse effects on the activity and stability of the FGF2 aptamer. As such active ingredients, VGEF inhibitors such as McGen (registered trademark), Lucentis (registered trademark), Airia (registered trademark), Avastin (registered trademark), etc., as therapeutic drugs for diseases accompanied by angiogenesis, anti-inflammatory agents such as steroids, human growth hormone preparations such as Norditropin (registered trademark), Genotropin (registered trademark), etc., as therapeutic drugs for bone diseases, analgesics and sedatives such as morphine may also be included. Diseases accompanied by angiogenesis such as age-related macular degeneration, osteoporosis, rheumatoid arthritis, osteoarthritis, bone / cartilage diseases such as fractures, and pharmaceutical compounds for the treatment or prevention of pain may be listed.

[0178] The aptamer preparation of the present invention is stable for more than 3 months at 4°C by having the above-mentioned structure. Here, "stable" means that when the preparation is sealed in a glass bottle and stored at 4°C as described above, the proportion of the monomeric aptamer present in the preparation is 70% or more. Preferably, the aptamer preparation of the present invention has a monomeric aptamer ratio of 80% or more in the preparation after storage at 4°C for 3 months. In addition, the aptamer preparation of the present invention is also stable when exposed to white fluorescent light or near-ultraviolet fluorescent light.

[0179] Therefore, the aptamer preparation of the present invention can be directly stored stably for a long period of time in the form of an aqueous liquid, preferably in a dosage form of an injection such as a filled syringe or cartridge, by refrigeration at 5° C. or less, and is extremely easy to handle.

[0180] The aptamer preparation of the present invention can be preferably used as a drug for treating or preventing diseases associated with angiogenesis such as age-related macular degeneration, bone / cartilage diseases such as osteoporosis, rheumatoid arthritis, osteoarthritis, fractures, and pain.

[0181] The aptamer preparation of the present invention can be administered parenterally (e.g., intravenous administration, subcutaneous administration, intramuscular administration, local administration, intraperitoneal administration, nasal administration, pulmonary administration, eye drop administration, etc.). The dosage of the aptamer preparation of the present invention varies depending on the type and activity of the FGF2 aptamer, the severity of the disease, the type of animal to be administered, the drug acceptance, body weight, age, etc. of the administered subject, and is generally about 0.0001 to about 100 mg / kg, for example, about 0.0001 to about 10 mg / kg, and preferably about 0.005 to about 1 mg / kg, based on the amount of the active ingredient (oligonucleotide portion of the aptamer) per day for an adult.

[0182] Example

[0183] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0184] Example 1 (Structural Analysis of FGF2 Aptamer in Solvent: Determination of Tm Value)

[0185] The structure of the aptamer indicated by aptamer ID1 is shown below. Capital letters represent RNA, lowercase letters represent DNA, and idT represents reverse dT. It should be noted that the brackets in each nucleotide represent the modification of the 2′ position, F represents a fluorine atom, and M represents an O-methyl group. C6 is a -(CH2)6-linker, and PEG40TS2 is a 2-branched TS-type polyethylene glycol with a molecular weight of 40,000 (SUNBRIGHT GL2-400TS, manufactured by NOF).

[0186] Aptamer ID1:

[0187] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A( M)A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-idT

[0188] The aptamer represented by aptamer ID1 was dissolved in water at 0.1 mg / mL, or in PBS or saline at 0.06 mg / mL. The resulting solution was heated at 95°C for 5 minutes, cooled to room temperature, and filled into a quartz glass cuvette. The temperature was changed from 20°C to 90°C, and UV absorption was measured using a spectrophotometer to determine the Tm value.

[0189] As a result, it was 60.1°C in PBS and 69.6°C in saline. On the other hand, no specific Tm value was observed in water. It is assumed that the aptamer represented by aptamer ID1 forms a higher-order structure due to intermolecular interactions in PBS and saline, which can be solvents for general injections. In addition, it is assumed that the aptamer represented by aptamer ID1 does not form base pairs between molecules or within molecules in water that does not contain electrolytes, and does not form a higher-order structure.

[0190] Example 2 (Structural Analysis of FGF2 Aptamer in Heavy Water: Measurement of NMR Spectrum)

[0191] 20 mg of the aptamer represented by aptamer ID 1 was filled into a glass vial and dissolved in about 1 mL of heavy water to prepare a measurement sample. The measurement was performed using a Bruker Avance 600 MHz NMR spectrometer. As a result, no imino proton signal from base pair formation that should be observed in a low magnetic field below 8 ppm was observed.

[0192] Therefore, it is assumed that the aptamer represented by aptamer ID1 does not form base pairs between molecules or within molecules in water containing no electrolyte, and does not constitute a higher-order structure.

[0193] Example 3 (Structural analysis of FGF2 aptamer in a solution containing electrolytes: SEC-MALS assay)

[0194] The aptamer represented by aptamer ID1 was dissolved in physiological saline at 20 mg / mL and incubated at 37°C for 2 weeks to artificially prepare an FGF2 aptamer preparation that formed a higher-order structure. This and a sample that was frozen immediately after preparation and minimized the formation of a higher-order structure were diluted with physiological saline at 0.2 mg / mL and analyzed.

[0195] The monomer and the polymer were separated by size exclusion chromatography, and the molecular weight of each was measured using a MALS (Multi Angle Light Scattering) detector manufactured by Wyatt Technology Co. Size exclusion chromatography was performed using a BEH200 SEC column using an ACQUITY UPLC manufactured by Waters Corporation.

[0196] As a result, the molecular weight of the peak considered to be a monomer was measured to be about 64000, and the molecular weight of the peak considered to be a higher-order structure composed of a polymer was measured to be about 122000. From this result, it was found that the higher-order structure formed by the aptamer represented by aptamer ID1 in the contained electrolyte water was a dimer.

[0197] Example 4 (Correlation between the monomer content rate of aptamers and binding activity)

[0198] The aptamer represented by aptamer ID1 was dissolved at 20 mg / mL or 2 mg / mL in PBS, physiological saline or 3.3% mannitol aqueous solution, and FGF2 aptamer preparations showing various monomer content rates were prepared under the storage conditions shown in Table 1. The monomer content of each prepared FGF2 aptamer preparation was determined by size exclusion chromatography. The results are also shown in Table 1.

[0199] The binding activity of each prepared FGF2 aptamer preparation to the FGF2 protein was measured by surface plasmon resonance (SPR) using Biacore T200 manufactured by GE. The sensor chip used CM4 that reacts with amino groups. Human FGF2 was dissolved in a fixation solution (10 mM sodium acetate, pH 6) to a concentration of 10 μg / mL. Ethyl-3-carbodiimide hydrochloride and N-hydroxysuccinimide were used in the reaction between the amino group on the protein side and the carboxyl group on the chip side. After the reaction, blocking was performed using ethanolamine. The fixed amount of FGF2 was about 1000 RU. The aptamer for analysis was prepared to 5 μM. 295 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM magnesium chloride, 1.8 mM calcium chloride, 20 mM Tris, 0.05% Tween 20, pH 7.6) was used in the running buffer, and 2 M sodium chloride was used as the regeneration solution. FGF2 was immobilized on flow cell FC2 or FC4, and the result of FC1 or FC3 was subtracted to form the final sensorgram. For activity, the relative value was evaluated relative to the FGF2 aptamer preparation standard, which was prepared at the time of use using a solution without electrolytes and had extremely low polymer content.

[0200] The preparation methods of each FGF2 aptamer preparation, the monomer content, and the results of the measurement of the binding activity to the FGF2 protein are shown in Table 1. From these results, it can be seen that the monomer content of the FGF2 aptamer preparation and the binding activity to the FGF2 protein are correlated.

[0201] [Table 1]

[0202] Monomer content of FGF2 aptamer preparations and binding activity to FGF2 protein

[0203] FGF2 aptamer preparation Preparation method (storage conditions) Monomer content (%) Binding activity (%) 2mg / mL 3.3% mannitol preparation Prepare when needed 98.7 98.1 20mg / mL 3.3% mannitol preparation Prepare when needed 91.3 91.6 20mg / mL 3.3% mannitol preparation 25℃ 1 week 80.5 75.7 20mg / mL 3.3% mannitol preparation 25℃ 2 weeks 71.5 65.2 20mg / mL 3.3% mannitol preparation 25℃ 1 month 63.4 57.2 20mg / mL 3.3% mannitol preparation 25℃ 3 months 50.2 50.2 2 mg / mL saline 37℃ 2 days 56.9 56.2 2mg / mL PBS 37℃ 2 days 57.2 60.6 20 mg / mL saline 37℃ 2 days 12.1 18.3 20mg / mL PBS 37℃ 2 days 11.3 13.1

[0204] Example 5 (Stability Test of FGF2 Aptamer Preparation)

[0205] The aptamer represented by aptamer ID1 was dissolved in a 3.3%, 3.6%, or 4.9% mannitol solution at 20 mg / mL or 2 mg / mL and stored for 3 months under various temperature conditions shown in Table 2. The monomer content and binding activity were measured by size exclusion chromatography and SPR. The results are shown in Table 2.

[0206] This result showed that the FGF2 aptamer preparation prepared using the mannitol aqueous solution containing no electrolytes was stable.

[0207] [Table 2]

[0208] Stability test results of FGF2 aptamer preparations

[0209]

[0210] Example 6 (Results in other FGF2 aptamer preparations)

[0211] Aptamers represented by aptamer IDs 2 to 6 were dissolved in water (mannitol aqueous solution), saline or PBS at appropriate concentrations to prepare FGF2 aptamer preparations. Each of the obtained FGF2 aptamer preparations was stored for several months under various temperature conditions and storage temperatures, and then the monomer content and binding activity were measured by size exclusion chromatography and SPR.

[0212] Aptamer ID2:

[0213] GL2-400TS-C6-C(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A( M)A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)G(M)-idT;

[0214] Aptamer ID3:

[0215] GL2-400TS-C6-C(M)C(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A( M)A(M)∪(M)G(M)∪(F)∪(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)G(M)G(M)-idT;

[0216] Aptamer ID4:

[0217] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)G(M)U(M)U(F)A( M)A(M)C(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)idT-;

[0218] Aptamer ID5:

[0219] GL2-400TS-C6-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)U(M )A(M)U(M)G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)idT-; and

[0220] Aptamer ID6:

[0221] idT-G(M)G(M)G(M)A(M)U(M)A(M)C(M)U(F)A(M)G(M)G(M)GC(M)A(M)U(M)U(F)A(M)A(M)U(M )G(M)U(F)U(M)A(M)C(M)C(M)A(M)GU(F)GU(F)A(M)G(M)U(M)C(M)C(M)C(M)-C6-GL2-400TS

[0222] These results show that, among other FGF2 aptamers, FGF2 aptamer preparations prepared using a mannitol aqueous solution containing no electrolytes are also stable.

[0223] Comparative Example 1 (Stability Test of FGF2 Aptamer Preparation)

[0224] The aptamer represented by aptamer ID1 was dissolved in physiological saline or PBS at 20 mg / mL to prepare FGF2 aptamer preparations. After each of the obtained FGF2 aptamer preparations was stored for 3 months under various temperature conditions shown in Table 3, the monomer content was measured by size exclusion chromatography. The results are shown in Table 3. From the results, it can be seen that the FGF2 aptamer preparations prepared using physiological saline and PBS containing electrolytes are unstable.

[0225] [Table 3]

[0226] Results of stability tests of FGF2 aptamer preparations prepared with solutions containing electrolytes

[0227]

[0228] Industrial Applicability

[0229] The aptamer preparation of the present invention is an aqueous liquid preparation such as an injection, and can be stably stored for a long time even when refrigerated. Therefore, it is extremely useful as a therapeutic or preventive agent for diseases that can exert a pharmacological effect by inhibiting FGF2 (for example, diseases accompanied by angiogenesis such as age-related macular degeneration, osteoporosis, rheumatoid arthritis, osteoarthritis, bone / cartilage diseases such as fractures, and pain), in terms of providing a preparation with excellent operability.

[0230] This application is based on Japanese Patent Application No. 2018-124390 (filing date: June 29, 2018) filed in Japan, and all contents thereof are incorporated herein by reference.

Claims

1. An aqueous liquid preparation comprising an aptamer or a salt thereof that binds to FGF2 and an osmotic pressure regulator that is a non-electrolyte, wherein the aptamer or the salt thereof is stable for a long time, The aptamer is composed of any one of the nucleotide sequences shown in SEQ ID NO: 3, 8, 9, 10 or 12: The osmotic pressure regulator is mannitol, The concentration of the aptamer is 2-20 mg / mL, The osmotic pressure regulator is prepared in a proportion of 3.3-4.9% w / v of the total amount of the aqueous liquid. Here, "long-term stability" means that when the aqueous solution is sealed in a glass bottle and stored at 4° C. for 3 months, the proportion of the monomeric aptamer is 70% or more.

2. The aqueous liquid preparation according to claim 1, wherein No inorganic salt is contained except the aptamer or a salt thereof.

3. The aqueous liquid preparation according to claim 1 or 2, which is stored at 5°C or lower.

4. The aqueous liquid preparation according to claim 1 or 2, wherein The ratio of monomeric aptamers after storage at 4°C for 3 months was 80% or more. The aqueous liquid preparation according to claim 1 or 2, which is an injection.

6. Use of the aqueous liquid preparation according to any one of claims 1 to 5 in the preparation of a medicament for preventing or treating a disease associated with angiogenesis, a bone / cartilage disease or pain, wherein: The disease accompanied by angiogenesis is age-related macular degeneration, and the bone / cartilage disease is osteoporosis, rheumatoid arthritis, osteoarthritis, and bone fracture.

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