Conjugate of aptamer and polyethyloxazoline

Abstract

To provide a method for enhancing the blood stability of an aptamer, and to provide a conjugate of an aptamer having improved stability by the method. The problem is solved by providing a conjugate of an aptamer and polyethyloxazoline, or a salt thereof.

Description

Aptamer and polyethyloxazoline conjugates

[0001] The present invention relates to a conjugate of an aptamer and polyethyloxazoline or a salt thereof, and a composition comprising the same. The present invention also relates to a method for producing an aptamer with an extended blood half-life, and a method for stabilizing the aptamer.

[0002] In recent years, the application of RNA aptamers to therapeutic drugs, diagnostic drugs, and reagents has been attracting attention, and several RNA aptamers have entered the clinical or practical application stage. In December 2004, Macugen, the world's first RNA aptamer drug, was approved in the United States as a treatment for age-related macular degeneration. An RNA aptamer is an RNA that specifically binds to a target substance such as a protein, and can be produced using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment) (see Patent Documents 1 to 3). The SELEX method is a method for generating RNA aptamers by combining RNA molecules with other target molecules. １４ This method selects RNA that specifically binds to a target substance from a pool of RNA with approximately 100 different nucleotide sequences. The RNA used has a structure in which a random sequence of approximately 40 residues is sandwiched between primer sequences. This RNA pool is then associated with the target substance, and only the RNA that binds to the target substance is recovered using a filter or other means. The recovered RNA is amplified by RT-PCR and used as a template for the next round. By repeating this process approximately 10 times, it may be possible to obtain an RNA aptamer that specifically binds to the target substance.

[0003] Although RNA aptamers are promising therapeutic agents, diagnostic agents, and reagents, they have the property of being easily excreted from the body due to their small molecular weight, and therefore, when used as therapeutic agents, it is necessary to extend the half-life of RNA aptamers in the blood.

[0004] To date, techniques using polyethylene glycol (hereinafter referred to as "PEG") have been reported as techniques for extending the blood half-life of substances used as medicines. For example, Non-Patent Document 1 describes that by binding PEG to an RNA aptamer, the pharmacokinetic properties of the RNA aptamer are significantly improved (the blood half-life is extended).

[0005] International Publication No. WO 91 / 19813 International Publication No. WO 94 / 08050 International Publication No. WO 95 / 07364

[0006] Shin Miyakawa, Masatoshi Fujiwara, Michihisa Nishiyama, Development of molecular targeted drugs using RNA aptamers, Drug Delivery System, 2008, Vol. 23, No. 5, pp. 534-543

[0007] As mentioned above, PEG is a substance that exhibits excellent effects in extending the blood half-life in the body, but it can cause allergic reactions, and in this respect there is room for improvement.

[0008] Therefore, an object of the present invention is to provide a method for increasing the stability of an aptamer in blood without using PEG, and an aptamer conjugate whose stability in blood has been increased by said method.

[0009] As a result of intensive research aimed at solving the above problems, the present inventors have found that the blood half-life can be extended by binding polyethyloxazoline (also referred to as "PEOZ") to an aptamer. The present inventors have also found that the use of a branched polyethyloxazoline (hereinafter simply referred to as "branched polyethyloxazoline") in which a linear polyethyloxazoline is bound to a branched linker in the above embodiment further extends the blood half-life, demonstrating a blood half-life equivalent to or superior to that when PEG is added, and have completed the present invention.

[0010] That is, one aspect of the present invention relates to the following: [1] A conjugate of an aptamer and a polyethyloxazoline, or a salt thereof. [2] The conjugate according to [1], wherein the polyethyloxazoline forms a branched chain, or a salt thereof. [3] The conjugate according to [1] or [2], wherein the polyethyloxazoline is bound to the 5'-end of the aptamer, or a salt thereof. [4] The conjugate according to any one of [1] to [3], wherein two polyethyloxazolines form a branched chain via a linker, or a salt thereof. [5] The conjugate according to any one of [2] to [4], wherein each branched chain of the polyethyloxazoline has a molecular weight of 20 to 60 kDa, or a salt thereof. [6] The conjugate according to [4], wherein the polyethyloxazoline is bound to the linker via a dipolar cycloaddition reaction, or a salt thereof. [7] The conjugate or salt thereof according to [6], wherein the dipolar cycloaddition reaction is a dipolar cycloaddition reaction between an azide on the polyethyloxazoline side and a dibenzocyclooctyne on the linker side. [8] The conjugate or salt thereof according to [1], wherein the polyethyloxazoline is bound to the 5'-end of the aptamer via a linker. [9] The conjugate or salt thereof according to [8], wherein the polyethyloxazoline-linker moiety has the following structure:

[0011]

[0012] (Here, l represents the degree of polymerization, and l is 200 to 600.)

[10] The conjugate or salt thereof according to [4], wherein the polyethyloxazoline-linker moiety has the following structure:

[0013]

[0014] (Here, m and n each represent a degree of polymerization. m is 200 to 600, and n is 200 to 600.)

[11] The conjugate or salt thereof according to any one of [1] to

[10] , wherein the aptamer is an anti-FGF2 aptamer or an anti-autotaxin aptamer.

[12] The conjugate or salt thereof according to any one of [1] to [9], wherein the aptamer is an aptamer consisting of a base sequence represented by the following formula (1): tC(M)U(M)G(M)A(M)G(M)G(M)gA(M)AsA(M)C(F)A(M)ggU(F)U(F)U(F)U(F)G(M)C(F)U(M)cC(M)U(M)cG(M)G(M)A(M)_idT (1) (Uppercase letters represent RNA, lowercase letters represent DNA, parentheses in nucleotides represent modification of the 2'-position of ribose, F represents a fluorine atom, and M represents an O-methyl group. In addition, idT at the end of the sequence represents modification with inverted-dT, and s in the sequence represents that the phosphate group linking the nucleotides has been phosphorothioated.)

[13] A composition comprising the conjugate according to any one of [1] to

[12] or a salt thereof.

[14] A method for producing an aptamer having an extended blood half-life, comprising a step of binding polyethyloxazoline to the aptamer.

[15] The method for production according to

[14] , wherein the polyethyloxazoline forms a branched chain.

[16] The method for production according to

[14] or

[15] , wherein the polyethyloxazoline is bound to the 5'-end of the aptamer.

[17] The method for production according to any one of

[14] to

[16] , wherein the branched polyethyloxazoline is bound to the aptamer via a linker.

[18] The method for production according to any one of

[15] to

[17] , wherein the polyethyloxazoline, each of whose branched chains has a molecular weight of 20 to 60 kDa, is bound to the aptamer.

[19] The method for producing the polyethyloxazoline and the linker according to

[17] or

[18] , wherein the polyethyloxazoline and the linker are bonded by a dipolar cycloaddition reaction.

[20] The method for producing the polyethyloxazoline and the linker according to

[19] , wherein the polyethyloxazoline and the linker are bonded by a dipolar cycloaddition reaction between an azide on the polyethyloxazoline side and a dibenzocyclooctyne on the linker side.

[21] The method for producing according to any one of

[14] to

[20] , wherein the polyethyloxazoline branched via a linker is bound to the aptamer by a Michael addition reaction between a maleimide group on the linker and a thiol group on the aptamer.

[22] The method for producing according to any one of

[14] to

[21] , wherein the polyethyloxazoline is bound to the 5' end of the aptamer via a linker.

[23] The method for producing according to

[22] , wherein the structure of the polyethyloxazoline-linker moiety is as follows:

[0015]

[0016] (Here, l represents the degree of polymerization, l is 200 to 600.)

[24] The method according to

[17] , wherein the structure of the polyethyloxazoline-linker moiety is the following structural formula:

[0017]

[0018] (Here, m and n each represent the degree of polymerization. m is 200 to 600, and n is 200 to 600.)

[25] A method for stabilizing an aptamer, comprising binding polyethyloxazoline to the aptamer.

[0019] According to the present invention, it is possible to provide an aptamer conjugate with improved stability in blood without using PEG, which can avoid the problem of allergic reactions caused by PEG and is useful in various therapies using the aptamer.

[0020] FIG. 1 shows a sensorgram illustrating the binding of a branched PEOZ (50 kDa x 2)-conjugated anti-FGF2 aptamer to human FGF2. FIG. 2 shows a sensorgram illustrating the binding of a PEG-conjugated anti-FGF2 aptamer to human FGF2. FIG. 3 shows the inhibitory effect of the addition of a branched PEOZ (50 kDa x 2)-conjugated anti-FGF2 aptamer and a PEG-conjugated anti-FGF2 aptamer on ERK phosphorylation in cells stimulated with FGF2. FIG. 4 shows the results of evaluating the blood retention of branched PEOZ (25 kDa x 2, 50 kDa x 2), linear PEOZ (50 kDa), PEG-conjugated anti-FGF2 aptamer, and unconjugated anti-FGF2 aptamer. FIG. 5 shows the results of evaluating the enzyme inhibitory activity of branched PEOZ (50 kDa x 2) and a PEG-conjugated anti-autotaxin aptamer. FIG. 6 shows the results of evaluating the blood retention of branched PEOZ (50 kDa×2) and PEG-added anti-autotaxin aptamer.

[0021] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0022] (1. Definitions) As used herein, the term "aptamer" refers to a nucleic acid molecule that has binding activity to a predetermined target molecule. An aptamer can inhibit the activity of a predetermined target molecule by binding to the target molecule.

[0023] In this specification, the term "polyethyloxazoline" has the meaning commonly used in the art, and is also referred to as poly(2-ethyl-2-oxazoline).

[0024] As used herein, the term "conjugate" refers to a complex composed of two or more substances. In the present invention, the conjugate is composed of at least an aptamer and polyethyloxazoline.

[0025] As used herein, the term "salt of a conjugate" refers to a conjugate (complex) that exists in the form of a neutralized salt. In the present invention, the salt of a conjugate is a salt of a conjugate composed of at least an aptamer and polyethyloxazoline. In one embodiment of the present invention, the salt of a conjugate may be a pharmaceutically acceptable salt of a conjugate. As used herein, "pharmaceutically acceptable" refers to a salt that is medically suitable for use in contact with the tissues of humans or other mammals without causing excessive toxicity, irritation, allergic reactions, etc. Examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, nitrate, sulfate, and phosphate; organic acid salts such as acetate, oxalate, maleate, fumarate, citrate, benzoate, and methanesulfonate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; aluminum salt; zinc salt; ammonium salt, tetramethylammonium salt; and addition salts of morpholine, piperidine, lysine, glycine, phenylalanine, aspartic acid, glutamic acid, and the like.

[0026] As used herein, the term "linker" refers to a linking moiety that links two or more substances. In the present invention, the linker links the aptamer and the polyethyloxazoline.

[0027] As used herein, the term "anti-FGF2 aptamer" refers to an aptamer that has binding activity to FGF2 and can inhibit the binding of FGF2 to an FGF receptor. That is, the anti-FGF2 aptamer has inhibitory activity against FGF2.

[0028] FGF2 is a protein that is strongly expressed during early development, differentiation, proliferation, and regeneration, and is a protein having an amino acid sequence represented by accession code EAX05222 or NP001997. FGF2 is also called bFGF (basic FGF), FGFB, or HBGF-2.

[0029] Inhibitory activity against FGF2 refers to the ability to inhibit any activity possessed by FGF2. For example, FGF2 acts on FGF receptor-expressing cells to activate signal transduction and induce the production of various cell growth factors and their receptors. Therefore, inhibitory activity against FGF2 can refer to the activity of inhibiting intracellular signal transduction mediated by FGF receptors. Furthermore, since the expression of these various cell growth factors and their receptors ultimately leads to enhanced cell proliferation and migration activity, inhibitory activity against FGF2 refers to the inhibition of these activities.

[0030] As used herein, the term "anti-autotaxin aptamer" refers to an aptamer that has binding activity to autotaxin and can inhibit the enzymatic activity of autotaxin. That is, the anti-autotaxin aptamer has inhibitory activity against autotaxin.

[0031] Autotaxin is a glycoprotein present in the blood and is an enzyme that breaks down lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline.

[0032] Inhibitory activity against autotaxin refers to the ability to inhibit any activity possessed by autotaxin. Autotaxin has phosphodiesterase activity, which cleaves phosphodiester bonds by hydrolysis, and inhibits this activity. Substrates acceptable for enzymatic activity are not limited to phosphodiester bond-containing substances present in living organisms (e.g., ATP, etc.), but also include substrates in which chromogenic or fluorescent substances have been added to compounds containing such substances. Chromogenic and fluorescent substances are well known to those skilled in the art. Autotaxin also possesses lysophospholipase D activity. This activity cleaves the bond opposite the glycerol backbone of the phosphodiester of lysophospholipids, primarily producing lysophosphatidic acid (LPA), and inhibiting this production is also included in inhibiting autotaxin activity.

[0033] As used herein, the term "blood half-life" has the meaning commonly used in the art, and refers to the time it takes for the blood concentration of a substance or drug component in a living body to be reduced by half. The blood half-life can be evaluated, for example, by the blood retention shown in the Examples. In the present invention, the blood half-life is extended by binding an aptamer to polyethyloxazoline. An extension of the blood half-life can also be expressed as an increase in blood retention, an increase in blood stability, or the like.

[0034] (2. Conjugate of Aptamer and Polyethyloxazoline) In one embodiment of the present invention, a conjugate of an aptamer and polyethyloxazoline (hereinafter also referred to as "the conjugate") or a salt thereof is provided. In this conjugate, by being bound to polyethyloxazoline, the effect of extending the half-life in blood is achieved. Furthermore, since PEG is not used, the problem of allergic reactions caused by PEG can be avoided.

[0035] In this conjugate, the polyethyloxazoline may be attached to either the 5'-end or the 3'-end of the aptamer.

[0036] In the conjugate, the polyethyloxazoline may be bound to the aptamer directly or via a linker, preferably via a linker.

[0037] The linker is not particularly limited, but examples thereof include alkyl chains, unsaturated fatty acid chains, cholesterol, polyethylene glycol, polypropylene glycol, polysaccharides, polypeptides, and polypeptoids.

[0038] The polyethyloxazoline may be in the form of a linear or branched chain. In one embodiment of the present invention, the conjugate may be a conjugate in which a linear polyethyloxazoline is bound to an aptamer via a branched linker (also referred to as a "branched polyethyloxazoline-added aptamer"). In another embodiment of the present invention, the conjugate may be a conjugate in which a linear polyethyloxazoline is bound to an aptamer via an unbranched linker (also referred to as a "linear polyethyloxazoline-added aptamer"). Since addition to an aptamer further extends the blood half-life, a branched form is preferred in this embodiment.

[0039] The molecular weight of the polyethyloxazoline is preferably 20 to 60 kDa. In the case of a branched polyethyloxazoline-added aptamer, the molecular weight of each branched chain of the polyethyloxazoline is preferably 20 to 60 kDa.

[0040] When polyethyloxazoline is bound to an aptamer via a linker, the binding mode between the polyethyloxazoline and the linker is not particularly limited, and examples thereof include binding via a dipolar cycloaddition reaction (also referred to as a "dipole cyclization reaction"), a transesterification reaction, a Michael addition reaction, an SN2 reaction, a dehydration condensation reaction, and the like.

[0041] When bonding is performed by a dipolar cycloaddition reaction, the bonding is preferably performed by a dipolar cycloaddition reaction between an azide on the polyethyloxazoline side and a dibenzocyclooctyne on the linker side.

[0042] When polyethyloxazoline is bound to the aptamer via a linker, the structure of the polyethyloxazoline-linker moiety is preferably as follows:

[0043]

[0044] In the above structure, m and n each represent the degree of polymerization, and are preferably 200 to 600, more preferably 220 to 550, and even more preferably 250 to 500.

[0045] The aptamer is preferably an anti-FGF2 aptamer or an anti-autotaxin aptamer. Examples of anti-FGF2 aptamers include, but are not limited to, aptamers described in International Publication Nos. 2015 / 147017 and 2011 / 099576. Examples of anti-autotaxin aptamers include, but are not limited to, aptamers described in International Publication Nos. 2015 / 163458 and 2015 / 147290. The anti-autotaxin aptamer is preferably a variant of an aptamer consisting of the base sequence shown in SEQ ID NO: 1 below (i.e., an aptamer consisting of the base sequence shown in formula (1)). SEQ ID NO: 1: 5'-tCUGAGGgAAACAggUUUUGCUcCUcGGA-3' (uppercase letters indicate RNA, lowercase letters indicate DNA). For convenience, in SEQ ID NO: 1 of the sequence listing, "RNA" is selected as the "molecular type." Formula (1): tC(M)U(M)G(M)A(M)G(M)G(M)gA(M)AsA(M)C(F)A(M)ggU(F)U(F)U(F)U(F)G(M)C(F)U(M)cC(M)U(M)cG(M)G(M)A(M)_idT (Uppercase letters represent RNA, lowercase letters represent DNA, parentheses in nucleotides represent modification of the 2'-position of ribose, F represents a fluorine atom, and M represents an O-methyl group. Furthermore, idT at the end of the sequence represents modification with inverted-dT, and s in the sequence represents that the phosphate group linking the nucleotides has been phosphorothioated. Furthermore, C and c represent cytosine, U represents uracil, G and g represent guanine, A represents alanine, and t represents thymine.)

[0046] The aptamer may be an RNA aptamer or a DNA aptamer. RNA aptamers, which are particularly expensive to produce, are more useful because they are stabilized in blood by the present invention.

[0047] (3. Composition) In one embodiment of the present invention, a composition (hereinafter also referred to as "the composition") containing the present conjugate or a salt thereof is provided.

[0048] The present composition can be used for various medical purposes, depending on the type of aptamer contained therein. Hereinafter, as an example of the use of the present composition, the case of use in a medical purpose will be described.

[0049] When the aptamer contained in the composition is an anti-FGF2 aptamer, the composition can be used as a pharmaceutical for treating or preventing, for example, diseases accompanied by angiogenesis, bone and cartilage diseases, or pain. More preferably, the composition can be used as a pharmaceutical for treating or preventing diseases accompanied by angiogenesis such as age-related macular degeneration, bone and cartilage diseases such as osteoporosis, rheumatoid arthritis, osteoarthritis, and fractures, and pain.

[0050] When the aptamer contained in the composition is an anti-autotaxin aptamer, the composition can be used as a pharmaceutical for treating or preventing diseases involving fibrosis of organs or tissues, particularly diseases involving fibrosis in various tissues. More preferably, the composition can be used as a pharmaceutical for treating or preventing pulmonary fibrosis, prostatic hyperplasia, myocardial fibrosis, myocardial fibrosis, musculoskeletal fibrosis, myelofibrosis, uterine fibroids, scleroderma, post-surgical adhesions, post-surgical scars, burn scars, hypertrophic scars, keloids, atopic dermatitis, peritoneal sclerosis, asthma, cirrhosis, chronic pancreatitis, scirrhous gastric cancer, hepatic fibrosis, renal fibrosis, fibrotic vascular disease, retinopathy due to fibrosing microangiitis, a complication of diabetes, neuropathy, nephropathy, glomerulonephritis, tubulointerstitial nephritis, hereditary kidney disease, arteriosclerotic peripheral arteritis, proliferative vitreoretinopathy.

[0051] The present composition may contain components other than the present conjugate or a salt thereof. Such components include, for example, pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol-starch, sodium bicarbonate, calcium phosphate, and calcium citrate; Examples of suitable additives include lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene.

[0052] The route of administration of the present composition is not particularly limited, and examples thereof include oral administration, parenteral administration, etc. Formulations suitable for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice, capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules, suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0053] Suitable formulations for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc.

[0054] The dose of the composition will vary depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the subject, but it will usually be 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, in terms of the active ingredient per day for an adult.

[0055] (4. Method for Producing Aptamers with Extended Blood Half-Life) In one embodiment of the present invention, there is provided a method for producing an aptamer with extended blood half-life (hereinafter also referred to as "this production method"), which comprises the step of binding polyethyloxazoline to the aptamer. In this production method, polyethyloxazoline is bound to the aptamer to form a conjugate between the aptamer and polyethyloxazoline, thereby making it possible to produce an aptamer with extended blood half-life.

[0056] In the case of a branched polyethyloxazoline-added aptamer, for example, it can be produced by adding a linker to the 5' end of the aptamer by Michael addition reaction, and then adding the branched polyethyloxazoline by dipolar cycloaddition reaction. More specifically, it can be produced, for example, by the method described in the Examples.

[0057] A linear polyethyloxazoline-added aptamer can be produced, for example, by attaching a linker to the 5' end of the aptamer using a crosslinking reaction between an NHS ester and a primary amine, and then adding the linear polyethyloxazoline by a dipolar cycloaddition reaction. More specifically, it can be produced, for example, by the method described in the Examples.

[0058] (5. Method for stabilizing an aptamer) In one embodiment of the present invention, there is provided a method for stabilizing an aptamer (hereinafter also referred to as "the present stabilization method"), characterized by binding polyethyloxazoline to the aptamer. In the present stabilization method, polyethyloxazoline is bound to the aptamer to form a conjugate between the aptamer and polyethyloxazoline, thereby extending the blood half-life. As a method for producing the conjugate, for example, the method described above (4. Method for producing an aptamer with an extended blood half-life) can be used.

[0059] (6. Others) In one embodiment of the present invention, the following inventions are provided that use a conjugate of an aptamer and polyethyloxazoline or a salt thereof. (1) A medicament for treating or preventing any disease, comprising as an active ingredient a conjugate of an aptamer and polyethyloxazoline or a salt thereof. (2) A method for treating or preventing any disease, comprising administering a therapeutically effective amount of a conjugate of an aptamer and polyethyloxazoline or a salt thereof to a subject. (3) Use of a conjugate of an aptamer and polyethyloxazoline or a salt thereof in the manufacture of a medicament for treating or preventing any disease. (4) A conjugate of an aptamer and polyethyloxazoline or a salt thereof for use in treating or preventing any disease.

[0060] In (1) to (4) of this embodiment, the disease to be treated or prevented can be appropriately determined depending on the type of conjugate of an aptamer and polyethyloxazoline or a salt thereof used. That is, the disease to be treated or prevented is not particularly limited as long as it can be treated or prevented by the conjugate of an aptamer and polyethyloxazoline or a salt thereof used.

[0061] In another embodiment of the present invention, the following inventions are provided: (1) A medicament for treating or preventing a disease accompanied by angiogenesis, a bone / cartilage disease, or pain, comprising as an active ingredient a conjugate of an anti-FGF2 aptamer and polyethyloxazoline or a salt thereof. (2) A method for treating or preventing a disease accompanied by angiogenesis, a bone / cartilage disease, or pain, comprising administering a therapeutically effective amount of a conjugate of an anti-FGF2 aptamer and polyethyloxazoline or a salt thereof to a subject. (3) Use of a conjugate of an anti-FGF2 aptamer and polyethyloxazoline or a salt thereof in the manufacture of a medicament for treating or preventing a disease accompanied by angiogenesis, a bone / cartilage disease, or pain. (4) A conjugate of an anti-FGF2 aptamer and polyethyloxazoline or a salt thereof for use in treating or preventing a disease accompanied by angiogenesis, a bone / cartilage disease, or pain. Note that the disease accompanied by angiogenesis, a bone / cartilage disease, or pain in this embodiment may be, for example, one described above in (3. Composition).

[0062] In another embodiment of the present invention, the following inventions are provided: (1) A pharmaceutical for treating or preventing diseases involving organ or tissue fibrosis, particularly diseases accompanied by fibrosis in various tissues, comprising as an active ingredient a conjugate of an anti-autotaxin aptamer and polyethyloxazoline or a salt thereof. (2) A method for treating or preventing diseases involving organ or tissue fibrosis, particularly diseases accompanied by fibrosis in various tissues, comprising administering a therapeutically effective amount of a conjugate of an anti-autotaxin aptamer and polyethyloxazoline or a salt thereof to a subject. (3) Use of a conjugate of an anti-autotaxin aptamer and polyethyloxazoline or a salt thereof in the manufacture of a pharmaceutical for treating or preventing diseases involving organ or tissue fibrosis, particularly diseases accompanied by fibrosis in various tissues. (4) A conjugate of an anti-autotaxin aptamer and polyethyloxazoline or a salt thereof for use in treating or preventing diseases involving organ or tissue fibrosis, particularly diseases accompanied by fibrosis in various tissues. In this embodiment, the disease associated with organ or tissue fibrosis may be, for example, one of those described above in (3. Composition).

[0063] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0064] (1. Synthesis of branched PEOZ, linear PEOZ, and PEG-attached aptamers) Branched PEOZ-attached aptamers were synthesized by attaching a Mal-bis-PEG3-DBCO linker (CP-2070, manufactured by Conju-Probe) to the Thiol C6 linker attached to the 5' end of the aptamer using a Michael addition reaction. This linker has DBCO at each end of a bi-branched PEG chain, and Poly(2-ethyl-2-oxazoline)-azide (HR12.0500 / 05.04A (molecular weight 50,000) or HR12.0250 / 05.04A (molecular weight 25,000), manufactured by AVROXA) to both DBCOs using a dipolar cycloaddition reaction. The linear PEOZ-conjugated aptamer was synthesized by conjugating a DBCO-PEG4-NHS linker (CP-2028, Conju-Probe) to the amino C6 linker at the 5' end of the aptamer using a crosslinking reaction between an NHS ester and a primary amine, followed by the addition of Poly(2-ethyl-2-oxazoline)-azide (HR12.0500 / 05.04A, AVROXA) via a dipolar cycloaddition reaction. The PEG-conjugated aptamer was synthesized by adding polyethylene glycol (SUNBRIGHT GL2-400TS01, NOF Corp.) with a molecular weight of 40,000 to the amino C6 linker at the 5' end of the aptamer. In the following, 25 kDa PEOZ was used (partly) only in (4. Evaluation of blood retention of branched PEOZ, linear PEOZ, and PEG-added anti-FGF2 aptamer), and 50 kDa PEOZ was used in other tests.

[0065] (2. Evaluation of the binding activity of branched PEOZ-conjugated anti-FGF2 aptamer to FGF2) The binding activity of branched PEOZ-conjugated aptamer to FGF2 was evaluated using the anti-FGF2 aptamer by surface plasmon resonance. A BIAcore™ T200 (manufactured by Cytiva) was used for the measurement. According to Cytiva's protocol, human FGF2 (AF-100-18B, manufactured by Peprotech) was immobilized at approximately 1000 RU on a Series S Sensor Chip CM4 (29104989, manufactured by Cytiva). The running buffer and aptamer solution for analyte were prepared using 300 mmol / L NaCl, 5.4 mmol / L KCl, 0.8 mmol / L MgCl. ２ , 1.8 mmol / l CaCl ２ , 20 mmol / L Tris (pH 7.6), 0.05% Tween 20 were used. The aptamer solution was prepared at free oligonucleotide concentrations of 2 μg / mL and 125 μg / mL and injected. A 2 mol / L NaCl solution was used to regenerate the sensor chip. The sensorgram obtained as a result of the measurement is shown in Figure 1. As with the positive control PEG-conjugated aptamer (Figure 2), a concentration-dependent increase in signal was observed, indicating that the branched PEOZ-conjugated aptamer binds to FGF2.

[0066] (3. Evaluation of In Vitro Inhibitory Activity of Branched PEOZ-Conjugated Anti-FGF2 Aptamer) Using an anti-FGF2 aptamer, the inhibitory activity of branched PEOZ-conjugated aptamers and PEG-conjugated aptamers against cell stimulation by FGF2 was compared. When NIH3T3 cells are stimulated with FGF2, the signal transduction system is activated, and the MAP kinase pathway and PIK3 / AKT1 pathway are activated via FRS2, Grb2, and SOS, ultimately inducing the expression of various growth factor and receptor genes such as VEGF-A, VEGF-C, HGF, angiopoietin-2, VEGFR, and PDGFR-α. During this process, ERK (Extracellular Signal-regulated Kinase) is known to be phosphorylated. NIH3T3 cells were stimulated with human FGF2 (AF-100-18B, Peprotech) (1 nmol / ml) and the aptamer was added to the culture medium. After 30 minutes, phosphorylated ERK was measured by enzyme-linked immunosorbent assay (ELISA). ELISA was performed using ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) + total ERK ELISA Kit (ab176660, Abcam). The amount of phosphorylated ERK (p-ERK) and total ERK in the sample was quantified by measuring the absorbance at 450 nm using a microplate reader system (SpectraMax (registered trademark) 190, Molecular Devices). The phosphorylation level of ERK is expressed as the ratio of the absorbance of p-ERK to the absorbance of total ERK (p-ERK / total ERK ratio). The p-ERK / total ERK ratio (R0) when no aptamer was added to the medium was set to 100%, and the inhibition rate of ERK phosphorylation by FGF2 stimulation was calculated from the p-ERK / total ERK ratio (R) at each concentration using the following formula. R0 and R were values ​​obtained by subtracting the p-ERK / total ERK ratio when no aptamer or FGF2 was added to the medium as a baseline. Phosphorylation rate of ERK by FGF2 = (R / R0) x 100 Phosphorylation inhibition rate of ERK by FGF2 = 100 - Phosphorylation rate of ERK by FGF2

[0067] The aptamer concentration required to inhibit ERK phosphorylation by 50% (IC ５０ ) was determined by nonlinear regression using the inhibition rate data. GraphPad Prism 7 (Graphpad Software) was used for the analysis. Figure 3 shows the inhibitory effect of adding branched PEOZ or PEG-attached aptamers on ERK phosphorylation in cells stimulated with FGF2. The addition of either aptamer showed concentration-dependent inhibitory activity, and the IC values ​​of branched PEOZ and PEG were ５０ The values ​​(mean ± standard deviation) were similar at 112.5 ± 25.9 and 127.1 ± 22.5 pmol / L, respectively. These results demonstrate that the branched PEOZ-conjugated aptamer of the present invention has the same level of inhibitory activity against living cells as the PEG-conjugated aptamer.

[0068] (4. Evaluation of blood retention of branched PEOZ, linear PEOZ, and PEGylated anti-FGF2 aptamer) Using the anti-FGF2 aptamer, the blood retention of branched PEOZ, linear PEOZ, and PEGylated aptamer in mice was compared. Each aptamer was administered intravenously to 6- or 7-week-old male C57BL / 6J mice (Japan SLC) at 0.5 mg / kg. 1, 3, 6, 24, 48, and 72 hours after administration, the mice were anesthetized with isoflurane (008803998, Viatris), and approximately 500 μL of blood was collected from the mice by cardiac bleed. The collected blood was placed in Capiject II (EDTA 2K) (CJ-2DK, Terumo Corporation) and centrifuged at 2,300 x g for 10 minutes at 4°C in a high-speed refrigerated microcentrifuge (MX-301, Tomy Seiko Co., Ltd.), and the resulting supernatant was collected as plasma. The aptamer concentration in the resulting plasma was measured using the enzyme-linked oligonucleotide sorbent assay (ELOSA). The method is described below. The detection probes used were an oligonucleotide labeled with fluorescein (FAM) at the 5'-end and an oligonucleotide labeled with an amino group at the 3'-end. A plasma sample diluted with 1 wt% SDS-containing PBS was mixed with a FAM-labeled detection probe, left to stand at 80°C for 10 minutes in the dark, and then rapidly cooled on ice to allow hybridization of the FAM-labeled detection probe and the aptamer. This was used as a pretreatment solution. The amino-labeled detection probe was immobilized on an active ester-coated 96-well (round) plate (manufactured by Sumitomo Bakelite Co., Ltd.), and the pretreatment solution was added. The plate was shaken at 60°C in the dark for 1 hour, then rapidly cooled on ice to allow hybridization of the amino-labeled detection probe and the aptamer. The plate was washed with a washing solution (PBS containing 0.05 v / v% Tween 20) and treated with 100 μL of a stock solution of horseradish peroxidase (HRP)-labeled anti-FITC antibody (6400-05, SouthernBiotech) diluted 8000-fold with PBS to bind the antibody to FAM.Thereafter, color development was carried out using a peroxidase color development kit T (ML-1120T, manufactured by Sumitomo Bakelite Co., Ltd.), and the absorbance at 450 nm was measured using a microplate reader system (SpectraMax (registered trademark) M2, SpectraMax (registered trademark) 190, manufactured by Molecular Devices Co., Ltd.).

[0069] The blood concentration profiles of each aptamer are shown in Figure 4. The blood half-lives of the branched 25 kDa x 2, branched 50 kDa x 2, and linear 50 kDa PEOZ-attached aptamers and PEG-attached aptamers were 5.55, 18.05, 6.35 hours, and 7.70 hours, respectively. This indicates that the blood retention of the branched 50 kDa x 2 PEOZ-attached aptamer was equivalent to or superior to that of the PEG-attached aptamer. Furthermore, the branched 25 kDa x 2 and 50 kDa x 2 PEOZ-attached aptamers exhibited blood retention equivalent to or superior to that of the linear 50 kDa aptamer, and both significantly increased blood retention compared to the aptamer alone (unattached).

[0070] (5. Evaluation of enzyme inhibitory activity of PEOZ-conjugated anti-autotaxin aptamer) Using the anti-autotaxin aptamer, the autotaxin enzyme inhibitory activity of the branched PEOZ-conjugated aptamer and the PEG-conjugated aptamer was compared. The anti-autotaxin aptamer used was an aptamer consisting of the base sequence shown in formula (1), which is a modified version of the aptamer having the nucleotide sequence shown in SEQ ID NO: 1. Formula (1): 5'-tC(M)U(M)G(M)A(M)G(M)G(M)gA(M)AsA(M)C(F)A(M)ggU(F)U(F)U(F)U(F)G(M)C(F)U(M)cC(M)U(M)cG(M)G(M)A(M)_idT-3' (Uppercase letters indicate RNA, lowercase letters indicate DNA, parentheses in nucleotides indicate modification of the 2'-position of ribose, F indicates a fluorine atom, and M indicates an O-methyl group. In addition, idT at the end of the sequence indicates modification with inverted-dT, and s in the sequence indicates that the phosphate group connecting the nucleotides has been phosphorothioated.)

[0071] Enzyme inhibitory activity was evaluated by the following method. As a substrate for autotaxin, a phosphodiester bond-containing synthetic substrate, p-nitrophenyl thymidine 5'-monophosphate (pNP-TMP) (T4510, manufactured by SIGMA) was selected (hereinafter referred to as NPP2 inhibition assay). The phosphodiester bond is cleaved by hydrolysis, liberating p-nitrophenol. This p-nitrophenol develops a yellow color and is detected. The assay was performed using a 96-well plate (UV-Star 96-well microplate, flat bottom, 655801, Greiner BIO-ONE) with a reaction volume of 100 μL. Solution A was used as the reaction solution. Here, solution A contained 145 mmol / l NaCl, 5.4 mmol / l KCl, 0.8 mmol / l MgCl. ２ , 1.8 mmol / l CaCl ２ , 20 mmol / L Tris (pH 7.6), 0.05% Tween 20. 10 μL of pNP-TMP prepared at 10 mmol / L in Solution A was added to 50 μL of aptamer solution prepared at each concentration in Solution A, mixed well, and then heated at 37°C for 5 minutes. Meanwhile, 40 μL of 6 ng of autotaxin diluted with Solution A was prepared and heated at 37°C for 5 minutes. After heating, the two were mixed to initiate the enzyme reaction. The final autotaxin concentration in the reaction solution was 0.3 nM, and the final substrate concentration was 1 mM. After heating the plate containing the reaction solution at 37°C for 6 hours, it was placed in a microplate reader system SpectraMax® M2 (Molecular Devices), and the absorbance was measured at a wavelength of 405 nm. The absorbance (A0) without aptamer was set to 100%, and the enzyme activity inhibition rate was calculated from the absorbance (A) at each concentration using the following formula. Note that A0 and A were values ​​obtained by subtracting the background value immediately after the start of the enzyme reaction (0 hours). Enzyme activity rate = (A / A0) x 100 Enzyme activity inhibition rate = 100 - enzyme activity rate

[0072] The aptamer concentration required to inhibit enzyme activity by 50% (IC ５０) was determined by nonlinear regression using the inhibition rate data. GraphPad Prism 7 (Graphpad Software) was used for the analysis. The enzyme activity inhibitory effects of branched PEOZ and PEG-added aptamers are shown in Figure 5. Both adducts showed concentration-dependent inhibitory activity, and the IIC of branched PEOZ and PEG ５０ The values ​​(mean ± standard deviation) were similar at 943.1 ± 105.0 and 634.2 ± 31.5 pmol / L, respectively. From the above, it was demonstrated that the branched PEOZ-conjugated anti-autotaxin aptamer of the present invention has the same level of enzyme inhibitory activity as the PEG-conjugated anti-autotaxin aptamer.

[0073] (6. Evaluation of Blood Retention of Branched PEOZ and PEGylated Anti-Autotaxin Aptamer) Using the anti-autotaxin aptamer, the blood retention of branched PEOZ and PEGylated aptamers in mice was compared. Each aptamer was administered intravenously to 6-week-old male C57BL / 6J mice (manufactured by Japan SLC Co., Ltd.) at 0.5 mg / kg. 1, 3, 6, 24, 48, and 72 hours after administration, the mice were anesthetized with isoflurane (008803998, manufactured by Viatris), and approximately 500 μL of blood was collected from the mice by cardiac sampling. The collected blood was placed in Capiject II (EDTA 2K) (CJ-2DK, Terumo Corporation) and centrifuged at 2,300 × g for 10 minutes at 4°C in a high-speed refrigerated microcentrifuge (MX-301, Tomy Seiko Co., Ltd.), and the resulting supernatant was collected as plasma.

[0074] The aptamer concentration in the obtained plasma was measured using the enzyme-linked oligonucleotide sorbent assay (ELOSA). The method is as follows. The detection probe was labeled at the 5'-end with fluorescein (FAM): 5'-U(M)C(M)C(M)G(M)A(M)G(M)G(M)A(M)G(M)C(M)A(M)A(M)A(M)A(M)C(M)-3' (SEQ ID NO: 2(1): 5'-UCCGAGGAGCAAAAC-3' (SEQ ID NO: 2), where the parentheses in the nucleotide indicate a modification at the 2' position of ribose and M indicates an O-methyl group. .) and an oligonucleotide labeled with biotin at the 3' end with the sequence: 5'-C(M)U(M)G(M)U(M)U(M)U(M)C(M)C(M)C(M)U(M)C(M)A(M)G(M)A(M)-3' (SEQ ID NO: 3(1): a modification of 5'-CUGUUUCCCUCAGA-3' (SEQ ID NO: 3), where the parentheses in the nucleotide indicate a modification at the 2' position of the ribose, and M represents an O-methyl group). A plasma sample diluted with 1 w / v% skim milk was mixed with the FAM-labeled detection probe, and the mixture was left to stand at 80°C for 10 minutes in the dark, then rapidly cooled on ice to allow hybridization of the FAM-labeled detection probe and the aptamer. This was used as a pretreatment solution. The biotin-labeled detection probe was immobilized on a streptavidin-coated 96-well plate (IMMOBILIZER STREPTAVIDIN F8 clear (436020, Thermo Fisher Scientific)) and then blocked with 1 w / v% skim milk. The pretreatment solution was added, and the plate was shaken at 50°C for 1 hour in the dark, then rapidly cooled on ice to allow hybridization of the biotin-labeled detection probe and aptamer. The plate was washed with a wash solution (PBS containing 0.05 v / v% Tween 20) and treated with 100 μL of a solution prepared by diluting a stock solution of horseradish peroxidase (HRP)-labeled anti-FITC antibody (6400-05, SouthernBiotech) 8000-fold with PBS, allowing the antibody to bind to FAM.Thereafter, color development was carried out using a peroxidase color development kit T (ML-1120T, manufactured by Sumitomo Bakelite Co., Ltd.), and the absorbance at 450 nm was measured using a microplate reader system (SpectraMax (registered trademark) M2, manufactured by Molecular Devices Co., Ltd.).

[0075] The blood concentration profiles of each aptamer are shown in Figure 6. The blood half-lives of the branched PEOZ-conjugated and PEG-conjugated aptamers were 6.30 and 3.75 hours, respectively. This indicates that the blood retention of the branched PEOZ-conjugated aptamer is superior to that of the PEG-conjugated aptamer.

[0076] The present invention can extend the blood half-life of aptamers, and is therefore useful in various medical applications using aptamers. This application is based on Japanese Patent Application No. 2024-012285 (filing date: January 30, 2024), the contents of which are incorporated in full herein.

Claims

1. A conjugate of an aptamer and polyethyloxazoline or a salt thereof.

2. The conjugate or salt thereof according to claim 1, wherein the polyethyloxazoline forms a branched chain.

3. The conjugate or salt thereof according to claim 1 or 2, wherein the polyethyloxazoline is bound to the 5' end of the aptamer.

4. The conjugate or salt thereof according to claim 1 or 2, wherein two of the polyethyloxazolines form a branched chain via a linker.

5. The conjugate or salt thereof according to claim 2, wherein each of the branched chains of the polyethyloxazoline has a molecular weight of 20 to 60 kDa.

6. The conjugate or salt thereof according to claim 4, wherein the polyethyloxazoline is bonded to the linker via a dipolar cycloaddition reaction.

7. The conjugate or salt thereof according to claim 6, wherein the dipolar cycloaddition reaction is a dipolar cycloaddition reaction between an azide on the polyethyloxazoline side and a dibenzocyclooctyne on the linker side.

8. The conjugate or salt thereof according to claim 1, wherein the polyethyloxazoline is bound to the 5' end of the aptamer via a linker.

9. The conjugate or salt thereof according to claim 8, wherein the structure of the polyethyloxazoline-linker moiety is: (where l represents the degree of polymerization, l being 200 to 600).

10. The conjugate or salt thereof according to claim 4, wherein the structure of the polyethyloxazoline-linker moiety is: (wherein m and n each represent the degree of polymerization, m being 200 to 600, and n being 200 to 600).

11. The conjugate or salt thereof according to claim 1 or 2, wherein the aptamer is an anti-FGF2 aptamer or an anti-autotaxin aptamer.

12. The conjugate or salt thereof according to claim 1 or 2, wherein the aptamer is an aptamer consisting of a base sequence represented by the following formula (1): tC(M)U(M)G(M)A(M)G(M)G(M)gA(M)AsA(M)C(F)A(M)ggU(F)U(F)U(F)U(F)G(M)C(F)U(M)cC(M)U(M)cG(M)G(M)A(M)_idT (1) (Uppercase letters represent RNA, lowercase letters represent DNA, parentheses in nucleotides represent modification of the 2'-position of ribose, F represents a fluorine atom, and M represents an O-methyl group. In addition, idT at the end of the sequence represents modification with inverted-dT, and s in the sequence represents that the phosphate group linking the nucleotides has been phosphorothioated.) 13. A composition comprising the conjugate of claim 1 or 2 or a salt thereof.

14. A method for producing an aptamer having an extended blood half-life, comprising the step of binding polyethyloxazoline to the aptamer.

15. The method of claim 14, wherein the polyethyloxazoline forms a branched chain.

16. The method of claim 14 or 15, wherein the polyethyloxazoline is attached to the 5' end of the aptamer.

17. The method of claim 14 or 15, wherein the branched polyethyloxazoline is bound to the aptamer via a linker.

18. The method of claim 15, wherein the polyethyloxazoline, each of whose branched chains has a molecular weight of 20 to 60 kDa, is bound to the aptamer.

19. The method of claim 17, wherein the polyethyloxazoline and the linker are bonded together by a dipolar cycloaddition reaction.

20. The method of claim 19, wherein the polyethyloxazoline and the linker are bonded by a dipolar cycloaddition reaction between an azide on the polyethyloxazoline side and a dibenzocyclooctyne on the linker side.

21. The manufacturing method described in claim 14 or 15, wherein the polyethyloxazoline branched via a linker is bound to the aptamer by a Michael addition reaction between a maleimide group on the linker side and a thiol group on the aptamer side.

22. The method of claim 14, wherein the polyethyloxazoline is bound to the 5' end of the aptamer via a linker.

23. The method of claim 22, wherein the structure of the polyethyloxazoline-linker moiety is: (where l represents the degree of polymerization, l being 200 to 600).

24. The method of claim 17, wherein the structure of the polyethyloxazoline-linker moiety is: (wherein m and n each represent the degree of polymerization, m being 200 to 600, and n being 200 to 600).

25. A method for stabilizing an aptamer, comprising binding polyethyloxazoline to the aptamer.

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