Therapeutic agent for hunner-type interstitial cystitis containing DNA oligonucleotide selectively binding to IFN-γ

Abstract

To provide a therapeutic agent for Hunner-type interstitial cystitis that can selectively inhibit IFN-γ, does not have a risk of biological contamination, and can be stored at a room temperature.SOLUTION: There is provided a therapeutic agent for Hunner-type interstitial cystitis or a reagent for test research, containing, as an active ingredient, a DNA oligonucleotide that has a base sequence indicated in any of SEQ ID NO:1-3 and selectively binds to IFN-γ. In the DNA oligonucleotide having the base sequence indicated by SEQ ID NO: 3, a base X in the sequence is an artificially produced base, and the artificially produced base may be chemically modified with a low-molecular-weight compound, a medium-molecular-weight compound, a polymer compound, a biopolymer or a biocompatible polymer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for Hanna-type interstitial cystitis, which comprises a DNA oligonucleotide that selectively binds to interferon-γ (IFN-γ). [Background technology]

[0002] Hanna type interstitial cystitis is a disease that was designated as an intractable disease by the Minister of Health, Labor and Welfare in May 2014 (designated intractable disease 226). The exact number of patients with Hanna type interstitial cystitis in Japan is unknown, but according to a Ministry of Health, Labor and Welfare health administrative report, the number of Hanna type interstitial cystitis patients who hold a certificate for specific medical expenses (designated intractable disease) has increased since 2015 after the designation of intractable disease, and was 863 in 2020 (Non-Patent Document 1). It is believed that there are many patients who do not hold the certificate, and according to a survey by the Japanese Interstitial Cystitis Research Group, the number of interstitial cystitis patients, including Hanna type and non-Hanna type, is approximately 4,500 in Japan, of which 45% (approximately 2,000 people) are Hanna type patients. The male-to-female ratio of Hanna type patients is 1:5.6, with more women than men, and it is known that they are particularly common in the middle-aged and elderly (Non-Patent Document 2).

[0003] Interstitial cystitis is a disease in which inflammation of the bladder occurs for unknown reasons, resulting in painful symptoms such as frequent urination and discomfort and pain in the bladder and urethra. Interstitial cystitis has been classified into Hanna type and non-Hanna type based on the type of disease. In Hanna type, characteristic abnormal inflammatory pathological images called Hanna lesions are seen on bladder endoscopy. On the other hand, in non-Hanna type, no inflammatory lesions are seen in the bladder, and it was found to be a completely different type of disease from Hanna type. Currently, when people talk about interstitial cystitis, they are referring to Hanna type.

[0004] No fundamental treatment for Hanna-type interstitial cystitis has yet been found. In the clinical practice guidelines (Non-Patent Document 3), conservative treatment (symptomatic treatment), oral medication, intravesical infusion therapy, and surgical treatment including endoscopic treatment are listed as symptom improvement methods. Until now, the only treatment covered by insurance for interstitial cystitis was bladder hydrodistension. However, bladder hydrodistension is highly invasive and carries the risk of bladder rupture as a side effect, and the physical and time burden on patients associated with the procedure has been a problem. In January 2021, Jimso (registered trademark) intravesical infusion solution 50% (injected into the bladder six times at two-week intervals) was newly approved as the only interstitial cystitis treatment drug in Japan. However, the mechanism of action of dimethyl sulfoxide (DMSO), the active ingredient of Jimso (registered trademark) intravesical infusion solution 50%, on interstitial cystitis has not been fully elucidated, and although it has a certain effect on pain and inflammation, there are still problems such as a short period of effectiveness and repeated recurrence.

[0005] Symptomatic treatment involves explaining the condition and providing dietary advice. Oral medications include analgesics, antidepressants, antiallergic drugs, and steroids. The above-mentioned hydrodistention of the bladder is widely used as an endoscopic treatment. If Hanna lesions are found in the bladder during the procedure, electrical or laser surgery (cauterization) is performed, but repeated surgery over a long period of time can cause the bladder to shrink. Hydrodistention of the bladder or cauterization of Hanna lesions results in symptom relief in about half of cases, but long-term remission is only seen in a minority of cases. For this reason, retreatment or additional treatment is required in many cases.

[0006] Drug injection therapy into the bladder includes 50% DMSO, heparin, steroids, etc. In some cases, botulinum toxin is injected into the bladder wall. In cases where the symptoms are intolerable and resistant to any of these treatments, total cystectomy and urinary diversion are indicated. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ministry of Health, Labor and Welfare Health Administration Report Number of recipients of specified medical expenses (designated intractable disease) certificates Data from FY2015 to FY2020<https: / / www.nanbyou.or.jp / entry / 5354> [Non-Patent Document 2] Rare Disease Center website<https: / / www.nanbyou.or.jp / entry / 4429> [Non-Patent Document 3] Guidelines for the Treatment of Interstitial Cystitis / Bladder Pain Syndrome (edited by the Japanese Interstitial Cystitis Study Group / Japan Urological Association), published April 25, 2019, revised May 2021 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, no effective treatment or cure has yet been found for Hanna-type interstitial cystitis. In order to improve the quality of life (QOL) of patients, it is necessary to develop highly effective treatments or cures.

[0009] In Hanna type interstitial cystitis, the pathological lesions are characterized by infiltration of lymphocytes, especially B cells and plasma cells, and show a typical inflammatory condition due to an autoimmune disease, but the cause of the disease remains unknown.

[0010] It has been revealed that Hanna-type interstitial cystitis often occurs concomitantly with systemic autoimmune diseases such as Sjögren's syndrome, autoimmune thyroiditis, and systemic lupus erythematosus, and autoantibodies against the urothelium have been reported in the blood of patients (Y. Akiyama, et. al., International Journal of Urology, 2020, 27, 491-503.). In addition, gene expression analysis of bladder samples taken from patients with Hanna-type interstitial cystitis has reported that gene expression of inflammation-related molecules such as IFN-γ, CXCR3, CXCL9, CXCL10, CXCL11, TNF-α, and TNFSF14 is significantly increased (T. Ogawa, et. al., The Journal of Urology, 2010, 183, 1206-1212.). Most of these molecules are IFN-γ and its cascade molecules. Considering that Hanna type interstitial cystitis is accompanied by a systemic autoimmune disease, it is believed that IFN-γ plays a central role in the pathogenesis of the disease, as in other autoimmune diseases. However, no therapeutic drug for Hanna type interstitial cystitis that targets IFN-γ has been developed to date.

[0011] To date, antibodies and Janus kinase inhibitors have been developed as drugs that inhibit the action of IFN-γ. However, for example, anti-IFN-γ antibodies have the following problems: (1) they are biological products, so there is a risk of biological contamination, (2) antigenicity is an issue with long-term administration, (3) they are protein products, so they require a cold chain for storage and transportation, and (4) their large molecular weight makes them unsuitable for local administration such as intravesical instillation.

[0012] Regarding the above-mentioned issue (2), the antibody production rate for general antibody drugs is said to be about 30%. Therefore, when long-term treatment is required, there are often cases where antibodies against antibodies are produced, causing anaphylactic reactions, making it difficult to continue treatment.

[0013] Regarding issue (1) above, serum and other substances are often used in the manufacturing process of biological products, raising concerns about the risk of biological contamination by viruses, etc. Regarding issue (3), the products must always be handled at low temperatures, which not only increases the costs of transportation and storage, but also reduces convenience for patients who use them.

[0014] As Janus kinase inhibitors, four types, Tofacitinib (product name: Xeljanz (registered trademark)), Baricitinib (product name: Olumiant (registered trademark)), Peficitinib (product name: Smilaf (registered trademark)), and Upadacitinib (product name: Rinvoq (registered trademark)), are applied to rheumatoid arthritis, an autoimmune disease, and are on the market. Since these Janus kinase inhibitors are low molecular weight compounds that can be produced by chemical synthesis, it is thought that they do not have the above-mentioned problems caused by being antibodies. On the other hand, there are multiple subtypes of Janus kinase, and they are activated by binding to the intracellular domains of multiple cytokine receptors such as not only IFN-γ receptors but also interleukin 2 (IL-2) receptors, interleukin 4 (IL-4) receptors, interleukin 7 (IL-7) receptors, and interferon α (IFN-α) receptors, and transmit receptor signals.

[0015] For this reason, Janus kinase inhibitors may inhibit signaling not only of IFN-γ, but also of IL-2, IL-4, IL-7, IFN-α, etc. (Yvan Jamilloux, et.al., Autoimmunity Reviews, 2019, 18, 11, 102390). This raises safety concerns with long-term administration, suggesting the possibility of unexpected side effects due to susceptibility to infection, etc.

[0016] The present invention has been made in consideration of the above circumstances, and aims to provide a therapeutic agent for Hanna type interstitial cystitis that can selectively inhibit IFN-γ as a target substance, is free of biological contamination, and can be stored at room temperature. [Means for solving the problem]

[0017] In order to solve the above problems, the therapeutic drug for Hanna type interstitial cystitis containing the DNA oligonucleotide of the present invention as an active ingredient adopts the following aspects.

[0018] The first aspect of the present invention provides a therapeutic agent for Hanna type interstitial cystitis, which comprises a DNA oligonucleotide that has a base sequence shown in any one of SEQ ID NOs: 1 to 3 and selectively binds to interferon-γ (IFN-γ). The DNA oligonucleotide according to this aspect selectively binds to IFN-γ and inhibits its activity, thereby exerting a therapeutic effect for Hanna type interstitial cystitis.

[0019] The base sequence shown in SEQ ID NO:2 is a sequence in which an oligonucleotide consisting of 9 natural base residues has been added to the 3' end of the base sequence shown in SEQ ID NO:1.

[0020] The base sequence shown in SEQ ID NO:3 is a sequence in which the 53rd base from the 5' end of the base sequence shown in SEQ ID NO:2 is replaced with an arbitrary base.

[0021] In the first aspect of the present invention, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO:3 is an artificially produced base, and the artificially produced base may be chemically modified with a low molecular weight compound.

[0022] The low molecular weight compound in the first aspect has a molecular weight of about 200 to 1000, and examples of candidates include anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporine, methotrexate, and leflunomide.

[0023] In the first aspect of the present invention, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 may be an artificially produced base, and the artificially produced base may be chemically modified with a medium molecular weight compound, a polymeric compound, a biopolymer, or a biocompatible polymer. The medium molecular weight compound in this aspect has a molecular weight of about 1000 to 20000, and the polymeric compound in this aspect has a molecular weight of about 20000 to 400000.

[0024] The polymer compound in the above embodiment may be any biocompatible polymer having a molecular weight of 20,000 or more. Examples of the medium molecular compound or polymer compound in this embodiment include, but are not limited to, PEG, dipolar polymer, oligosaccharide, lipophilic polymer, peptide, oligonucleotide, antibody, etc. Antibodies belong to the polymer compound, while PEG, dipolar polymer, oligosaccharide, lipophilic polymer, peptide, and oligonucleotide belong to the medium molecular compound or polymer compound depending on their molecular weight. The molecular weight of the medium molecular compound or polymer compound is expressed by the average molecular weight defined by the number average molecular weight (Mn) or weight average molecular weight (Mw).

[0025] The second aspect of the present invention is a therapeutic agent for a disease selected from a disease associated with IFN-γ in dogs and cats, a lower urinary tract disease caused by the bladder in dogs and cats, or an autoimmune disease in dogs and cats, which comprises, as an active ingredient, a DNA oligonucleotide having a base sequence shown in any one of SEQ ID NOs: 1 to 3 and selectively binding to IFN-γ in dogs and cats. An example of a lower urinary tract disease caused by the bladder in dogs and cats is idiopathic cystitis.

[0026] In the second embodiment, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 may be an artificially produced base, and the artificially produced base may be chemically modified with a low molecular weight compound. The low molecular weight compound in this embodiment has a molecular weight of about 200 to 1000, and examples of the low molecular weight compound include anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporine, methotrexate, and leflunomide.

[0027] In the second embodiment, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 may be an artificially produced base, and the artificially produced base may be chemically modified with a medium molecular weight compound, a polymeric compound, a biopolymer, or a biocompatible polymer. The medium molecular weight compound in this embodiment has a molecular weight of about 1000 to 20000, and the polymeric compound in this embodiment has a molecular weight of about 20000 to 400000. The polymeric compound in the above embodiment may be any polymer having a molecular weight of 20000 or more and a biocompatible polymer. Examples of the medium molecular weight compound or polymeric compound in this embodiment include, but are not limited to, PEG, a dipolar polymer, an oligosaccharide, a lipophilic polymer, a peptide, an oligonucleotide, an antibody, and the like. An antibody belongs to a polymeric compound, while PEG, a dipolar polymer, an oligosaccharide, a lipophilic polymer, a peptide, and an oligonucleotide belong to a medium molecular weight compound or a polymeric compound depending on their molecular weight.

[0028] A third aspect of the present invention is a reagent for research use, which comprises as an active ingredient a DNA oligonucleotide having a base sequence shown in any one of SEQ ID NOs: 1 to 3 and selectively binding to IFN-γ.

[0029] In the third embodiment, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 may be an artificially produced base, and the artificially produced base may be chemically modified with a low molecular weight compound. The low molecular weight compound in this embodiment has a molecular weight of about 200 to 1000, and examples of candidates include anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporine, methotrexate, and leflunomide.

[0030] In the third embodiment, the base X in the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 may be an artificially produced base, and the artificially produced base may be chemically modified with a medium molecular weight compound, a polymeric compound, a biopolymer, or a biocompatible polymer. The medium molecular weight compound in this embodiment has a molecular weight of about 1000 to 20000, and the polymeric compound in this embodiment has a molecular weight of about 20000 to 400000. The polymeric compound in the above embodiment may be any polymer having a molecular weight of 20000 or more and a biocompatible polymer. Examples of the medium molecular weight compound or polymeric compound in this embodiment include, but are not limited to, PEG, a dipolar polymer, an oligosaccharide, a lipophilic polymer, a peptide, an oligonucleotide, an antibody, and the like. An antibody belongs to a polymeric compound, while PEG, a dipolar polymer, an oligosaccharide, a lipophilic polymer, a peptide, and an oligonucleotide belong to a medium molecular weight compound or a polymeric compound depending on their molecular weight. Effect of the Invention

[0031] The DNA oligonucleotide having the base sequence according to the present invention selectively binds to IFN-γ. This allows selective inhibition of IFN-γ activity. In addition, according to the Hanna type interstitial cystitis therapeutic drug containing the DNA oligonucleotide having the base sequence according to the present invention, since it is not necessary to use serum or the like in its production, it can be produced without the risk of biological contamination by viruses or the like. In addition, the DNA oligonucleotide having the base sequence according to the present invention can be stored at room temperature. Therefore, it is more advantageous than the conventional method in terms of transportation and storage costs, and can improve the convenience of the patient to use. In addition, the Hanna type interstitial cystitis therapeutic drug containing the DNA oligonucleotide having the base sequence according to the present invention can be administered by intravesical injection due to its molecular weight.

[0032] The DNA oligonucleotide of the present invention does not inhibit the signaling of IL-2, IL-4, IL-7, IFN-α, etc., but inhibits only the action of IFN-γ. As a result, the Hanna type interstitial cystitis treatment drug containing the DNA oligonucleotide having the base sequence of the present invention as an active ingredient can reduce unexpected side effects due to susceptibility to infection, etc., even when administered for a long period of time, compared to Janus kinase inhibitors, etc. In addition, compared to anti-IFN-γ antibodies, the DNA oligonucleotide of the present invention has lower antigenicity than antibodies, making it a drug that can be used for a long period of time. Furthermore, the DNA oligonucleotide of the present invention can be stored at room temperature without the risk of biological contamination during its production. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows the change in the number of hairs on a grafted skin tissue before and after administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 2A] FIG. 1 shows the experimental results regarding the expression of MHC class I in the hair bulb root sheath by administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 2B]FIG. 1 shows the experimental results regarding the expression of MHC class I in the outer root sheath by administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 3A] FIG. 1 shows the experimental results regarding the expression of MHC class II in the connective tissue root sheath by administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 3B] FIG. 1 shows the experimental results regarding the expression of MHC class II in the outer root sheath by administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 4A] FIG. 13 shows the results of the effect on mouse IFN-γ-induced STAT1 phosphorylation when a surrogate aptamer according to one embodiment of the present invention was added to L929 mouse fibroblast cells. [Figure 4B] FIG. 1 shows the results of the effect of addition of negative control DNA to L929 mouse fibroblasts on mouse IFN-γ-induced STAT1 phosphorylation. [Figure 5A] FIG. 1 shows the suppression of an increase in urination frequency in an experiment on the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model. [Figure 5B] FIG. 13 shows the suppression of increased sensitivity to pelvic pain in an experiment on the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model. [Figure 6] FIG. 1 shows the results of histopathological evaluation of the bladder regarding the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model. [Figure 7A] 1 is a diagram showing a comparison of mRNA expression levels in bladder tissues in an experiment on the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model. In the surrogate aptamer administration group, the expression levels of inflammatory cytokines (IFN-γ, TNF-α) and pain-causing substances (pre-SP, NGF) in bladder tissues were suppressed to near normal levels similar to those in the normal group. [Figure 7B]FIG. 7B is a graph showing the quantification of the expression levels of the mRNAs shown in FIG. 7A. [Figure 8] FIG. 1 shows the binding of a DNA oligonucleotide according to one embodiment of the invention to canine and feline IFN-γ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] An embodiment of a therapeutic agent for Hanna type interstitial cystitis containing the DNA oligonucleotide according to the present invention as an active ingredient will be described below.

[0035] As for drugs targeting IFN-γ, the anti-IFN-γ antibody Emapalumab was approved by the FDA in 2018 for the treatment of hemophagocytic lymphohistiocytosis, a refractory autoimmune disease, and is marketed under the brand name Gamifant.

[0036] However, as mentioned above, anti-IFN-γ antibodies have issues such as the risk of biological contamination due to being a biological drug, antigenicity during long-term administration, inadequacy for local administration such as transdermal or transmucosal administration due to their large molecular weight, and storage and transportation conditions due to being a protein drug. Therefore, there is a need to develop a therapeutic drug that can solve these issues and effectively inhibit IFN-γ.

[0037] The present inventors have attempted to develop an IFN-γ inhibitor using the DNA oligonucleotide according to the present invention as a DNA aptamer as a means for solving the above-mentioned problems. A DNA aptamer is a ligand molecule in which complementary sequences in a DNA oligonucleotide molecule form complementary strands, causing a single-stranded DNA oligonucleotide to form a secondary or tertiary structure, and the three-dimensional structure causes the single-stranded DNA oligonucleotide to specifically and strongly bind to a target molecule. By binding a DNA aptamer having a specific sequence, the activity of a target molecule can be inhibited or suppressed, but it can also be enhanced. Although the molecular weight of a DNA aptamer is about 1 / 10 or less smaller than that of an antibody, it has high affinity equivalent to that of an antibody and has high target selectivity. For this reason, an IFN-γ inhibitor using a DNA aptamer can minimize the occurrence of side effects due to off-targets. In addition, since the DNA aptamer can be produced by chemical synthesis, it is considered to be a suitable modality as a means for solving problems. In this specification, "selectively binds to IFN-γ" includes that the DNA oligonucleotide according to this embodiment binds to the target substance IFN-γ as a DNA aptamer strongly and specifically.

[0038] DNA aptamers form three-dimensional structures with a compact size compared to antibodies, allowing for local administration such as transdermal and transmucosal administration.

[0039] DNA aptamers have the following advantages: (1) their molecular weight is relatively small, and they may be administered as transdermal or transmucosal preparations such as ointments and patches; (2) they are chemically synthesized, so there is a low risk of biological contamination; (3) they are generally low in antigenicity; (4) they are DNA, so they are sufficiently stable at room temperature under near-neutral conditions free of nucleic acid degrading enzymes (nucleases); and (5) they have almost no inhibitory activity against cytochrome P450, a drug-metabolizing enzyme, so they do not affect concomitant drugs. DNA aptamers are also expected to be useful because they do not cause problems such as the production of antibodies against antibody drugs, which is one of the issues when long-term treatment using antibodies is required.

[0040] A specific approach to treating diseases using DNA aptamers is to neutralize IFN-γ by administering the aptamer itself or a modified version, thereby treating autoimmune diseases or diseases thought to be primarily caused by excessive production of IFN-γ.

[0041] The inventors have discovered a DNA aptamer having the sequence of SEQ ID NO: 1 in Table 1, which contains two artificial bases Ds (7-(2-thienyl)imidazo[4,5-b]pyridine) in its base sequence, that can specifically bind to human IFN-γ with high affinity and inhibit its activity.

[0042] Since DNA aptamers are rapidly degraded by nucleic acid degrading enzymes (nucleases) in biological tissues, even if they show strong activity in vitro, their activity does not necessarily manifest in vivo. Therefore, the present inventors have found that a DNA aptamer having the base sequence of SEQ ID NO: 2 in Table 1, in which a 9-residue natural base sequence is bound to the 3' end, acquires nuclease resistance and becomes stable in biological tissues. It has been confirmed that a DNA aptamer having the base sequence of SEQ ID NO: 2 containing two Ds in the sequence is effective in an autoimmune humanized mouse model of alopecia areata transplanted with human scalp tissue, and is stable in biological tissues and can inhibit IFN-γ (Patent Application No. 2021-166794). In addition, for a DNA aptamer having the sequence of SEQ ID NO:3, which is a sequence in which the 53rd base from the 5' end of the sequence shown in SEQ ID NO:2 is replaced with an arbitrary base X, it has been confirmed that a PEG-modified form in which polyethylene glycol (PEG) is added to the base moiety of X retains its binding ability to IFN-γ by SPR (Surface Plasmon Resonance). From this, it was found that modification to an arbitrary base X does not affect the IFN-γ binding activity of the DNA aptamer having the sequence of SEQ ID NO:3, that is, the DNA aptamer having the sequence of SEQ ID NO:3 retains the same IFN-γ inhibitory activity as the DNA aptamer having the sequence of SEQ ID NO:2.

[0043] In this embodiment, a DNA oligonucleotide having a base sequence shown in Table 1 is used as a DNA aptamer. This embodiment includes the use of a DNA oligonucleotide having a base sequence shown in Table 1 as a therapeutic agent for Hanna type interstitial cystitis.

[0044] [Table 1]

[0045] The base sequence shown in SEQ ID NO: 2 in Table 1 is a sequence in which an oligonucleotide (mini-hairpin sequence) consisting of 9 natural base residues (5'-CGCGAAGCG-3') is added to the 3' end of the sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 3 in Table 1 is a sequence in which the 53rd base from the 5' end of the sequence shown in SEQ ID NO: 2 is replaced with any base X. X represents any natural base, any non-natural base, or a modified base, or a modified base to which a polymer compound (biopolymer) used in living organisms, such as a low molecular weight compound, a peptide, an oligonucleic acid, an oligosaccharide, or a protein, or a polymer with biocompatibility is bound. Details of the base sequence of SEQ ID NO: 4 will be described later.

[0046] Examples of polymeric compounds to be bound to modified bases include polyethylene glycol (PEG) with a molecular weight of 20,000 or more, and any polymer with biocompatibility with a molecular weight of 20,000 or more. A biocompatibility polymer is a chemically synthesized product that is not normally used in the body, and is safe even when placed in the body without causing inflammation or toxic reactions. Examples of medium molecular compounds include peptides, oligonucleic acids, oligosaccharides, proteins, PEG, and any polymer with biocompatibility with a molecular weight of more than 1,000 and less than 20,000.

[0047] Functional groups that can be used for modification include azide groups (-N3), amino groups (-NH2), carboxyl groups (-COOH) or their active esters, alkynyl groups (-CC) or cyclic structures containing an alkynyl structure, formyl groups (-CHO), hydrazide groups (-NH-NH2), hydroxyl groups (-OH), thiol groups (-SH), cyano groups (-CN), vinyl groups (-CHCH2), and maleimide groups.

[0048] In the present specification, the term "natural base" refers to any of adenine, guanosine, cytosine, and thymine. In the present specification, the term "unnatural base" refers to a base that is artificially synthesized and has properties similar to those of a natural base, and may be referred to as an "artificial base" in the present specification. In the present specification, the term "modified base" refers to a base to which a side chain structure having one or more activated functional groups for modification has been added, and is a type of "artificially produced base". Examples of modifications include natural bases that have been methylated, deaminated, swapped atomic positions, thiolated oxygen at the phosphate site, and introduced water-soluble or lipophilic substituents into the base portion. Specific examples include modified pyrimidines, modified purines, and other heterocyclic bases. Ds in the sequences of SEQ ID NOs: 1 to 3 represents 7-(2-thienyl)imidazo[4,5-b]pyridine, which is an artificial base. In addition to Ds itself, a base with a side chain introduced into Ds may be used as the artificial base. In the present embodiment, the DNA aptamers having the sequences shown in SEQ ID NOs: 1, 2, 3, and 4 in Table 1 are referred to as "aptamer 1," "aptamer 2," "aptamer 3," and "aptamer 4," respectively, hereinafter.

[0049] Regarding the usefulness of the DNA aptamer according to the present embodiment, in this specification, a DNA aptamer (aptamer 2) shown in Table 1 was intradermally injected into an autoimmune alopecia model of an immune-tolerant mouse to which a human scalp tissue fragment had been transplanted, and the results showed that hair regeneration was promoted and further hair loss was suppressed (Example 4). Details will be described later.

[0050] As a result of pathological analysis of the mechanism of aptamer 2 activity expression in this model, aptamer 2 almost completely suppressed the expression of MHC class I and II. Detailed results will be described later. In other words, aptamer 2 is considered to have improved the symptoms of alopecia areata by inhibiting the activity of IFN-γ, thereby suppressing the production of MHC class I and II, which are the basis of the expression of autoimmunity, and improving autoimmunity. This indicates that the provision of an IFN-γ inhibitor using a DNA aptamer can be a means of solving the problem not only of alopecia areata, but also of Hanna-type interstitial cystitis, which is presumed to be mainly caused by excessive production of IFN-γ.

[0051] Whether or not a DNA aptamer that inhibits IFN-γ activity is effective in treating Hanna-type interstitial cystitis must be confirmed in an animal model of the disease before its effectiveness in humans can be confirmed. The animal model that is closest to the pathology of human Hanna-type interstitial cystitis that has been established to date is the mouse autoimmune interstitial cystitis model (a model using a transgenic mouse (URO-OVA mouse) that expresses OVA antigen in the bladder) (Y. Akiyama, et. al., Am. J. Physiol. Renal Physiol., 2021, 320, F174-182: hereinafter referred to as "Reference 1"). However, the above-mentioned DNA aptamer that specifically binds to human IFN-γ did not show binding activity to mouse IFN-γ. For this reason, the effectiveness of the above-mentioned DNA aptamer cannot be confirmed in a mouse model.

[0052] Therefore, the present inventors investigated obtaining an aptamer that exhibits high similarity in physical properties to the above-mentioned DNA aptamer and that binds to mouse IFN-γ and inhibits its activity as a surrogate aptamer.

[0053] The criteria for a surrogate aptamer to exhibit high similarity to the physical properties of the above-mentioned DNA aptamer are as follows. (1) It is a DNA aptamer. (2) The base sequence of the aptamer contains two artificial bases Ds, and the other bases are natural bases. (3) The number of bases in the aptamer is within ±10% (51 to 62 residues) of the 57 residues of the above-mentioned DNA aptamer. (4) The aptamer has a nine-residue mini-hairpin sequence at the 3' end of its base sequence. Aptamers that satisfy the above criteria are structurally similar to the above-mentioned DNA aptamers, and therefore are expected to have similar physical properties.

[0054] As a result of searching for a surrogate aptamer that satisfies the above criteria, a DNA aptamer (aptamer 4) having a sequence described in SEQ ID NO: 4 in Table 1, which has 62 base residues, contains two Ds, and has a mini-hairpin sequence at the 3' end, was obtained (Example 5). Details will be described later. The obtained DNA aptamer had a KD value indicating the binding ability to mouse IFN-γ of 2.47 nM, and had a high binding ability of about 1 / 100 of the binding ability of aptamer 2 to human IFN-γ (KD value: 33 pM). In addition, by adding 5 times the molar concentration of mouse IFN-γ, the aptamer competitively inhibited and almost completely suppressed the activity of mouse IFN-γ. From this result, it was confirmed that the DNA aptamer obtained by the search had sufficient activity to function as a surrogate aptamer.

[0055] The obtained surrogate aptamer was administered intravesically to mice with autoimmune interstitial cystitis model (URO-OVA model) described in Reference 1. As a result, the surrogate aptamer significantly suppressed the increase in urination frequency and the increase in sensitivity to pelvic pain in the mice compared to the control PBS administration group, and showed a high effect of suppressing the onset of cystitis (Example 6).

[0056] Histopathological evaluation of the bladder showed that the bladder inflammation score was significantly lower in the aptamer-administered group than in the PBS-administered group, and no difference was observed between the aptamer-administered group and the normal group, indicating that bladder inflammation was suppressed by administration of the surrogate aptamer. Furthermore, as a result of mRNA expression analysis in the bladder tissue, the expression of inflammatory cytokines induced by IFN-γ stimulation, such as IFN-γ and TNF-α, as well as the expression of pre-SP and NGF, which are substances that cause pain, were suppressed by administration of the surrogate aptamer. It was strongly suggested that the suppressive effect of the surrogate aptamer on bladder inflammation was expressed by the activity inhibitory effect of IFN-γ in the tissue (Example 6).

[0057] Because the DNA aptamer of the present invention exhibits an inhibitory effect against human IFN-γ in human tissue and because the physical properties of the DNA aptamer and the surrogate aptamer are highly similar, it is inferred that, like the surrogate aptamer, the DNA aptamer has an inhibitory effect on the onset of human Hanna type interstitial cystitis and is useful as a therapeutic agent for Hanna type interstitial cystitis.

[0058] As the DNA aptamer, a DNA oligonucleotide having any of the sequences listed in Table 1 can be used as it is, or a modified site that does not affect the activity of the DNA aptamer can be used. Examples of modified DNA aptamers include those in which a medium molecule or polymer compound such as PEG, peptide, or oligonucleotide is bonded by a chemical method, those in which the same DNA aptamers are multimerized by a chemical method, and those in which a part of the sequence of the DNA aptamer is converted or modified. When the DNA oligonucleotide according to this embodiment is modified and used as a DNA aptamer, the base portion is preferably used as the modified portion. Using existing methods, an artificial base or modified base can be modified, and the 3' end and 5' end can also be modified.

[0059] PEG-modified DNA aptamers are used to improve the pharmacokinetics (PK)-pharmacodynamics (PD) profile of proteins, peptides, and oligonucleotides, including aptamers, for the purpose of improving in vivo kinetics, and many PEG-modified aptamers have been developed. When the binding activity of a PEG-modified aptamer to a target protein is maintained, it is known that the aptamer exhibits the same activity in the body as the aptamer before PEG modification, and there is almost no toxicity due to PEG modification (C. Simone Fishburn, Journal of Pharmaceutical Sciences, 2008, 97, 10, 4167-4183; Katarina D. Kovacevic, et.al., Advanced Drug Delivery Reviews, 2018, 134, 36-50).

[0060] Formulations for systemic administration can be formulated as injectable formulations, such as vials containing lyophilized powder, vials containing an aptamer solution, or pre-filled syringes.

[0061] The DNA aptamer of this embodiment can be manufactured into an inhalation formulation by placing nanoparticles that adsorb or encapsulate the DNA aptamer or a solution thereof, or a powder of the DNA aptamer granulated to an appropriate size together with a granulating material, into an inhalation device.

[0062] The DNA aptamer of this embodiment can be used as an eye drop by taking advantage of its high water solubility and dissolving it directly in an appropriate solvent such as a buffer solution.

[0063] One of the local administration methods is via mucous membrane. The DNA aptamer of the present embodiment can be dissolved in a solvent such as a buffer solution having high biocompatibility and used as a transmucosal administration agent such as intravesical administration.

[0064] As for injectable preparations, inhaled preparations, and eye drops, the DNA aptamer of this embodiment can be encapsulated or attached to lipid nanoparticles, nanoparticles of biodegradable polymers such as PLGA (Polylactic-co-Glycolic Acid), gold nanoparticles, etc., and dispersed or dissolved in physiological saline, physiological buffer solution, etc., and used.

[0065] The DNA aptamer of this embodiment can be applied as a transdermal topical administration agent such as a solution, ointment, cream, lotion, milky lotion, emulsion, gel, biodegradable microneedle, or patch.

[0066] In the process of producing a transdermal preparation, it is possible to use, as an absorption enhancer, lower alcohols such as ethanol, polyhydric alcohols such as ethylene glycol, fatty acids, esters such as ethyl acetate, surfactants, ionic liquids, etc. In addition, in the production of a transdermal preparation, a production process of nanoparticle formation using a biodegradable polymer such as polylactic acid or liposomes can be applied, and these processes can be appropriately combined depending on the purpose.

[0067] The DNA aptamer of this embodiment can also be used as an administration formulation using a device compatible with physical percutaneous absorption promotion methods such as iontophoresis, electroporation, thermal poration, sonophoresis, microneedle array patch, needleless syringe, micropump, etc.

[0068] It was confirmed that the DNA aptamer of this embodiment binds to canine IFN-γ and feline IFN-γ in addition to human IFN-γ (Example 7). Details will be described later. Here, it is presumed that idiopathic cystitis, which is one of the lower urinary tract diseases caused by the bladder in dogs and cats, is involved in the overproduction of IFN-γ in dogs and cats. From the results of Example 7, it can be said that the DNA aptamer of this embodiment has an activity inhibitory effect on canine IFN-γ and feline IFN-γ. From this, it is considered to have an effect of suppressing the onset of idiopathic cystitis, which is one of the lower urinary tract diseases caused by the bladder in dogs and cats. Therefore, the DNA aptamer of this embodiment can be used as a therapeutic agent for idiopathic cystitis in dogs and cats, diseases related to IFN-γ, autoimmune diseases, and the like.

[0069] Since the DNA aptamer according to the present embodiment can selectively inhibit IFN-γ, it can be used as a research reagent for experiments involving IFN-γ. For example, whether in vitro or in vivo, the possibility that IFN-γ is involved in a physiological phenomenon of interest can be evaluated and examined by a test in which the DNA aptamer according to the present embodiment is applied, and the cause of the physiological phenomenon can be considered. In addition, the DNA aptamer according to the present embodiment can be added as a reagent to a cell culture medium or administered to an animal, and can be used in a wide range of tests including a reaction system that inhibits IFN-γ. EXAMPLES

[0070] Example 1: Synthesis of DNA aptamers Aptamer 1 and aptamer 2 were chemically synthesized according to the methods described in WO 2013 / 073602 and WO 2016 / 143700.

[0071] Example 2: Synthesis of aptamer 3 Using the methods described in WO 2013 / 073602 and WO 2016 / 143700, an amino-modifier C6-dT amide was introduced into the X position of the sequence of SEQ ID NO: 3 to synthesize aptamer 3. Other X substitutions can be synthesized by using commercially available artificial base or modified base amidites.

[0072] Example 3: Synthesis of PEG-modified DNA aptamers Aptamer 3 (1 eq) having a primary amine side chain at the base moiety of X produced in Example 2 and commercially available NHS-PEG (40000) (1.5 eq) were mixed in a phosphate buffer of pH 7 to 8 and stirred at room temperature for 1 day. The reaction solution was concentrated, and the resulting modified product was purified by reversed-phase HPLC to obtain a PEG-modified version of aptamer 3. It was confirmed by SPR (Surface Plasmon Resonance) that the resulting PEG-modified version of aptamer 3 retained its binding ability to IFN-γ.

[0073] Example 4: Confirmation of therapeutic effect using a humanized mouse model of alopecia areata Step 1: Preparation of a humanized mouse model of alopecia areata According to the method described in A. Gilhar, et. al., Journal of Investigative Dermatology, 2013, 133, 3, 844-847, a humanized mouse model of alopecia areata induced by intradermal injection of human activated lymphocytes into human scalp tissue transplanted into the mouse was prepared.

[0074] Process 2 The humanized mice of the alopecia areata model were divided into three groups, and each group was administered with vehicle (PBS), Dexamethasone + Minoxidil (positive control), and Aptamer 2. For the vehicle group, 15 μL of PBS was administered intradermally to the transplanted skin once every two days. For the group administered with Aptamer 2 (hereinafter referred to as the "aptamer administration group"), 15 μL of PBS solution of Aptamer 2 was administered intradermally to the transplanted skin once every two days, and the concentration of the aptamer 2 solution was gradually increased from 12 nM to 300 nM over 143 days. For the group administered with Dexamethasone + Minoxidil, 40 μL of the administration solution containing Dexamethasone 2 mg and 5% Minoxidil was applied to the transplanted skin once a day.

[0075] The results after administration in Example 4 are shown in Figure 1. Figure 1 shows the change in the number of hairs on the grafted skin tissue before administration and 143 days after the start of administration, with the vertical axis showing the change in the number of hairs per grafted skin tissue. In the vehicle group ("Vehicle" in Figure 1), hair loss further progressed during the PBS administration period, but in the positive control group ("Dexamethasone + Minoxidil" in Figure 1) and the aptamer administration group ("Aptamer" in Figure 1), further hair loss was suppressed and hair regrowth was observed.

[0076] Pathological analysis of hair follicle tissue showed a significant suppression of CD8+ T cell infiltration in both the positive control group and the aptamer-administered group. This suggests that the suppression of inflammatory responses in the positive control group and the aptamer-administered group inhibited the progression of hair loss and promoted hair regeneration.

[0077] Furthermore, the expression of MHC was examined by pathological analysis of hair follicle tissue after administration in Example 4. Figure 2A shows the results of MHC class I expression in the hair bulb root sheath, Figure 2B shows the results of MHC class I expression in the outer root sheath, and Figure 3A shows the results of MHC class II expression in the connective tissue root sheath and MHC class II expression in the outer root sheath. The vertical axis shows the expression level of MHC class I (Figures 2A and 2B) or class II (Figures 3A and 3B) as a relative value when the expression level in each vehicle group ("Vehicle" in Figures 2A, 2B, 3A, and 3B) is set to 1.

[0078] Both MHC class I and II were suppressed in expression only in the aptamer-administered group ("Aptamer" in Figures 2A, 2B, 3A, and 3B). This indicates that the mechanism by which inflammation was suppressed and hair regeneration was promoted is different between the positive control group ("Dexamethasone + Minoxidil" in Figures 2A, 2B, 3A, and 3B) and the aptamer-administered group. In the positive control group, direct inflammation suppression by activation of the glucocorticoid receptor was observed, while in the aptamer-administered group, inflammation was suppressed by suppressing the production of MHC class I and class II, which cause autoimmunity. In other words, in the aptamer-administered group, hair follicle tissue recovered from the breakdown of immune tolerance, and a more fundamental therapeutic effect was obtained. These results indicate that the DNA aptamer of this embodiment may be able to suppress inflammatory responses through a similar mechanism not only in autoimmune skin diseases, but also in autoimmune diseases that occur in other tissues and diseases such as Hanna type interstitial cystitis, which are thought to be primarily caused by excessive production of IFN-γ.

[0079] It was confirmed that the use of the therapeutic agent containing the DNA aptamer according to the present embodiment can suppress hair loss and promote hair regeneration.In addition, the results of pathological analysis confirmed that the administration of the DNA aptamer according to the present embodiment can almost completely suppress the expression of MHC class I and II.

[0080] Example 5: Production of mouse IFN-γ aptamer (surrogate aptamer) Process 1 The SELEX method was performed targeting mouse IFN-γ to obtain an aptamer. The obtained mouse IFN-γ aptamer is a DNA aptamer (aptamer 4 in Table 1) with 62 base residues, containing two Ds, and having a mini-hairpin sequence at the 3' end, and having the sequence shown in SEQ ID NO: 4. The binding ability to mouse IFN-γ was measured by the SPR method, and the KD value was 2.47 nM.

[0081] Process 2 We verified that the obtained aptamer inhibited the activity of mouse IFN-γ. Various molar concentrations of aptamer 4 were added simultaneously with 2 ng / mL mouse IFN-γ to L929 mouse fibroblasts, and after incubation at 37°C for 15 minutes, the inhibition of STAT1 phosphorylation by aptamer 4 was confirmed by flow cytometry using an anti-phosphorylated STAT1 antibody. The results are shown in Figures 4A and 4B. Figure 4A shows the results when aptamer 4 was added to L929 mouse fibroblasts, and Figure 4B shows the results when negative control DNA was added to L929 mouse fibroblasts. "Aptamer" in Figure 4A represents the surrogate aptamer according to this embodiment, and "Nc DNA" in Figure 4B represents the negative control DNA. "1eq", "5eq", "10eq", ​​"50eq", ​​and "100eq" in Figures 4A and 4B represent the molar concentration of the aptamer relative to the molar concentration of mouse IFN-γ (for example, 100eq represents aptamer:IFN-γ=100:1). By adding aptamer 4 in a molar concentration five times that of mouse IFN-γ, phosphorylation of STAT1 was almost completely inhibited (Figure 4A). In contrast, in the system where negative control DNA was added, STAT1 phosphorylation was not inhibited even when the negative control DNA was added in an amount 100 times that of mouse IFN-γ (Figure 4B). This result confirmed that the obtained aptamer 4 inhibited the activity of mouse IFN-γ and had sufficient activity as a surrogate aptamer.

[0082] Example 6: Examination of the effect of surrogate aptamers in a mouse autoimmune interstitial cystitis model Step 1: Preparation of autoimmune interstitial cystitis model mice (URO-OVA model) Autoimmune interstitial cystitis model mice (URO-OVA model) were created using the method described in Reference 1. 100μg of OVA antigen was subcutaneously injected into normal mice, and splenocytes were collected from the mice two weeks later. These splenocytes were transplanted intravenously at 5x10^7 cells per mouse into transgenic mice (URO-OVA mice) expressing OVA antigen in the bladder epithelium. In the URO-OVA mice transplanted with splenocytes, an immune response specific to the bladder epithelium was induced, causing inflammation.

[0083] Process 2 The surrogate aptamer was administered intravesically (10 nmol / time) to autoimmune interstitial cystitis model mice (URO-OVA model) once every 2 days for 3 weeks. The PBS-administered group (interstitial cystitis model mice) and the normal group (normal mice) were used as control groups. The normal group (normal mice) was not administered the surrogate aptamer or PBS. Figure 5A shows the results of measuring the number of urinations in a 24-hour period once a week. The PBS-administered group (interstitial cystitis model mice) (■) showed a significant increase in the number of urinations compared to the normal group (◆), whereas the increase in the number of urinations in the aptamer-administered group (●) was completely suppressed and was comparable to that of normal mice. Figure 5B shows the results of evaluating sensitivity to pelvic pain using an Electronic Von Frey once every 7 days. In the PBS-administered group (interstitial cystitis model mice) (■), sensitivity to pelvic pain was significantly increased (sensory threshold was decreased) compared to the normal group (◆), whereas the increase in sensitivity was significantly suppressed in the aptamer-administered group (●). From the above, it was found that the aptamer-administered group significantly suppressed the increase in urination frequency (Figure 5A) and the increase in sensitivity to pelvic pain (Figure 5B) in mice compared to the PBS-administered group, demonstrating a high effect in suppressing the onset of cystitis.

[0084] Process 3 After 3 weeks of observation from the start of administration of the aptamer or PBS, the mice were sacrificed and the bladders were histopathologically evaluated. The results are shown in Figure 6 and Table 2. As a result of HE staining, infiltration of immune cells (→), angiogenesis (*), mucosal congestion (>), and interstitial edema (**) were observed in the PBS-administered group, whereas these changes were hardly observed in the aptamer-administered group. Table 2 shows the inflammation scores of the bladder after the observation period. Grade 0 indicates a state without inflammatory findings, and from Grade 1 to 3, inflammatory findings such as infiltration of immune cells, angiogenesis, mucosal congestion, and interstitial edema are frequently observed. The inflammation score of the aptamer-administered group ("Cystitis / Aptamer" in Table 2) was significantly lower than that of the PBS-administered group ("Cystitis / PBS" in Table 2), and no difference was observed between the aptamer-administered group and the normal group ("Normal" in Table 2), indicating that bladder inflammation was suppressed by administration of the aptamer.

[0085] [Table 2]

[0086] The results of mRNA expression analysis in bladder tissue are shown in Figures 7A and 7B. Figure 7A shows the results of 1% agarose gel electrophoresis of the RT-PCR products, and Figure 7B is a graph quantifying the expression levels of the mRNA shown in Figure 7A. In the surrogate aptamer administration group ("Cystitis / Aptamer" in Figures 7A and 7B), the expression levels of IFN-γ and TNF-α, which are inflammatory cytokines induced by IFN-γ stimulation, and the expression levels of pre-SP and NGF, which are substances causing pain, in the bladder tissue were suppressed to near normal levels similar to those in the Normal group. This strongly suggests that the effects of surrogate aptamer administration on urination frequency, sensitivity to pelvic pain, and bladder inflammation are due to the inhibitory effect of IFN-γ activity in the tissue.

[0087] The results in the above-mentioned Example 4 and Example 6 are considered to be caused by the DNA aptamer according to the present embodiment strongly inhibiting the activity of IFN-γ.Therefore, by using the DNA aptamer according to the present embodiment, it is possible to provide an unprecedented effective therapeutic drug and treatment for the disease that is considered to be mainly caused by the overproduction of IFN-γ, such as alopecia areata and other autoimmune diseases and Hannah type interstitial cystitis.

[0088] In addition, from the results of the above examples, it is considered that the DNA aptamer according to this embodiment binds with high specificity to IFN-γ. Compared to Janus kinase inhibitors (Yvan Jamilloux, et.al., Autoimmunity Reviews, 2019, 18, 102390), which inevitably suppress multiple cytokine signals due to their pharmacological properties, the DNA aptamer according to this embodiment, which selectively suppresses IFN-γ activity, can reduce the possibility of side effects.

[0089] DNA aptamers generally have a low possibility of generating anti-DNA aptamer antibodies, and therefore the DNA aptamer according to this embodiment can be administered over a long period of time in the treatment of chronic inflammatory diseases.

[0090] Example 7: Confirmation of binding of the DNA aptamer according to this embodiment to canine and feline IFN-γ In order to confirm that the DNA aptamer according to this embodiment binds to canine and feline IFN-γ, an electrophoretic mobility shift assay (EMSA) was performed. 100 nM of aptamer 2 was mixed with 400 nM of canine IFN-γ or feline IFN-γ and left to stand at room temperature for 15 to 30 minutes. The obtained sample was subjected to 8 to 10% polyacrylamide gel electrophoresis and detected with SYBR Gold. The results are shown in FIG. 8. A shift band indicating a complex between aptamer 2 and canine IFN-γ (left in FIG. 8) and a shift band indicating a complex between aptamer 2 and feline IFN-γ (right in FIG. 8) were detected, confirming that aptamer 2 binds to both canine IFN-γ and feline IFN-γ. In addition, as a positive control for the EMSA test, a binding confirmation test was performed using aptamer 2 and human IFN-γ by the above-mentioned test method. As a result, a shifted band indicating a complex was detected, confirming the binding of aptamer 2 to human IFN-γ.

[0091] From the results of Example 7, it is presumed that the DNA aptamer according to this embodiment inhibits the activity of IFN-γ in dogs and cats. Therefore, by using this DNA aptamer, it is possible to provide an effective therapeutic agent and treatment method for idiopathic cystitis, which is one of the lower urinary tract diseases caused by the bladder in dogs and cats, diseases related to IFN-γ, and autoimmune diseases.

[0092] The DNA aptamer according to the present embodiment can be produced by chemical synthesis, and therefore can be provided as a safe drug with stable quality and low risk of biological contamination.

[0093] The DNA aptamer according to the present embodiment can be produced at a low cost compared to biological preparations. Furthermore, while biological preparations require low temperature conditions for storage and transportation, DNA aptamers are stable even at room temperature. Therefore, a cold chain is not necessarily required for transportation and storage of the preparation containing the DNA aptamer according to the present embodiment.

[0094] By preparing the DNA aptamer of this embodiment as a transdermal preparation, it is possible to provide a therapeutic drug that is easy to administer, has low risk of side effects, and is non-invasive.

[0095] By administering the therapeutic agent containing the DNA aptamer according to the present embodiment as an injection, it is possible to treat diseases such as systemic autoimmune diseases and Hanna-type interstitial cystitis, which are believed to be mainly caused by excessive production of IFN-γ. In addition, when the therapeutic agent is an injection, it is possible to prepare a formulation that is easy for patients to use, such as a prefilled syringe that can be stored at room temperature.

[0096] Since the DNA aptamer according to the present embodiment can selectively inhibit IFN-γ, it can be used as a research reagent for experiments on systems in which IFN-γ may be involved. For example, whether in vitro or in vivo, the possibility that IFN-γ may be involved in a physiological phenomenon of interest can be clarified by applying the DNA aptamer according to the present embodiment, and the cause of the physiological phenomenon can be considered. In addition, it can be used in a wide range of tests including reaction systems that inhibit IFN-γ by adding it to cell culture medium as a reagent or administering it to animals.

Claims

1. A therapeutic agent for Hanna-type interstitial cystitis, comprising as an active ingredient a DNA oligonucleotide having a base sequence shown in any one of SEQ ID NOs: 1 to 3 and selectively binding to interferon-γ (IFN-γ).

2. 2. The therapeutic agent for Hanna type interstitial cystitis according to claim 1, wherein the base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificially produced base, and the artificially produced base is chemically modified with a low molecular weight compound.

3. The therapeutic agent for Hanna-type interstitial cystitis according to claim 2, wherein the low molecular weight compound is an anti-inflammatory compound selected from the group consisting of glucocorticoid, tacrolimus, sirolimus, cyclosporine, methotrexate, and leflunomide.

4. 2. The therapeutic agent for Hanna type interstitial cystitis according to claim 1, wherein the base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificially produced base, and the artificially produced base is chemically modified with a medium molecular compound, a high molecular compound, a biopolymer, or a biocompatible polymer.

5. The Hanna-type interstitial cystitis therapeutic agent according to claim 4, wherein the polymer compound is polyethylene glycol (PEG) having a molecular weight of 20,000 or more, or any biocompatible polymer having a molecular weight of 20,000 or more.

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