Therapeutic agent for disease selected from IFN-γ-related diseases in dogs and cats, lower urinary tract diseases caused by the urinary bladder in dogs and cats, or autoimmune diseases in dogs and cats, comprising DNA oligonucleotide that selectively binds to IFN-γ

Abstract

To provide a therapeutic agent for Hanna-type interstitial cystitis that can selectively inhibit IFN-γ, is free from biological contamination, and can be stored at room temperature.SOLUTION: Provided is a therapeutic agent or research reagent for Hannah-type interstitial cystitis, which contains as an active ingredient a DNA oligonucleotide that has a base sequence set forth in any one of SEQ ID NOs: 1 to 3 and selectively binds to IFN-γ. In the DNA oligonucleotide having the base sequence set forth in SEQ ID NO: 3, 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 high-molecular-weight 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 contains a DNA oligonucleotide that selectively binds to interferon-γ (IFN-γ). [Background technology]

[0002] Hanna type interstitial cystitis (Hanna type interstitial cystitis) was designated an intractable disease by the Minister of Health, Labor and Welfare in May 2014 (designated intractable disease 226). While the exact number of patients with Hanna type interstitial cystitis in Japan is unknown, according to a Ministry of Health, Labor and Welfare health administrative report, the number of Hanna type interstitial cystitis patients holding certificates for specific medical expenses (designated intractable disease) has increased since fiscal year 2015, after the designation, reaching 863 in fiscal year 2020 (Non-Patent Document 1). It is believed that many patients do not hold such certificates. According to a survey by the Japan Interstitial Cystitis Study Group, the total number of interstitial cystitis patients in Japan, including Hanna type and non-Hanna type, is estimated to be approximately 4,500, of which 45% (approximately 2,000) are Hanna type patients. The male-to-female ratio of Hanna type patients is 1:5.6, with a female predominance, and it is known that the disease is particularly prevalent among middle-aged and elderly people (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 or pain in the bladder or urethra. Interstitial cystitis has been classified into Hanna type and non-Hanna type based on the type of disease. In Hanna type, a unique abnormal inflammatory pathology called Hanna lesions is seen on bladder endoscopy. On the other hand, in non-Hanna type, no inflammatory lesions are seen in the bladder, and it has been determined that this is a completely different type of disease from Hanna type. Currently, when people talk about interstitial cystitis, they are referring to Hanna type.

[0004] A fundamental treatment for Hanna-type interstitial cystitis has yet to be found. Clinical practice guidelines (Non-Patent Document 3) list conservative treatment (symptomatic treatment), oral medication, intravesical instillation, and surgical treatment, including endoscopic therapy, as methods for symptomatic improvement. Until now, the only treatment covered by insurance for interstitial cystitis was hydrodistention. However, hydrodistention is highly invasive, carries the risk of bladder rupture as a side effect, and the procedure places significant physical and time burdens on patients. In January 2021, Dimso® Intravesical Solution 50% (injected into the bladder six times every two weeks) was approved as the only treatment for interstitial cystitis in Japan. However, the mechanism of action of dimethyl sulfoxide (DMSO), the active ingredient in Dimso® Intravesical Solution 50%, on interstitial cystitis remains unclear. While it has some efficacy against pain and inflammation, problems remain, such as a short duration of response and repeated recurrence.

[0005] Symptomatic treatment involves explaining the condition and providing dietary advice. Oral medications include analgesics, antidepressants, antiallergic drugs, and steroids. The aforementioned hydrodistention of the bladder is widely used as an endoscopic treatment. If Hanna lesions are found in the bladder during this procedure, electrical or laser surgery (cauterization) is performed, but repeated surgery over a long period of time can lead to bladder atrophy. Hydrodistention of the bladder or cauterization of Hanna lesions results in symptomatic relief in approximately half of cases, but long-term relief 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. Intramural injection of botulinum toxin may also be performed. In cases where these treatments are resistant and intolerable symptoms persist, radical cystectomy and urinary diversion may be indicated. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ministry of Health, Labour 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] Clinical Practice Guidelines for Interstitial Cystitis and Bladder Pain Syndrome (edited by the Japanese Interstitial Cystitis Study Group / Japanese 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 patients' quality of life (QOL), the development of highly effective treatments or cures is necessary.

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

[0010] It has been reported that Hanna-type interstitial cystitis frequently coexists with systemic autoimmune diseases such as Sjögren's syndrome, autoimmune thyroiditis, and systemic lupus erythematosus. Furthermore, autoantibodies against the urothelium have been reported in the blood of patients with Hanna-type interstitial cystitis (Y. Akiyama, et al., International Journal of Urology, 2020, 27, 491-503). Furthermore, gene expression analysis of bladder samples collected from patients with Hanna-type interstitial cystitis has shown significant increases in the expression of inflammation-related molecules, such as IFN-γ, CXCR3, CXCL9, CXCL10, CXCL11, TNF-α, and TNFSF14 (T. Ogawa, et al., The Journal of Urology, 2010, 183, 1206-1212). Most of these molecules are IFN-γ and its cascade components. Considering that Hanna type interstitial cystitis is accompanied by a systemic autoimmune disease, it is thought that IFN-γ plays a central role in the pathogenesis, just 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, anti-IFN-γ antibodies, for example, have the following issues: (1) they are biological products, which means there is a risk of biological contamination, (2) antigenicity is an issue with long-term administration, (3) they are protein products, which means 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 around 30%. Therefore, when long-term treatment is required, antibodies against antibodies are produced, which can cause anaphylactic reactions, making it difficult to continue treatment.

[0013] Regarding issue (1), 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 increases transportation and storage costs and reduces convenience for patients who use them.

[0014] Four Janus kinase inhibitors, namely, tofacitinib (product name: Xeljanz®), baricitinib (product name: Olumiant®), peficitinib (product name: Smilaf®), and upadacitinib (product name: Rinvoq®), are commercially available for use in the treatment of rheumatoid arthritis, an autoimmune disease. These Janus kinase inhibitors are small molecules that can be produced by chemical synthesis, and are therefore thought to be free of the aforementioned problems associated with antibodies. Meanwhile, Janus kinases exist in multiple subtypes, and are activated by binding to the intracellular domains of multiple cytokine receptors, including not only the IFN-γ receptor but also the interleukin-2 (IL-2) receptor, interleukin-4 (IL-4) receptor, interleukin-7 (IL-7) receptor, and interferon-α (IFN-α) receptor, thereby transducing receptor signals.

[0015] Therefore, 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 and suggests the possibility of unexpected side effects such as increased susceptibility to infection.

[0016] The present invention has been made in view 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 from the risk 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] A first reference aspect of the present invention provides a therapeutic agent for Hanna type interstitial cystitis, which comprises a DNA oligonucleotide that has a base sequence set forth 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 exerts a therapeutic effect for Hanna type interstitial cystitis by selectively binding to IFN-γ and inhibiting its activity.

[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 embodiment 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 reference 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.

[0023] In the first embodiment 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 medium-sized compound, a high-molecular-weight compound, a biopolymer, or a biocompatible polymer. In this embodiment, the medium-sized compound has a molecular weight of about 1,000 to 20,000, and the high-molecular-weight compound has a molecular weight of about 20,000 to 400,000.

[0024] The polymer compound in the above embodiment may be any biocompatible polymer with a molecular weight of 20,000 or more. Examples of medium-sized or high-molecular-weight compounds in this embodiment include, but are not limited to, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, and antibodies. While antibodies belong to the polymer category, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides belong to the medium-sized or high-molecular-weight compounds depending on their molecular weight. The molecular weight of a medium-sized or high-molecular-weight compound is expressed as the average molecular weight, defined as the number-average molecular weight (Mn) or the weight-average molecular weight (Mw).

[0025] A first aspect of the present invention is a therapeutic agent for a disease selected from canine and feline IFN-γ-related diseases, canine and feline lower urinary tract diseases caused by the bladder, and canine and feline autoimmune diseases, which comprises as an active ingredient a DNA oligonucleotide having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 3 and selectively binding to canine and feline IFN-γ. An example of a canine and feline lower urinary tract disease caused by the bladder is idiopathic cystitis.

[0026] In the first aspect, 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 aspect 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 first aspect, 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-sized compound, a high-molecular-weight compound, a biopolymer, or a biocompatible polymer. The medium-sized compound in this aspect has a molecular weight of approximately 1,000 to 20,000, and the high-molecular-weight compound in this aspect has a molecular weight of approximately 20,000 to 400,000. The high-molecular-weight compound in the above aspect may be any biocompatible polymer having a molecular weight of 20,000 or more. Examples of medium-sized or high-molecular-weight compounds in this aspect include, but are not limited to, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, and antibodies. While antibodies are classified as high-molecular-weight compounds, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides are classified as medium-sized or high-molecular-weight compounds depending on their molecular weight.

[0028] A second embodiment of the present invention is a reagent for research and testing containing, as an active ingredient, a DNA oligonucleotide that has a base sequence shown in any one of SEQ ID NOs: 1 to 3 and selectively binds to IFN-γ.

[0029] 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.

[0030] 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-sized compound, a high-molecular-weight compound, a biopolymer, or a biocompatible polymer. The medium-sized compound in this embodiment has a molecular weight of approximately 1,000 to 20,000, and the high-molecular-weight compound in this embodiment has a molecular weight of approximately 20,000 to 400,000. The high-molecular-weight compound in the above embodiment may be any biocompatible polymer having a molecular weight of 20,000 or more. Examples of medium-sized or high-molecular-weight compounds in this embodiment include, but are not limited to, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, and antibodies. While antibodies are classified as high-molecular-weight compounds, PEG, dipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides are classified as medium-molecular-weight or high-molecular-weight compounds depending on their molecular weight. [Effects of the Invention]

[0031] The DNA oligonucleotide having the base sequence of the present invention selectively binds to IFN-γ, thereby selectively inhibiting the activity of IFN-γ. Furthermore, a therapeutic agent for Hanna-type interstitial cystitis containing a DNA oligonucleotide having the base sequence of the present invention does not require the use of serum or the like in its production, and therefore can be produced without the risk of biological contamination by viruses or the like. Furthermore, the DNA oligonucleotide having the base sequence of the present invention can be stored at room temperature. This not only offers advantages over conventional methods in terms of transportation and storage costs, but also improves patient convenience. Furthermore, due to its molecular weight, a therapeutic agent for Hanna-type interstitial cystitis containing a DNA oligonucleotide having the base sequence of the present invention can be administered by intravesical infusion.

[0032] The DNA oligonucleotides of the present invention do not inhibit the signaling of IL-2, IL-4, IL-7, IFN-α, etc., but only inhibit the activity of IFN-γ. Therefore, a therapeutic agent for Hanna-type interstitial cystitis containing a 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 over a long period of time, compared to Janus kinase inhibitors, etc. Furthermore, compared to anti-IFN-γ antibodies, the DNA oligonucleotides of the present invention have lower antigenicity, making them suitable for long-term use. Furthermore, the DNA oligonucleotides of the present invention can be stored at room temperature without risk of biological contamination during production. [Brief explanation 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 experimental results regarding the expression of MHC class I in the hair bulb root sheath following administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 2B]FIG. 1 shows experimental results regarding the expression of MHC class I in the outer root sheath following administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 3A] FIG. 1 shows experimental results regarding the expression of MHC class II in the connective tissue root sheath following administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 3B] FIG. 1 shows experimental results regarding the expression of MHC class II in the outer root sheath following administration of a DNA oligonucleotide according to one embodiment of the present invention. [Figure 4A] FIG. 1 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 fibroblasts. [Figure 4B] FIG. 1 shows the results of the effect of adding 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 model of interstitial cystitis. [Figure 5B] FIG. 10 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 model of interstitial cystitis. [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 model of interstitial cystitis. [Figure 7A] 1 is a diagram showing a comparison of mRNA expression levels in bladder tissue 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 tissue were suppressed to near normal levels, similar to those in the normal group. [Figure 7B]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 present invention to canine and feline IFN-γ. DETAILED DESCRIPTION OF THE INVENTION

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

[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 product, antigenicity during long-term administration, inadequacy for topical administration such as transdermal or transmucosal administration due to their large molecular weight, and storage and transportation conditions due to being protein products. Therefore, there is a need to develop a therapeutic drug that can resolve these issues and effectively inhibit IFN-γ.

[0037] To solve the above-mentioned problems, the present inventors attempted to develop an IFN-γ inhibitor using the DNA oligonucleotide of the present invention as a DNA aptamer. A DNA aptamer is a ligand molecule that specifically and strongly binds to a target molecule through the formation of a secondary or tertiary structure by complementary sequences within the DNA oligonucleotide molecule. By binding a DNA aptamer with a specific sequence, the activity of a target molecule can be inhibited or suppressed, but can also be enhanced. Despite being approximately one-tenth the molecular weight of an antibody, DNA aptamers possess high affinity and target selectivity comparable to antibodies. Therefore, IFN-γ inhibitors using DNA aptamers can minimize the occurrence of off-target side effects. Furthermore, because DNA aptamers can be produced by chemical synthesis, they are considered to be a suitable modality for solving these problems. As used herein, "selectively binds to IFN-γ" refers to the strong and specific binding of the DNA oligonucleotide of the present invention to the target substance IFN-γ as a DNA aptamer.

[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 and are expected to be useful: (1) their relatively small molecular weight allows them to be administered as transdermal or transmucosal preparations such as ointments and patches; (2) because they are chemically synthesized, there is a low risk of biological contamination; (3) they are generally low in antigenicity; (4) because they are DNA, they are sufficiently stable at room temperature under near-neutral conditions in the absence of nucleic acid degrading enzymes (nucleases); and (5) they have almost no inhibitory activity against cytochrome P450, a drug-metabolizing enzyme, and therefore do not affect concomitant medications. Furthermore, DNA aptamers do not pose the problem of antibody production against antibody drugs, which is one of the issues that arise when long-term antibody-based treatments are required, making them suitable for long-term administration.

[0040] A specific method for treating diseases using DNA aptamers is expected to be 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] Because DNA aptamers are rapidly degraded by nucleic acid-degrading enzymes (nucleases) in biological tissues, even if they exhibit strong activity in vitro, this does not necessarily mean that they will exhibit the same activity in vivo. Therefore, the present inventors discovered that a DNA aptamer having the base sequence of SEQ ID NO: 2 in Table 1, which has a nine-residue natural base sequence attached to the 3' end, acquires nuclease resistance and is stable in biological tissues. A DNA aptamer having the base sequence of SEQ ID NO: 2, which contains two Ds in its sequence, demonstrated efficacy in an autoimmune humanized mouse model of alopecia areata transplanted with human scalp tissue, and was confirmed to exist stably in biological tissues and to be able to inhibit IFN-γ (Patent Application No. 2021-166794). Furthermore, for a DNA aptamer having the sequence of SEQ ID NO: 3, 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 by SPR (Surface Plasmon Resonance) that the PEG-modified version, in which polyethylene glycol (PEG) is added to the base moiety of X, retains its IFN-γ binding ability. This indicates that modification of the arbitrary base X does not affect the IFN-γ binding activity of the DNA aptamer having the sequence of SEQ ID NO: 3, i.e., 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 the base sequence shown in Table 1 is used as a DNA aptamer. This embodiment includes the use of a DNA oligonucleotide having the 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 nine 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 substituted with an arbitrary base X. X represents any natural base, any unnatural base, or modified base, or a modified base to which a polymeric compound (biopolymer) used in living organisms, such as a low molecular weight compound, peptide, oligonucleic acid, oligosaccharide, or protein, or a biocompatible polymer, is bound. Details of the base sequence of SEQ ID NO: 4 will be described later.

[0046] Examples of polymeric compounds that can be bound to modified bases include polyethylene glycol (PEG) with a molecular weight of 20,000 or more, and any biocompatible polymer with a molecular weight of 20,000 or more. Biocompatible polymers are chemically synthesized products that are not normally used in vivo and are safe enough to be introduced into the body without causing inflammation or toxic reactions. Examples of medium-sized molecules include peptides, oligonucleotides, oligosaccharides, proteins, PEG, and any biocompatible polymers with a molecular weight greater 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 activated esters, alkynyl groups (-CC) or cyclic structures containing alkynyl structures, formyl groups (-CHO), hydrazide groups (-NH-NH2), hydroxyl groups (-OH), thiol groups (-SH), cyano groups (-CN), vinyl groups (-CHCH2), and maleimide groups.

[0048] As used herein, "natural base" refers to any of adenine, guanosine, cytosine, and thymine. As used herein, "unnatural base" refers to a base artificially synthesized and possessing properties similar to those of natural bases, and is sometimes referred to herein as "artificial base." As used herein, "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 subjected to methylation, deamination, atomic position swapping, thiolation of the oxygen at the phosphate moiety, and introduction of water-soluble or lipophilic substituents into the base moiety. Specific examples include modified pyrimidines, modified purines, and other heterocyclic bases. Ds in the sequences of SEQ ID NOs: 1 to 3 represents the artificial base 7-(2-thienyl)imidazo[4,5-b]pyridine. In addition to Ds itself, bases with side chains added to Ds may also be used as artificial bases. Hereinafter, in this embodiment, the DNA aptamers having the sequences shown in SEQ ID NOs: 1, 2, 3, and 4 in Table 1 will be referred to as "aptamer 1," "aptamer 2," "aptamer 3," and "aptamer 4," respectively.

[0049] Regarding the usefulness of the DNA aptamer according to this embodiment, in this specification, it was confirmed that intradermal injection of the DNA aptamer (aptamer 2) shown in Table 1 into an autoimmune alopecia model of immunotolerant mice transplanted with a human scalp tissue fragment promoted hair regeneration and inhibited further hair loss (Example 4). Details will be described later.

[0050] Pathological analysis of the mechanism of aptamer 2 activity in this model revealed that aptamer 2 almost completely suppressed the expression of MHC class I and II. Detailed results are described below. In other words, aptamer 2 inhibits the activity of IFN-γ, thereby suppressing the production of MHC class I and II, which are the basis for the expression of autoimmunity, thereby improving the symptoms of alopecia areata. This suggests that aptamer 2 may be a means of solving problems not only in alopecia areata but also in Hanna-type interstitial cystitis, which is thought to be primarily caused by excessive production of IFN-γ, by providing an IFN-γ inhibitor using a DNA aptamer.

[0051] To determine whether DNA aptamers that inhibit IFN-γ activity are effective in treating Hanna-type interstitial cystitis, their efficacy must be confirmed in animal models of the disease before their efficacy can be confirmed in humans. The currently established animal model that most closely resembles the pathology of human Hanna-type interstitial cystitis is the mouse autoimmune interstitial cystitis model (a model using transgenic mice (URO-OVA mice) expressing 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 DNA aptamer specifically binding to human IFN-γ did not exhibit binding activity to mouse IFN-γ. Therefore, the efficacy of the DNA aptamer cannot be confirmed in mouse models.

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

[0053] The criteria for a surrogate aptamer to exhibit high similarity in physical properties to 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 similar in structure 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 met the above criteria, a DNA aptamer (aptamer 4) was obtained, which has the sequence set forth in SEQ ID NO: 4 in Table 1, with 62 bases, two Ds, and a mini-hairpin sequence at the 3' end (Example 5). Details are described below. The resulting DNA aptamer had a KD value of 2.47 nM, indicating its binding ability to mouse IFN-γ, approximately 1 / 100 of the binding ability of aptamer 2 to human IFN-γ (KD value: 33 pM). Furthermore, when added at a molar concentration five times higher than that of mouse IFN-γ, the aptamer competitively inhibited and almost completely suppressed the activity of mouse IFN-γ. These results confirmed that the DNA aptamer obtained by this search possessed sufficient activity to function as a surrogate aptamer.

[0055] The obtained surrogate aptamer was intravesically administered to mice with the 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 sensitivity to pelvic pain in the mice compared to the control PBS-administered group, demonstrating a high effect in suppressing the onset of cystitis (Example 6).

[0056] Histopathological evaluation of the bladder revealed that the aptamer-treated group had significantly lower bladder inflammation scores than the PBS-treated group, with no significant difference from the normal group, indicating that administration of the surrogate aptamer suppressed bladder inflammation. Furthermore, mRNA expression analysis in bladder tissue revealed that administration of the surrogate aptamer suppressed the expression of IFN-γ-induced inflammatory cytokines such as IFN-γ and TNF-α, as well as the expression of pain-causing substances pre-SP and NGF. This strongly suggests that the inhibitory effect of the surrogate aptamer on bladder inflammation is due to its inhibitory effect on IFN-γ activity in the tissue (Example 6).

[0057] The DNA aptamer of the present invention exhibits an inhibitory effect against human IFN-γ in human tissue, and given the high similarity in physical properties between the DNA aptamer and the surrogate aptamer, it is inferred that, like the surrogate aptamer, the DNA aptamer has the effect of suppressing 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 is, or a modified version of the DNA aptamer at a site that does not affect its activity can also be used. Examples of modified DNA aptamers include those to which a medium-sized or high-molecular compound such as PEG, peptide, or oligonucleotide is chemically bonded; those to which the same DNA aptamers are chemically multimerized; and those to which a portion of the DNA aptamer sequence has been converted or modified. When the DNA oligonucleotide according to this embodiment is modified and used as a DNA aptamer, the base moiety is preferably used as the modified moiety. Modification with artificial or modified bases can be performed using existing methods, and the 3' and 5' ends can also be modified.

[0059] PEG-modified DNA aptamers have been used to improve the pharmacokinetic (PK)-pharmacodynamic (PD) profiles of proteins, peptides, and oligonucleotides, including aptamers, for the purpose of improving their in vivo disposition. Many PEG-modified aptamers have been developed to date. When PEG-modified aptamers retain their binding activity to their target proteins, they exhibit activity equivalent to that of unmodified aptamers in vivo, and PEG-modified aptamers are known to be largely toxic (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 injection preparations, such as vials containing lyophilized powder, vials containing aptamer solutions, or pre-filled syringes.

[0061] The DNA aptamer of this embodiment can be manufactured as 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 possible local administration method is administration via the mucosa. The DNA aptamer of this embodiment can be dissolved in a solvent such as a buffer solution with high biocompatibility and used as a transmucosal administration agent, such as intravesical administration.

[0064] For injectable formulations, inhaled formulations, and eye drops, the DNA aptamer of this embodiment can be encapsulated or attached to fatty 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.

[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 poultice.

[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 using a biodegradable polymer such as polylactic acid or liposomes to form nanoparticles can be applied, and these processes can be combined appropriately depending on the purpose.

[0067] The DNA aptamer of this embodiment can also be used as an administration formulation using devices compatible with physical transdermal absorption enhancement methods such as iontophoresis, electroporation, thermalporation, sonophoresis, microneedle array patch, needleless syringe, and micropump.

[0068] The DNA aptamer of this embodiment was confirmed to bind not only to human IFN-γ, but also to canine IFN-γ and feline IFN-γ (Example 7). Details will be described later. Here, idiopathic cystitis, a type of lower urinary tract disease caused by the bladder in dogs and cats, is presumed to be related to 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 inhibitory effect on the activity of canine IFN-γ and feline IFN-γ. This suggests that it has an inhibitory effect on the onset of idiopathic cystitis, a type of lower urinary tract disease 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, IFN-γ-related diseases, autoimmune diseases, and the like in dogs and cats.

[0069] Because 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 by tests using the DNA aptamer according to the present embodiment, and the cause of the physiological phenomenon can be considered. Furthermore, the DNA aptamer according to the present embodiment can be added as a reagent to cell culture media or administered to animals, and used in a wide range of tests, including reaction systems for inhibiting IFN-γ. [Example]

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

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

[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, as 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 reverse-phase HPLC to obtain a PEG-modified version of aptamer 3. The resulting PEG-modified version of aptamer 3 was confirmed to retain its binding ability to IFN-γ using SPR (Surface Plasmon Resonance).

[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 was created by intradermal injection of human activated lymphocytes into human scalp tissue transplanted into the mouse.

[0074] Process 2 The humanized mice model of alopecia areata were divided into three groups and administered vehicle (PBS), dexamethasone + minoxidil (positive control), or aptamer 2. The vehicle group received 15 μL of PBS intradermally administered to the grafted skin every two days. The aptamer 2 group (hereafter referred to as the "aptamer group") received 15 μL of aptamer 2 in PBS intradermally administered to the grafted skin every two days, with the aptamer 2 solution concentration gradually increasing from 12 nM to 300 nM over 143 days. The dexamethasone + minoxidil group received 40 μL of a solution containing 2 mg of dexamethasone and 5% minoxidil applied to the grafted skin every 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-administered 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 slowed the progression of hair loss and promoted hair regeneration.

[0077] Furthermore, MHC expression was examined by pathological analysis of hair follicle tissue after administration in Example 4. Figure 2A shows the results for MHC class I expression in the hair bulb root sheath, Figure 2B shows the results for MHC class I expression in the outer root sheath, and Figure 3A shows the results for 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, with the expression level in the respective vehicle group ("Vehicle" in Figures 2A, 2B, 3A, and 3B) set to 1.

[0078] Suppression of both MHC class I and II expression was observed 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 promoted differed between the positive control group ("Dexamethasone + Minoxidil" in Figures 2A, 2B, 3A, and 3B) and the aptamer-administered group. The positive control group demonstrated direct inflammation suppression through activation of the glucocorticoid receptor, whereas the aptamer-administered group demonstrated inflammation suppression through the suppression of MHC class I and class II production, which are the cause of autoimmunity. In other words, the hair follicle tissue in the aptamer-administered group recovered from the breakdown of immune tolerance, resulting in a more fundamental therapeutic effect. 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 a therapeutic agent containing the DNA aptamer according to the present embodiment suppresses hair loss and promotes hair regeneration. Furthermore, pathological analysis confirmed that 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 We performed SELEX targeting mouse IFN-γ to obtain an aptamer. The resulting mouse IFN-γ aptamer is a DNA aptamer with the sequence set forth in SEQ ID NO: 4 (aptamer 4 in Table 1), which has 62 bases, two Ds, and a mini-hairpin sequence at the 3' end. The binding ability to mouse IFN-γ was measured by SPR, and the KD value was 2.47 nM.

[0081] Process 2 The obtained aptamer was verified to inhibit the activity of mouse IFN-γ. L929 mouse fibroblasts were incubated with 2 ng / mL mouse IFN-γ and various molar concentrations of aptamer 4 at the same time. After 15 minutes of incubation at 37°C, 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 of this embodiment, and "Nc DNA" in Figure 4B represents the negative control DNA. In Figures 4A and 4B, "1 eq," "5 eq," "10 eq," "50 eq," and "100 eq" represent the molar concentration of the aptamer relative to the molar concentration of mouse IFN-γ (for example, 100 eq represents an aptamer:IFN-γ ratio of 100:1). Addition of aptamer 4 at a molar concentration five times that of mouse IFN-γ almost completely inhibited STAT1 phosphorylation (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 greater than 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 (URO-OVA model) mice A mouse model of autoimmune interstitial cystitis (URO-OVA model) was generated using the method described in Reference 1. Normal mice were subcutaneously injected with 100 μg of OVA antigen, and splenocytes were collected from the mice two weeks later. These splenocytes were then transplanted intravenously at a rate of 5 × 10^7 cells per mouse into transgenic mice (URO-OVA mice) expressing OVA antigen in the bladder epithelium. In the URO-OVA mice that received the splenocyte transplant, an immune response specific to the bladder epithelium was induced, resulting in inflammation.

[0083] Process 2 The surrogate aptamer was administered intravesically (10 nmol / dose) to mice with an autoimmune interstitial cystitis model (URO-OVA model) every other day for three weeks. A PBS-administered group (interstitial cystitis model mice) and a normal group (normal mice) served as controls. The normal group (normal mice) received no surrogate aptamer or PBS. Figure 5A shows the results of measuring the number of urinations per 24 hours once a week. The PBS-administered group (interstitial cystitis model mice) (■) showed a significant increase in urination frequency compared to the normal group (◆), whereas the aptamer-administered group (●) completely suppressed the increase in urination frequency, reaching levels comparable to those of normal mice. Figure 5B shows the results of assessing pelvic pain sensitivity using an Electronic Von Frey monitor every seven days. The PBS-administered group (interstitial cystitis model mice) (■) showed a significantly increased sensitivity to pelvic pain (decreased sensory threshold) 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 a 3-week observation period following the start of aptamer or PBS administration, mice were sacrificed and their bladders were subjected to histopathological evaluation. The results are shown in Figure 6 and Table 2. HE staining revealed immune cell infiltration (→), angiogenesis (*), mucosal congestion (>), and interstitial edema (**) in the PBS-treated group, whereas these changes were largely absent in the aptamer-treated group. Table 2 shows the bladder inflammation scores after the observation period. Grade 0 indicates no inflammatory findings, while Grades 1 to 3 frequently show inflammatory findings such as immune cell infiltration, angiogenesis, mucosal congestion, and interstitial edema. The aptamer-treated group (Table 2, "Cystitis / Aptamer") had significantly lower inflammation scores than the PBS-treated group (Table 2, "Cystitis / PBS") and showed no difference from the normal group (Table 2, "Normal"), indicating that aptamer administration suppressed bladder inflammation.

[0085] [Table 2]

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

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

[0088] Furthermore, based on the results of the above examples, it is believed that the DNA aptamer according to this embodiment binds to IFN-γ with high specificity. 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 likelihood of side effects.

[0089] DNA aptamers generally have a low possibility of generating anti-DNA aptamer antibodies, which makes it possible to administer the DNA aptamer of this embodiment 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 To confirm the binding of the DNA aptamer according to this embodiment 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 allowed to stand at room temperature for 15 to 30 minutes. The resulting sample was subjected to 8-10% polyacrylamide gel electrophoresis and detected with SYBR Gold. The results are shown in Figure 8. A shifted band indicating a complex between aptamer 2 and canine IFN-γ (Figure 8, left) and a shifted band indicating a complex between aptamer 2 and feline IFN-γ (Figure 8, right) were detected, confirming that aptamer 2 binds to both canine IFN-γ and feline IFN-γ. Additionally, as a positive control for the EMSA test, a binding confirmation test was performed using aptamer 2 and human IFN-γ using the aforementioned 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, the DNA aptamer can be used to provide effective therapeutic agents and treatments for idiopathic cystitis, a type of lower urinary tract disease caused by the bladder in dogs and cats, as well as IFN-γ-related diseases and autoimmune diseases.

[0092] The DNA aptamer according to this 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 this embodiment can be produced at a lower cost than biological preparations. Furthermore, while biological preparations require low-temperature conditions for storage and transportation, DNA aptamers are stable at room temperature. Therefore, a cold chain is not necessarily required for the transportation and storage of a preparation containing the DNA aptamer according to this embodiment.

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

[0095] By systemically administering a therapeutic agent containing the DNA aptamer according to the present embodiment as an injection, it can be applied to diseases in which excessive production of IFN-γ is thought to be the main cause, such as systemic autoimmune diseases and Hanna-type interstitial cystitis. Furthermore, even when used as an injection, it can be made into a formulation that is easy for patients to use, such as a prefilled syringe that can be stored at room temperature.

[0096] Because 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 involvement of IFN-γ in a physiological phenomenon of interest can be clarified by applying the DNA aptamer according to the present embodiment, allowing for consideration of the cause of the physiological phenomenon. Furthermore, the aptamer can be added as a reagent to cell culture media or administered to animals for use in a wide range of tests involving reaction systems that inhibit IFN-γ.

Claims

1. A therapeutic agent for a disease selected from diseases associated with IFN-γ in dogs and cats, lower urinary tract diseases caused by the bladder in dogs and cats, and autoimmune diseases in dogs and cats, 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 IFN-γ in dogs and cats.

2. 2. The therapeutic agent 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. 2. The therapeutic agent 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.

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