Hunner type interstitial cystitis treatment medicine containing DNA oligonucleotide selectively combined with IFN-gamma

Abstract

Provided is a drug for the treatment of Hunner-type interstitial cystitis, which can selectively inhibit I FN-gamma, has no risk of biological contamination, and can be stored at room temperature. Provided is a drug for the treatment of Hunner-type interstitial cystitis or a reagent for experimental research, which contains, as an active ingredient, a DNA oligonucleotide that has a base sequence depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 3 and that selectively binds to I FN-[gamma]. In the DNA oligonucleotide containing the base sequence represented by SEQ ID NO: 3, the base X in the sequence is an artificially produced base, and the artificially produced base can be chemically modified by a low-molecular compound, a middle-molecular compound, a high-molecular compound, a biopolymer or a biocompatible polymer.

Description

Technical Field

[0001] The present invention relates to a therapeutic drug for Hunner type interstitial cystitis, which contains a DNA oligonucleotide which selectively combines with interferon gamma (IFN-gamma). Background Art

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

[0003] Interstitial cystitis is a disease in which inflammation of the bladder occurs due to unknown causes, resulting in uncomfortable symptoms such as frequent urination, discomfort or pain in the bladder and urethra. Interstitial cystitis is divided into Hunner type and non-Hunner type according to the type of disease. In the Hunner type, endoscopy of the bladder can reveal a unique abnormal inflammatory pathological pattern called Hunner lesions. On the other hand, in the non-Hunner type, no inflammatory lesions are found in the bladder, indicating that this is a completely different type of disease from the Hunner type. Today, the term interstitial cystitis refers to the Hunner type.

[0004] No radical treatment for Hunner type interstitial cystitis has been found so far. The diagnosis and treatment guidelines (non-patent document 3) list conservative treatment (symptomatic treatment), oral therapeutic drugs, surgical treatment including intravesical instillation therapy, and endoscopic treatment as methods to improve symptoms. So far, the only treatment for interstitial cystitis covered by insurance is bladder hydrodilatation. However, bladder hydrodilatation has the following problems: it is highly invasive, there is a risk of bladder rupture as a side effect, and the operation will impose a greater burden on the patient's body and time. In January 2021, Zymso (registered trademark) intravesical instillation solution 50% (6 times instilled into the bladder every 2 weeks) was newly approved in Japan as the only interstitial cystitis treatment drug. However, the mechanism of action of dimethyl sulfoxide (DMSO), the active ingredient of Zymso (registered trademark) intravesical instillation solution 50%, on interstitial cystitis has not been fully elucidated. Although it has a certain effect on pain and inflammation, it has problems such as short duration of efficacy and repeated recurrence.

[0005] Symptomatic treatment uses explanation of the condition and dietary guidance. Oral medications include painkillers, antidepressants, antiallergic drugs, and steroids. The above-mentioned hydrodilatation of the bladder is widely used as an endoscopic treatment. In this operation, if Hunner's lesions are found in the bladder, surgery using electricity or laser (cautery) is required, but there is a problem that long-term repeated surgery may cause bladder atrophy. Regarding hydrodilatation of the bladder or cautery of Hunner's lesions, symptoms can be relieved in about half of the cases, but only a few cases can achieve long-term relief. Therefore, many cases require retreatment or additional treatment.

[0006] As intravesical drug instillation therapy, 50% DMSO, heparin, steroids, etc. are used. Sometimes botulinum toxin is also instilled into the bladder wall. For cases where all treatments are ineffective and unbearable symptoms persist, radical cystectomy and urinary diversion are recommended.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: Ministry of Health, Labor and Welfare Health Administration Report Example Specific Medical Expenses (Specified Intractable Diseases) Number of Patients with Acceptance Certificates Data from 2015 to 2020<https: / / www.nanbyou.or.jp / entry / 5354>

[0010] Non-patent literature 2: Refractory Disease Center website<https: / / www.nanbyou.or.jp / entry / 4429>

[0011] Non-patent document 3: Guidelines for the diagnosis and treatment of interstitial cystitis and bladder pain syndrome (edited by the Japan Interstitial Cystitis Research Society / Japan Urological Society), issued on April 25, 2019, revised in May 2021. Summary of the invention

[0012] Problems to be Solved by the Invention

[0013] As described above, effective therapeutic drugs and treatment methods for Hunner's interstitial cystitis have not yet been found. It is necessary to develop highly effective therapeutic drugs or treatment methods to improve the QOL (Quality of Life) of patients.

[0014] The pathological feature of Hunner's interstitial cystitis is the infiltration of lymphocytes, especially B cells and plasma cells in the lesions. Although this shows an inflammatory condition caused by a typical autoimmune disease, its cause is still unclear.

[0015] Regarding Hunner type interstitial cystitis, it is also known that it is complicated with systemic autoimmune diseases such as Sjögren's syndrome, autoimmune thyroiditis, and systemic lupus erythematosus. In addition, it is reported that autoantibodies against urothelium were detected from the patient's blood (Y.Akiyama, et.al., International Journal of Urology, 2020, 27, 491-503). In addition, the results obtained by gene expression analysis of bladder samples collected from patients with Hunner type interstitial cystitis reported that the gene expression of inflammation-related molecules such as IFN-γ, CXCR3, CXCL9, CXCL10, CXCL11, TNF-α, and TNFSF14 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. Therefore, considering that the concurrent disease is a systemic autoimmune disease, it is believed that IFN-γ plays a core role in the etiology of Hunner's interstitial cystitis, just like other autoimmune diseases. However, so far, no therapeutic drug for Hunner's interstitial cystitis targeting IFN-γ has been developed.

[0016] So far, 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) Since they are biological preparations, there are risks such as biological contamination; (2) There are antigenicity issues when they are administered for a long time; (3) Since they are protein preparations, cold chain is required for storage and transportation; (4) Due to their large molecular weight, they are not suitable for local administration such as intravesical instillation.

[0017] Regarding the above-mentioned problem (2), it is generally believed that the rate of antibody production for common antibody drugs is about 30%. Therefore, in cases where long-term treatment is required, antibodies against the antibodies are often produced, leading to allergic reactions and making it difficult to continue.

[0018] In addition, regarding the above-mentioned problem (1), since serum and the like are often used in the manufacturing process of biological preparations, there is a risk of biological contamination such as viruses. Regarding problem (3), since it is necessary to always handle at low temperatures, the cost of transportation and storage increases, and the convenience of patients using it is also reduced.

[0019] As Janus kinase inhibitors, Tofacitinib (product name: XELJANZ (registered trademark)), Baricitinib (product name: OLUMIANT (registered trademark)), Peficitinib (product name: Smyraf (registered trademark)), and Upadacitinib (product name: RINVOQ (registered trademark)) are used for rheumatoid arthritis, which is an autoimmune disease, and are already on the market. These Janus kinase inhibitors are low molecular weight compounds that can be produced by chemical synthesis, and therefore, it is generally believed that they do not have the above-mentioned problems caused by antibodies. On the other hand, there are many subtypes of Janus kinases, which bind not only to IFN-γ receptors but also to the intracellular domains of various cytokine receptors such as interleukin 2 (IL-2) receptor, interleukin 4 (IL-4) receptor, interleukin 7 (IL-7) receptor, and interferon α (IFN-α) receptor to be activated and transmit receptor signals.

[0020] Therefore, Janus kinase inhibitors may not only inhibit the signal transduction of IFN-γ, but also inhibit the signal transduction of IL-2, IL-4, IL-7, IFN-α, etc. (Yvan Jamilloux, et.al., Autoimmunity Reviews, 2019, 18, 11, 102390). This means that the material has safety risks when administered for a long time, and may show unexpected side effects such as susceptibility to infection.

[0021] The present invention has been completed in view of these circumstances, and its purpose is to provide a therapeutic drug for Hunner's type interstitial cystitis, which can selectively inhibit IFN-γ by targeting it, has no risk of biological contamination, and can be stored at room temperature.

[0022] Means for solving problems

[0023] In order to solve the above-mentioned problems, the therapeutic agent for Hunner's type interstitial cystitis containing a DNA oligonucleotide as an active ingredient of the present invention adopts the following configuration.

[0024] The first embodiment of the present invention provides a therapeutic drug for Hunner's interstitial cystitis, which contains a DNA oligonucleotide having a base sequence shown in any one of SEQ ID NOs. 1 to 3 and selectively binds to interferon gamma (IFN-γ). The DNA oligonucleotide involved in this embodiment selectively binds to IFN-γ and inhibits its activity to exert a therapeutic effect on Hunner's interstitial cystitis.

[0025] The base sequence shown in SEQ ID NO: 2 is a sequence obtained by adding an oligonucleotide composed of 9 residues of natural bases to the 3′ end of the base sequence shown in SEQ ID NO: 1.

[0026] 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 substituted with an arbitrary base.

[0027] In the first embodiment of the present invention, 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 may be chemically modified with a low molecular weight compound.

[0028] The molecular weight of the low molecular weight compound in the first embodiment is about 200 to 1000, and candidates thereof include anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporin, methotrexate, and leflunomide.

[0029] In the first embodiment of the present invention, the base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificial base, and the artificial base may be chemically modified by a medium molecular compound, a high molecular compound, a biopolymer, or a biocompatible polymer. The molecular weight of the medium molecular compound in this embodiment is about 1,000 to 20,000, and the molecular weight of the high molecular compound in this embodiment is about 20,000 to 400,000.

[0030] The polymer compound in the above scheme can be any biocompatible polymer with a molecular weight of 20,000 or more. Examples of the middle molecular compound or polymer compound in this scheme include, but are not limited to, PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, antibodies, etc. Antibodies are polymer compounds, but PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides are middle molecular compounds or polymer compounds according to their molecular weights. The molecular weight of a middle molecular compound or polymer compound is represented by an average molecular weight defined by a number average molecular weight (Mn) or a weight average molecular weight (Mw).

[0031] The second embodiment of the present invention is a therapeutic drug for a disease selected from the group consisting of 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, comprising as an active ingredient a DNA oligonucleotide having a base sequence represented by 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.

[0032] In the second embodiment, the base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificial base, and the artificial base can be chemically modified with a low molecular compound. The molecular weight of the low molecular compound in this embodiment is about 200 to 1000, and candidates include: anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporin, methotrexate, and leflunomide.

[0033] In the above second embodiment, the base X in the sequence of the DNA oligonucleotide having the base sequence shown in sequence number 3 is an artificially manufactured base, and the artificially manufactured base can be chemically modified by a medium molecular compound, a high molecular compound, a biopolymer or a polymer with biocompatibility. The molecular weight of the medium molecular compound in this embodiment is about 1000 to 20000, and the molecular weight of the high molecular compound in this embodiment is about 20000 to 400000. The high molecular compound in the above embodiment can be any high molecule with a molecular weight of 20000 or more and biocompatible. As the medium molecular compound or high molecular compound in this embodiment, PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, antibodies, etc. can be cited, but are not limited to this. Antibodies belong to high molecular compounds, but PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides belong to medium molecular compounds or high molecular compounds according to their molecular weight.

[0034] A third embodiment of the present invention is a reagent for experimental research, comprising as an active ingredient a DNA oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 3 and selectively binding to IFN-γ.

[0035] In the third embodiment, the base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificial base, and the artificial base can be chemically modified with a low molecular compound. The molecular weight of the low molecular compound in this embodiment is about 200 to 1000, and candidates include: anti-inflammatory compounds selected from glucocorticoids, tacrolimus, sirolimus, cyclosporin, methotrexate, and leflunomide.

[0036] In the third embodiment, the base X in the sequence of the DNA oligonucleotide having the base sequence shown in sequence number 3 is an artificially manufactured base, and the artificially manufactured base can be chemically modified by a medium molecular compound, a high molecular compound, a biopolymer or a polymer with biocompatibility. The molecular weight of the medium molecular compound in this embodiment is about 1000 to 20000, and the molecular weight of the high molecular compound in this embodiment is about 20000 to 400000. The high molecular compound in the above embodiment can be any high molecule with a molecular weight of 20000 or more and biocompatible. As the medium molecular compound or high molecular compound in this embodiment, PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, oligonucleotides, antibodies, etc. can be cited, but are not limited to this. Antibodies belong to high molecular compounds, but PEG, bipolar polymers, oligosaccharides, lipophilic polymers, peptides, and oligonucleotides belong to medium molecular compounds or high molecular compounds according to their molecular weight.

[0037] Effects of the Invention

[0038] The DNA oligonucleotide with a base sequence involved in the present invention selectively binds to IFN-γ. Thus, the activity of IFN-γ can be selectively inhibited. In addition, the Hunner type interstitial cystitis therapeutic drug containing a DNA oligonucleotide with a base sequence according to the present invention does not require the use of serum, etc., and therefore can be manufactured without the risk of biological contamination such as viruses. In addition, the DNA oligonucleotide with a base sequence involved in the present invention can be stored at room temperature. Therefore, it is advantageous compared to the existing method in terms of transportation and storage costs, and in addition, the convenience of patients using it can also be improved. In addition, the Hunner type interstitial cystitis therapeutic drug containing a DNA oligonucleotide with a base sequence involved in the present invention can be administered by intravesical instillation due to its molecular weight.

[0039] The DNA oligonucleotide of the present invention does not inhibit the signal transduction of IL-2, IL-4, IL-7, IFN-α, etc., but only inhibits the action of IFN-γ. Therefore, the therapeutic drug for Hunner type interstitial cystitis containing a DNA oligonucleotide having a base sequence as an active ingredient according to the present invention can reduce unexpected side effects caused by susceptibility to infection, etc., even in the case of long-term administration, compared with Janus kinase inhibitors, etc. In addition, even when compared with anti-IFN-γ antibodies, the antigenicity of the DNA oligonucleotide of the present invention is lower than that of antibodies, so it is a drug that can be used for a long time. In addition, the DNA oligonucleotide of the present invention has no risk of biological contamination during manufacture and can be stored at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing changes in the number of hairs on a transplanted skin tissue piece before and after administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0041] Figure 2A This is a diagram showing the experimental results regarding the expression of MHC class I in dermal sheath cup cells caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0042] Figure 2B This is a diagram showing the experimental results regarding the expression of MHC class I in the outer root sheath caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0043] Figure 3A This is a diagram showing the experimental results regarding the expression of MHC class II in the connective tissue root sheath caused by the administration of the DNA oligonucleotide according to one embodiment of the present invention.

[0044] Figure 3B This is a diagram showing the experimental results regarding the expression of MHC class II in the outer root sheath caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0045] Figure 4A This is a diagram showing the results of the effect of mouse IFN-γ on STAT1 phosphorylation when the surrogate aptamer according to one embodiment of the present invention was added to L929 mouse fibroblasts.

[0046] Figure 4B The graph shows the results of the effect of mouse IFN-γ on STAT1 phosphorylation when negative control DNA was added to L929 mouse fibroblasts.

[0047] Figure 5AThis is a graph showing the suppression of an increase in the frequency of urination in an experiment on the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model.

[0048] Figure 5B This is a graph showing the suppression of increased pelvic pain sensitivity in an experiment on the effect of a surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model.

[0049] Figure 6 This figure shows the results of histopathological evaluation of the bladder regarding the effect of the surrogate aptamer according to one embodiment of the present invention on a mouse interstitial cystitis model.

[0050] Fig. 7A This is a graph 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 controlled to near the normal level, the same as that of the normal group.

[0051] Figure 7B Yes Fig. 7A The graph is a quantitative graph of the mRNA expression levels shown.

[0052] Figure 8 This is a diagram showing the binding of a DNA oligonucleotide according to one embodiment of the present invention to dog and cat IFN-γ. DETAILED DESCRIPTION

[0053] Hereinafter, embodiments of the therapeutic agent for Hunner's type interstitial cystitis containing a DNA oligonucleotide as an active ingredient according to the present invention will be described.

[0054] As for drugs targeting IFN-γ, emalumab, an anti-IFN-γ antibody, was approved by the FDA in 2018 for the treatment of hemophagocytic lymphohistiocytosis, a refractory autoimmune disease, and is marketed under the brand name of Gamifant.

[0055] However, as mentioned above, anti-IFN-γ antibodies have the following problems: since they are biological preparations, there are risks such as biological contamination; long-term administration will produce antigenicity; due to their molecular weight, they are not suitable for topical administration such as transdermal administration and transmucosal administration; since they are protein preparations, there are storage and transportation conditions. Therefore, it is expected that a therapeutic drug that can effectively inhibit IFN-γ and solve these problems will be developed.

[0056] As a means to solve the above-mentioned problems, the inventors have tried to develop an IFN-γ inhibitory drug using the DNA oligonucleotide involved in the present invention as a DNA aptamer. DNA aptamers refer to ligand molecules that form complementary chains with each other through complementary sequences in DNA oligonucleotide molecules, so that single-stranded DNA oligonucleotides form secondary structures and tertiary structures, and specifically and firmly bind to target molecules through their three-dimensional structures. By binding to DNA aptamers with specific sequences, the activity of target molecules can be hindered and inhibited, and at the same time, it can also be hyperactive. Although the molecular weight of DNA aptamers is about 1 / 10 less than that of antibodies, they have the same high affinity as antibodies and have high target selectivity. Therefore, IFN-γ inhibitory drugs using DNA aptamers can minimize the side effects caused by off-target. In addition, since DNA aptamers can be produced by chemical synthesis, it is considered to be a mode suitable for solving the problem. In this specification, "selectively binding to IFN-γ" includes that the DNA oligonucleotide involved in this embodiment is firmly and specifically bound to IFN-γ as a target substance as a DNA aptamer.

[0057] DNA aptamers form a compact three-dimensional structure compared to antibodies, and therefore can be administered topically, such as transdermally or through mucosally.

[0058] DNA aptamers have the following advantages and are expected to be useful: (1) They have a small molecular weight and can be administered as transdermal preparations such as ointments and patches or transmucosal preparations; (2) Since they are chemically synthesized products, there is no risk of biological contamination; (3) They are generally low in antigenicity; (4) Since they are DNA, they have sufficient stability at room temperature under conditions near neutrality in the absence of nucleases (nucleases); (5) Since they have almost no inhibition of the activity of cytochrome P450, which is a drug-metabolizing enzyme, they will not affect the combined drug. In addition, DNA aptamers can also be administered for a long time because they do not produce antibodies to antibody drugs, which is one of the problems when antibodies are required for long-term treatment.

[0059] As a specific method for treating diseases using DNA aptamers, a method is envisioned in which an aptamer itself or a modified form thereof is administered to neutralize IFN-γ, thereby treating autoimmune diseases or diseases in which excessive production of IFN-γ is believed to be the main cause.

[0060] As a DNA aptamer that can specifically bind to human IFN-γ with high affinity and inhibit its activity, the present inventors have discovered a DNA aptamer having a sequence of sequence number 1 in Table 1, wherein the sequence of sequence number 1 includes two artificial bases Ds (7-(2-thienyl)imidazo[4,5-b]pyridine) in the base sequence.

[0061] DNA aptamers are rapidly degraded by nucleases (nucleases) in biological tissues, so even if they show strong activity in vitro, they may not show activity in vivo. Therefore, the inventors found that a DNA aptamer having a base sequence of sequence number 2 in Table 1 acquires nuclease resistance and is stable in biological tissues, and the base sequence of sequence number 2 is a natural base sequence with 9 residues bonded to the 3′ end. It was confirmed that a DNA aptamer having a base sequence of sequence number 2 including two Ds in the sequence showed effectiveness in an autoimmune humanized mouse alopecia areata model transplanted with human scalp tissue, and also existed stably in biological tissues and was able to inhibit IFN-γ (instructions for use of Japanese Patent Application No. 2021-166794). In addition, regarding the DNA aptamer having 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, i.e., the sequence of SEQ ID NO. 3, the PEG-modified body in which polyethylene glycol (PEG) is added to the base portion of X was confirmed to retain the ability to bind to IFN-γ in surface plasmon resonance (SPR). From this, it can be seen that modification with 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 maintains the same IFN-γ inhibitory activity as the DNA aptamer having the sequence of SEQ ID NO. 2.

[0062] In this embodiment, a DNA oligonucleotide having a base sequence shown in Table 1 is used as a DNA aptamer. This embodiment includes using a DNA oligonucleotide having a base sequence shown in Table 1 as a therapeutic agent for Hunner's type interstitial cystitis.

[0063]

Table 1

[0064] Serial Number Sequence (5′→3′) 1 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGG 2 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGGGCGCGAAGCG 3 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGGGCCGXAGCG 4 GGCCGGTACCCGADsCCACAGTTTATDsGTTGTACTAGTTTTGCAGGGTCTGGCCCGCGAAGCG

[0065] The base sequence represented by sequence number 2 in Table 1 is a sequence formed by adding an oligonucleotide (mini-hairpin sequence) consisting of 9 residues of natural bases (5′-CGCGAAGCG-3′) to the 3′ end of the sequence represented by sequence number 1. The base sequence represented by sequence number 3 in Table 1 is a sequence formed by replacing the 53rd base from the 5′ end of the sequence represented by sequence number 2 with an arbitrary base X. X is an arbitrary natural base, an arbitrary non-natural base or a modified base, or represents a substance formed by combining a low molecular weight compound, peptide, oligonucleotide, oligosaccharide, protein, or other high molecular weight compound (biopolymer) used in biology or a biocompatible polymer with a modified base. The base sequence of sequence number 4 will be described in detail later.

[0066] Examples of polymer compounds bound to modified bases include polyethylene glycol (PEG) with a molecular weight of 20,000 or more or any biocompatible polymer with a molecular weight of 20,000 or more. Biocompatible polymers refer to safe chemical synthetic products that are not usually used in organisms, and even if they enter the organism, they will not cause inflammation or toxic reactions. Examples of medium molecular compounds include peptides, oligonucleotides, oligosaccharides, proteins, PEG, and any biocompatible polymers with a molecular weight greater than 1,000 and less than 20,000.

[0067] As functional groups for modification, an azido group (-N3), an amino group (-NH2), a carboxyl group (-COOH) or an active ester thereof, a cyclic structure containing an alkynyl group (-CC) or an alkynyl structure, a formyl group (-CHO), a hydrazide group (-NH-NH2), a hydroxyl group (-OH), a thiol group (-SH), a cyano group (-CN), a vinyl group (-CHCH2), or a maleimide group can be used.

[0068] In this specification, "natural base" refers to any one of adenine, guanine, cytosine, and thymine. In this specification, "non-natural base" refers to a base that is artificially synthesized and has a property similar to that of a natural base, and is sometimes referred to as "artificial base" in this specification. In this specification, "modified base" refers to a base with a side chain structure added, and the side chain structure has one or more functional groups activated for modification, which is a kind of "artificially manufactured base". As an example of modification, it can be cited: methylation, deamination, replacement of atomic position, sulfurization of oxygen at the phosphate site, and introduction of water-soluble or fat-soluble substituents to the base part can be cited. Specifically, it can be cited: modified pyrimidine, modified purine, other heterocyclic bases, etc. Ds in the sequence of sequence numbers 1 to 3 shows 7-(2-thienyl)imidazo[4,5-b]pyridine as an artificial base. As an artificial base, in addition to Ds itself, a base having a side chain introduced into Ds can also be used. Hereinafter, in this embodiment, the DNA aptamers having the sequences shown by sequence numbers 1, 2, 3, and 4 in Table 1 are respectively referred to as "aptamer 1", "aptamer 2", "aptamer 3", and "aptamer 4".

[0069] Regarding the usefulness of the DNA aptamer involved in this embodiment, in this specification, the DNA aptamer (aptamer 2) described in Table 1 was intradermally injected into an autoimmune hair loss model of immune-tolerant mice transplanted with human scalp tissue pieces, and the results of promoting hair regeneration and inhibiting further hair loss were confirmed (Example 4). This will be described in detail later.

[0070] By pathologically analyzing the mechanism of active expression of aptamer 2 in the model, the results showed that aptamer 2 almost completely inhibited the expression of MHC class I and class II. The detailed results will be described later. That is, it can be considered that aptamer 2 inhibits the production of MHC class I and class II, which are the basis of autoimmune expression, by inhibiting the activity of IFN-γ, thereby improving autoimmunity and thus improving the symptoms of alopecia areata. This shows that aptamer 2 can not only be used as a means to solve the problem of alopecia areata, but also, even in Hunner type interstitial cystitis, which is presumed to be mainly caused by excessive production of IFN-γ, providing an IFN-γ inhibitor using a DNA aptamer can also be used as a means to solve the problem.

[0071] Regarding whether the DNA aptamer that inhibits the activity of IFN-γ is effective for the treatment of Hunner's interstitial cystitis, before confirming its effectiveness in humans, it is necessary to confirm its effectiveness through a disease animal model. As the animal model that is closest to the condition of human Hunner's interstitial cystitis established so far, there is a known mouse autoimmune interstitial cystitis model (a model using transgenic mice (URO-OVA mice) that express 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 does not show binding activity to mouse IFN-γ. Therefore, the effectiveness of the above-mentioned DNA aptamer cannot be confirmed in the mouse model.

[0072] Therefore, the present inventors have studied the acquisition of an aptamer that shows high similarity in physical properties to the above-mentioned DNA aptamer and binds to mouse IFN-γ to inhibit its activity as a substitute (surrogate) aptamer.

[0073] The criteria for the surrogate aptamer to show high similarity in physical properties to the above-mentioned DNA aptamer are as follows.

[0074] (1) DNA aptamer.

[0075] (2) The base sequence of the aptamer contains two artificial bases Ds, and the other bases are natural bases.

[0076] (3) The base number of the aptamer is within ±10% of the 57 residues of the above-mentioned DNA aptamer (51 residues to 62 residues).

[0077] (4) A 9-residue mini-hairpin sequence is present at the 3′ end of the aptamer base sequence.

[0078] The structure of the aptamer satisfying the above criteria is similar to that of the above-mentioned DNA aptamer, and therefore it is estimated that the physical properties thereof are also similar.

[0079] The alternative aptamer that meets the above criteria was explored, and as a result, a DNA aptamer (aptamer 4) having the sequence described in sequence number 4 in Table 1 (Example 5) was obtained. The sequence described in sequence number 4 in Table 1 has 62 bases, contains 2 Ds, and has a mini-hairpin sequence at the 3′ end. It will be described in detail later. The KD value of the obtained DNA aptamer indicating the binding ability to mouse IFN-γ is 2.47nM, which has a high binding ability of about 1 / 100 of the binding ability of aptamer 2 to human IFN-γ (KD value; 33pM). In addition, by adding 5 times the amount of mouse IFN-γ in molar concentration, the aptamer competitively inhibited, thereby almost completely inhibiting the activity of mouse IFN-γ. From this result, it can be confirmed that the DNA aptamer obtained by exploration has sufficient activity to be used as an alternative aptamer.

[0080] The obtained substituted aptamer was intravesically administered to mice of the autoimmune interstitial cystitis model (URO-OVA model) described in Reference 1. As a result, the substituted aptamer showed a high effect, i.e., significantly inhibited the increase in urination frequency and sensitivity to pelvic pain in mice, thereby inhibiting the onset of cystitis (Example 6), compared with the control PBS administration group.

[0081] After pathological histological evaluation of the bladder, as the inflammation score of the bladder, in the aptamer-administered group, the inflammation score was significantly lower than that of the PBS-administered group, and there was no difference with the normal group, indicating that the administration of the surrogate aptamer inhibited bladder inflammation. In addition, after analyzing the mRNA expression in the bladder tissue, the expression of inflammatory cytokines such as IFN-γ and TNF-α induced by IFN-γ stimulation, and even the expression of pre-SP and NGF as substances that cause pain were also inhibited by the administration of the surrogate aptamer. This fully shows that the inhibitory effect of the surrogate aptamer on bladder inflammation is expressed by the inhibitory effect of IFN-γ activity in the tissue (Example 6).

[0082] By analogy, the DNA aptamer of the present invention exhibits an inhibitory effect on human IFN-γ in human tissues, and since the physical properties of the DNA aptamer and the surrogate aptamer are highly similar, the DNA aptamer also has the effect of inhibiting the onset of human Hunner's type interstitial cystitis, similarly to the surrogate aptamer, and can be used as a therapeutic drug for Hunner's type interstitial cystitis.

[0083] As a DNA aptamer, a DNA oligonucleotide having any of the sequences described in Table 1 may be directly used, or a DNA oligonucleotide modified at a site that does not affect the activity of these DNA aptamers may be used. Examples of modified forms of DNA aptamers include: modified forms formed by chemically combining middle molecular or high molecular compounds such as PEG, peptides, oligonucleotides, modified forms formed by chemically polymerizing the same DNA aptamers, and modified forms formed by transforming or modifying a portion of the sequence of the DNA aptamer. When the DNA oligonucleotide involved in the present embodiment is modified and used as a DNA aptamer, the base portion is preferably used as the modified portion. Artificial bases or modified bases can be modified using existing methods, and the 3' end and 5' end can also be modified.

[0084] PEGylated DNA aptamers are used to improve the PK (Pharmacokinetics)-PD (Pharmacodynamics) curve commonly used in oligonucleotides containing proteins, peptides, and aptamers to improve in vivo kinetics. So far, many PEGylated aptamers have been developed. It is known that while maintaining the binding activity of the PEGylated aptamer to the target protein, it exhibits the same activity as the aptamer before PEGylation in vivo, and PEGylation hardly causes toxicity (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).

[0085] In the case of preparations for systemic administration, as injection preparations, they can be prepared in the form of vials containing lyophilized powders, vials containing aptamer solutions, or prefilled syringes.

[0086] The DNA aptamer of the present embodiment can be made into an inhalation preparation by loading nanoparticles adsorbing or containing the DNA aptamer or a solution thereof, or powder obtained by granulating the DNA aptamer and a granulating material into a suitable size into an inhalation device.

[0087] The DNA aptamer of the present embodiment can be used as an eye drop by directly dissolving it in an appropriate solvent such as a buffer solution by utilizing its high water solubility.

[0088] As one of the local administration methods, administration via mucosa is conceivable. The DNA aptamer of the present embodiment can be dissolved in a solvent such as a buffer having high biocompatibility and used as a transmucosal administration agent such as intravesical administration.

[0089] As an injectable preparation, inhalation preparation, and eye drop, the DNA aptamer of this embodiment can be encapsulated in or bonded to adipose nanoparticles, nanoparticles of biodegradable polymers such as PLGA (Polylactic-co-Glycolic Acid), gold nanoparticles, etc., and then dispersed or dissolved in physiological saline, physiological buffer, etc. for use.

[0090] The DNA aptamer of this embodiment can be used as a transdermal topical drug delivery agent such as a solution, an ointment, a plaster, a lotion, an emulsion, a milk, a gel, a biodegradable microneedle, or a papule.

[0091] In the process of manufacturing a transdermal drug, it is conceivable to use, as an absorption enhancer, lower alcohols such as ethanol, polyols such as ethylene glycol, fatty acids, esters such as ethyl acetate, surfactants, ionic liquids, etc. In addition, when manufacturing a transdermal drug, a process of manufacturing nanoparticles using a biodegradable polymer such as polylactic acid or liposomes can be applied, and these processes can be appropriately combined according to the purpose.

[0092] The DNA aptamer of this embodiment can also be used as a preparation for administration using equipment corresponding to methods such as iontophoresis, electroporation, thermal poration, sonophoresis, microneedle array patch, needleless syringe, micropump, etc., which are physical transdermal absorption promotion methods.

[0093] It was confirmed that the DNA aptamer of the present embodiment can bind to dog IFN-γ and cat IFN-γ in addition to human IFN-γ (Example 7). This will be described in detail later. Among them, it is speculated that idiopathic cystitis, which is one of the lower urinary tract diseases caused by the bladder in dogs and cats, is related to the excessive production of IFN-γ in dogs and cats. From the results of Example 7, it can be seen that the DNA aptamer of the present embodiment has the effect of inhibiting the activity of dog IFN-γ and cat IFN-γ. It is therefore believed that it has an inhibitory effect on 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 the present embodiment can be used as a therapeutic drug for idiopathic cystitis in dogs and cats, diseases related to IFN-γ, and autoimmune diseases.

[0094] The DNA aptamer involved in this embodiment can be used as a research reagent for experiments related to IFN-γ because it can selectively inhibit IFN-γ. For example, whether in vitro or in vivo, the possibility of IFN-γ participating in the physiological phenomenon of interest can be evaluated and discussed by experiments in which the DNA aptamer involved in this embodiment is activated, thereby investigating the cause of the physiological phenomenon. In addition, by adding the DNA aptamer involved in this embodiment as a reagent to a cell culture medium or administering it to an animal, it can be used in many experiments including a reaction system that inhibits IFN-γ.

[0095] Example 1: Synthesis of DNA aptamers

[0096] Aptamer 1 and aptamer 2 were chemically synthesized by the methods described in International Publication Nos. 2013 / 073602 and 2016 / 143700.

[0097] Example 2: Synthesis of Aptamer 3

[0098] Using the methods described in International Publication Nos. 2013 / 073602 and 2016 / 143700, amino-modifier C6-dT phosphoramidite was introduced at position X of the sequence of SEQ ID NO: 3 to synthesize aptamer 3. Other X-substituted forms can be synthesized by using commercially available artificial bases or phosphoramidites of modified bases.

[0099] Example 3: Synthesis of PEG-modified DNA aptamers

[0100] The aptamer 3 (1 eq) having a primary amine side chain in the base part of X prepared 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 body was purified by reverse phase HPLC to obtain a PEG-modified body of aptamer 3. The obtained PEG-modified body of aptamer 3 was confirmed to retain its ability to bind to IFN-γ by SPR (Surface Plasmon Resonance).

[0101] Example 4: Confirmation of therapeutic effect using a humanized mouse alopecia areata model

[0102] Step 1 Preparation of humanized mouse alopecia areata model

[0103] According to the method described in A. Gilhar, et al., Journal of Investigative Dermatology, 2013, 133, 3, 844-847, alopecia areata was induced by intradermal injection of human activated lymphocytes into human scalp tissue transplanted into mice, thereby producing humanized mice of alopecia areata model.

[0104] Process 2

[0105] The humanized mice with alopecia areata model were divided into 3 groups and administered with vehicle (PBS), dexamethasone + minoxidil (positive control), and aptamer 2, respectively. For the vehicle group, 15 μL of PBS was intradermally administered to the transplanted skin once every 2 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 intradermally administered to the transplanted skin once every 2 days, and the concentration of the solution of aptamer 2 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 2 mg of dexamethasone and 5% minoxidil was applied to the transplanted skin, once a day.

[0106] The results after administration in Example 4 are shown in Figure 1 . Figure 1 The figure shows the change in the number of hair roots on the transplanted skin tissue piece before administration and 143 days after the start of administration, and the vertical axis represents the change in the number of hair roots per transplanted skin tissue piece. Figure 1 In the positive control group ( Figure 1 "dexamethasone + minoxidil") and aptamer administration group ( Figure 1 In the presence of an "aptamer" ("aptamer"), further hair loss was inhibited and hair regrowth was observed.

[0107] According to the results of pathological analysis of hair follicle tissue, significant suppression of CD8-positive T cell infiltration was observed in the positive control group and the aptamer-administered group. It is believed that in the positive control group and the aptamer-administered group, the progression of hair loss was suppressed by suppressing the inflammatory response and promoting hair regeneration.

[0108] Furthermore, the expression of MHC was examined by pathological analysis of hair follicle tissue after administration in Example 4. Figure 2A Results showing the expression of MHC class I in dermal sheath cup cells, Figure 2B Results showing the expression of MHC class I in the outer root sheath, Figure 3A The results of the expression of MHC class II in the connective tissue root sheath and the expression of MHC class II in the outer root sheath are shown. Figure 2A and Figure 2B ) or Class II ( Figure 3A and Figure 3B ) as the expression level of each vector group ( Figure 2A , Figure 2B , Figure 3A , Figure 3B The relative values ​​are shown when the expression level of the "vector" is 1.

[0109] The expression of MHC class I and class II was only in the aptamer administration group ( Figure 2A , Figure 2B , Figure 3A , Figure 3B This means that the positive control group ( Figure 2A , Figure 2B , Figure 3A , Figure 3B The mechanism of inhibiting inflammation and promoting hair regeneration in the "dexamethasone + minoxidil" group and the aptamer administration group is different. In the positive control group, it can be seen that the activation of the glucocorticoid receptor directly inhibits inflammation, and in the aptamer administration group, it is believed that inflammation is inhibited by inhibiting the production of MHC class I and class II that lead to autoimmunity. In other words, in the aptamer administration group, it is believed that the immune tolerance destruction of the hair follicle tissue is restored, and a more radical therapeutic effect is obtained. This result shows that the DNA aptamer involved in this embodiment is not only for autoimmune skin diseases, but also for autoimmune diseases occurring in other tissues or Hunner's type interstitial cystitis, which are generally believed to be caused by excessive production of IFN-γ. The disease may be able to inhibit the inflammatory response through the same mechanism.

[0110] When the therapeutic drug containing the DNA aptamer according to the present embodiment is used, it is confirmed that hair loss can be suppressed and hair regeneration can be promoted. In addition, after pathological analysis, it is confirmed that the expression of MHC class I and class II can be almost completely suppressed by administering the DNA aptamer according to the present embodiment.

[0111] Example 5: Production of mouse IFN-γ aptamer (surrogate aptamer)

[0112] Process 1

[0113] The SELEX method targeting mouse IFN-γ was performed to obtain an aptamer. The obtained mouse IFN-γ aptamer is a DNA aptamer having a sequence described in sequence number 4 (aptamer 4 in Table 1), the sequence described in sequence number 4 has 62 bases, contains 2 Ds, and has a mini-hairpin sequence at the 3′ end. The binding ability to mouse IFN-γ was determined by the SPR method, and the result was a KD value of 2.47nM.

[0114] Process 2

[0115] The obtained aptamer was verified to inhibit the activity of mouse IFN-γ. 2 ng / mL mouse IFNγ was added to L929 mouse fibroblasts, and aptamer 4 at various molar concentrations was added, and after incubation at 37°C for 15 minutes, flow cytometry using an anti-phospho-STAT1 antibody was used to confirm that aptamer 4 inhibited STAT1 phosphorylation.

[0116] The results are shown in Figure 4A and Figure 4B . Figure 4A is a diagram showing the results when aptamer 4 was added to L929 mouse fibroblasts, Figure 4B This is a graph showing the results when negative control DNA was added to L929 mouse fibroblasts. Figure 4A The "aptamer" in the embodiment refers to the replacement aptamer involved in this embodiment. Figure 4B "Nc DNA" in the table indicates negative control DNA. Figure 4A and Figure 4B "1eq", "5eq", "10eq", ​​"50eq", ​​"100eq" in the table represent the molar concentration of the aptamer relative to the molar concentration of mouse IFN-γ (as an example, 100eq represents aptamer:IFN-γ=100:1). By adding aptamer 4 at a molar concentration 5 times that of mouse IFN-γ, the phosphorylation of STAT1 was almost completely inhibited ( Figure 4A ). In contrast, in the system to which negative control DNA was added, even when 100 times the amount of negative control DNA was added to mouse IFN-γ, the phosphorylation of STAT1 was not inhibited ( Figure 4B ). From this result, it was confirmed that the obtained aptamer 4 inhibited the activity of mouse IFN-γ and had sufficient activity as a surrogate aptamer.

[0117] Example 6: Investigation of the Effects of Alternative Aptamers in a Mouse Autoimmune Interstitial Cystitis Model

[0118] Step 1 Preparation of Autoimmune Interstitial Cystitis Model (URO-OVA Model) Mice

[0119] Autoimmune interstitial cystitis model (URO-OVA model) mice were prepared by the method described in Reference 1. 100 μg of OVA antigen was injected subcutaneously into normal mice, and spleen cells were collected from the mice 2 weeks later. The spleen cells were divided into 5×10 7 The researchers implanted transgenic mice expressing OVA antigen into the bladder epithelium (URO-OVA mice) via intravenous injection. In URO-OVA mice implanted with spleen cells, an immune response was specifically triggered on the bladder epithelium, resulting in inflammation.

[0120] Process 2

[0121] The surrogate aptamer was intravesically administered to mice with an autoimmune interstitial cystitis model (URO-OVA model) once every 2 days for 3 weeks (10 nmol / time). As a control group, a PBS-administered group (interstitial cystitis model mice) and a normal group (normal mice) were used. The normal group (normal mice) was not administered with the surrogate aptamer and PBS. Figure 5A The results of the frequency of urination within 24 hours measured once a week are shown. In the PBS-administered group (interstitial cystitis model mice) (■), the frequency of urination increased significantly compared with the normal group (◆), while in the aptamer-administered group (●), the increase in the frequency of urination was completely suppressed and was the same as that of normal mice. Figure 5B The results of evaluating the sensitivity to pelvic pain using an electronic analgesic (Electronic Von Frey) once every 7 days are shown. In the PBS-administered group (interstitial cystitis model mice) (■), the sensitivity to pelvic pain was significantly increased (the sensory threshold was reduced) compared with the normal group (◆), while in the aptamer-administered group (●), the increase in sensitivity was significantly suppressed. As can be seen from the above, the aptamer-administered group has a higher effect than the PBS-administered group, that is, the increase in the frequency of urination of mice is significantly suppressed ( Figure 5A ) and increased sensitivity to pelvic pain ( Figure 5B ), inhibit the onset of cystitis.

[0122] Process 3

[0123] After a 3-week observation period from the administration of the aptamer or PBS, the mice were killed and the bladder pathological histology was evaluated. Figure 6 And Table 2. After HE staining, in the PBS-administered group, immune cell infiltration (→), angiogenesis (*), mucosal congestion (>), and interstitial edema (**) were observed, while in the aptamer-administered group, these changes were hardly observed. Table 2 shows the inflammation score of the bladder after the observation period. Level 0 indicates a state with no signs of inflammation, and from levels 1 to 3, many signs of inflammation such as infiltration of immune cells, angiogenesis, mucosal congestion, and interstitial edema can be seen. In the aptamer-administered group (“Cystitis / aptamer” in Table 2), the inflammation score was significantly lower than that in the PBS-administered group (“Cystitis / PBS” in Table 2), and no difference was found with the normal group (“Normal” in Table 2), indicating that bladder inflammation was suppressed by administering the aptamer.

[0124]

Table 2

[0125]

[0126] In addition, the results of analyzing mRNA expression in bladder tissue are shown in Fig. 7A , Figure 7B . Fig. 7Ais a diagram showing the results of 1% agarose gel electrophoresis of RT-PCR products. Figure 7B Yes Fig. 7A The graph is a quantitative graph of the mRNA expression level shown in FIG. Fig. 7A , Figure 7B In the "Cystitis / aptamer" in the bladder tissue, the expression levels of inflammatory cytokines IFN-γ and TNF-α induced by IFN-γ stimulation, and the expression levels of pain-causing substances pre-SP and NGF were suppressed to about the same normal level as the normal group. This clearly shows that the effect of administering the surrogate aptamer on urination frequency, sensitivity to pelvic pain, and bladder inflammation is brought about by the action of suppressing the activity of IFN-γ in the tissue.

[0127] It is believed that the results in Examples 4 and 6 are due to the fact that the DNA aptamer according to the present embodiment potently inhibits the activity of IFN-γ. Therefore, the use of the DNA aptamer according to the present embodiment can provide an effective therapeutic drug and a therapeutic method that are not currently available for diseases such as autoimmune diseases represented by alopecia areata and Hunner's interstitial cystitis, which are believed to be mainly caused by excessive production of IFN-γ.

[0128] In addition, according to the results of the above examples, it is believed that the DNA aptamer involved in this embodiment binds to IFN-γ with high specificity. In terms of pharmacological properties, compared with Janus kinase inhibitors (YvanJamilloux, et.al., Autoimmunity Reviews, 2019, 18, 102390) that inevitably inhibit multiple cytokine signals, the DNA aptamer involved in this embodiment that selectively inhibits IFN-γ activity can reduce the possibility of side effects.

[0129] DNA aptamers are generally less likely to produce anti-DNA aptamer antibodies. Therefore, the DNA aptamers involved in this embodiment can be administered for a long time in the treatment of chronic inflammatory diseases.

[0130] Example 7: Confirmation of the binding of the DNA aptamer according to the present embodiment to IFN-γ of dogs and cats

[0131] In order to confirm the binding of the DNA aptamer involved in this embodiment to dog and cat IFN-γ, an electrophoretic mobility shift assay (EMSA) was performed. 100nM aptamer 2 and 400nM dog IFN-γ or cat IFN-γ were mixed and allowed to stand at room temperature for 15 to 30 minutes. The obtained sample was subjected to 8 to 10% polyacrylamide gel electrophoresis and detected by SYBR Gold. The results are shown in Figure 8A shifted band representing the complex of aptamer 2 and dog IFN-γ was detected ( Figure 8 Left) and the shifted band representing the complex of aptamer 2 and feline IFN-γ ( Figure 8 Right), thereby confirming that aptamer 2 binds to dog IFN-γ and cat IFN-γ, respectively. In addition, as a positive control for the EMSA experiment, a binding confirmation test using aptamer 2 and human IFN-γ was performed by the aforementioned test method. As a result, a shifted band representing a complex was detected, confirming that aptamer 2 binds to human IFN-γ.

[0132] Based on the results in Example 7, it is speculated that the DNA aptamer according to this embodiment inhibits the activity of IFN-γ in dogs and cats. Therefore, using this DNA aptamer, an effective therapeutic drug and method can be provided 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.

[0133] The DNA aptamer according to the present embodiment can be produced by chemical synthesis, and thus can be provided as a safe drug with stable quality and low risk of biological contamination.

[0134] Compared with biological preparations, the DNA aptamer involved in this embodiment can be manufactured at a low price. In addition, the storage and transportation of biological preparations require low temperature conditions, while DNA aptamers are very stable even at room temperature. Therefore, the transportation and storage of the preparation containing DNA aptamers involved in this embodiment do not necessarily require a cold chain.

[0135] By preparing the DNA aptamer according to the present embodiment into a transdermal drug preparation, it is possible to provide a therapeutic drug that is non-invasive in administration, has a low risk of side effects, and is easy to use.

[0136] By administering the therapeutic drug containing DNA aptamers according to the present embodiment systemically as an injection, it can be applied to diseases such as systemic autoimmune diseases and Hunner's interstitial cystitis, which are believed to be mainly caused by excessive production of IFN-γ. In addition, even in the case of an injection, it can be made into a preparation that is easy for patients to use, such as a prefilled syringe that can be stored at room temperature.

[0137] Since the DNA aptamer involved in this embodiment can selectively inhibit IFN-γ, it can be used as a research reagent for experiments on systems with the possibility of IFN-γ involvement. For example, whether in vitro or in vivo, the possibility of IFN-γ involvement in a physiological phenomenon of interest can be clarified by allowing the DNA aptamer involved in this embodiment to act, thereby investigating the cause of the physiological phenomenon. In addition, by adding it as a reagent to a cell culture medium or administering it to an animal, it can be used in many experiments including a reaction system that inhibits IFN-γ.

Claims

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

2. The therapeutic drug for Hunner 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 artificial base, and the artificial base is chemically modified with a low molecular compound.

3. The therapeutic drug for Hunner type interstitial cystitis according to claim 2, wherein: The low molecular weight compound is an anti-inflammatory compound selected from glucocorticoids, tacrolimus, sirolimus, cyclosporin, methotrexate and leflunomide.

4. The therapeutic drug for Hunner 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 polymer having biocompatibility.

5. The therapeutic drug for Hunner type interstitial cystitis 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.

6. A therapeutic drug for a disease selected from a disease of dogs and cats associated with IFN-γ, a lower urinary tract disease of dogs and cats caused by the bladder, or an autoimmune disease of dogs and cats, comprising as an active ingredient a DNA oligonucleotide having a base sequence as shown in any one of SEQ ID NOs: 1 to 3 and selectively binding to IFN-γ of dogs and cats.

7. The therapeutic drug according to claim 6, wherein The base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificial base, and the artificial base is chemically modified with a low molecular compound.

8. The therapeutic drug according to claim 6, 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 polymer having biocompatibility.

9. A reagent for experimental research, comprising as an active ingredient a DNA oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 3 and selectively binding to IFN-γ.

10. The test research reagent according to claim 9, wherein: The base X in the sequence of the DNA oligonucleotide having the base sequence shown in SEQ ID NO: 3 is an artificial base, and the artificial base is chemically modified with a low molecular compound.

11. The test research reagent according to claim 9, 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 polymer having biocompatibility.

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