Medicine for treating xerophthalmia containing DNA oligonucleotide selectively binding to IFN-gamma

Abstract

Provided is a xerophthalmia treatment drug which can selectively inhibit I FN-gamma, has no risk of biological contamination, and can be stored at room temperature. Provided is a therapeutic drug for xerophthalmia, 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 having 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 invention relates to a drug for treating dry eye disease, which contains a DNA oligonucleotide which selectively combines with IFN-γ. Background Art

[0002] Dry eye is a general term for eye diseases caused by dryness of the ocular surface. Dry eye is defined as a disease in which the stability of the tear layer is reduced due to various factors, which can cause eye discomfort and visual dysfunction, and is sometimes accompanied by symptoms on the ocular surface (non-patent document 1). If the symptoms of this disease worsen further, it may even lead to corneal transplantation or blindness. According to the Japanese Ophthalmological Society, there are more than 22 million dry eye patients in Japan (non-patent document 2), and there is an increasing trend. In the pathogenesis of dry eye, "reduced stability of the tear layer" is crucial. Factors that make tears unstable include reduced tear volume, increased tear surface tension, hyperevaporation of tears, and reduced hydrophilicity (wetting) of the corneal and conjunctival epithelium. Clinically, these are considered to be tear-deficient dry eye, dry eye caused by meibomian gland dysfunction, dry eye caused by environmental factors such as IT eye disease or video display terminal work, and dry eye caused by abnormal mucin on the ocular surface.

[0003] Tear-deficient dry eye is further divided into a type with organic abnormalities of the lacrimal glands (the so-called Sjögren's syndrome (SS) type) and a type without obvious tissue abnormalities (non-SS type). SS-type dry eye is an autoimmune disease characterized by chronic inflammation of the systemic exocrine glands, mainly the lacrimal glands and salivary glands. Inflammation damages the lacrimal glands, leading to severe dry eye. Even in non-SS-type dry eye, the stability of the tear layer is reduced, leading to epithelial damage to the cornea and conjunctiva due to dryness. As a result, the moisture of the epithelial surface is reduced, and the stability of the tear layer is also reduced. This vicious cycle can cause inflammation and aggravate epithelial damage (Non-Patent Literature 3).

[0004] No radical treatment for dry eye has been found yet. In the Guidelines for the Diagnosis and Treatment of Dry Eye (Non-Patent Document 1), as methods for improving symptoms, symptomatic treatments such as artificial tears, hyaluronic acid eye drops, rebamipide (Mucosta (registered trademark)) and diquafosol sodium (Diquas (registered trademark)) eye drops for replenishing tears, and surgical treatments such as inserting punctal plugs, that is, plugging the lacrimal glands with plugs to prevent tear discharge, thereby maintaining the tear volume. Although these treatments can improve the symptoms of dry eye, they are not radical treatments, so there is a problem that symptoms will recur if treatment is stopped.

[0005] In Japan, dry eye is defined as "a disease in which the stability of the tear layer is reduced due to various factors, causing eye discomfort, visual dysfunction, and may be accompanied by damage to the surface of the eye" (Non-Patent Document 4). Although it is not recommended to use anti-inflammatory drugs to treat dry eye, in the United States, dry eye is believed to be caused by inflammation. It is reported that in dry eye, inflammatory cytokines increase in tears and corneal epithelium, especially the increase in IFN-γ, which is associated with a decrease in conjunctival goblet cells and dry eye symptoms (Non-Patent Document 5). Based on the above, dry eye is currently believed to be related to inflammation.

[0006] In the United States and Europe, as anti-inflammatory drugs, eye drops include 0.05% cyclosporine (Restasis (registered trademark)) eye drops and LFA-1 antagonist Lifitegrast (Xiidra (registered trademark)), and their effectiveness has been reported. However, these eye drops have problems such as a long time required for the efficacy to manifest and difficulty in continuing the treatment due to side effects.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent literature 1: Guidelines for the diagnosis and treatment of dry eye disease (Editor: Diagnosis and Treatment Guidelines Writing Committee of the Dry Eye Research Society), issued on May 10, 2019;

[0010] Non-patent literature 2: Materials of the 21st Japan Ophthalmology Press Conference, June 2, 2022;

[0011] Non-patent document 3: N. Yokoi, et al., American Jornal Ophthamology, 2015, 159, 748-754;

[0012] Non-patent literature 4: Japanese dry eye syndrome definition and diagnostic criteria revised edition (2016 edition), Dry Eye Syndrome Research Group, Dry Eye Syndrome Definition and Diagnostic Criteria Committee;

[0013] Non-patent document 5: Stephen C Pflugfelder, et al., Investigative Ophthalmology & Visual Science, 2015, 56, 7545-7550. Summary of the invention

[0014] Problems to be Solved by the Invention

[0015] As described above, no radical therapeutic drug or treatment method has been found for dry eye disease, and it is necessary to develop a highly effective therapeutic drug or treatment method to improve the QOL (Quality of Life) of patients.

[0016] In dry eye, pathological features of lymphocyte infiltration and goblet cell reduction can be seen in the lesions, which shows a chronic inflammatory condition, but its cause is still unclear.

[0017] It is known that dry eye is often complicated by systemic autoimmune diseases such as Sjögren's syndrome, rheumatism, and systemic lupus erythematosus (Shindo Yumiko, Ohno Shigeaki, Department of Allergy, 2003, 52, 518-521). It has been reported that in the tears of patients with dry eye, inflammatory-related molecules such as IFN-γ, IL-1β, IL-6, IL-8, and TNF-α are significantly increased (Matilde Roda, et.al., International Jornal of Molecular Sciences, 2020, 21, 3111). In addition, it has been reported that the amount of IFN-γ in tears is related to the symptoms of dry eye (non-patent document 4), and it has also been reported that the amount of IFN-γ in tears can be used as a biomarker for dry eye (David Charles Jackson, et.al., Investigative Ophthalmology & Visual Science, 2016, 57, 4824-4830). It is generally believed that when dry eye occurs, IFN-γ plays a major role, just like other autoimmune diseases. In addition, it is reported that when IFN-γ knockout mice are used to make dry eye model mice, the onset of dry eye is mild. When IFN-γ is administered to the mice, the number of goblet cells decreases and CD4 positive T cell infiltration is enhanced as symptoms of dry eye (Xiabo Zhang, et.al., Investigative Ophthalmology & Visual Science, 2011, 52, 6279-6285). It is also reported that by administering anti-IFN-γ antibodies to dry eye mouse models, these dry eye symptoms can be suppressed (Xiaobo Zhang, et.al., Experimental Eye Research, 2014, 118, 117-124). These reports show that IFN-γ plays an important role in the condition of dry eye. However, so far, there is no therapeutic drug for dry eye that targets IFN-γ.

[0018] 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, and they are not suitable for eye drops that need to be administered multiple times a day.

[0019] 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.

[0020] 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.

[0021] As Janus kinase inhibitors, four types, namely, 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.

[0022] 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 during long-term administration and may show unexpected side effects caused by susceptibility to infection. In addition, Janus kinase inhibitors require buffers due to their low water solubility, so they are also worried about their own irritation.

[0023] The present invention is completed in view of these circumstances, and its purpose is to provide a dry eye treatment drug that can selectively inhibit IFN-γ as a target substance, has no risk of biological contamination, can be stored at room temperature, has high water solubility, and is less irritating.

[0024] Means for solving problems

[0025] In order to solve the above problems, the dry eye therapeutic agent of the present invention, which contains a DNA oligonucleotide as an active ingredient, adopts the following configuration.

[0026] The first embodiment of the present invention provides a drug for treating dry eye disease, 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 for dry eye disease.

[0027] The base sequence shown in SEQ ID NO: 2 is a sequence obtained by adding an oligonucleotide composed of 9-residue natural bases to the 3′ end of the base sequence shown in SEQ ID NO: 1.

[0028] 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.

[0029] In the first embodiment of the present invention, the base X in the sequence of the DNA oligonucleotide is an artificial base, and the artificial base can be chemically modified with a low molecular compound.

[0030] 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.

[0031] 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.

[0032] 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).

[0033] Effects of the Invention

[0034] 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 dry eye treatment 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, it can also improve the convenience of patients using it. In addition, the dry eye treatment drug containing a DNA oligonucleotide with a base sequence involved in the present invention can be administered by eye drops due to its molecular weight.

[0035] 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 dry eye treatment drug 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 and can be stored at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the change 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, wherein the vertical axis represents the change in the number of hairs per transplanted skin tissue piece.

[0037] Figure 2A This is a graph showing the suppression of MHC class I expression in dermal sheath cup cells caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0038] Figure 2B This is a diagram showing the suppression of MHC class I expression in the outer root sheath caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0039] Figure 3A This is a diagram showing the suppression of MHC class II expression in the connective tissue root sheath caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0040] Figure 3B This is a diagram showing the suppression of MHC class II expression in the outer root sheath caused by administration of a DNA oligonucleotide according to one embodiment of the present invention.

[0041] 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.

[0042] 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.

[0043] Figure 5 This is a diagram showing the results of suppressing corneal disorders in an experiment on the preventive effect of the surrogate aptamer in one embodiment of the present invention on a mouse dry eye model.

[0044] Figure 6 This is a diagram showing the results of suppressing corneal disorders in an experiment on the therapeutic effect of the surrogate aptamer in one embodiment of the present invention on a mouse dry eye model.

[0045] Fig. 7A This is a diagram showing the results of an experiment on the therapeutic effect of a surrogate aptamer in one embodiment of the present invention on a mouse dry eye model, which showed that the surrogate aptamer inhibited the decrease of goblet cells on the conjunctiva of the ocular surface.

[0046] Figure 7BThis is a diagram showing the results of an experiment on the therapeutic effect of a surrogate aptamer in one embodiment of the present invention on a mouse dry eye model, showing that the surrogate aptamer inhibited the infiltration of CD4-positive T cells into the conjunctiva of the ocular surface. DETAILED DESCRIPTION

[0047] Hereinafter, embodiments of the dry eye therapeutic agent containing a DNA oligonucleotide as an active ingredient according to the present invention will be described.

[0048] 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.

[0049] 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.

[0050] 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 a DNA aptamer with a specific sequence, the activity of the target molecule can be hindered and inhibited, and it can also be hyperactive. Although the molecular weight of the DNA aptamer is about 1 / 10 less than that of the antibody, it has the same high affinity as the antibody and has high target selectivity. Therefore, the IFN-γ inhibitory drug 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.

[0051] DNA aptamers form a compact three-dimensional structure compared to antibodies, so they can be administered topically, such as by eye drops, transdermal administration, and transmucosal administration.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] DNA aptamers are rapidly degraded by nucleases (nucleases) in biological tissues, so even if they show strong activity in vitro, they may not necessarily 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.

[0056] In this embodiment, a DNA oligonucleotide having the base sequence described in Table 1 is used as a DNA aptamer. This embodiment includes using a DNA oligonucleotide having the base sequence described in Table 1 as a therapeutic agent for dry eye disease.

[0057]

Table 1

[0058] Serial Number Sequence (5′→3′) 1 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGG 2 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGGGCGCGAAGCG 3 CCCGCCCGGGTCCGCGAAGCGGTAGGTDsTGGGCTAGGCDsGCTGGCGGGCCGXAGCG 4 GGCCGGTACCCGADsCCACAGTTTATDsGTTGTACTAGTTTTGCAGGGTCTGGCCCGCGAAGCG

[0059] 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.

[0060] 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.

[0061] As the functional group for modification, an azido group (-N 3 ), amino group (-NH 2 ), carboxyl (-COOH) or its active ester, alkynyl (-CC) or alkynyl structure containing cyclic structure, formyl (-CHO), hydrazide (-NH-NH 2 ), hydroxyl (-OH), thiol (-SH), cyano (-CN), vinyl (-CHCH 2 ), maleimide group.

[0062] 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".

[0063] 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.

[0064] 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 dry eye, where one of the reasons is presumed to be the excessive production of IFN-γ, which is the subject of the present invention, providing an IFN-γ inhibitor using a DNA aptamer can also be used as a means to solve the problem.

[0065] Regarding whether the DNA aptamer that inhibits the activity of IFN-γ is effective in treating dry eye, 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 dry eye in humans established so far, a universal mouse dry eye model is known (a dry eye induction model induced by a dry environment with a humidity of 20% and administration of scopolamine) (Terry G. Coursey, et.al., Translational Vision Science & Technology, 2018, 7, 24: 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.

[0066] 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.

[0067] The criteria for the surrogate aptamer to show high similarity in physical properties to the above-mentioned DNA aptamer are as follows.

[0068] (1) DNA aptamer.

[0069] (2) The base sequence of the aptamer contains two artificial bases Ds, and the other bases are natural bases.

[0070] (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).

[0071] (4) A 9-residue mini-hairpin sequence is present at the 3′ end of the aptamer base sequence.

[0072] 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.

[0073] 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.

[0074] The obtained aptamer was administered to mice with dry eye induced by 20% humidity in a dry environment or scopolamine administration (reference 1) at the same time or 3 days after the induction of dry eye. The result showed that the aptamer suppressed the increase of corneal lesion score in mice compared with the non-administered group (Examples 6 and 7).

[0075] After pathological histological evaluation of ocular tissues, the density of goblet cells on the conjunctiva of the ocular surface was higher in the aptamer-administered group compared with the non-administered group, and there was no difference from the normal group. In addition, the density of CD4-positive T cells on the conjunctiva of the ocular surface was lower in the aptamer-administered group compared with the non-administered group, and there was no difference from the normal group. The above indicates that inflammation of the conjunctiva of the ocular surface is suppressed by administering a replacement aptamer (Example 7).

[0076] 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 dry eye in humans, similarly to the surrogate aptamer, and can be used as a drug for the treatment of dry eye.

[0077] 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.

[0078] 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, 4167-4183; Katarina D. Kovacevic, et.al., Advanced Drug Delivery Reviews, 2018, 134, 36-50).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] Example 1: Synthesis of DNA aptamers

[0089] Aptamer 1 and aptamer 2 were chemically synthesized by the methods described in International Publication Nos. 2013 / 073602 and 2016 / 143700.

[0090] Example 2: Synthesis of Aptamer 3

[0091] 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.

[0092] Example 3: Synthesis of PEG-modified DNA aptamers

[0093] 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).

[0094] Example 4: Confirmation of therapeutic effect using a humanized mouse alopecia areata model

[0095] Step 1 Preparation of humanized mouse alopecia areata model

[0096] 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.

[0097] Process 2

[0098] 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.

[0099] The results after administration in Example 4 are shown in Figure 1 . Figure 1The 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.

[0100] 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.

[0101] 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.

[0102] 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 3BThe 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.

[0103] 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.

[0104] Example 5: Production of mouse IFN-γ aptamer (surrogate aptamer)

[0105] Process 1

[0106] 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.

[0107] Process 2

[0108] 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.

[0109] 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.

[0110] Example 6: Investigation of the preventive effect of alternative aptamers in a mouse dry eye model

[0111] Step 1 Preparation of dry eye mice

[0112] Mice with dry eye induced by dry environment and scopolamine administration were prepared (Terry G.Coursey, et.al., Translational Vision Science & Technology, 2018, 7, 24). Normal mice were placed in a cage with a humidity of 20%, and dry stress was given, and scopolamine hydrobromide hydrate was intraperitoneally administered 0.5 mg / mL 4 times a day to cause a dry state on the surface of the eye, thereby causing corneal epithelial disease and inflammation of the corneal and conjunctival epithelium.

[0113] Process 2

[0114] The aptamer was administered to both eyes of the mice by eye drops from the day of induction of dry eye, 3 times a day for 5 days (0.01 mg / mL or 1 mg / mL, 3 μL / time). The control group of dry eye mice used a non-administered group (dry eye mice), and the control group of normal mice used a normal group (normal mice), and each control group did not receive the aptamer. Figure 5The results are shown in which Oregon Green Dextran, a fluorescein derivative, was dripped into the eyes of mice in each group 5 days after inducing dry eye, and the eyes were washed 1 minute later, and the residual Oregon Green Dextran in the corneal epithelium was scored to evaluate the corneal epithelial disease. In the non-administered group (dry eye mice), the corneal epithelial disease score increased significantly compared with the normal group (normal mice), while in the aptamer-administered group (1 mg / mL), the corneal epithelial disease score was lower than that of the non-administered group. It can be seen that the aptamer-administered group showed an effect of inhibiting the corneal epithelial disease of mice and thus inhibiting the onset of dry eye compared with the non-administered group.

[0115] Example 7: Investigation of the therapeutic effects of alternative aptamers in a mouse dry eye model

[0116] Process 1

[0117] Dry eye was induced in mice by the same method as in step 1 of Example 6. Three days after the induction of dry eye, the replacement aptamer or the existing drugs Restasis and Xiidra as a comparative control were administered to the dry eye mice by eye drops three times a day for 7 days (1 mg / mL, 3 μL / time). As a control group for dry eye mice, a non-administered group (dry eye mice) was used, and as a control group for normal mice, a normal group (normal mice) was used. Figure 6 The results are obtained by dropping Oregon Green Dextran, a fluorescein derivative, into the eyes of mice in each group 10 days after inducing dry eye (i.e., 7 days after administration of the alternative aptamer or the existing drug), washing the eyes 1 minute later, and scoring the Oregon Green Dextran remaining in the corneal epithelium to evaluate corneal epithelial disease. In the non-drug group (dry eye mice), the corneal epithelial disease score increased significantly compared with the normal group (normal mice), while the aptamer-administered group and the Xiidra-administered group, one of the existing drug-administered groups, had lower values ​​of corneal epithelial disease scores compared with the non-drug group (dry eye mice). It can be seen that the aptamer-administered group improved the corneal epithelial disease of mice compared with the non-drug group (dry eye mice), showing a therapeutic effect for dry eye.

[0118] Process 2

[0119] After the evaluation of corneal epithelial disorders in mice (7 days after administration of the alternative aptamer or the existing drug, ie, 10 days after induction of dry eye), the eyes were removed and histopathological evaluation of the corneal and conjunctival epithelium was performed. Fig. 7AThe results are shown by staining the sections of mouse eye tissue made 10 days after inducing dry eye with periodic acid Schiff (PAS) to evaluate the goblet cell density of the conjunctival epithelium. In the non-drug group (dry eye mice), the goblet cell density after PAS staining was significantly reduced compared with the normal group (normal mice), while in the aptamer-administered group, the goblet cell density was at the same level as the normal group (normal mice). Even in the existing drug-administered groups (Restasis and Xiidra), the decrease in goblet cell density was suppressed compared with the non-drug group, but the inhibitory effect was weaker than that of the aptamer-administered group. In addition, the results obtained by evaluating eye tissue sections using immunohistochemical staining with anti-CD4 antibodies are shown. Figure 7B In the non-drug group (dry eye mice), the infiltration of CD4-positive T cells into the conjunctival epithelium increased significantly compared with the normal group (normal mice), while in the aptamer-administered group, the infiltration of CD4-positive T cells was suppressed. In addition, even in the existing drug-administered groups (Restasis and Xiidra), the infiltration of CD4-positive T cells was suppressed compared with the non-drug group, but the inhibitory effect was weaker than that of the aptamer-administered group.

[0120] 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 dry eye, which are believed to be mainly caused by excessive production of IFN-γ.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] By administering the therapeutic drug containing DNA aptamers according to the present embodiment as an injection, the drug can be applied to diseases such as systemic autoimmune diseases and dry eye syndrome, which are believed to be mainly caused by excessive production of IFN-γ. In addition, even when the drug is made into 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.

Claims

1. A drug for treating dry eye, 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 dry eye treatment drug 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 dry eye treatment drug 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 dry eye treatment drug 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 dry eye treatment drug according to claim 4, wherein: The polymer compound is polyethylene glycol (PEG) having a molecular weight of 20,000 or more, or any biocompatible polymer having a molecular weight of 20,000 or more.

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