Glial-cell-dependent neurite outgrowth promoter

Abstract

The present invention addresses the problem of providing a novel technique for promoting neurite outgrowth activity. The present invention for solving the aforementioned e problem is a glial-cell-dependent neurite outgrowth promoter comprising an NF-κB decoy as an active ingredient, the glial-cell-dependent neurite outgrowth promoter being used in order to promote neurite outgrowth of nerve cells via glial cells.

Description

Glial cell-dependent neurite outgrowth promoter

[0001] The present invention relates to the use of an oligonucleotide decoy capable of binding to the DNA binding site of NF-κB.

[0002] Nuclear factor κB (NF-κB) is a homo- or hetero-protein dimer complex composed of any two of a group of five proteins (Rel proteins), namely p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel, and RelB, in mammals, and is a general term for a group of transcription factors that regulate (repress or activate) the transcription of target genes. The p50 / p65 (RelA) heterodimer was first identified in 1986 by David Baltimore et al. as a transcription factor that binds to the enhancer of the κ light chain of immunoglobulin selectively expressed in B cells, and thus it is called so.

[0003] NF-κB is activated by stimuli such as stress, cytokines, and ultraviolet rays. NF-κB is one of the transcription factors that play a central role in the immune response and is involved in many physiological phenomena such as acute and chronic inflammatory responses, cell proliferation, and apoptosis. Poor control of NF-κB activity causes inflammatory diseases such as Crohn's disease and rheumatoid arthritis, as well as cancer and septic shock, and constitutive activation of NF-κB is often observed in malignant tumors in particular. Furthermore, NF-κB is also involved in the growth of cytomegalovirus (CMV) and human immunodeficiency virus (HIV).

[0004] The NF-κB signaling pathway has been reported to play a central role in promoting neurite outgrowth, a crucial process in the development of the nervous system. For example, Non-Patent Literature 1 discloses that brain-derived neurotrophic factor (BDNF)-mediated neurite outgrowth of nodular ganglion neurons is suppressed by inhibiting the phosphorylation of the inhibitor of kappa B alpha (IκBα) protein, which is involved in the NF-κB signaling pathway, or by inhibiting the transcriptional activity of NF-κB.

[0005] On the other hand, current research trends in nerve function, including nerve regeneration, aimed at improving pain and itching, are exploring both methodologies that suppress neurite outgrowth and methodologies that promote neurite outgrowth. Currently, researchers are exploring how to extract clinical effects from these two methodologies. For example, Patent Document 1 discloses an anti-pain agent containing an aptamer that binds to nerve growth factor (NGF) and inhibits the neurite outgrowth activity and / or cell proliferation activity of NGF. Conversely, Non-Patent Document 2 discloses that Maresin 1 promotes neurite outgrowth of dorsal root ganglion (DRG) neurons and suppresses neuropathic pain.

[0006] International Publication No. 2013 / 047844

[0007] H. Gutierrez et al. Development 132, 1713-1726 (2005) J. Wei et al. J. Neuroinflammation 19, 32 (2022)

[0008] The problem that this invention aims to solve is to provide a novel technology that promotes neurite outgrowth.

[0009] As suggested in Non-Patent Document 1, the NF-κB signaling pathway plays a crucial role in the development of the nervous system. This document shows that inhibiting the NF-κB signaling pathway suppresses neurite outgrowth. However, in order to more clearly elucidate the role of the NF-κB signaling pathway, the inventors diligently conducted research using an NF-κB decoy that binds to NF-κB in the NF-κB signaling pathway and antagonistically inhibits the binding of NF-κB to target DNA. They discovered that the NF-κB decoy acts on glial cells to promote neurite outgrowth of nerve cells via glial cells. The present invention was completed based on the above discovery and is described below.

[0010] [Item 1] A glial cell-dependent neurite extension promoter, comprising NF-κB decoy as an active ingredient, used to promote neurite extension of nerve cells via glial cells. [Item 2] The glial cell-dependent neurite extension promoter according to Item 1, wherein the nerve cells are sensory nerve cells. [Item 3] The glial cell-dependent neurite extension promoter according to Item 2, wherein the sensory nerve cells are sensory nerve cells of a patient suffering from intervertebral disc degeneration. [Item 4] The glial cell-dependent neurite extension promoter according to Item 3, wherein the sensory nerve cells of a patient suffering from intervertebral disc degeneration are sensory nerve cells of a patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain. [Item 5] The glial cell-dependent neurite extension promoter according to Item 4, wherein the sensory nerve cells of a patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain are sensory nerve cells in the sensory nerve root area of ​​a patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain. [Item 6] A glial cell-dependent neurite extension promoter according to any one of items 1 to 5, wherein the glial cells are Schwann cells or satellite glial cells. [Item 7] A glial cell-dependent neurite extension promoter according to any one of items 1 to 6, used by local administration to the site of glial cells or its vicinity. [Item 8] A glial cell-dependent neurite extension promoter according to any one of items 1 to 7, used by local administration to the spinal cord dorsal horn or its vicinity. [Item 9] A glial cell-dependent neurite extension promoter according to any one of items 1 to 8, used by local administration into the intervertebral disc such that the NF-κB decoy reaches the site of glial cells by infiltration or diffusion through non-hematogenous distribution. [Item 10] A glial cell-dependent neurite extension promoter according to any one of items 1 to 9, used by local administration into the intervertebral disc so that the NF-κB decoy reaches the spinal cord dorsal horn by infiltration or diffusion through non-hematogenous distribution. [Item 11] A glial cell-dependent neurite extension promoter according to any one of items 1 to 10, used by administration to patients suffering from anti-inflammatory drug-resistant intervertebral disc degeneration. [Item 12] A glial cell-dependent neurite extension promoter according to any one of items 1 to 11, used for the treatment of anti-inflammatory drug-resistant intervertebral disc degeneration.[Item 13] A glial cell-dependent neurite extension promoter according to any one of Items 1 to 12, to be administered to patients suffering from intervertebral disc degeneration without signs of inflammation. [Item 14] A glial cell-dependent neurite extension promoter according to any one of Items 1 to 13, to be used for the treatment of intervertebral disc degeneration without signs of inflammation. [Item 15] A glial cell-dependent neurite extension promoter according to any one of Items 1 to 14, wherein the NF-κB decoy is one of the following (a) to (c): (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form complementary base pairs. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, wherein a mutant of the oligonucleotide represented by SEQ ID NO: 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by SEQ ID NO: 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and these mutants form complementary base pairs. (c) A double-stranded oligonucleotide having complementary base pairs formed between a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA. 5'-GGGPuNNPyPyCC-3' (κB sequence) [Item 16] A glial cell-dependent neurite extension promoter according to any one of Items 1 to 14, wherein the NF-κB decoy is (a) below. (a) A double-stranded oligonucleotide having complementary base pairs formed between the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID 3).

[0011] [Item 17] Use of NF-κB decoy in the manufacture of a therapeutic agent for intervertebral disc degeneration that promotes neurite outgrowth of nerve cells via glial cells. [Item 18] The use according to Item 17, wherein the nerve cells are nerve cells of sensory nerves. [Item 19] The use according to Item 18, wherein the therapeutic agent is a therapeutic agent for intervertebral disc degeneration accompanied by chronic neuropathic pain. [Item 20] The use according to Item 19, wherein the nerve cells of sensory nerves are nerve cells of sensory nerves in the sensory nerve root region. [Item 21] The use according to Item 20, wherein the glial cells are Schwann cells or satellite glial cells. [Item 22] The use according to any one of Items 17 to 21, wherein the therapeutic agent is filled in a syringe for local administration to or near the site of glial cells. [Item 23] The use according to any one of Items 17 to 22, wherein the therapeutic agent is filled in a syringe for local administration to or near the spinal cord dorsal horn. [Item 24] The use according to any one of items 17 to 23, wherein the therapeutic agent is filled in a syringe for local administration into the intervertebral disc, and the amount of the therapeutic agent in the syringe is sufficient to allow the NF-κB decoy to reach the site of glial cells by osmosis or diffusion through non-hematogenous distribution after local administration into the intervertebral disc. [Item 25] The use according to any one of items 17 to 24, wherein the therapeutic agent is filled in a syringe for local administration into the intervertebral disc, and the amount of the therapeutic agent in the syringe is sufficient to allow the NF-κB decoy to reach the spinal cord dorsal horn by osmosis or diffusion through non-hematogenous distribution after local administration into the intervertebral disc. [Item 26] The use according to any one of items 17 to 25, wherein the therapeutic agent is administered to a patient suffering from anti-inflammatory drug-resistant intervertebral disc degeneration. [Item 27] ​​The use described in any one of Items 17 to 26, wherein the therapeutic agent is a therapeutic agent for degenerative disc disease resistant to anti-inflammatory drugs. [Item 28] The use described in any one of Items 17 to 27, wherein the therapeutic agent is administered to a patient suffering from degenerative disc disease without signs of inflammation. [Item 29] The use described in any one of Items 17 to 28, wherein the therapeutic agent is a therapeutic agent for degenerative disc disease without signs of inflammation.[Item 30] The use described in any one of items 17 to 29, wherein the NF-κB decoy is one of the following (a) to (c): (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form complementary base pairs: 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTTCCCTAAAAGGGAGG-5' (Sequence ID No. 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, in which a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, forming complementary base pairs. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA. 5'-GGGPuNNPyPyCC-3' (κB sequence) [Item 31] The use according to any one of Items 17 to 29, wherein the NF-κB decoy is (a) below: (a) A double-stranded oligonucleotide having complementary base pairs formed by the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID 3).

[0012] [Item 32] A method for treating intervertebral disc degeneration, wherein the method comprises the following steps: (1) identifying a patient suffering from intervertebral disc degeneration; (2) administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of NF-κB decoy, wherein the NF-κB decoy promotes neurite outgrowth of nerve cells via glial cells. [Item 33] The method for treating intervertebral disc degeneration according to Item 32, wherein the nerve cells are nerve cells of sensory nerves. [Item 34] The method for treating intervertebral disc degeneration according to Item 33, wherein the patient suffering from intervertebral disc degeneration is a patient suffering from intervertebral disc degeneration with chronic neuropathic pain. [Item 35] The method for treating intervertebral disc degeneration according to Item 34, wherein the nerve cells of sensory nerves are nerve cells of sensory nerves in a sensory nerve root area. [Item 36] A method for treating a patient suffering from degenerative disc disease according to Item 35, wherein the glial cells are Schwann cells or satellite glial cells. [Item 37] A method for treating a patient suffering from degenerative disc disease according to any one of Items 32 to 36, wherein in step (2), the pharmaceutical composition is locally administered to the site of or near the site of the glial cells. [Item 38] A method for treating a patient suffering from degenerative disc disease according to any one of Items 32 to 37, wherein in step (2), the pharmaceutical composition is locally administered to the dorsal horn of the spinal cord or near the site of the spinal cord. [Item 39] A method for treating a patient suffering from degenerative disc disease according to any one of Items 32 to 38, wherein in step (2), the pharmaceutical composition is locally administered into the intervertebral disc, and the NF-κB decoy is delivered to the site of the glial cells by infiltration or diffusion through non-hematogenous distribution. [Item 40] A method for treating a patient suffering from degenerative disc disease according to any one of items 32 to 39, wherein in step (2) above, the pharmaceutical composition is administered locally into the intervertebral disc, and the NF-κB decoy is brought to the spinal cord dorsal horn by non-hematogenous distribution, penetration or diffusion. [Item 41] A method for treating a patient suffering from degenerative disc disease according to any one of items 32 to 40, wherein the patient is a patient suffering from anti-inflammatory drug-resistant degenerative disc disease.[Item 42] A method for treating a patient suffering from intervertebral disc degeneration as described in any one of items 32 to 41, wherein the intervertebral disc degeneration is resistant to anti-inflammatory drugs. [Item 43] A method for treating a patient suffering from intervertebral disc degeneration as described in any one of items 32 to 42, wherein the patient suffers from intervertebral disc degeneration without signs of inflammation. [Item 44] A method for treating a patient suffering from intervertebral disc degeneration as described in any one of items 32 to 43, wherein the intervertebral disc degeneration is resistant to inflammation. [Item 45] A method for treating a patient suffering from intervertebral disc degeneration as described in any one of items 32 to 44, wherein the NF-κB decoy is one of the following (a) to (c): (a) A double-stranded oligonucleotide in which the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO: 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, wherein a mutant of the oligonucleotide represented by SEQ ID NO: 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by SEQ ID NO: 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and these mutants form complementary base pairs. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, which antagonistically inhibits the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA. 5'-GGGPuNNPyPyCC-3' (κB sequence) [Item 46] A method for treating a patient suffering from degenerative disc disease as described in any one of Items 32 to 44, wherein the NF-κB decoy is (a) below.(a) A double-stranded oligonucleotide formed by complementary base pairing of the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3. 5'-CCTTGAAGGGGAATTTCCCCCC-3' (Sequence ID No. 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID No. 3).

[0013] [Item 47] An NF-κB decoy for use in the treatment of intervertebral disc degeneration, which promotes neurite outgrowth of nerve cells via glial cells. [Item 48] The NF-κB decoy according to Item 47, wherein the nerve cells are nerve cells of sensory nerves. [Item 49] The NF-κB decoy according to Item 48, wherein the intervertebral disc degeneration is intervertebral disc degeneration accompanied by chronic neuropathic pain. [Item 50] The NF-κB decoy according to Item 49, wherein the nerve cells of sensory nerves are nerve cells of sensory nerves in the sensory nerve root region. [Item 51] The NF-κB decoy according to Item 50, wherein the glial cells are Schwann cells or satellite glial cells. [Item 52] The NF-κB decoy according to any one of Items 47 to 51, for use by local administration to or near the site of glial cells. [Item 53] An NF-κB decoy according to any one of items 47 to 52, for use by local administration to the spinal cord dorsal horn or its vicinity. [Item 54] An NF-κB decoy according to any one of items 47 to 53, for use by local administration into the intervertebral disc such that the NF-κB decoy reaches the site of glial cells by osmosis or diffusion through non-hematogenous distribution. [Item 55] An NF-κB decoy according to any one of items 47 to 54, for use by local administration into the intervertebral disc such that the NF-κB decoy reaches the spinal cord dorsal horn by osmosis or diffusion through non-hematogenous distribution. [Item 56] An NF-κB decoy according to any one of items 47 to 55, for use by administration to patients suffering from anti-inflammatory drug-resistant intervertebral disc degeneration. [Item 57] An NF-κB decoy according to any one of items 47 to 56, wherein the intervertebral disc degeneration is anti-inflammatory drug resistant intervertebral disc degeneration. [Item 58] An NF-κB decoy according to any one of items 47 to 57, for use in patients suffering from intervertebral disc degeneration without signs of inflammation. [Item 59] An NF-κB decoy according to any one of items 47 to 58, wherein the intervertebral disc degeneration is intervertebral disc degeneration without signs of inflammation. [Item 60] An NF-κB decoy according to any one of items 47 to 59, wherein the NF-κB decoy is one of the following (a) to (c): (a) A double-stranded oligonucleotide in which the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO: 3 form a complementary base pair.5'-CCTTGAAGGGGAATTTCCCCTC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, wherein a mutant of the oligonucleotide represented by SEQ ID NO: 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by SEQ ID NO: 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and these mutants form complementary base pairs. (c) A double-stranded oligonucleotide in which a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, forms a complementary base pair, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA. 5'-GGGPuNNPyPyCC-3' (κB sequence) [Item 61] An NF-κB decoy according to any one of Items 47 to 59, wherein the NF-κB decoy is (a) below. (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID 3).

[0014] According to the present invention, a therapeutic effect on intervertebral disc degeneration can be obtained through the action of nerve cell neurite outgrowth mediated by glial cells.

[0015] The effects of administering NF-κB decoy via the soles of the feet on a rat model of brewer's yeast-induced acute pain (pain threshold ratio). Data are shown as mean ± standard error. A graph showing the effects of administering NF-κB decoy via the soles of the feet on a rat model of brewer's yeast-induced acute pain (sum of pain threshold ratios from 0 to 30h). Data are shown as mean ± standard error. The effects of administering NF-κB decoy intrathecally on a rat model of brewer's yeast-induced acute pain (pain threshold ratio). Data are shown as mean ± standard error. The effects of administering NF-κB decoy intrathecally on a rat model of brewer's yeast-induced acute pain (sum of pain threshold ratios). Data are shown as mean ± standard error. The effects of administering NF-κB decoy intrathecally on a rat model of SNL (Synchronized Non-Neutral Lung) on ​​a rat model. Data are shown as mean ± standard error. Effects of NF-κB decoys on rat dorsal root ganglion neurons via glial cells (N=9). Data are presented as mean ± standard error. N=4

[0016] In this application, "NF-κB (Nuclear Factor-kappa B)" refers to a group of transcription factors that regulate (repress or activate) the transcription of target genes. These are homozygous or heterozygous protein dimer complexes composed of any two of the five types of proteins (Rel proteins) in mammals: p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel, and RelB.

[0017] The five Rel proteins mentioned above can be classified into two types based on their carboxyl group end (C-terminus): (1) Class I (p50, p52) having a transcriptional repression domain (TRD) (2) Class II (p65, c-Rel, RelB) having a transcriptional activation domain (TAD) Therefore, NF-κB composed only of Class I Rel proteins with TRDs, such as a homodimer of p50, a homodimer of p52, and a heterodimer of p50 and p52, has the function of repressing gene transcription. Furthermore, NF-κB composed of class I Rel proteins with TRD and class II Rel proteins with TAD, and NF-κB composed solely of class II Rel proteins with TAD, have the function of activating gene transcription.

[0018] In this application, NF-κB may be a protein dimer complex of any combination of the five types of proteins (Rel proteins) (i.e., any combination from the 15 possible combinations). In this application, from the viewpoint of activating gene transcription in relation to glial cell-dependent neurite extension promotion, NF-κB is preferably a p50 / p65 heterodimer, a p52 / RelB heterodimer, or a c-Rel / p65 heterodimer, and more preferably a p50 / p65 heterodimer or a c-Rel / p50 heterodimer. Furthermore, in this application, from the viewpoint of repressing gene transcription in relation to glial cell-dependent neurite extension promotion, NF-κB is preferably a p50 / p50 homodimer.

[0019] On the other hand, the five types of Rel proteins have a common structure at their amino group terminal (N-terminus) consisting of approximately 300 amino acid residues called the Rel homology domain (RHD). Dimerization between the Rel proteins, binding between NF-κB and IκB proteins, and binding between NF-κB and DNA occur via the RHD. The binding between NF-κB and DNA is necessary for NF-κB to regulate (repress or activate) the transcription of target genes, and depends on NF-κB recognizing a specific common recognition DNA sequence present in the target chromosomal DNA. Examples of such common recognition DNA sequences include, but are not limited to, the following sequence (hereinafter referred to as the κB sequence in this application): 5'-GGGPuNNPyPyCC-3' (κB sequence)

[0020] Here, G represents DNA with guanine, C with cytosine, Pu with a purine ring (adenine (A) or guanine (G)), Py with a pyrimidine ring (cytosine (C) or thymine (T)), and N with any base.

[0021] In this application, NF-κB preferably recognizes and binds to 5'-GGGPuNNPyPyCC-3' (κB sequence) present in the target chromosomal DNA, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells. In this application, NF-κB preferably recognizes and binds to the following sequence (hereinafter referred to as κB sequence 1 in this application) present in the target chromosomal DNA, from the viewpoint of further promoting neurite outgrowth of nerve cells via glial cells: 5'-GGGATTTCCC-3' (Sequence ID 1)

[0022] In this application, "decoy" refers to a compound that (1) mimics a target chromosomal DNA region recognized by a DNA-binding protein, (2) binds to the DNA-binding protein and antagonistically inhibits the binding of the DNA-binding protein to the target chromosomal DNA region, and (3) regulates the transcription of a target gene via the DNA-binding protein. Here, the compound has a double-stranded structure in which one or two oligonucleotides, consisting of nucleic acids, nucleic acid analogs, and / or modified thereof, form complementary base pairs in opposite directions within or between molecules.

[0023] Furthermore, the decoy in this application is preferably a compound that, from the viewpoint of efficiently regulating the transcription of the target gene, (1) mimics the target chromosomal DNA region recognized by the DNA-binding protein which is a transcription factor, (2) binds to the DNA-binding protein which is a transcription factor and antagonistically inhibits the binding of the DNA-binding protein which is a transcription factor to the target chromosomal DNA region, and (3) regulates the transcription of the target gene via the DNA-binding protein which is a transcription factor. Here, the compound has a double-stranded structure in which one or two oligonucleotides, consisting of nucleic acids, nucleic acid analogs, and / or modified thereof, form complementary base pairs in opposite directions within or between molecules.

[0024] The decoy in this application is a compound that (1) mimics a target chromosomal DNA region recognized by a DNA-binding protein, (2) binds to the DNA-binding protein and antagonistically inhibits the binding of the DNA-binding protein to the target chromosomal DNA region, and (3) regulates the transcription of a target gene via the DNA-binding protein, wherein the compound has any structure, as long as it consists of one or two oligonucleotides, comprising nucleic acids, nucleic acid analogs, and / or modified thereof, having a double-stranded structure in which complementary base pairs in opposite directions are formed within or between molecules.

[0025] In this application, the decoy is preferably a linear decoy (a decoy in which two oligonucleotides form complementary base pairs in opposite directions between their molecules), a staple-type decoy (a decoy in the shape of a staple after pressing, in which a single oligonucleotide has a 5' terminal sequence that forms a complementary base pair in the opposite direction to the intermediate sequence, a 3' terminal sequence that also forms a complementary base pair in the opposite direction to the intermediate sequence, and loop portions consisting of 3 to 10 base sequences that do not form complementary base pairs within the molecule at both ends of the intermediate portion), or a dumbbell-type decoy (a dumbbell-shaped annular decoy having an intermediate portion in which complementary base pairs are formed, and loop portions consisting of 3 to 10 base sequences that do not form complementary base pairs within the molecule at both ends of the intermediate portion).

[0026] Furthermore, the decoy in this application is more preferably a linear decoy, from the viewpoint of keeping synthesis costs low and having the flexibility to adapt to structural changes of the target molecule.

[0027] As stated above, the decoy in this application consists of nucleic acids, nucleic acid analogs, and / or modified forms thereof.

[0028] In this application, the nucleic acids constituting the decoy refer to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The DNA includes DNA containing one base selected from the four base groups consisting of adenine (A), cytosine (C), guanine (G), and thymine (T). The RNA includes RNA containing one base selected from the four base groups consisting of adenine (A), cytosine (C), guanine (G), and uracil (U).

[0029] In this application, the nucleic acid analogs constituting the decoy refer to artificially synthesized molecules having a structure similar to natural nucleic acids (DNA and RNA). These nucleic acid analogs may be produced, for example, by replacing the sugar-phosphate backbone of a nucleic acid with a different chemical structure. Examples of these nucleic acid analogs, for example, but not limited to, include acyclovir, valacyclovir, famciclovir, 5-fluorouracil (5-FU), cytarabine, gemcitabine, azidothymidine (AZT), lamivudine, emtricitabine, and tenofovir.

[0030] In this application, a modified nucleic acid or modified nucleic acid analog constituting a decoy refers to a molecule whose structure has been chemically modified from nucleic acids (DNA and RNA) or nucleic acid analogs. These modifications may be carried out, for example, to improve the stability of the decoy in this application, reduce immunogenicity, improve target specificity, promote intracellular uptake, improve pharmacokinetics, and / or add function.

[0031] Modifications performed in this application to improve the stability of the decoy include, but are not limited to, phosphorothioate modification, 2'-O-methyl modification, pseudouridine (Ψ) modification, 2'-fluoro modification, and introduction of a cross-linking structure to the sugar moiety.

[0032] Modifications performed in this application for the purpose of reducing the immunogenicity of the decoy include, but are not limited to, pseudouridine (Ψ) modification, 5-methylcytidine (m5C) modification, 2'-O-methyl modification, phosphorothioate modification, and N1-methylpsoiduridine (m1Ψ) modification.

[0033] Modifications performed in this application for the purpose of improving the target specificity of the decoy include, but are not limited to, 2'-O-methoxyethyl (2'-MOE) modification, locked nucleotide acid (LNA) modification, phosphorothioate modification, 2'-fluoro (2'-F) modification, and gapmer design.

[0034] Modifications performed in this application for the purpose of promoting intracellular uptake of the decoy include, but are not limited to, galactose modification, peptide modification, lipid modification, polyethylene glycol (PEG) modification, and aptamer modification.

[0035] Modifications performed in this application for the purpose of improving the pharmacokinetics of the decoy include, but are not limited to, 2'-O-methoxyethyl (2'-MOE) modification, phosphorothioate modification, N-acetylgalactosamine (GalNAc) modification, polyethylene glycol (PEG), and locked nucleotide acid (LNA) modification.

[0036] Modifications performed for the purpose of adding decoy functionality in this application include, but are not limited to, modifications by adding fluorescent dyes, modifications by adding radioisotopes, and modifications by adding other drugs.

[0037] In this application, the nucleic acids, nucleic acid analogs, and / or modified thereof constituting the decoy are preferably deoxyribonucleic acid (DNA) from the viewpoint of mimicking the target chromosomal DNA region recognized by DNA-binding proteins and being more stable in vivo. Furthermore, from the viewpoint of comprehensively improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function, the nucleic acids, nucleic acid analogs, and / or modified thereof constituting the decoy in this application are preferably phosphorothioate modified nucleic acids or nucleic acid analogs. Furthermore, the nucleic acids, nucleic acid analogs, and / or their modifications constituting the decoy in this application are more preferably phosphorothioate modified deoxyribonucleic acid (DNA) from the viewpoint of mimicking the target chromosomal DNA region recognized by DNA-binding proteins and being more stable in vivo, as well as from the viewpoint of comprehensively improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function.

[0038] In this application, the decoy preferably has a double-stranded structure in which one or two oligonucleotides, consisting of nucleic acids, nucleic acid analogs, and / or phosphorothioate-modified thereof, form complementary base pairs in opposite directions within or between molecules, from a comprehensive viewpoint of objectives such as improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function.

[0039] Furthermore, from a comprehensive viewpoint of objectives such as improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function, it is more preferable that the decoy in this application has a double-stranded structure in which one or two oligonucleotides, each containing at least one phosphorothioate-modified nucleic acid or nucleic acid analog, form complementary base pairs in opposite directions within or between molecules.

[0040] Furthermore, from a comprehensive viewpoint of objectives such as improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function, it is even more preferable that the decoy in this application has a double-stranded structure in which two oligonucleotides, each containing at least one phosphorothioate-modified nucleic acid or nucleic acid analog, form complementary base pairs opposite to each other at the intermolecular level.

[0041] Furthermore, from a comprehensive viewpoint of the objectives of improving stability, reducing immunogenicity, improving target specificity, promoting intracellular uptake, improving pharmacokinetics, and / or adding function, it is even more preferable that the decoy in this application has a double-stranded structure in which one or two oligonucleotides (i.e., in the oligonucleotides, all phosphodiester bonds between bases are replaced with phosphorothioate bonds) consisting of a nucleic acid or a phosphorothioate-modified nucleic acid analog form complementary base pairs in opposite directions within or between molecules.

[0042] The decoys in this application may be prepared using chemical oligonucleotide synthesis methods known in the art (e.g., phosphoramidite methods, etc.) or biochemical synthesis methods (e.g., polymerase chain reaction (PCR) methods, cloning vector methods, etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc., etc.). The decoys in this application may be prepared using chemical oligonucleotide synthesis methods using a DNA synthesizer, etc.

[0043] The decoy in the present application may be created by placing one type or two or more types of single-stranded oligonucleotides having complementary sequences with each other in a solution with an appropriate salt concentration at an appropriate temperature for an appropriate time, so as to form complementary base pairs intramolecularly and / or intermolecularly.

[0044] Also, from the perspective of creating a linear decoy, it is preferable that two or more types of single-stranded oligonucleotides having complementary sequences with each other are placed in a solution with an appropriate salt concentration at an appropriate temperature for an appropriate time in equimolar amounts, so as to form complementary base pairs intramolecularly and / or intermolecularly.

[0045] The "NF-κB decoy" in the present application refers to a compound that (1) mimics the target chromosomal DNA region recognized by the transcription factor NF-κB, (2) binds to the transcription factor NF-κB and antagonistically inhibits the binding of the transcription factor NF-κB to the target chromosomal DNA region, and (3) regulates the transcription of target genes through the transcription factor NF-κB. Here, the compound is composed of nucleic acids, nucleic acid analogs, and / or their modified forms, and a single or double-stranded oligonucleotide has a double-stranded structure in which complementary base pairs in opposite directions are formed intramolecularly or intermolecularly.

[0046] The NF-κB decoy in the present application may be any compound as long as it is a compound as described above. However, from the perspective of specifically inhibiting the function of the transcription factor NF-κB, it is preferable that the NF-κB decoy antagonistically inhibits the recognition and binding of the transcription factor NF-κB to the sequence 5'-GGG PuNNPyPyCC-3' (κB sequence) present in the target chromosomal DNA. Examples of such NF-κB decoys include, but are not limited to, NF-κB decoys containing the sequence 5'-GGG PuNNPyPyCC-3' (κB sequence).

[0047] Furthermore, from the viewpoint of specifically inhibiting the function of the transcription factor p50 / p65(RelA) heterodimer NF-κB, it is more preferable that the NF-κB decoy in this application is one that antagonistically inhibits the recognition and binding of the sequence 5'-GGGATTTCCC-3' (κB sequence 1) present in the target chromosomal DNA by the transcription factor p50 / p65(RelA) heterodimer NF-κB. Examples of such an NF-κB decoy include, but are not limited to, an NF-κB decoy containing the sequence 5'-GGGATTTCCC-3' (SEQ ID NO: 1) (κB sequence 1).

[0048] In this application, if the NF-κB decoy contains a κB sequence or κB sequence 1, it may further have an extension sequence consisting of nucleic acids, nucleic acid analogs, and / or modified versions thereof of any base length on its 5' side and / or 3' side, as long as the NF-κB decoy does not become unable to bind to the transcription factor NF-κB. The extension sequence may include a double-stranded structure forming complementary base pairs in opposite directions. The extension sequence may also include a single strand that does not form a complementary base pair. Furthermore, the extension sequence may include a single-stranded loop that does not form a complementary base pair.

[0049] From the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1, and stabilizing its binding to the transcription factor NF-κB, it is preferable that the extended sequence includes a double-stranded structure in which complementary base pairs are formed in opposite directions. Furthermore, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1, and stabilizing its binding to the transcription factor NF-κB, it is even more preferable that the extended sequence has a double-stranded structure in which complementary base pairs are formed in opposite directions.

[0050] In this application, if the NF-κB decoy includes a κB sequence or κB sequence 1, it is preferable that it further has an extended sequence on its 5' side and / or 3' side, respectively, that is at least 1 nucleotide and no more than 40 nucleotides in length, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1 and stabilizing its binding to the transcription factor NF-κB.

[0051] Furthermore, if the NF-κB decoy in this application includes a κB sequence or κB sequence 1, it is more preferable that it further has an extended sequence on its 5' side and / or 3' side, respectively, which is at least 2 nucleotides long and no more than 20 nucleotides long, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1 and stabilizing its binding to the transcription factor NF-κB.

[0052] Furthermore, if the NF-κB decoy in this application includes a κB sequence or κB sequence 1, it is even more preferable that it further has extension sequences of 4 nucleotides or more and 10 nucleotides or less on its 5' side and / or 3' side, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1 and stabilizing its binding to the transcription factor NF-κB.

[0053] Furthermore, if the NF-κB decoy in this application includes a κB sequence or κB sequence 1, it is most preferable that it further has extension sequences of 7 nucleotides and / or 4 nucleotides in length on its 5' side and / or 3' side, respectively, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1 and stabilizing its binding to the transcription factor NF-κB.

[0054] In this application, the NF-κB decoy may be a decoy in which the extended sequence includes any sequence, as long as the NF-κB decoy does not become unable to bind to the transcription factor NF-κB.

[0055] In this application, if the NF-κB decoy includes a κB sequence or κB sequence 1, it is preferable that it further includes an extension sequence 5'-CCTTGAA-3' and / or 5'-CTCC-3' on its 5' side and / or 3' side, respectively, from the viewpoint of stabilizing the double-stranded structure of the κB sequence or κB sequence 1 and stabilizing its binding to the transcription factor NF-κB.

[0056] The NF-κB decoy in this application more preferably includes the following sequence: 5'-CCTTGAAGGGATTTCCCCTC-3' (Sequence ID 2)

[0057] In this application, the NF-κB decoy is more preferably a double-stranded linear decoy containing the following sequence: 5'-CCTTGAAGGGGATTTCCCTCCC-3' (Sequence ID 2). Examples of such NF-κB decoys include, but are not limited to, AMG0103.

[0058] In this application, AMG0103 is a double-stranded oligonucleotide in which the following 20-base APK and APL chains form complementary base pairs.

[0059] Here, the sequence of the APK chain is as follows: 5'-CCTTGAAGGGGATTTCCCTCCC-3' (Sequence ID 2)

[0060] Furthermore, the sequence of the APL chain is as follows: 3'-GGAACTTCCCTAAAAGGGAGG-5' (Sequence ID 3)

[0061] Here, the APK chain and the APL chain are phosphorothioate oligonucleotides, each composed of phosphorothioate DNA, in which one of the oxygen atoms in all the phosphate groups in the nucleic acid is replaced with a sulfur atom (S-modified).

[0062] The aforementioned APK chain is represented by the following name in the International Union of Pure and Applied Chemistry (IUPAC) naming convention:

[0063]

[0064] The APK chain can be represented by the following structural formula.

[0065]

[0066] The aforementioned APL chain, when represented by its IUPAC name, is as follows:

[0067]

[0068] The APL chain can be represented by the following structural formula.

[0069] Therefore, the structural formula of AMG0103 in this application is as follows.

[0070] In the standard DNA skeleton found in nature, phosphodiester bonds are included between the bases. However, in AMG0103, in order to stabilize the oligonucleotide skeleton against nuclease degradation and extend the half-life of the oligonucleotide in the biological environment, all phosphodiester bonds between the bases are replaced with phosphorothioate bonds, as shown in the figure above. Each phosphorothioate bond introduced into the oligonucleotide creates a chiral center at each bond designated as "Sp" or "Rp" conformation. This can lead to the synthesis of oligonucleotides, ultimately resulting in the production of multiple isomers. However, AMG0103 in this application may be any optical isomer of any conformation, or a mixture of them in any proportion, as long as it is represented by the above structural formula.

[0071] The NF-κB decoy of this application may also be (a) below: (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTTCCCTAAAAGGGAGG-5' (Sequence ID No. 3)

[0072] The NF-κB decoy of this application may also be (b) below: (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, wherein a mutant having 20% ​​or less, preferably 10% or less, more preferably 5% or less of the base sequence mutated, substituted, inserted, and / or deleted relative to the oligonucleotide represented by SEQ ID NO: 2 and a mutant having 20% ​​or less, preferably 10% or less, more preferably 5% or less of the base sequence mutated, substituted, inserted, and / or deleted relative to the oligonucleotide represented by SEQ ID NO: 3 5'-CCTTGAAGGGGAATTTCCCCTC-3' (SEQ ID NO: 2) 3'-GGAACTTCCCTAAAAGGGAGG-5' (SEQ ID NO: 3)

[0073] Whether or not a double-stranded oligonucleotide has the ability to bind to NF-κB can be easily determined, for example, by a transcription factor binding assay. Examples of transcription factor binding assays include a gel shift assay, in which the double-stranded oligonucleotide and NF-κB are incubated and the band shift observed when electrophoresis is performed on a non-denaturing gel, and an enzyme immunoassay (ELISA), in which a sample containing NF-κB is added to the wells of a microplate on which the double-stranded oligonucleotide is immobilized, incubated, washed, and then detected using an antibody against NF-κB. If binding between the double-stranded oligonucleotide and NF-κB is detected by at least one of these assays, the double-stranded oligonucleotide can be determined to have the ability to bind to NF-κB.

[0074] The NF-κB decoy of this application may also be (c) below: (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant in which 20% or less, preferably 10% or less, more preferably 5% or less of the base sequence of the oligonucleotide represented by SEQ ID NO: 2 is mutated, substituted, inserted, and / or deleted, and a mutant in which 20% or less, preferably 10% or less, more preferably 5% or less of the base sequence of the oligonucleotide represented by SEQ ID NO: 3, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA: 5'-CCTTGAAGGGGAATTTCCCCTCCC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) 5'-GGGPuNNPyPyCC-3' (κB sequence)

[0075] Whether a double-stranded oligonucleotide antagonistically inhibits the binding of NF-κB to the κB sequence present in chromosomal DNA can be easily determined, for example, by chromatin immunoprecipitation. Specifically, samples obtained from cells into which the double-stranded oligonucleotide has been introduced and control cells without the introduction are subjected to immunoprecipitation using an antibody against NF-κB, and the κB sequence present in the chromosomal DNA is analyzed and detected using methods such as qPCR, sequencing, or DNA chips. If the amount of κB sequence present in the chromosomal DNA is detected lower in cells into which the double-stranded oligonucleotide has been introduced compared to control cells, it can be determined that the binding of NF-κB is antagonistically inhibited.

[0076] Furthermore, whether a double-stranded oligonucleotide competitively inhibits the binding of NF-κB to the κB sequence present in chromosomal DNA can be determined, for example, by ELISA. Specifically, this can be determined by incubating a cell extract of NF-κB-expressing cells on an ELISA plate coated with an NF-κB binding sequence (κB sequence) in the presence of an NF-κB decoy, and detecting the competitive reaction between the NF-κB binding sequence and the NF-κB decoy (competitive binding assay).

[0077] In this application, the NF-κB decoy having the ability to bind to NF-κB is preferred, in terms of having high NF-κB binding ability, to have 10 times higher NF-κB binding ability, more preferably 100 times higher NF-κB binding ability, and even more preferably 200 times higher NF-κB binding ability, compared to the negative control single-stranded decoy (single-stranded NF-κB decoy oligodeoxynucleotide) in the competitive binding assay.

[0078] If at least one assay detects that a double-stranded oligonucleotide antagonistically inhibits the binding of NF-κB to a κB sequence, then it can be determined that the double-stranded oligonucleotide antagonistically inhibits the binding of NF-κB to a κB sequence present in chromosomal DNA.

[0079] In (c), the κB sequence can be specifically identified as the sequence represented by sequence number 1: 5'-GGGATTTCCC-3' (sequence number 1)

[0080] The double-stranded oligonucleotides described in (a) to (c) above may be DNA in which nucleotides are linked by phosphodiester bonds, or they may be phosphorothioate DNA in which one or more phosphate groups in the DNA (for example, but not limited to, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-two, twenty-two, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty, thirty-one, thirty-two, thirty-two, thirty-two, thirty-two, thirty-two, thirty-two, thirty-five, thirty-five, thirty-two The double-stranded oligonucleotides described above in (a) to (c) are preferably phosphorothioate DNAs in which one of the oxygen atoms in all phosphate groups is substituted with a sulfur atom (S-modified), from the viewpoint of promoting glial cell-dependent neurite outgrowth.

[0081] In this application, a “naked” NF-κB decoy refers to an NF-κB decoy prepared without using delivery aids commonly used in the art to introduce nucleic acids, nucleic acid analogs, and / or modified versions thereof into cells or tissues. Examples of such delivery aids include, but are not limited to, lipid nanoparticles, cationic lipids, liposomes, and viruses (e.g., but are not limited to, adenovirus vectors, lentiviral vectors, retrovirus vectors, and adeno-associated virus (AAV) vectors). Since these delivery aids have problems such as cytotoxicity, immunogenicity, low stability, limited target specificity, and high manufacturing costs, it is preferable that the NF-κB decoy in this application is a naked NF-κB decoy.

[0082] The agents containing NF-κB decoy as an active ingredient in this application may contain NF-κB decoy alone. Furthermore, the agents containing NF-κB decoy as an active ingredient in this application may further contain, in addition to NF-κB decoy, solvents, diluents, stabilizing compounds, solvents, suspending agents, carriers, additives, or other active ingredients or agents.

[0083] The drug containing NF-κB decoy as an active ingredient in this application contains an effective amount of NF-κB decoy for the treatment of intervertebral disc degeneration. The effective amount for the treatment of intervertebral disc degeneration is determined as appropriate depending on the patient's symptoms, route of administration, etc. From the viewpoint of efficacy, side effects, and / or cost, the effective amount for the treatment is preferably 0.03 mg to 300 mg per day for adults, more preferably 0.1 mg to 100 mg, and even more preferably 0.3 mg to 30 mg per day.

[0084] The drug containing NF-κB decoy as an active ingredient in this application may be administered dissolved in any solvent. Examples of such solvents include, but are not limited to, water, physiological saline (e.g., Ringer's solution, etc.), and buffered physiological saline (e.g., but are not limited to, phosphate-buffered saline (PBS), Hanks' solution, Ringer's lactate solution, Engel's acetate solution, etc.).

[0085] The drug containing NF-κB decoy as the active ingredient in this application may be administered after being diluted with any diluent. Examples of such diluents include, but are not limited to, water, physiological saline (e.g., Ringer's solution, etc.), and buffered physiological saline (e.g., but are not limited to, phosphate-buffered saline (PBS), Hanks' solution, Ringer's lactate solution, Engel's acetate solution, etc.).

[0086] The pharmaceutical agent containing NF-κB decoy as an active ingredient in this application may further contain any pharmaceutically acceptable additive in addition to the active ingredient. The pharmaceutically acceptable additive may be any pharmaceutically acceptable additive commonly used in the pharmaceutical art. Examples of pharmaceutically acceptable additives include, but are not limited to, excipients (e.g., lactose, starch, etc.), binders (e.g., hydroxypropylcellulose, polyvinylpyrrolidone, etc.), disintegrants (e.g., croscarmellose sodium, crospovidone, etc.), lubricants (e.g., magnesium stearate, talc, etc.), preservatives (e.g., parabens, benzyl alcohol, etc.), colorants (e.g., iron oxide, titanium dioxide, etc.), flavorings (e.g., vanillin, menthol, etc.), sweeteners (e.g., aspartame, saccharin, etc.), solubilizers (e.g., polysorbate 80, propylene glycol, etc.), stabilizers (e.g., sodium edetate, ascorbic acid, etc.), surfactants (e.g., sorbitan monolaurate, sodium lauryl sulfate, etc.), and thickeners (e.g., carboxymethylcellulose, etc.). Examples of additives include cellulose (e.g., xanthan gum), preservatives (e.g., potassium sorbate, sodium benzoate), emulsifiers (e.g., lecithin, monoglycerides), buffers (e.g., sodium phosphate, sodium citrate), fillers (e.g., microcrystalline cellulose, mannitol), wetting agents (e.g., glycerin, sorbitol), foaming agents (e.g., sodium bicarbonate, citric acid), bulking agents (e.g., calcium phosphate, cellulose), coating agents (e.g., hypromellose, ethylcellulose), antioxidants (e.g., ascorbic acid, tocopherol), preservatives (e.g., phenol, chlorobutanol), solvents (e.g., water, ethanol), buffers (e.g., phosphate buffer, acetate buffer), and osmotic pressure regulators (e.g., sodium chloride, glycerin).

[0087] The NF-κB decoy active ingredient in this application may be provided as a powder obtained by synthesizing and drying the NF-κB decoy. The NF-κB decoy active ingredient in this application may be provided as a solution dissolved in any solvent at any concentration. The NF-κB decoy active ingredient in this application may be provided as an aerosol dispersed in any gas medium at any concentration. The NF-κB decoy active ingredient in this application may be provided as a suspension dispersed in any liquid medium at any concentration. The NF-κB decoy active ingredient in this application may be provided as a solid dispersion in any solid medium at any concentration.

[0088] The drug containing NF-κB decoy as an active ingredient in this application may be any dosage form commonly used in the field of pharmaceutical technology. The drug containing NF-κB decoy as an active ingredient in this application may be, for example, but not limited to, an injection, an intravenous infusion, an oral preparation, a sublingual preparation, a transdermal patch, a transdermal patch, an inhalant, an eye drop, a nasal spray, a suppository, a vaginal preparation, a topical cream, a topical gel, a topical ointment, a topical lotion, a microneedle patch, a liposome preparation, a nanoparticle preparation, a microsphere preparation, a depot preparation, an implant, an enteral preparation, a transdermal gel, a transdermal spray, a transdermal film, and a transdermal microemulsion.

[0089] The agent containing NF-κB decoy as the active ingredient in this application is administered orally or parenterally. Parenteral administration includes, but is not limited to, local administration (e.g., intramuscular, subcutaneous, intrathecal, subarachnoid, intraventricular, etc.), intravenous, or intranasal administration. From the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells, the agent containing NF-κB decoy as the active ingredient in this application is preferably administered locally by parenteral administration.

[0090] Furthermore, the drug containing NF-κB decoy as the active ingredient in this application is preferably administered locally to the site of glial cells or its vicinity, from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells and from the viewpoint of more efficiently treating patients suffering from intervertebral disc degeneration. Here, "the vicinity of the site of glial cells" refers to the range in which the locally administered drug can reach the site of glial cells by infiltration or diffusion through non-hematogenous distribution. In other words, when a drug is locally administered to a specific site, if the drug reaches the site of glial cells from that specific site by infiltration or diffusion through non-hematogenous distribution, that specific site falls under the category of "the vicinity of the site of glial cells."

[0091] In this application, the drug containing NF-κB decoy as the active ingredient is preferably administered locally to the site of or near glial cells that affect nerve cells of sensory nerves projecting from the periphery to the dorsal roots of spinal nerves, from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells and more efficiently treating patients suffering from intervertebral disc degeneration.

[0092] Furthermore, when a humoral factor secreted by a glial cell acts on nerve cells of sensory nerves projecting from the periphery to the dorsal roots of spinal nerves, that glial cell is referred to as a "glial cell that affects nerve cells of sensory nerves projecting from the periphery to the dorsal roots of spinal nerves."

[0093] Furthermore, the drug containing NF-κB decoy as the active ingredient in this application is preferably administered locally to the spinal cord dorsal horn or its vicinity, from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells, from the viewpoint of targeting glial cells that affect nerve cells of sensory nerves projecting from the periphery to the dorsal roots of spinal nerves, and from the viewpoint of more efficiently treating patients suffering from intervertebral disc degeneration. Here, "near the spinal cord dorsal horn" refers to the area in which the locally administered drug can reach the spinal cord dorsal horn by infiltration or diffusion through non-hematogenous distribution. In other words, when a drug is locally administered to a specific site, if the drug reaches the spinal cord dorsal horn from that specific site by infiltration or diffusion through non-hematogenous distribution, that specific site falls under the category of "near the spinal cord dorsal horn."

[0094] Furthermore, from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells and more efficiently treating patients suffering from intervertebral disc degeneration, it is preferable to administer the drug containing NF-κB decoy as an active ingredient in this application locally into the intervertebral disc so that the NF-κB decoy reaches the site of glial cells by infiltration or diffusion through non-hematogenous distribution.

[0095] Furthermore, from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells and more efficiently treating patients suffering from intervertebral disc degeneration, it is preferable to administer the drug containing NF-κB decoy as an active ingredient in this application locally into the intervertebral disc so that the NF-κB decoy reaches the spinal cord dorsal horn by infiltration or diffusion through non-hematogenous distribution.

[0096] Furthermore, the drug containing NF-κB decoy as the active ingredient in this application is preferably administered locally into or near the spinal cavity from the viewpoint of more efficiently promoting neurite outgrowth of nerve cells via glial cells and from the viewpoint of more efficiently treating patients suffering from intervertebral disc degeneration. Here, "near the spinal cavity" refers to the area in which the administered drug can reach the spinal cavity by infiltration or diffusion through non-hematogenous distribution. In other words, when a drug is administered locally to a specific site, if the drug reaches the spinal cavity from that specific site by infiltration or diffusion through non-hematogenous distribution, that specific site falls under the category of "near the spinal cavity."

[0097] When administering a drug containing NF-κB decoy as the active ingredient parenterally to a local area, it may be used in the form of being filled into a syringe.

[0098] When administering a drug containing NF-κB decoy as the active ingredient to the dorsal horn of the bone marrow, intravertebral disc, intramedullary cavity, or near these locations, it is preferable to use a syringe equipped with an 18-25G needle with a length of 100-250 mm, and more preferably a syringe equipped with a 21-23G needle with a length of 120-210 mm, from the viewpoint of needle strength and the likelihood of reaching the target tissue.

[0099] In this application, "nerve cell" refers to a basic constituent cell of the nervous system that transmits information between cells using electrical and / or chemical signals. Nerve cells are sometimes also called "neurons." Typically, a nerve cell has the following three substructures: (1) the cell body (the central part of the cell, including the nucleus and other organelles); (2) the axon (a long fibrous structure that transmits signals to other nerve cells, cells that make up muscles and glands, etc.); and (3) the dendrites (branch-like projections that receive signals from other nerve cells). Information transmission between nerve cells typically takes place at structures called synapses, where the axon and dendrites are coupled and connected. Damage to nerve cells can occur in any of the structures of the nerve cell, such as the cell body, axon, and dendrites. When damage to nerve cells occurs, intercellular information transmission at synapses is typically impaired.

[0100] In this application, "nerve cells" are classified, based on their location within an organism, into, for example, (1) central nervous system cells (located in the central nervous system such as the brain and spinal cord, and responsible for higher-order functions such as cognition, thought, consciousness, and command), and (2) peripheral nerve cells (connecting various tissues and parts of the body to the central nervous system). In this application, "nerve cells" are preferably peripheral nerve cells from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys and from the viewpoint of being involved in regulating the pathogenesis of intervertebral disc degeneration accompanied by chronic neuropathic pain.

[0101] In this application, "peripheral nerve cells" are classified, based on their function, for example, but are not limited to, (1) sensory nerve cells (receiving external stimuli [light, sound, temperature, etc.] and sending signals to the brain and spinal cord), (2) motor nerve cells (transmitting signals from the brain and spinal cord to muscles and glands and controlling movement and secretion), (3) autonomic nerve cells (further classified into sympathetic nerve cells and parasympathetic nerve cells, which regulate the functions of internal organs, blood vessels, glands, etc. through endocrine secretions such as hormones), and (4) interneurons (located between nerve cells of (1), (2), and (3), which process and transmit information). In this application, "nerve cells" are preferably sensory nerve cells from the viewpoint of efficiently receiving neurite outgrowth promotion by NF-κB decoys and from the viewpoint of being involved in the pathogenesis of intervertebral disc degeneration accompanied by chronic neuropathic pain.

[0102] In this application, "nerve cells" are classified, based on their morphology, into, for example, (1) unipolar nerve cells (nerve cells with one projection extending from the cell body), (2) bipolar nerve cells (nerve cells with two projections extending from the cell body), and (3) multipolar nerve cells (nerve cells with three or more projections extending from the cell body). The aforementioned bipolar nerve cells include pseudounipolar nerve cells as a subtype. Pseudounipolar nerve cells are nerve cells in which a single projection extending from one point on the cell body branches into two midway, with one branch leading to, for example, peripheral organs (various organs, skin, etc.) and the other branch leading to, for example, the central nervous system (brain, spinal cord). In this application, "nerve cells" are preferably bipolar nerve cells and / or pseudounipolar nerve cells, from the viewpoint of efficiently receiving neurite outgrowth promotion by NF-κB decoys and from the viewpoint of being involved in the pathogenesis regulation of intervertebral disc degeneration accompanied by chronic neuropathic pain.

[0103] In this application, "neuronal process" includes both the axon and the dendrite of the nerve cell.

[0104] In this application, "neurite elongation" refers to the extension of neurites to a greater length. In this application, neurite elongation also includes the case where new neurites arise from areas on nerve cells where no neurites previously existed. In this case, the neurite elongation may be accompanied by an increase in the number of neurites. Furthermore, the neurite elongation may lead to plastic regulation of neurotransmission through synapse formation, regeneration of damaged synapses, and modulation of existing synapses. In this application, neurite elongation is preferably neurite elongation of peripheral nerve cells, from the viewpoint of efficiently promoting neurite elongation by NF-κB decoys and from the viewpoint of being involved in the regulation of the pathogenesis of intervertebral disc degeneration accompanied by chronic neuropathic pain. In addition, in this application, neurite elongation is preferably neurite elongation of sensory nerve cells, from the viewpoint of efficiently promoting neurite elongation by NF-κB decoys and from the viewpoint of being involved in the regulation of the pathogenesis of intervertebral disc degeneration accompanied by chronic neuropathic pain. Furthermore, in this application, neurite outgrowth is preferably that of bipolar neurons and / or pseudounipolar neurons, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys and from the viewpoint of being involved in the regulation of the pathophysiology of intervertebral disc degeneration accompanied by chronic neuropathic pain.

[0105] In this application, "glial cells" refer to a group of cell types that make up the central and peripheral nervous systems but are not nerve cells. In other words, glial cells in this application are cells that are distinct from nerve cells among the various cell types that make up the nervous system. Glial cells are sometimes also called "neural glial cells." In this application, glial cells can be further classified into several types of cells based on their location, cell morphology, expressed proteins, function, etc. For example, but are not limited to, they may be classified into microglia, astrocytes, oligodendrocytes, ependymal cells, Schwann cells, and satellite glial cells.

[0106] Furthermore, glial cells in this application may exist in two states: resting glial cells in a normal state, and activated glial cells activated by pathological stimuli or damaging stimuli. Moreover, these activated glial cells are classified into functionally opposing types: M1 type glial cells that function damagingly (inflammatoryly) and M2 type glial cells that function protectively (anti-inflammatoryly).

[0107] In this application, "microglia" refers to a type of glial cell containing integrin alpha-M (ITGAM) / differentiation cluster 11b (CD11b), cell surface glycoprotein F4 / 80, differentiation cluster 45 (CD45), ionized calcium binding adapter protein 1 (Iba1), and transmembrane protein 119. Microglia refer to glial cells identified by the expression of proteins such as 119; TMEM119). The roles of microglia in the nervous system include, but are not limited to, maintaining homeostasis of nerve tissue, removing pathogens, and repairing damaged sites. Microglia are also known to protect nerve tissue by recognizing pathogens and damaged cells and phagocytosing them. Furthermore, microglia may secrete signaling molecules that regulate inflammatory responses and support the survival and regeneration of nerve cells.

[0108] In this application, "astrocyte" refers to a type of glial cell that has numerous projections and exhibits a star-shaped morphology when stained with an anti-GFAP antibody or the like. The role of astrocytes in the nervous system includes, but is not limited to, linking nerve cells to the vascular system, uptake of neurotransmitters, formation of the blood-brain barrier, maintenance of homeostasis of neural circuits, promotion of synaptic plasticity, and regulation of microcirculation. More specifically, co-culturing astrocytes and nerve cells in a culture vessel, or adding the culture supernatant of astrocytes (containing proteins secreted by astrocytes) to nerve cells, can promote the extension of nerve processes in nerve cells. Therefore, astrocytes can provide nutrients to nerve cells. Furthermore, astrocytes can rapidly remove excess ions and neurotransmitters present outside the nerve cells. Therefore, astrocytes can play a role in maintaining the survival and function of nerve cells.

[0109] In this application, "Schwann cells" refers to a type of glial cell that surrounds the axon of a peripheral nerve cell. Schwann cells contribute to the survival of developing nerve cells, axonal projection, and the regeneration of neural circuits after injury. Schwann cells are classified into myelin-forming Schwann cells (e.g., those identified by the expression of proteins such as SRY-Box transcription factor 10 (SOX10), S100 calcium-binding protein (S100), Early Growth Response 2 (EGR2), Myelin Basic Protein (MBP), and Myelin Protein Zero (MPZ) as markers) and non-myelin-forming Schwann cells (e.g., those that do not form a myelin sheath, such as SOX10 and Growth Associated Protein 43). Myelin formation is characterized by the expression of proteins such as 43 (GAP43), S100, neural cell adhesion molecule (NCAM), and p75 nerve growth factor receptor (P75NTR), which are identified as markers, and myelin formation is responsible for saltatory conduction in peripheral nerve cells.

[0110] In this application, "satellite glial cells" refers to a type of glial cell that is present in sensory ganglia (e.g., spinal ganglia and trigeminal ganglia) or autonomic ganglia. These cells are arranged to surround the cell bodies of nerve cells within the ganglia and are closely related to the nerve cells. Satellite glial cells can sometimes be identified using protein expression markers such as Glial Fibrillary Acid Protein (GFAP), S100 Calcium Binding Protein B (S100B), Glutamine Synthase (GS), Cluster of Difference 57 (CD57) / Human Natural Killer-1 (HNK-1), SOX10, and Vimentin. Functions of satellite glial cells include, for example, structural support (physically supporting nerve cells), protection and repair of nerve cells, regulation of neurotransmitters, and inflammatory responses.

[0111] In this application, glial cells are preferably microglia, and / or astrocytes, and / or Schwann cells, and / or satellite glial cells, from the viewpoint of promoting neurite outgrowth of nerve cells. Furthermore, in this application, glial cells are more preferably astrocytes, from the viewpoint of promoting neurite outgrowth of nerve cells.

[0112] In this application, glial cells may be peripheral nervous system glial cells present in the peripheral nervous system, or central nervous system glial cells present in the central nervous system.

[0113] In this application, the peripheral nervous system glial cells are preferably Schwann cells and / or satellite glial cells, from the viewpoint of promoting neurite outgrowth of peripheral nerve cells.

[0114] In this application, the central nervous system glial cells are preferably microglia and / or astrocytes, from the viewpoint of promoting neurite outgrowth of peripheral nerve cells.

[0115] In this application, "neurite outgrowth of nerve cells mediated by glial cells" refers to neurite outgrowth induced in nerve cells by the action of humoral factors secreted from glial cells on said nerve cells.

[0116] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via activated glial cells," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0117] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via M2-type glial cells, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0118] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via microglia," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0119] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via activated microglia," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0120] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via M2-type microglia, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0121] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via astrocytes," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0122] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via activated astrocytes," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0123] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via M2-type astrocytes, from the viewpoint of efficiently receiving neurite outgrowth promotion by NF-κB decoys.

[0124] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via Schwann cells," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0125] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via activated Schwann cells," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0126] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via satellite glial cells, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0127] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably "neurite outgrowth of nerve cells via activated satellite glial cells," from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0128] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via microglia and astrocytes, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0129] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via activated microglia and activated astrocytes, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0130] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via M2-type microglia and M2-type astrocytes, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0131] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via Schwann cells and satellite glial cells, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0132] In this application, "neurite outgrowth of nerve cells via glial cells" is preferably neurite outgrowth of nerve cells via activated Schwann cells and activated satellite glial cells, from the viewpoint of efficiently promoting neurite outgrowth by NF-κB decoys.

[0133] In this application, "intervertebral disc" refers to the cartilage located between the vertebral bodies that make up the spine. The functions of the intervertebral disc include, but are not limited to, acting as a cushion to absorb shock and acting as a joint in the spine to allow movement of the entire spine. When viewed in a horizontal cross-section, the intervertebral disc consists of a nucleus pulposus located in the center and an annulus fibrosus that concentrically surrounds it.

[0134] The nucleus pulposus is mainly composed of chondrocytes and fibroblasts. The chondrocytes produce a jelly-like matrix in the nucleus pulposus, which plays a role in distributing the pressure applied to the intervertebral disc. The fibroblasts produce extracellular matrix components such as collagen and proteoglycans, which maintain the elasticity and flexibility of the nucleus pulposus.

[0135] The annulus fibrosus is mainly composed of fibroblasts and collagen fibers. The fibroblasts produce the collagen fibers, which maintain the strength and elasticity of the annulus fibrosus. As a result, the annulus fibrosus firmly holds the nucleus pulposus inside and maintains the overall shape of the intervertebral disc.

[0136] On the other hand, bundles of nerves that branch off from the spinal cord and send signals to various parts of the body, or bundles of nerves that transmit signals from various parts of the body, are called nerve roots. They emerge from between the vertebrae of the spine and pass adjacent to the intervertebral discs. These nerve roots are classified into motor nerve roots (anterior roots), which are bundles of motor nerve cells, and sensory nerve roots (posterior roots), which are bundles of sensory nerve cells. After emerging from the anterior and posterior root portions of the spinal cord, they merge to form a single spinal nerve. The nerve cells of the nerve roots extend nerve processes as nerve fibers into the annulus fibrosus in the intervertebral disc, especially in the outer layer. Examples of nerve cells in the nerve roots that extend nerve fibers into the annulus fibrosus include, but are not limited to, sensory nerve cells and autonomic nerve cells. Examples of nerve fibers of sensory nerve cells include, but are not limited to, Aδ fibers (myelinated nerve fibers that transmit acute pain and temperature changes; these fibers have a relatively fast conduction velocity) and C fibers (unmyelinated nerve fibers that transmit dull pain and temperature changes; these fibers have a slow conduction velocity but play a role in sensing persistent pain). Examples of nerve fibers of autonomic nerve cells include, but are not limited to, B fibers (myelinated nerve fibers that function as part of the autonomic nervous system and transmit visceral sensations and reflexes). In addition to nerve cells, various glial cells are also present in the nerve root.

[0137] In this application, "sensory nerve cells" are preferably sensory nerve cells located in the sensory nerve root region of the intervertebral disc, from the viewpoint of efficiently receiving neurite outgrowth promotion by NF-κB decoy. Furthermore, in this application, "sensory nerve cells" are preferably sensory nerve cells located in the sensory nerve root region of the intervertebral disc that extend neurites to the annulus fibrosus of the intervertebral disc, from the viewpoint of efficiently receiving neurite outgrowth promotion by NF-κB decoy.

[0138] In this application, "intervertebral disc degeneration" refers to a disease involving structural and functional changes in the intervertebral disc. Causes of intervertebral disc degeneration in this application include, but are not limited to, a decrease in the water content of the intervertebral disc, damage to the annulus fibrosus, degeneration of the nucleus pulposus, etc., due to aging, excessive exercise, or the influence of the work environment, and these changes impair the stability and mobility of the spine. Main symptoms of intervertebral disc degeneration include, but are not limited to, pain, herniated disc, spinal stenosis, spondylolisthesis, numbness, muscle weakness, limited range of motion, changes in posture, and decreased reflexes. In this application, intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by pain or numbness, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells. In this application, intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by pain and numbness, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0139] In this application, "patients suffering from degenerative disc disease" may be male or female, and may be of any age. From the viewpoint of promoting neurite outgrowth of nerve cells via glial cells, patients suffering from degenerative disc disease in this application are preferably 40 years of age or older, more preferably 50 years of age or older, and even more preferably 60 years of age or older.

[0140] In this application, "patients suffering from intervertebral disc degeneration" may refer to patients with intervertebral disc degeneration who have lower back pain that has persisted for at least one month, more preferably two months, and even more preferably three months.

[0141] In this application, "patients suffering from intervertebral disc degeneration" may also refer to patients with intervertebral disc degeneration whose Numerical Rating Scale (NRS) score for lower back pain is higher than their NRS score for buttock pain or lower limb pain.

[0142] In this application, "patients suffering from intervertebral disc degeneration" may refer to patients with intervertebral disc degeneration whose condition is not adequately treated with conservative therapies for lower back pain. Here, conservative therapy refers to methods of managing symptoms without surgery. Examples of the aforementioned conservative treatments include, but are not limited to, rest, drug therapy (for example, but are not limited to, anti-inflammatory drugs, neuromodulators [for example, but are not limited to, gabapentin, pregabalin, opioid agonists, etc.], local anesthetics [for example, but are not limited to, lidocaine, bupivacaine, mepivacaine, etc.], muscle relaxants [for example, but are not limited to, eperisone hydrochloride, tizanidine hydrochloride, methocarbamol, etc.]), physical therapy (for example, but are not limited to, exercise therapy [for example, but are not limited to, stretching, lumbar spine exercise programs, etc.], manual therapy [for example, but are not limited to, massage, acupressure, etc.], physical therapy [for example, but are not limited to, acupuncture, thermotherapy, low-frequency therapy, ultrasound therapy, etc.]), orthotic therapy, etc.

[0143] In this application, "patients suffering from intervertebral disc degeneration" may also refer to patients with intervertebral disc degeneration whose NRS score is between 4 and 9.

[0144] In this application, "patients suffering from intervertebral disc degeneration" are patients with intervertebral disc degeneration who have lower back pain that lasts for at least one month, more preferably two months or more, and even more preferably three months or more, whose NRS score for lower back pain is higher than that for buttock pain or lower limb pain, who have not responded adequately to conservative treatment for lower back pain, and whose NRS score is between 4 and 9.

[0145] The means of identifying "patients suffering from intervertebral disc degeneration" in this application are not particularly limited and can be carried out by conventional methods. Generally, "patients suffering from intervertebral disc degeneration" can be identified by collecting information on the patient's subjective symptoms, medical history, occupation, and physical burden in daily life, conducting a physical examination including interviews to understand the characteristics of the symptoms, checking posture, measuring range of motion, and neurological examinations, and appropriately combining physical examinations to evaluate the degree of pain and motor impairment, and imaging diagnoses using MRI (magnetic resonance imaging) or CT (computed tomography).

[0146] In this application, "pain" refers to an unpleasant sensory and emotional experience associated with, or described in relation to, substantial or potential injury to a tissue, as defined by the International Association for the Study of Pain (IASP). In this application, "pain" and "soreness" are synonymous. Examples of such pain include, but are not limited to, sharp pain, dull pain, burning pain, stabbing pain, throbbing pain, etc. In this application, "pain" is classified into pain with a physical or organic cause and pain without a physical or organic cause, based on its pathogenesis.

[0147] The aforementioned pain with organic causes refers to pain that results from organic damage to the body, and is classified into nociceptive pain and neuropathic pain.

[0148] The aforementioned pain without a physical or organic cause refers to pain conditions in which no physical or organic disorder is medically identified, and is classified into psychogenic pain and primary pain.

[0149] The aforementioned nociceptive pain refers to pain that is perceived when a stimulus (noxious stimulus) that actually damages or has the potential to damage normal tissue excites nociceptors at the terminals of peripheral nerve fibers, and this excitation is transmitted via peripheral nerves from the spinal cord to the cerebrum. The aforementioned nociceptive pain includes pain that does not have a pathological significance, such as "pain when pinched" or "pain when touching something hot," as well as pain that occurs in conjunction with inflammation (inflammatory pain).

[0150] The aforementioned neuropathic pain refers to pain caused by lesions or diseases of the somatosensory nervous system. Unlike the aforementioned nociceptive pain, neuropathic pain is generally resistant to pain suppressants such as non-steroidal anti-inflammatory drugs (NSAIDs) and opioids. Clinically, neuropathic pain may be accompanied by spontaneous pain, as well as allodynia (pain triggered by tactile stimuli) and hyperalgesia (pain perceived as excessively high compared to nociceptive pain). Neuropathic pain is further classified into acute neuropathic pain (less than one month), subacute neuropathic pain (one to three months), and chronic neuropathic pain (more than three months).

[0151] In this application, painful intervertebral disc degeneration is preferably painful intervertebral disc degeneration with a physical organic cause, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0152] In this application, the intervertebral disc degeneration accompanied by pain is preferably intervertebral disc degeneration accompanied by neuropathic pain, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0153] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by neuropathic pain with allodynia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0154] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by neuropathic pain accompanied by hyperalgesia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0155] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by neuropathic pain with allodynia and hyperalgesia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0156] In this application, the intervertebral disc degeneration accompanied by pain is preferably intervertebral disc degeneration accompanied by chronic neuropathic pain, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0157] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by chronic neuropathic pain with allodynia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0158] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by chronic neuropathic pain accompanied by hyperalgesia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0159] In this application, the painful intervertebral disc degeneration is preferably intervertebral disc degeneration accompanied by chronic neuropathic pain with allodynia and hyperalgesia, from the viewpoint of promoting neurite outgrowth of nerve cells via glial cells.

[0160] In this application, the intervertebral disc degeneration is preferably anti-inflammatory drug-resistant intervertebral disc degeneration. Here, anti-inflammatory drug-resistant intervertebral disc degeneration refers to intervertebral disc degeneration in which pain suppression is not observed even with the administration of anti-inflammatory drugs.

[0161] The term "anti-inflammatory drug" here is not limited. For example, azapropazone, aspirin, aceclofenac, acetylsalicylic acid, acemetacin, amtolmethin guayl, amlodipine, amorolphin, aliclofenac, aluminoprofen, ampiroxicam, isonixin, ibuproxam, ibuprofen, imidazole salicylate, indomethacin, indomethacin farnesil, etodolac, etoricoxib, emorphazon, oxaprozin, oxycodone, otenaproxesul, chlorsalicylic acid, clonfenac, guaymesal, ke Toprofen, Ketrolac, Sodium Salicylate, Zaltoprofen, Synmethacin, Diaselein, Diclofenac, Diflunisal, Sulindac, Sulfapyrizole, Celecoxib, Zetrolac, Talniflumart, Tiaprofenic Acid, Tiaramide, Tenoxicam, Dexibprofen, Dexketoprofen, Denaverine Hydrochloride, Tolmetin, Tropinindomethacinate, Nabumeton, Navumeton, Naproxinod, Naproxen, Nimesluide, Nepafenac, Valdecoxib, Paracetamol L, Parsalmid, Parecoxib, Parecoxib sodium, Hydrocodone, Piketoprofen, Piroxicam, Phenylbutazone, Felbinac, Fenthiazac, Fenbufen, Phosphosal, Fluticasone, Flupyric acid, Flurbiprofen, Floctafenin, Butibufen, Bromfenac, Pranoprofen, Proquazone, Proglumitacin, Propionic acid derivatives, Benzydamine, Benzydamine salicylate, Perbiprofen, Polmacoxib, Misoprostol, Metocafenamine, Nonsteroidal anti-inflammatory drugs (NSAIDs) such as mefenamic acid, meloxicam, mofezolac, lysine clonixinate, lysine salicylate, lipoflurbiprofen, limazolium methyl sulfate, lufenamic acid, lumiracoxib, loxoprofen, lofecoxib, lornoxicam, calcium acetate, (S)-flurbiprofen, 3D-1002, AAT-076, ASA, ATB-352, EVT-401, FB-3001, HR-1801, JLP-2004, JMHJ-01, PDX-02, etc.Steroidal anti-inflammatory drugs (glucocorticoids) such as prednisolone, methylprednisolone, hydrocortisone, dexamethasone, betamethasone, triamcinolone, deflazacort, beclomethasone, budesonide, mometasone, fluticasone, clobetasol, alclomethasone, desoxymethasone, hydrocortisone acetate, methyldesonide, fluocinolone acetonide, deoxycorticosterone, prednisone, etc.; infliximab, adalimumab, etanercept, golimumab, certolizumab pegol, tocilizumab, sarilumab, anakinra, canakinumab, ustekinumab, secukinumab, ixekizumab, risankizumab Biological agents such as dupilumab, brodalumab, teseperumab, bedalizumab, omalizumab, abatacept, and natalizumab; immunosuppressants such as tacrolimus, cyclosporine, azathioprine, mycophenolate mofetil, sirolimus, everolimus, temsirolimus, leflunomide, baricitinib, tofacitinib, upadacitinib, filgotinib, anagrelide, trichlormethiazide, and ibrutinib; antirheumatic drugs such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, minocycline, bucillamine, penicillamine, sodium aurthiomalate, tafcitinib, and baricitinib (Disease Modifying Anti-Rheumatic Drugs (DMARDs); Antihistamines such as chlorpheniramine, diphenhydramine, clemastine, loratadine, desloratadine, cetirizine, levocetirizine, fexofenadine, bilastine, epinastine, olopatadine, ketotifen, azelastine, ranitidine, cimetidine, famotidine, etc.; curcumin, catechin, flavonoids (rutin, quercetin), resveratrol, boswellic acid, gingerol, berberine, alpha-lipoic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (EPA);Examples include anti-inflammatory components derived from plants and natural sources such as DHA, polyphenols, propolis, apigenin, rosmarinic acid, caffeic acid, isoflavones, anthocyanins, blueberry extract, herbal extracts (rosemary, thyme), glycyrrhizic acid, arbutin, esculin, hyaluronic acid, and tranexamic acid.

[0162] In this application, patients suffering from intervertebral disc degeneration may have intervertebral disc degeneration without signs of inflammation. Here, "without signs of inflammation" means that there is no swelling or fever in the lumbar region, and / or the concentration of C-reactive protein (CRP) in the blood is less than 1 mg / dL, preferably 0.5 mg / dL or less, and more preferably 0.3 mg / dL or less.

[0163] In this application, "to treat" a disease means to improve the pathology and / or symptoms of the disease, to stop and / or slow the progression of the pathology and / or symptoms of the disease, and to prevent the onset of the pathology and / or symptoms of the disease.

[0164] The above-mentioned description of "agents containing NF-κB decoy as an active ingredient" applies directly to the "therapeutic agents for intervertebral disc degeneration" and "pharmaceutical compositions containing a therapeutically effective amount of NF-κB decoy" in this application.

[0165] Example 1: Effects on a rat model of brewer's yeast-induced acute pain. Brewer's yeast is a potent inflammatory agent, and when administered subcutaneously to the hind leg soles of rats, inflammation is induced at the administration site, resulting in inflammatory pain, which is a type of nociceptive pain. Here, we investigated the effects of AMG0103 on inflammatory pain induced subcutaneously to the hind leg soles, and the effects of AMG0103 on the sensory nervous system via inflammatory pain induced by intrathecal administration. This model is widely used to evaluate the pain-suppressing effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and is also a pathological model of acute pain.

[0166] Animal materials: Sprague Dawley (SD) rats (Nippon SLC) Brewer's yeast: #Y4625 (Sigma-Aldrich) NF-κB decoy: AMG0103 Pressure stimulation analgesic effect measuring device: Analysy meter 7200 (Ugo Basile)

[0167] Methods: Two studies were conducted in which AMG0103 was administered subcutaneously to the right hind paw of rats, along with PBS(-) as a control. Another study involved administering AMG0103 and PBS(-) as a control into the spinal cavity of rats (the space between the spinal cord and the dura mater; the spinal cavity is adjacent to the spinal cord and is also called the spinal canal). In both studies, an AMG0103 group was established to receive AMG0103, and a control group was established to receive PBS(-). The dose of AMG0103 was 100 μg (administration volume: 20 μL), and the dose of PBS(-) was the same as that of AMG0103 (20 μL) (see group composition).

[0168] Group composition of the rat right hind limb subcutaneous administration study.

[0169] Group composition of the rat intrathecal administration study

[0170] In both studies, 0.1 mL of 20% w / v brewer's yeast physiological saline suspension was administered subcutaneously to the right hind paw of rats one hour after administration of AMG0103 or PBS(-). After administration of the 20% w / v brewer's yeast physiological saline suspension, a pressure stimulation analgesia measurement device was used to measure the pressure over time when the rats exhibited avoidance behavior in response to the pain induced by the pressure stimulation, with the right hind paw placed between a rounded cone-shaped pusher and a small base. This pressure was used as an indicator of the pain threshold. In the study where AMG0103 was administered subcutaneously to the right hind paw, the pain threshold was measured 2, 4, 6, 24, and 30 hours after administration of the 20% w / v brewer's yeast physiological saline suspension. In the study where AMG0103 was administered intrathecally, the pain threshold was measured 1, 2, 4, 6, 24, and 30 hours after administration of the 20% w / v brewer's yeast physiological saline suspension. Furthermore, the pain threshold before administration of AMG0103, PBS(-), and 20% w / v brewer's yeast physiological saline suspension was set to 1. From the pain thresholds measured at each time point after administration of AMG0103, PBS(-), and 20% w / v brewer's yeast physiological saline suspension, the pain threshold ratio, mean value, and standard error at each time point were calculated. In addition, the area below the line on the graph for each group was calculated as the sum of pain threshold ratios (0→30h), and the mean value and standard error of the sum were calculated. A t-test was used for statistical analysis.

[0171] In a rat subcutaneous administration study of AMG0103 to the right hind paw, the PBS (-) administration group showed a decrease in the pain threshold ratio upon administration of 20% w / v brewer's yeast physiological salt suspension, with a decrease of approximately 60% after 6 hours of administration, and this decrease was observed up to 30 hours after administration. On the other hand, the AMG0103 group showed a significant suppression of the decrease in the pain threshold ratio after 2 hours of administration (PBS (-) group: 0.47 ± 0.01, AMG0103 group: 0.53 ± 0.02, p < 0.05, Student t-test), but showed similar behavior to the PBS (-) group at subsequent measurement points (Figure 1a, Table 3a).

[0172] Effects of AMG0103 administration via the soles of the feet on a rat model of brewer's yeast-induced acute pain (pain threshold ratio).

[0173] Furthermore, when comparing the summation of pain threshold ratios (0→30h) between the PBS(-) group and the AMG0103 group, no significant difference was observed between the two groups (PBS(-) group: 15.24±0.44, AMG0103 group: 15.31±0.40, Figure 1b, Table 3b).

[0174] Effects of AMG0103 administration via the soles of the feet on a rat model of brewer's yeast-induced acute pain (sum of pain threshold ratios from 0 to 30h).

[0175] In a study in which AMG0103 was administered intrathecally to rats, the PBS(-) group showed a decrease in pain threshold ratio upon administration of a 20% w / v brewer's yeast physiological saline suspension. Similar to the study in which AMG0103 was administered subcutaneously to the right hind paw of rats, the pain threshold ratio decreased by approximately 60% 6 hours after administration, and this decrease was observed up to 30 hours after administration. In contrast, the AMG0103 group showed suppression of the decrease in the pain threshold ratio from 2 hours after administration to 30 hours after administration, and demonstrated significant suppression of the decrease in the pain threshold at 4 hours after administration (PBS(-) group: 0.58±0.03, AMG0103 group: 0.68±0.03) and 24 hours after administration (PBS(-) group: 0.66±0.03, AMG0103 group: 0.77±0.03) (p < 0.05 at all time points, Student t-test, Figure 2a, Table 4a).

[0176] Effects of intrathecal administration of AMG0103 on a rat model of brewer's yeast-induced acute pain (pain threshold ratio)

[0177] Furthermore, when the sum of pain threshold ratios (0→30h) was calculated and compared between the PBS(-) group and the AMG0103 group (PBS(-) group: 17.73±0.34, AMG0103 group: 20.58±0.55), a significant difference (p<0.01, Student t-test) was observed (Figure 2b, Table 4b).

[0178] Effects of intrathecal administration of AMG0103 on a rat model of brewer's yeast-induced acute pain (summary of pain threshold ratios)

[0179] Subcutaneous administration of AMG0103 to the right hind limb sole showed a transient pain-suppressing effect. However, no significant difference was observed in the summation of pain threshold ratios (0 to 30h) between the PBS(-) group and the AMG0103 group, suggesting that AMG0103 does not have a pain-suppressing effect when administered subcutaneously. On the other hand, a significant difference was observed in the summation of pain threshold ratios (0 to 30h) between the PBS(-) group and the AMG0103 group when administered intrathecally. This suggests that AMG0103 did not reduce stimulation of nociceptors by suppressing the production of pain-causing substances generated by inflammation of the sole, but rather suppressed pain by acting on glial cells that affect sensory nerves (such as spinal nerve dorsal roots) from within the spinal cavity.

[0180] Example 2: Effects of partial spinal nerve ligation on a neuropathic pain model. As a different pathological model from Example 1, we investigated using a rat model of neuropathic pain induced by partial spinal nerve ligation (SNL) (SNL model). This SNL model is created by tightly ligating the L5 and L6 spinal nerves, which are part of the 4th to 6th lumbar (L4-6) spinal nerves that form the sciatic nerve, with 6-0 silk sutures (Folia Pharmacol. Jpn., 127, 151-155, 2006). Furthermore, since it has been reported that mechanical allodynia (pain caused by external stimuli) persists for 10 weeks in this SNL model (Kim SH, et al. Pain. 1992; 50: 355-363.), it can be considered a pathological model of chronic neuropathic pain in human intervertebral disc degeneration.

[0181] Materials: Sprague Dawley (SD) rat (Nippon SLC) Catheter: Polyethylene tube PE (Nippon Becton Dickinson) Nesco suture silk suture: 6-0 (Alfresa Pharma) NF-κB decoy: AMG0103 Dynamic planter estesiometer: #37550 (Ugo Basile)

[0182] After anesthetizing the animals with isoflurane, the head and neck were shaved and the animals were fixed to a brain-positioning device. An incision was made in the midline skin of the head to expose the arachnoid membrane between the occipital bone and the first cervical vertebra. A hole was made in the arachnoid membrane with an 18G needle, and a catheter was inserted and left in place on the lumbar side. One week later, the lumbar spine of the anesthetized animals was shaved, and an incision was made between the lumbar vertebrae and the pelvic bone. The transverse process of the sixth lumbar vertebra was removed with a drill, and the spinal nerves of the fifth and sixth lumbar vertebrae were tightly ligated with Nesco suture silk sutures (6-0). The onset of pathogenic pain due to spinal nerve ligation, and the state of pain, were assessed using the pain threshold as an indicator. A dynamic planter estesiometer was used to push up a plastic filament onto the sole of the rat's foot at a set rate of increasing force, and the force applied when the rat retracted its foot was measured (Von Frey method).

[0183] Before intrathecal administration (Day 0), the pain threshold was measured using the Von Frey method to confirm that the pain threshold had sufficiently decreased. Then, based on animals that recovered their weight well after intrathecal cannulation and their pain thresholds, the animals were divided into a control group that received PBS(-) intrathecally and an AMG0103 group that received AMG0103 intrathecally. Pain threshold measurement and intrathecal administration were carried out according to Table 5. AMG0103 was administered in gradual increments: 3 μg / body for the first dose (Day 0), 10 μg / body for the second dose (Day 3), and 30 μg / body for the third dose (Day 6). The pain threshold was measured before AMG0103 administration.

[0184] Group composition

[0185] Treatment details The day of the first administration was designated as Day 0.

[0186] One day after intrathecal administration (Day 1), the AMG0103 group showed improvement in the lowered pain threshold compared to the control group, although no statistically significant difference was observed (PBS(-) group: 15.06±0.64, AMG0103 group: 17.16±1.15). Three days after intrathecal administration (Day 3), the pain threshold of the AMG0103 group (19.29±0.80) showed a significant improvement compared to the PBS(-) group (15.79±0.75) (p<0.01, Student t-test). Six days after intrathecal administration (Day 6, after the second dose of AMG0103), the pain threshold (19.44 ± 0.93) in the AMG0103 group showed a significant improvement compared to the PBS(-) group (15.25 ± 0.80) (p < 0.01, Student test). Furthermore, nine days after intrathecal administration (Day 9, after the third dose of AMG0103), the pain threshold (19.59 ± 0.80) in the AMG0103 group showed sustained improvement compared to the PBS(-) group (15.94 ± 0.99), and this improvement was significantly greater than that of the PBS(-) group (p < 0.05, Student test). Fourteen days after intrathecal administration (Day 14), the pain threshold (17.75 ± 1.22) in the AMG0103 group showed an improving trend compared to the PBS(-) group (16.16 ± 0.86), although the difference was not statistically significant (Figure 3, Table 7).

[0187] Effects of intrathecal administration of AMG0103 on a rat SNL model For animals number 109 and 203, the third dose on Day 6 could not be administered because the catheter became dislodged. However, in the statistical analysis for Day 9 and Day 14, the measurements from these animals were not excluded.

[0188] These results confirmed that AMG0103 has the effect of improving neuropathic pain in a spinal nerve partial ligation neuropathic pain model (SNL model). Therefore, similar to the brewer's yeast-induced acute pain model mentioned above, it is thought that pain was suppressed by acting on glial cells affecting sensory nerves (such as spinal nerve dorsal roots) from within the spinal cavity. Furthermore, since this model is a good pathological model of chronic neuropathic pain, AMG0103 is considered useful for the treatment of chronic neuropathic pain in humans (for example, intervertebral disc degeneration accompanied by chronic neuropathic pain).

[0189] Example 3: Effects of AMG0103 on rat dorsal root ganglion neurons via glial cells

[0190] Materials: Rat astrocytes: #AST01C (Cosmo Bio) Culture medium for rat astrocytes: Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (low glucose) Rat dorsal root ganglion neurons - embryonic: #R-eDRG-515 (Lonza) Culture medium for rat dorsal root ganglion neurons - embryonic: Primary Neuron Basal Medium (PNBM) with 100 ng / mL NGF NF-κB decoy: AMG0103 Anti-βIII tubulin (Tuj1) antibody: #ab18207 (Abcam) Fluorescently labeled antibody: Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 555, #A-21428 (Thermo Fisher Scientific) CO ２ Incubator: SCA-165DRS (Astec) Electroporation system: 4D-nucleofector (Lonza) High-content analysis system: Operetta CLS (PerkinElmer Japan)

[0191] Method: Using rat astrocyte culture medium and culture vessels, CO2 was used. ２Rat astrocytes cultured in an incubator were detached and collected from the culture vessel using 0.05% Trypsin-EDTA, and then AMG0103 was introduced using an electroporation system. The final concentrations of AMG0103 introduced into the rat astrocytes were 10 and 50 nM. The AMG0103-introduced rat astrocytes were then subjected to CO2 injection using rat astrocyte culture medium and culture vessels. ２ The cells were cultured in an incubator for 72 hours. After the culture period, the culture supernatant was collected and centrifuged to remove cell residue, and this supernatant was used as the conditioned medium. Next, the culture medium of rat dorsal root ganglion neurons-embryonic cells, which had been cultured in culture vessels, was replaced with conditioned medium derived from rat astrocytes and cultured for 5 days. For the control, conditioned medium derived from astrocytes that had not been introduced with AMG0103 was used. After the culture period, the rat dorsal root ganglion neurons-embryonic cells were fixed with 4% paraformaldehyde, and Tuj1 was detected using anti-βIII tubulin (Tuj1) antibody and fluorescently labeled antibody. Based on the detected Tuj1, the neurite length was measured using a high-content analysis system. Tuj1 is one of the β-tubulins and is commonly used as a neuronal marker because it is present in the cell body, dendrites, and axons. The mean and standard error were calculated based on the neurite length of each group. Dunnett's test was used for the statistical analysis.

[0192] When comparing the length of dorsal root ganglion neurons and embryonic neurites in the control group (175.5 ± 10.6 μm) and the AMG0103 group (10 nM: 164.4 ± 4.7 μm, 50 nM: 229.1 ± 9.4 μm), a significant increase was observed in the 50 nM AMG0103 group (p < 0.01, Dunnett's test). The results are shown in Figure 4 and Table 8.

[0193] The effect of AMG0103 on rat dorsal root ganglion neurons via glial cells.

[0194] These results suggest that AMG0103, when introduced only into astrocytes, directly acts on rat dorsal root ganglion neurons-embryonic, indicating that it promotes neurite extension via glial cells, i.e., it has a glial cell-dependent neurite extension promoting effect.

[0195] Studies using the aforementioned disease model suggest that AMG0103 administered intrathecally acts on glial cells such as the dorsal roots of spinal nerves in the spinal cord, which are present in the spinal cavity. Furthermore, studies using the aforementioned glial cells (astrocytes) and rat dorsal root ganglion neurons suggest that AMG0103 suppresses pain by inhibiting NF-κB activity in astrocytes, thereby enhancing nerve extension of rat dorsal root ganglion neurons.

[0196] The present invention is applicable to the pharmaceutical industry and / or the medical industry that provide pharmaceuticals and / or medical treatments for diseases in which glial cell-dependent neurite outgrowth promoters are effective (e.g., intervertebral disc degeneration).

Claims

1. A glial cell-dependent neurite extension promoter containing NF-κB decoy as the active ingredient, used to promote neurite extension of nerve cells via glial cells.

2. The glial cell-dependent neurite outgrowth promoter according to claim 1, wherein the nerve cells are sensory nerve cells.

3. The glial cell-dependent neurite outgrowth promoter according to claim 2, wherein the nerve cells of the sensory nerve are nerve cells of a patient suffering from intervertebral disc degeneration.

4. The glial cell-dependent neurite outgrowth promoter according to claim 3, wherein the nerve cells of the sensory nerves of a patient suffering from intervertebral disc degeneration are nerve cells of a patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain.

5. The glial cell-dependent neurite outgrowth promoter according to claim 4, wherein the nerve cells of the sensory nerves of the patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain are nerve cells of the sensory nerves in the sensory nerve root area of ​​the patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain.

6. The glial cell-dependent neurite extension promoter according to claim 5, wherein the glial cells are astrocytes and / or microglia and / or Schwann cells and / or satellite glial cells.

7. The glial cell-dependent neurite extension promoter according to claim 1, which is used by local administration to or near the site where glial cells are located.

8. The glial cell-dependent neurite extension promoter according to claim 1, which is used by local administration to the spinal cord dorsal horn or its vicinity.

9. The glial cell-dependent neurite extension promoter according to claim 1, which is used by local administration into the intervertebral disc such that the NF-κB decoy reaches the site of glial cells by infiltration or diffusion through non-hematogenous distribution.

10. The glial cell-dependent neurite extension promoter according to claim 1, which is used by local administration into the intervertebral disc such that the NF-κB decoy reaches the spinal cord dorsal horn by infiltration or diffusion via non-hematogenous distribution.

11. The glial cell-dependent neurite extension promoter according to claim 1, for use in patients suffering from anti-inflammatory drug-resistant intervertebral disc degeneration.

12. The glial cell-dependent neurite outgrowth promoter according to claim 1, used for the treatment of anti-inflammatory drug-resistant intervertebral disc degeneration.

13. The glial cell-dependent neurite extension promoter according to claim 1, which is used by administering it to patients suffering from intervertebral disc degeneration without signs of inflammation.

14. The glial cell-dependent neurite outgrowth promoter according to claim 1, used for the treatment of intervertebral disc degeneration without signs of inflammation.

15. The glial cell-dependent neurite extension promoter according to any one of claims 1 to 14, wherein the NF-κB decoy is any of (a) to (c) below: (a) A double-stranded oligonucleotide in which an oligonucleotide represented by Sequence ID No. 2 and an oligonucleotide represented by Sequence ID No. 3 form a complementary base pair: 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTTCCCTAAAAGGGAGG-5' (Sequence ID No. 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, wherein a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and these two mutants form a complementary base pair. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA: 5'-GGGPuNNPyPyCC-3' (κB sequence) 16. The glial cell-dependent neurite extension promoter according to any one of claims 1 to 14, wherein the NF-κB decoy is (a) below: (a) A double-stranded oligonucleotide in which the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO: 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (SEQ ID NO: 2) 3'-GGAACTTCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) 17. Use of NF-κB decoy in the manufacture of a therapeutic agent for intervertebral disc degeneration that promotes neurite outgrowth of nerve cells via glial cells.

18. The use according to claim 17, wherein the nerve cell is a nerve cell of a sensory nerve.

19. The use according to claim 18, wherein the therapeutic agent is a therapeutic agent for intervertebral disc degeneration accompanied by chronic neuropathic pain.

20. The use according to claim 19, wherein the nerve cells of the sensory nerve are nerve cells of the sensory nerve in the sensory nerve root region.

21. The use according to claim 20, wherein the glial cells are astrocytes and / or microglia and / or Schwann cells and / or satellite glial cells.

22. The use according to claim 17, wherein the therapeutic agent is filled in a syringe for local administration to or near the site of glial cells.

23. The use according to claim 17, wherein the therapeutic agent is filled in a syringe for local administration to the spinal cord dorsal horn or its vicinity.

24. The use according to claim 17, wherein the therapeutic agent is filled in a syringe for local administration into the intervertebral disc, and the amount of the therapeutic agent in the syringe is sufficient to allow the NF-κB decoy to reach the site of glial cells by osmosis or diffusion through non-hematogenous distribution after local administration into the intervertebral disc.

25. The use according to claim 17, wherein the therapeutic agent is filled in a syringe for local administration into the intervertebral disc, and the amount of the therapeutic agent in the syringe is sufficient to allow the NF-κB decoy to reach the spinal cord dorsal horn by infiltration or diffusion through non-hematogenous distribution after local administration into the intervertebral disc.

26. The use according to claim 17, wherein the therapeutic agent is administered to a patient suffering from anti-inflammatory drug-resistant intervertebral disc degeneration.

27. The use according to claim 17, wherein the therapeutic agent is a therapeutic agent for anti-inflammatory drug-resistant intervertebral disc degeneration.

28. The use according to claim 17, wherein the therapeutic agent is administered to a patient suffering from intervertebral disc degeneration without signs of inflammation.

29. The use according to claim 17, wherein the therapeutic agent is a therapeutic agent for intervertebral disc degeneration without signs of inflammation.

30. The use according to any one of claims 17 to 29, wherein the NF-κB decoy is any of (a) to (c) below. (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID No. 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, in which a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, form a complementary base pair. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA: 5'-GGGPuNNPyPyCC-3' (κB sequence) 31. The use according to any one of claims 17 to 29, wherein the NF-κB decoy is (a) below. (a) A double-stranded oligonucleotide formed by complementary base pairing of the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO:

3. 5'-CCTTGAAGGGGAATTTCCCCCC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) 32. A method for treating intervertebral disc degeneration, wherein the method comprises the following steps: (1) identifying a patient suffering from intervertebral disc degeneration; (2) administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of NF-κB decoy, wherein the NF-κB decoy promotes neurite outgrowth of nerve cells via glial cells.

33. The method for treating intervertebral disc degeneration according to claim 32, wherein the nerve cells are nerve cells of sensory nerves.

34. The method for treating intervertebral disc degeneration according to claim 33, wherein the patient suffering from intervertebral disc degeneration is a patient suffering from intervertebral disc degeneration accompanied by chronic neuropathic pain.

35. The method for treating intervertebral disc degeneration according to claim 34, wherein the nerve cells of the sensory nerve are nerve cells of the sensory nerve in the sensory nerve root region.

36. The method for treating a patient suffering from intervertebral disc degeneration according to claim 35, wherein the glial cells are astrocytes and / or microglia and / or Schwann cells and / or satellite glial cells.

37. The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein in step (2) above, the pharmaceutical composition is administered locally to the site of or near the site of glial cells.

38. The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein in step (2) above, the pharmaceutical composition is administered locally to the spinal cord dorsal horn or its vicinity.

39. A method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein in step (2) above, the pharmaceutical composition is administered locally into the intervertebral disc, and the NF-κB decoy is made to reach the site of glial cells by infiltration or diffusion through non-hematogenous distribution.

40. A method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein in step (2) above, the pharmaceutical composition is administered locally into the intervertebral disc, and the NF-κB decoy is delivered to the spinal cord dorsal horn by infiltration or diffusion through non-hematogenous distribution.

41. The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the patient is a patient suffering from anti-inflammatory drug-resistant intervertebral disc degeneration.

42. A method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the intervertebral disc degeneration is resistant to anti-inflammatory drugs.

43. The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the patient is a patient suffering from intervertebral disc degeneration without signs of inflammation.

44. A method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the intervertebral disc degeneration is intervertebral disc degeneration without findings of inflammation.

45. The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the NF-κB decoy is any of (a) to (c) below. (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID No. 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, in which a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, form a complementary base pair. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA: 5'-GGGPuNNPyPyCC-3' (κB sequence) 46. ​​The method for treating a patient suffering from intervertebral disc degeneration according to claim 32, wherein the NF-κB decoy is (a) below. (a) A double-stranded oligonucleotide formed by complementary base pairing of the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO:

3. 5'-CCTTGAAGGGGAATTTCCCCCC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3) 47. NF-κB decoy for use in the treatment of intervertebral disc degeneration, promoting neurite outgrowth of nerve cells via glial cells.

48. The NF-κB decoy according to claim 47, wherein the nerve cell is a nerve cell of a sensory nerve.

49. The NF-κB decoy according to claim 48, wherein the intervertebral disc degeneration is intervertebral disc degeneration accompanied by chronic neuropathic pain.

50. The NF-κB decoy according to claim 49, wherein the nerve cells of the sensory nerve are nerve cells of the sensory nerve in the sensory nerve root region.

51. The NF-κB decoy according to claim 50, wherein the glial cells are astrocytes and / or microglia and / or Schwann cells and / or satellite glial cells.

52. The NF-κB decoy according to claim 47, for use by local administration to or near the site of glial cells.

53. The NF-κB decoy according to claim 47, for use by local administration to the spinal cord dorsal horn or its vicinity.

54. The NF-κB decoy according to claim 47, for use by local administration into an intervertebral disc so that the NF-κB decoy reaches the site of glial cells by infiltration or diffusion through non-hematogenous distribution.

55. The NF-κB decoy according to claim 47, for use by local administration into an intervertebral disc so that the NF-κB decoy reaches the spinal cord dorsal horn by infiltration or diffusion via non-hematogenous distribution.

56. The NF-κB decoy according to claim 47, for use in patients suffering from anti-inflammatory drug-resistant intervertebral disc degeneration.

57. The NF-κB decoy according to claim 47, wherein the intervertebral disc degeneration is anti-inflammatory drug resistant intervertebral disc degeneration.

58. The NF-κB decoy according to claim 47, for use by administration to a patient suffering from intervertebral disc degeneration without signs of inflammation.

59. The NF-κB decoy according to claim 47, wherein the intervertebral disc degeneration is intervertebral disc degeneration without signs of inflammation.

60. The NF-κB decoy according to any one of claims 47 to 59, wherein the NF-κB decoy is any of (a) to (c) below. (a) A double-stranded oligonucleotide in which the oligonucleotide represented by Sequence ID No. 2 and the oligonucleotide represented by Sequence ID No. 3 form a complementary base pair. 5'-CCTTGAAGGGGAATTTCCCCTC-3' (Sequence ID No. 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (Sequence ID No. 3) (b) A double-stranded oligonucleotide having the ability to bind to NF-κB, in which a mutant of the oligonucleotide represented by Sequence ID No. 2 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 has 20% or less of its base sequence mutated, substituted, inserted, and / or deleted, form a complementary base pair. (c) A double-stranded oligonucleotide having complementary base pairs formed by a mutant of the oligonucleotide represented by Sequence ID No. 2 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, and a mutant of the oligonucleotide represented by Sequence ID No. 3 having 20% ​​or less of its base sequence mutated, substituted, inserted, and / or deleted, thereby antagonistically inhibiting the binding of NF-κB to the κB sequence specified below, which is present in chromosomal DNA: 5'-GGGPuNNPyPyCC-3' (κB sequence) 61. The NF-κB decoy according to any one of claims 47 to 59, wherein the NF-κB decoy is (a) below. (a) A double-stranded oligonucleotide formed by complementary base pairing of the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO:

3. 5'-CCTTGAAGGGGAATTTCCCCCC-3' (SEQ ID NO: 2) 3'-GGAACTCCCCTAAAAGGGAGG-5' (SEQ ID NO: 3)

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