阅读说明：本技术 用嵌合体诱饵治疗脊柱病症 (Treatment of spinal disorders with chimeric baits ) 是由 舛田浩一 中泽隆弘 于 2019-12-24 设计创作，主要内容包括：本文提供用于治疗脊柱病症的方法和组合物。具体而言,能够结合于两种转录因子(NF-κB和STAT6)的DNA结合位点的双链寡核苷酸诱饵用于治疗椎间盘退化、再生软骨细胞细胞外基质、脊柱疼痛和促进椎间盘细胞中的蛋白聚糖合成的用途。(Provided herein are methods and compositions for treating spinal disorders. In particular, the use of a double-stranded oligonucleotide decoy capable of binding to the DNA binding sites of two transcription factors (NF- κ B and STAT6) for the treatment of disc degeneration, regeneration of chondrocyte extracellular matrix, spinal pain and promotion of proteoglycan synthesis in intervertebral disc cells.)

1. A method for treating disc degeneration comprising administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby treating disc degeneration in the subject.

2. The method of claim 1, wherein the bait has a size of 13-mer to 15-mer.

3. The method of claim 2, wherein the bait has a sequence represented by SEQ ID NO 1 or 6.

4. The method of claim 1, wherein at least a portion of the linkages between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate linkages.

5. The method of claim 1, wherein the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly.

6. The method of claim 1, wherein the bait is administered directly into the subject's facet joint.

7. The method of claim 1, wherein the bait is administered by intradiscal injection or epidural injection.

8. A method for regenerating chondrocyte extracellular matrix, comprising administering to a subject in need thereof a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby regenerating chondrocyte extracellular matrix in the subject.

9. The method of claim 8, wherein the chondrocyte extracellular matrix is an intervertebral disc extracellular matrix.

10. The method of claim 8, wherein the bait has a size of 13-mer to 15-mer.

11. The method of claim 10, wherein the bait has a sequence represented by SEQ ID No. 1 or 6.

12. The method of claim 8, wherein at least a portion of the linkages between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate linkages.

13. The method of claim 8, wherein the bait is administered directly into the subject's facet joint.

14. The method of claim 8, wherein the bait is administered by intradiscal injection or epidural injection.

15. A method for promoting proteoglycan synthesis in a subject's intervertebral disc cells, comprising administering to a subject in need thereof a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby promoting proteoglycan synthesis in said subject's intervertebral disc cells.

16. The method of claim 15, wherein the intervertebral disc cells comprise nucleus pulposus cells and/or annulus fibrosus cells.

17. The method of claim 15, wherein the bait has a size of 13-mer to 15-mer.

18. The method of claim 17, wherein the bait has a sequence represented by SEQ ID No. 1 or 6.

19. The method of claim 15, wherein at least a portion of the linkages between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate linkages.

20. The method of claim 15, wherein the bait is administered directly into the subject's facet joint.

21. The method of claim 15, wherein the bait is administered by intradiscal injection or epidural injection.

22. A method for treating spinal pain, comprising administering to a subject in need thereof a double-stranded oligonucleotide decoy capable of binding to a DNA binding site of NF-kb and a DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby treating spinal pain in the subject.

23. The method of claim 22, wherein the bait has a size of 13-mer to 15-mer.

24. The method of claim 23, wherein the bait has a sequence represented by SEQ ID No. 1 or 6.

25. The method of claim 22, wherein at least a portion of the linkages between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate linkages.

26. The method of claim 22, wherein the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly.

27. The method of claim 22, wherein the bait is administered directly into the subject's facet joint.

28. The method of claim 1, wherein the bait is administered by intradiscal injection or epidural injection.

29. A therapeutic agent for intervertebral disc degeneration, comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT 6).

30. A double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for use in treating disc degeneration.

31. An agent for regenerating chondrocyte extracellular matrix, comprising a double-stranded oligonucleotide decoy capable of binding to a DNA binding site of NF- κ B and a DNA binding site of signal transducer and activator of transcription 6 (STAT 6).

32. A double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for use in regenerating chondrocyte extracellular matrix.

33. An agent for promoting proteoglycan synthesis in a subject's intervertebral disc cells, comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of Signal transducer and activator of transcription 6 (STAT 6).

34. A double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for use in promoting proteoglycan synthesis in a intervertebral disc cell of a subject.

35. A therapeutic agent for spinal pain comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT 6).

36. A double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for use in the treatment of spinal pain.

Technical Field

The present invention relates generally to oligonucleotide decoys, and more particularly to the use of double-stranded oligonucleotide decoys capable of binding to the DNA binding sites of two transcription factors for the treatment of spinal pain.

Background

Lumbago (LBP) is the leading cause of disability in the united states today. In 3 years, 15-20% of americans need medical care for back pain. Back pain causes more than five hundred (500) billion dollars of loss to society due to lost productivity and medical care costs. LBP can be caused by many components of the lumbar spine, particularly the intervertebral disc (IVD), paraspinal muscles, and the articular processes of the vertebrae or facet joints.

Recent studies have shown that as much as 30% of LBP is produced by facet joints. Although it has recently been proposed that facet joint pathology causes LBP, historically, degeneration of IVD has been associated with back pain and is thought to precede facet Osteoarthritis (OA). However, the relationship between the facet joints and degenerative changes in IVD is largely unknown.

Currently, the treatment of most facet joint OA is limited to physical therapy, medial branch block, intra-articular local anesthesia, steroid injection, or radio frequency denervation. Based on the US Preventive Service Task Force (USPSTF) standard, the evidence level of the medial branch block is I-II-1. The evidence grades of the articular facet joint block and the radio frequency denervation are II-1-2 and II-2-II-3 respectively. Surgery is also occasionally performed, although there is no clear evidence to support surgical intervention. Recently, the effect of hyaluronic acid on facet joint arthropathy has been studied, but the efficacy of this treatment remains controversial.

Inflammatory features are known to play a key role in the progression of facet joint degeneration and pain production [2,3 ]. A one-year follow-up of a double-blind, controlled clinical trial using lumbar facet joint nerve block to treat chronic LBP produced in facet joints indicates that non-surgical therapy can be used to manage these patients. Facet joint injection is a common minimally invasive procedure involving injection of corticosteroids; however, steroids are known to have side effects such as inhibition of matrix synthesis of chondrocytes [4] and infection.

Therefore, there is a need for a non-invasive therapy to treat LBP, such as facet joint pain.

Summary of The Invention

The present invention is based on the following findings: injection of chimeric decoy oligodeoxynucleotides into the facet joints showed similar or better efficacy than dexamethasone in ameliorating thrombin-induced facet joint pain in rats. Accordingly, the present invention provides a method for treating spinal pain. The method comprises administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby treating spinal pain in the subject. In various embodiments, the bait has a size of 13-15 mer. In various embodiments, the bait has a sequence represented by SEQ ID NO 1 or 6. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly. In various embodiments, the bait is administered directly into the facet joint of the subject. In various embodiments, the bait is administered by intradiscal injection or epidural injection. In a similar aspect, the present invention provides a therapeutic agent for spinal pain comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT 6); and a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for treating spinal pain. The above description of the method for treating spinal pain applies equally to these similar aspects.

In another aspect, the invention provides a method of treating disc degeneration in a subject. The method comprises administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby treating disc degeneration in the subject. In various embodiments, the bait has a size of 13-15 mer. In various embodiments, the bait has a sequence represented by SEQ ID NO 1 or 6. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly. In various embodiments, the bait is administered directly into the facet joint of the subject. In various embodiments, the bait is administered by intradiscal injection or epidural injection. In a similar aspect, the present invention provides a therapeutic agent for intervertebral disc degeneration, comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT 6); and a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-. kappa.B and the DNA binding site of Signal transducer and activator of transcription 6 (STAT6) for the treatment of disc degeneration. The above description of the method of treating disc degeneration applies equally to these similar aspects.

In another aspect, the invention provides a method for regenerating chondrocyte extracellular matrix in a subject. The method comprises administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby regenerating chondrocyte extracellular matrix in the subject. In various embodiments, the bait has a size of 13-15 mer. In various embodiments, the bait has a sequence represented by SEQ ID NO 1 or 6. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly. In various embodiments, the bait is administered directly into the facet joint of the subject. In various embodiments, the bait is administered by intradiscal injection or epidural injection. In a similar aspect, the present invention provides an agent for regenerating chondrocyte extracellular matrix, comprising a double-stranded oligonucleotide decoy capable of binding to a DNA binding site of NF- κ B and a DNA binding site of signal transducer and activator of transcription 6 (STAT 6); and a double-stranded oligonucleotide decoy for regenerating chondrocyte extracellular matrix, capable of binding to a DNA binding site of NF-. kappa.B and a DNA binding site of signal transducer and activator of transcription 6 (STAT 6). The above description of the method for regenerating a chondrocyte extracellular matrix in a subject is equally applicable to these similar aspects.

In another aspect, the present invention provides a method for promoting proteoglycan synthesis in a disc cell of a subject. The method comprises administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby promoting proteoglycan synthesis in the intervertebral disc cells of the subject. In various embodiments, the bait has a size of 13-15 mer. In various embodiments, the bait has a sequence represented by SEQ ID NO 1 or 6. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, at least a portion of the bonds between nucleotides in the double-stranded oligonucleotide decoy comprise phosphorothioate bonds. In various embodiments, the 5' end of the bait is bound to the PLGA nanoparticle via a linker or directly. In various embodiments, the bait is administered directly into the facet joint of the subject. In various embodiments, the bait is administered by intradiscal injection or epidural injection. In a similar aspect, the invention provides a therapeutic agent for promoting proteoglycan synthesis in a intervertebral disc cell of a subject, comprising a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF- κ B and the DNA binding site of signal transducer and activator of transcription 6 (STAT 6); and a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6) for promoting proteoglycan synthesis in the intervertebral disc cells of the subject. The above description of methods for promoting proteoglycan synthesis in a subject's intervertebral disc cells applies equally to these similar aspects.

Brief Description of Drawings

Figure 1 is a graph showing the results of assessing rat mechanical allodynia using both injected and contralateral hind paws for 50% paw withdrawal threshold response to mechanical stimulation by von Frey capillary.

Fig. 2A and 2B are graphs showing results from behavioral assessment of general conditions and activities caused by facet joint pain. To assess the general condition of the rats, changes in body weight were analyzed (fig. 2A). To assess the changes in general activity caused by facet joint pain, a commercially available device consisting of a camera and behavioral analysis software (homeage secan, Clever Sys Inc, Reston, VA) was performed before and 1 week after surgery (preoperative and postoperative) (fig. 2B).

Fig. 3A and 3B are graphs showing the immunohistochemical data for the dorsal root ganglion for Iba and CGRP (fig. 3A) and the correlation of immunohistochemical staining and von frey test results (fig. 3B).

FIG. 4 is a graph showing the results of proteoglycan turnover in human intervertebral disc cells stimulated by interleukin-1.

FIG. 5 shows the residual S-PG in the tissue (ratio to control).

Fig. 6 shows the disc height index.

FIG. 7 shows an MRI T2 spin echo weighted image.

FIG. 8 shows T2 MRI (L3/4 control, L2/3, L4/5 injection).

Modes for carrying out the invention

The present invention provides chimeric baits useful in various embodiments to treat disc degeneration, regenerate chondrocyte extracellular matrix, promote proteoglycan synthesis, and treat spinal pain.

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

The term "comprising" used interchangeably with "comprising", "containing" or "characterized by" is an inclusive or open language and does not exclude additional, unrecited elements or method steps. The phrase "consisting of … …" does not include any element, step, or ingredient not specified in the claims. The phrase "consisting essentially of … …" limits the scope of the claims to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the invention corresponding in scope to each of these phrases. Thus, a composition or method that comprises the enumerated elements or steps contemplates specific embodiments in which the composition or method consists essentially of, or consists of, those elements or steps.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

The term "bait" as used herein refers to a structure that is similar to the structure to which a substance should initially bind or act upon. A decoy for a transcription factor as provided herein can be a double-stranded oligonucleotide having the same DNA sequence as the binding region for the transcription factor on a genomic gene. In the presence of such oligonucleotide decoys, some transcription factor molecules bind to the decoy oligonucleotide, rather than to the binding region on the genomic gene to which they would otherwise bind. This results in a reduction in the number of transcription factor molecules that bind to the binding region of the genomic gene to which they should bind, resulting in a reduction in the activity of the transcription factor. The chimeric bait contains a DNA sequence for binding more than one transcription factor.

The term "allodynia" as used herein refers to central pain sensitization (increased response of neurons) following a normally painless, often repetitive stimulus. Allodynia can result in the triggering of a pain response by a stimulus that does not normally cause pain.

"discogenic pain" as used herein refers to pain originating from a damaged intervertebral disc, particularly due to degenerative disc disease.

The intervertebral discs (IVD) of the spine consist of a collagen-rich outer Annulus Fibrosus (AF) leading to its tensile strength and an inner Nucleus Pulposus (NP) containing large Proteoglycans (PG) that can retain moisture to resist compressive loads. Biologically, intervertebral disc cells in both AF and NP maintain a balance or steady state metabolism between the anabolism and catabolism of their extracellular matrix (ECM), and are regulated by a variety of substances, including cytokines, enzymes, inhibitors thereof, and growth factors, such as insulin-like growth factor (IGF), transforming growth factor beta (TGF- β), and Bone Morphogenic Protein (BMP). Various enzymes such as Matrix Metalloproteinases (MMPs) and cytokines mediate catabolic processes or breakdown of the matrix. The degeneration of IVD is thought to be due to an imbalance between the anabolic and catabolic processes maintained in normal intervertebral discs or a loss of homeostasis metabolism.

As used herein, "facet joint" refers to a joint in the spine that makes the back flexible and enables the subject to bend and twist. Nerves exit the spinal cord through these joints on their way to other parts of the body. Healthy facet joints have cartilage that allows the vertebrae to move smoothly with respect to each other without grinding.

The term "subject" or "host organism" as used herein refers to any individual or patient on which the subject methods are performed. Typically the subject is a human, although as will be appreciated by those skilled in the art, the subject may be an animal. Thus, other animals, including mammals (such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, and the like), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of subject.

The term "therapeutically effective amount" or "effective amount" means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Thus, the term "therapeutically effective amount" is used herein to refer to any amount of a formulation that causes a significant improvement in a disease condition when repeatedly administered to an affected area over a period of time. This amount will vary with the condition being treated, the stage of development of the condition, and the type and concentration of the formulation being administered. The appropriate amounts in any given case will be readily apparent to those skilled in the art or can be determined by routine experimentation.

The terms "reduce" and "inhibit" as used herein are used together, as it is recognized that in some cases the reduction may be reduced below the detection level of a particular assay. Thus, it may not always be clear whether an expression level or activity is "reduced below" the level of detection of the assay or is completely "inhibited". However, it will be clearly determinable after treatment according to the present method.

As used herein, "treatment" or "treating" means administering a composition to a subject or system suffering from an undesirable disorder. A condition can include a condition, disease, or disorder. By "preventing" or "preventing" is meant administering the composition to a subject or system at risk of the disorder. Conditions may include a predisposition to a disease or disorder. The effect (treatment and/or prevention) of administering the composition to a subject can be, but is not limited to, cessation of one or more symptoms of the disorder, alleviation or prevention of one or more symptoms of the disorder, reduction in severity of the disorder, complete ablation of the disorder, stabilization or delay in the development or progression of a particular event or feature, or minimization of the chance that a particular event or feature will occur.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, α -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., the alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as a naturally occurring amino acid, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (IUPAC-IUB Biochemical Nomenclature Commission). Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The term "gene" as used herein means a deoxyribonucleotide sequence comprising the coding region of a structural gene. "Gene" can also include untranslated sequences located near the coding region on both the 5 'and 3' ends, such that the gene corresponds to the length of a full-length mRNA. Sequences located 5 'to the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3 'or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" includes both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are gene segments transcribed into heterogeneous nuclear rna (hnrna); introns may contain regulatory elements, such as enhancers. Introns are removed or "clipped" from the nucleus or primary transcript; thus introns are not present in messenger rna (mrna) transcripts. The mRNA functions during translation to specify the sequence or order of amino acids in the nascent polypeptide.

"sequence" of a nucleic acid refers to the order and identity of the nucleotides in the nucleic acid. Sequences are typically read in the 5 'to 3' direction. The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to the skilled artisan or by visual inspection. An exemplary algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST program, it is described, for example, in Altschul et al (1990) "Basic local alignment search tool" J. mol. biol. 215: 403. 410, Gish et al (1993) "Identification of protein coding regions by database similarity search" Nature Gene. 3: 266. 272, Madden et al (1996) "Applications of network BLAST server" meth. Enzymol. 266: 131. 141, Altschul et al (1997) "Gapped and PSI-BLAST: a new generation of protein database search programs "Nucleic Acids Res. 25:3389-3402, and Zhang et al (1997)" PowerBLAST: a new network BLAST application for interactive or automatic sequence analysis and interpretation "Genome Res.7: 649-656, each of which is incorporated by reference. Many other optimal alignment algorithms are also known in the art and are optionally used to determine percent sequence identity.

The terms "functionally linked" and "operably linked" as used herein are used interchangeably and refer to the functional relationship between two or more DNA segments, particularly the gene sequences to be expressed and those sequences controlling their expression. For example, a promoter/enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Promoter regulatory sequences operably linked to transcribed gene sequences are physically contiguous with the transcribed sequences.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or to essentially identical sequences when the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one of the conservatively modified variations. Each nucleic acid sequence herein encoding a polypeptide also describes each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid, except AUG (which is typically the only codon for methionine) and TGG (which is typically the only codon for tryptophan), may be altered to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the coding sequence is a "conservatively modified variant" in which the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents.

Various double-stranded oligonucleotide decoys that show binding affinity for transcription factors are known to treat or prevent diseases caused by transcription factors by administering transcription factor decoys to reduce the activity of the transcription factor of interest. Recently, double-stranded oligonucleotide decoys comprising a first binding site for a first transcription factor and a second binding site for a second transcription factor have been described (see, e.g., U.S. publication No. 2018/0298381 and international publication No. WO2017/043639, both of which are incorporated herein by reference). Briefly, a first strand of the sense strand comprising the first binding site and a second strand of the sense strand comprising the second binding site hybridize to form a double strand. Further, the sense strand of the first binding site and the sense strand of the second binding site hybridize at least partially.

An exemplary double-stranded oligonucleotide decoy described in U.S. publication No. 2018/0298381 is an NF-. kappa.B/STAT 6-15mer-B decoy, which is represented by formula I (SEQ ID NO: 1) below:

thus, for NF-. kappa.B/STAT 6-15mer-B having a structure represented by the formula [ I ], the first transcription factor was NF-. kappa.B, and the second transcription factor was STAT 6. The first and second strands are complementary, and thus the second strand is the complementary strand of the first strand. The sequence GGGATTTCCT (SEQ ID NO: 2) indicated by capital letters in the first strand is the binding site for NF-. kappa.B, and the sequence TTCCCAGGAAA (SEQ ID NO: 3) indicated by capital letters in the second strand (which is written on the left side with its 3' end in formula [ I ] because it is a complementary strand; and thus is practically identical to the sequence represented in formula [ I ], although they are written in the opposite direction) is the binding site for STAT 6.

Consensus sequences of transcription factors are generally represented by a general formula. The consensus sequence for NF-. kappa.B was GGGRHTYYHC (SEQ ID NO: 4) (wherein R represents A or G, Y represents C or T, and H represents A, C or T), and the consensus sequence for STAT6 was TTCNNNNGAA (SEQ ID NO: 5) (wherein N represents A, G, T or C). Thus, the NF-. kappa.B binding site GGGATTTCCT (SEQ ID NO: 2) in the first strand is identical to the consensus sequence of NF-. kappa.B, except that the only base at the 3' end is mismatched to the consensus sequence of NF-. kappa.B. Binding site TTCCCAGGAAA (SEQ ID NO: 3) of STAT6 in the second strand contains the entire consensus sequence of STAT 6. When describing the base sequence, the base sequence of the sense strand is described, although the binding site of the transcription factor and the consensus sequence are double-stranded. Thus, the base sequences of the binding site and the consensus sequence as described above are both base sequences of the sense strand. Thus, the first strand contains the sense strand of the NF- κ B binding site, and the second strand contains the sense strand of the STAT6 binding site.

Another exemplary oligonucleotide decoy NF-. kappa.B/STAT 6-15mer-A) is provided as formula II (SEQ ID NO: 6):

thus, for NF-. kappa.B/STAT 6-15mer-B having a structure represented by the formula [ II ], the first transcription factor was NF-. kappa.B, and the second transcription factor was STAT 6. The first and second strands are complementary, and thus the second strand is the complementary strand of the first strand. The sequence GGGACTTCCC (SEQ ID NO: 7) indicated in capital letters in the first strand is the binding site for NF-. kappa.B, and the sequence TTCATGGGAAG (SEQ ID NO: 8) indicated in capital letters in the second strand (which is written on the left side at its 3' end in formula [ II ] because it is a complementary strand; and thus is practically identical to the sequence represented in formula [ II ], although they are written in the opposite direction) is the binding site for STAT 6.

The chimeric baits of the invention may be simple double-stranded, or may be hairpin or dumbbell (staple) baits in which one or both ends of each strand are bound via a spacer. Hairpin and dumbbell baits are preferred because of their greater stability. Hairpin decoys are most preferred when comprehensively assessing binding activity and stability to transcription factors. Methods for making such hairpin double-stranded chimeric baits and other nuclease-resistant modifications are well known, as described in U.S. publication No. 2018/0298381.

Although the role of cytokines in cartilage matrix metabolism and cartilage degradation has been recognized in cartilage studies, preliminary studies have shown that facet joint chondrocytes in non-surgical samples can produce large amounts of cytokines IL-1 and TNF- α; this may be a phenomenon specific to facet joints. Interestingly, the presence of cytokines was higher in the early stages of degeneration (grade 2/3) than in the late stages of degeneration (grade 4/5); this may indicate that cytokines are involved in the progression of facet joint cartilage degradation (2/3 → 4/5). In addition, cytokine blocking studies have shown that Prostaglandin (PG) synthesis is inhibited by constitutively expressed cytokines (see, e.g., U.S. patent No. 7,585,848, incorporated herein by reference).

In vitro studies showed that chimeric bait (NF-. kappa.B/STAT 6) significantly inhibited cytokine gene expression from synovial explants from knee joints of osteoarthritis patients. Additional results indicate that injection of an initial bait (AMG0101, formula I) into the facet joint reduces allodynia in a rat thrombin-induced facet joint arthritis model. These results indicate that chimeric baits can reverse the negative balance of facet cartilage homeostasis and suppress pain induced by cytokine pathways.

Accordingly, the present invention provides a method for treating spinal pain. The method comprises administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide decoy capable of binding to the DNA binding site of NF-kb and the DNA binding site of signal transducer and activator of transcription 6 (STAT6), thereby treating spinal pain in the subject.

As demonstrated herein, it was shown that chimeric decoy oligodeoxynucleotides (chimeric decoys) developed to bind to both NF-kB and signal transducer and activator of transcription 6 (STAT6) binding sites, reduce gene expression of pro-inflammatory cytokines and pain-associated molecules of synovial tissue [5] and slow IL-1 β accelerated proteoglycan turnover [6 ]. Because such chimeric baits do not inhibit matrix synthesis or increase the risk of infection, chimeric baits are novel topical treatments for facet joint pain. Inhibition of the cytokine pathway by chimeric decoys would reduce pain production by blocking several NF-. kappa.B-driven cytokine pathways (such as IL-1 and TNF). In a recently developed rat thrombin-induced facet joint pain model [7], the present invention describes clinically relevant efficacy data of the effects of chimeric decoys on facet joint pain compared to that of dexamethasone.

As described herein, chimeric baits and dexamethasone showed similar significant analgesic effects on mechanical allodynia; these pain outcome measurements are supported by IHC data for pain-related molecules in DRGs. Importantly, only chimeric baits showed significant effects on post-operative general activity (i.e., distance traveled and weight change (known as the sensitivity index for post-operative pain [10 ])). It was found that body weight increases significantly even during high activity, which further supports the safety and efficacy of chimeric baits. The strong correlation between pain status results obtained from von frey test and IHC staining indicates the importance of pain marker analysis in DRG as an additional confirmation of efficacy results for afferent nerve pain production. Therefore, the generation of facet pain induced by thrombin injection can be improved by injecting chimeric decoys, which can be used as a new therapeutic approach for facet joint pain.

The chimeric baits of the present invention may be administered as such or may also be administered after conjugation with a substance constituting an appropriate Drug Delivery System (DDS). Examples of DDS for oligonucleotides include liposomes containing cationic substances, cell membrane-penetrating peptides, polymers containing them, and atelopeptide collagen. In addition to these, chimeric baits can also be conjugated to PLGA (polylactic acid/glycolic acid copolymer) nanoparticles for administration. PLGA nanoparticles are particles composed of PLGA and having a diameter of tens to hundreds of nanometers. When the chimeric bait is conjugated to a PLGA nanoparticle, the 5' end of the first strand of the chimeric bait is preferably conjugated to the PLGA nanoparticle via a disulfide linker and an amino linker. This can be done, for example, by reacting PLGA-NHS ester with a chimeric bait to obtain a PLGA-conjugated chimeric bait and further making it into nano-sized particles by using the Marangoni (Marangoni) effect.

After administration of the bait, the method comprises determining or measuring the level of LBP in order to determine the efficacy of the treatment. In some embodiments, the method may comprise determining or measuring the level of the intervertebral disorder prior to treatment in order to determine the amount of bait required to adequately treat the LBP in the subject. In various embodiments, the level of a fibrocartilage degradation factor or a precursor thereof (e.g., zymogen, mRNA, etc.) may be measured to determine the amount of fibrocartilage degradation. In general, the fibrocartilage degradation factor includes any compound that, when present, will cause degradation of fibrocartilage tissue in the intervertebral disc. The fibrocartilage degradation factor may act directly on fibrocartilage cells or fibrocartilage tissue to cause degradation, affect a compound that directly degrades fibrocartilage tissue, or affect a modulator of a compound that degrades fibrocartilage tissue. Fibrocartilage degradation factors include enzymes that directly degrade cartilage matrix and other chemicals that stimulate cartilage degradation, including cytokines such as IL-1. IL-1 appears to cause fibrocartilage degradation indirectly by at least upregulating matrix metalloproteinase activity. Non-limiting examples of methods of measuring fibrocartilage degradation factors include measuring Nitric Oxide (NO) production, protease detection, or both.

Proteases occupying specific sets of fibrocartilage degradation factors can be detected in both normal and pathological discs. These proteases include, but are not limited to, Matrix Metalloproteinases (MMPs) and members of the ADAMTS family. Fibrocartilage degradation factors, including proteases, may be detected by any method known in the art. These methods include Western blot analysis, immunohistochemistry, RNA transcript detection, and zymography. Fibrocartilage or fibrochondrocytes from an intervertebral disc may be treated with a fibrochondroprotective agent prior to measurement of fibrocartilage degradation factors. Detection may also be performed before, after, or both exposure to fibrocartilage degradation factors. In various embodiments, the fibrocartilage degradation factor will be a native factor.

The route of administration of the chimeric bait is not particularly limited, but may preferably be parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, dermal administration or direct administration to a target organ or tissue. The dose is appropriately selected depending on, for example, the target disease, the symptoms of the patient and the administration route, but usually 0.1 to 10000 nmol, preferably 1 to 1000 nmol, and more preferably 10 to 100 nmol per day may be administered to an adult.

Pharmaceutical compositions of transcription factor inhibiting compounds, such as chimeric decoys, can be prepared by mixing one or more transcription factor inhibiting compounds with pharmaceutically acceptable carriers, excipients, binders, diluents, and the like, to therapeutically treat, reverse, or ameliorate various intervertebral disc disorders and/or LBP. A therapeutically effective dose refers to an amount of one or more transcription factor inhibiting compounds sufficient to result in an improvement in the symptoms of a disc disorder. An effective dose may also refer to an amount of one or more transcription factor inhibiting compounds sufficient to result in the prevention of disc disorders and/or LBP. In some embodiments, the effective dose will only partially prevent disc disorders and/or LBP. In these cases, the disc disorder, although it may still be present, will be less than would be expected if no treatment were given.

Pharmaceutical compositions may be manufactured by processes well known in the art, such as conventional granulation, mixing, dissolution, encapsulation, lyophilization, emulsification or grinding processes and the like. In certain embodiments, the transcription factor inhibiting compound can be administered locally rather than systemically, such as by injection as a sustained release formulation. In some embodiments, an effective amount of a transcription factor inhibiting compound can be administered to the pathological disc in any satisfactory physiological buffer, such as Phosphate Buffered Saline (PBS) or in a 5% lactose solution. The dosage forms disclosed in this specification are given as examples and should not be construed as limiting the invention.

As described below, formulations of transcription factor inhibiting compounds can be designed for short-acting, rapid-release, long-acting, and sustained-release. Thus, the pharmaceutical formulations may also be formulated for controlled or slow release, for example, by inclusion in a biodegradable matrix or carrier.

The transcription factor inhibitor/bait in the present compositions may also be present in micelles or liposomes or some other encapsulated form, or may be administered in an extended release form to provide an extended storage and/or delivery effect. Thus, the pharmaceutical formulation may be compressed into pellets or cylinders and implanted as a dose. Such implants may employ known inert materials such as silicone and biodegradable polymers.

The therapeutically effective dose of the transcription factor inhibitor may vary depending on the route of administration and the dosage form. The exact dosage is selected by the physician in view of the condition of the patient to be treated. The dosage and administration are adjusted to provide a sufficient level of active moiety or to maintain the desired effect. The specific dosage can be adjusted according to the disease condition, age, body weight, general health condition, sex and diet of the subject, dosage interval, administration route, excretion rate and drug combination. Depending on the half-life and clearance rate of a particular formulation, a sustained action pharmaceutical composition may be administered repeatedly at intervals, such as every 3-4 days, weekly, or biweekly (twice monthly). Guidance on specific dosages and delivery methods is provided in publications known in the art. It is well within the scope of routine experimentation to include an effective amount of any of the above dosage forms and, therefore, well within the scope of the present invention.

The invention also provides kits for carrying out the methods described herein. The kit may further comprise one or more reagents, buffers, media, proteins, analytes, labels, cells, computer programs for analyzing the results and/or disposable laboratory equipment, such as petri dishes or multi-well plates, in order to facilitate the performance of the method. Solid supports may include beads, petri dishes, multi-well plates, and the like.

The following examples are intended to illustrate, but not limit, the present invention.

Example 1

Rat thrombin-induced facet arthritis model

In this study, a rat model of thrombin-induced facet arthritis was developed. Thrombin is an essential protein of the coagulation cascade, but it also has the ability to cleave PG and produce fragments of fibronectin and other matrix components. Thrombin has also been shown to stimulate cytokines and proteases. Injection of thrombin induces cartilage degradation both due to PG loss by direct enzymatic action and damage due to matrix fragment driven inflammatory processes. Importantly, this rat facet arthritis model has been shown to be associated with sensorimotor dysfunction, allodynia, and gait changes.

80 female Sprague-Dawley rats (11 weeks, 220 g) were used in this study. Under general anesthesia, the right L4/5 facet joint was carefully exposed without damaging the joint capsule. To induce facet joint pain and test the efficacy of the therapeutic agents, bovine thrombin (20U/2 μ L saline) (thrombin group) or thrombin + dexamethasone (dexamethasone group) (20U/5 μ G/2 μ L saline) or thrombin + chimera bait (chimera group) (20U/10 μ G/2 μ L saline) was slowly injected into the facet joint space using an MS05 (5 μ L) syringe (Ito Corporation, Japan) with an 33/28G double gauge needle. Half of the rats were sacrificed on day 10 (D10) and the other half at 4 weeks (4W).

All data are expressed as mean ± Standard Error (SE). Statistical analysis of the data was performed using two-way repeated analysis of variance (ANOVA) for gross analysis or using one-way ANOVA for comparison at each time point. Pearson correlation coefficients were used to evaluate the relationship between von frey test results and IHC staining. P values less than 0.05 were considered statistically different.

Example 2

Behavioral assessment of mechanical allodynia

It is believed that facet joint damage can lead to changes in DRG and/or spinal function, leading to sensitization and events that underlie the promotion of the state of pain management. Mechanical allodynia in rats was assessed using a 50% paw withdrawal threshold response to mechanical stimulation by von frey capillary filaments in both the injected and contralateral hindpaws [8 ]. Von frey hairs were presented perpendicular to the face with sufficient force to cause slight flexion of the paw, and held for approximately 6-8 seconds (sec). Paw withdrawal and withdrawal were considered positive reactions. The results of the von frey test on the injection side were used for analysis.

(FIG. 1):

two-way repeated ANOVA revealed a significant effect on time and treatment. Although the paw withdrawal thresholds were significantly reduced for all groups until day 10 after surgery (P <0.01), values for the chimera and dexamethasone groups were significantly higher than those for the thrombin group (P < 0.01). On day 10, paw withdrawal thresholds were significantly higher for both dexamethasone (11.83 g) and chimera (11.91 g) groups than for thrombin group (8.60 g) (P < 0.01). These significant differences were observed at most time points from day 10 to day 28 (P <0.05, one-way ANOVA).

To evaluate the thermal nociceptive response, a radiant heat source was focused on the toe surface of the hind paw. The time (PWL) from the start of the radiant heat until the paw is retracted was measured. Rats were acclimated for at least 3 consecutive days before starting the experiment (baseline behavioral testing). The behavioral testing was performed in a quiet dedicated room at the same time each day. Each paw was tested 4 times, alternating between paws, with at least 1 minute intervals between tests. The interval between two tests on the same paw is at least 5 minutes. A significant reduction in PWL is defined as thermal hyperalgesia. A cut-off value of 20 seconds was used to avoid tissue damage.

Example 3

General conditions and activities

To assess the general condition of the rats, changes in body weight were analyzed. To assess changes in general activity caused by facet joint pain, a commercially available device consisting of a camera and behavioral analysis software (homeage can, Clever Sys Inc, Reston, VA) was used before and 1 week after surgery (preoperative and postoperative).

The camera is used for shooting gait films, and a mirror system is used for shooting the position of each foot when the rat walks. Rats were acclimated to the device for one week prior to starting the project. During this time, they were trained to walk on a moving treadmill for 20 seconds at a constant speed of 15 cm/sec. A high speed digital camera was placed under the treadmill to monitor the movement of the rat and recorded at a rate of 100 frames/sec. Once the rats were habituated to the treadmill, they walked at 15 cm/sec for 20 seconds, resting for 60 seconds between the duration of the study. The results were analyzed using Clever sys. inc. software to see over 35 gait measurements. The system has been validated in the evaluation of gait abnormalities in various disease states, including the OA rat model.

(FIGS. 2A and 2B):

on day 10, the weight gain (+9.66%) of the chimera group was significantly higher than that of the thrombin group (6.72%, P < 0.05). At 1 week post-surgery, the distance traveled was significantly reduced for all groups (P < 0.01). The relative distance traveled (post/pre) of the chimera group was significantly higher than the thrombin group (0.84 versus 0.73, P < 0.05).

Example 4

Immunohistochemistry for DRG

After sacrifice, at least 6 rats in each group were analyzed for ionic calcium binding adaptor-1 (Iba-1; a microglia/macrophage specific calcium binding protein) and calcitonin gene-related peptide (CGRP; a pain-related neuropeptide) expression in DRG neurons. Semi-quantitative analysis was performed as previously published [9 ]. The correlation between von frey test results and IHC staining was also evaluated.

(FIGS. 3A and 3B): iba-1: iba-1 positive microglia/mm in chimera group at 4 week time point²The mean number of (C) was significantly lower than that of the thrombin group (-33%, P)<0.05). No significant difference was observed on day 10. CGRP: on day 10, the average percentage of CGRP positive neurons in the dexamethasone and chimera groups was significantly lower than in the thrombin group (14.3%, P, respectively)<0.05, -22.9%, P<0.01). At 4 weeks, these differences were maintained. Importantly, the IHC data for Iba-and CGRP are inversely related to the von Frey test results (P)<0.05, see fig. 3B).

Example 5

Chimeric baits to regulate degeneration of human intervertebral disc cells

A study was conducted to assess whether chimeric baits could modulate Proteoglycan (PG) turnover in human intervertebral disc cells stimulated by interleukin-1.

As previously reported [11]]Human Nucleus Pulposus (NP) cells from a scoliotic spine of a 16 year old patient were expanded in monolayer culture, passaged and embedded in alginate beads. The beads were cultured for 4 weeks to obtain a chondrocyte phenotype. Then use³⁵S Probeled proteoglycans of human NP cells embedded in alginate beads, washed and further cultured for up to 6 days in the presence of IL-1 β (5 ng/ml) and in the presence/absence of either chimera bait (10. mu.M) or NF- κ B bait. Measurement in collected Medium (days 2, 4 and 6)³⁵S-PG and remaining in beads after 6 days incubation period³⁵S-PG, and calculate the total³⁵The percentage of residual PG of S-PG (media + beads) to reveal the extent of PG degradation.

The effect of chimeric baits on the rate of proteoglycan loss from beads was studied using a pulse-chase procedure. As shown in figure 4, treatment with IL-1 β significantly accelerated PG turnover of alginate beads (IL-1 β, 49%, day 6 control 72%, P <0.05, two-way ANOVA). On the other hand, the chimera decoy reversed the effect of IL-1 β (chimera + IL 1: 76% at day 6, P <0.05) and showed no significant difference compared to the control group. Although the NF-. kappa.B decoy (10. mu.M) group showed similar trends (NF-. kappa.B + IL-1 decoy; 69% on day 6), the differences did not reach a statistically significant level.

The results show that chimeric baits counteract the acceleration of catabolism in human intervertebral disc cells stimulated with IL-1. This suggests that chimeric baits may delay or reverse disc degeneration and thus reduce pain in patients with disc degeneration.

The objective of this study was to investigate the effect of chimeric decoy ODNs on PG degradation in human Annulus Fibrosis (AF) tissue from patients undergoing discectomy.

Example 6

Effect of chimeric decoy oligodeoxynucleotides on degradation of proteoglycans in human fibrous Ring tissues

Materials and methods:

AF tissue was taken from 7 patients undergoing lumbar surgery [67 ± 9 years; 6 men, 1 woman ]. The tissue was washed, cut into 3 mm pieces and incubated as explant cultures (3-5 pieces per group).

PG turnaround

AF tissue was treated with 20. mu. Ci/ml as described previously³⁵S pre-mark for 16 hours. Explants were cultured in DMEM/F-12 medium containing 20% FBS for up to 6 days in the presence or absence of NFkB decoy ODN (10 μ M) or chimera decoy ODN (10 μ M). The medium was replaced every other day with the same treatment medium and the medium was collected. At the end of the incubation period the tissue was collected and digested with papain. In culture medium and digests³⁵The amount of S-PG was measured by a rapid Alcian (Alcian) blue filtration assay. The total amount of synthesis was evaluated³⁵Residual in S-PG³⁵And (4) S-PG. Data were also normalized by the control group of each patient.

Statistical analysis:

two-way ANOVA with Fisher PSLD test was used as post hoc test. Results are expressed as mean ± sem.

As a result:

the control level of PG loss was significantly different in these 7 patients (table 1, P < 0.01). PG degradation was significantly inhibited in the chimeric decoy ODN group compared to the control group (p = 0.025). (control, NFkB, chimera; day 2: 1.00. + -. 0.01, 1.00. + -. 0.02, 1.04. + -. 0.02, day 4: 1.00. + -. 0.02, 1.04. + -. 0.05, 1.15. + -. 0.07, day 6: 1.00. + -. 0.05, 1.18. + -. 0.12, 1.34. + -. 0.13) (FIG. 5). After 6 days of culture, the chimera group retained 34% more proteoglycan compared to the control. PG loss was not significantly different between the NFkB decoy group and the other groups.

And (4) conclusion:

addition of chimeric decoy ODN to human AF explant cultures from degenerated discs significantly inhibited PG loss compared to the control group. NFkB baits that inhibit only the NFkB pathway showed a tendency to inhibit PG loss, but were not significant in this experiment.

TABLE 1 residual PG on day 6 (percentage of marked PG)

[ Table 1]

Example 7

Efficacy of chimeric baits on disc degeneration in rabbit circular puncture model

In this model, degenerated Nucleus Pulposus (NP) from rabbits were transplanted as xenograft tissues onto the Dorsal Root Ganglia (DRG) of RNU nude rats to determine whether these tissues would induce functional and sensory dysfunction.

The specific objective of this experiment was to determine whether (1) intra-discal injection of chimeric baits inhibited the overexpression of pro-inflammatory cytokines and matrix degrading enzymes, and (2) intra-discal injection into rabbit intervertebral discs using different doses of chimeric baits reduced degenerative disc tissue-induced pain in nude rats.

The method comprises the following steps:

rabbit puncture disc degeneration model and injection of chimeric bait: female new zealand white rabbits (n = 80) were used in this study. Under general anesthesia, the annulus fibrosus was punctured with an 18 gauge needle in two non-continuous intervertebral discs (L2/3 and L4/5). 4 weeks after the initial puncture, the center of Nucleus Pulposus (NP) was injected with vehicle phosphate buffered saline (10. mu.l), chimeric bait (10, 100. mu.g in 10. mu.l saline) or NF κ B bait (100. mu.g in 10. mu.l saline) using a 26 gauge needle.

Radiographic analysis of disc height:

disc height (IVD) was obtained from lateral radiography and is expressed as Disc Height Index (DHI). DHI was normalized to preoperative DHI (% DHI) and further normalized to DHI of the L3/4 non-punctured disc.

MRI analysis:

the degradation level of IVD is classified according to Pfirrman using T2 weighted images.

As a result:

DHI (fig. 6):

repeated two-way ANOVA revealed that treatment significantly affected% DHI (P < 0.05). Post hoc analysis showed that the% DHI of the chimera 100 ug group was significantly higher than the PBS group. At week 16, the chimera 100 ug and decoy 100 ug groups showed a significant increase in% DHI compared to the PBS group (p <0.01 and p <0.05, respectively).

MRI T2 spin echo weighted images (fig. 7) and Pfirrmann scale (fig. 8).

The Pfirrmann rating of the chimera 100 μ g group tended to be lower compared to the PBS group (P < 0.088).

Discussion:

injection of chimeric bait into NPs 4 weeks after circular puncture restored disc height and slightly improved MRI grade. The data indicate that chimeric bait injection can induce structural changes at 100 μ g.

Injection of the chimeras into the intervertebral disc reduced pain production in the xenograft rat model of radiculopathy. These results indicate that injection of chimeric baits alters the pathological state of degenerated intervertebral discs and reduces the development of pain, and thus can be used as a new treatment for degenerative disc diseases.

Although the present invention has been described with reference to the above examples, it should be understood that modifications and variations are included within the spirit and scope of the present invention. Accordingly, the invention is not limited except as by the following claims.

Reference to the literature

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[11] Aerota, et al, Differential effects of fibrous fragments on fibrous media cells, a complex with particulate chlorine units, Spine (Phila Pa 1976. 2005;30(7): 722) 728.

[12] WO 2017/043639 A1。