WO2019176901A1 - Soulagement de la douleur de l'arthrose par ciblage de micro-arn -21 - Google Patents

Soulagement de la douleur de l'arthrose par ciblage de micro-arn -21 Download PDF

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WO2019176901A1
WO2019176901A1 PCT/JP2019/009867 JP2019009867W WO2019176901A1 WO 2019176901 A1 WO2019176901 A1 WO 2019176901A1 JP 2019009867 W JP2019009867 W JP 2019009867W WO 2019176901 A1 WO2019176901 A1 WO 2019176901A1
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mir
pain
osteoarthritis
inhibitor
tlr7
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Japanese (ja)
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直哉 星川
敦 坂井
鈴木　秀典
信朗 高井
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学校法人日本医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a pain relieving drug for osteoarthritis.
  • Osteoarthritis is a disease with a high prevalence and chronic progression. According to the 2013 National Life Basic Survey, joint pain is the number one cause of the need for nursing care for those who need support in nursing care insurance. ing. Non-steroidal anti-inflammatory drugs, acetaminophen, opioids, etc. are used as pharmacotherapy for pain relief of osteoarthritis, but surgical treatment such as artificial joint replacement is necessary due to insufficient efficacy The number of cases is increasing. In addition, many of existing pharmacotherapy have acute adverse effects, and there are also adverse effects associated with chronic use, so the development of new analgesics is desired. The cause of pain in osteoarthritis is still unclear, and it is necessary to investigate the cause of pain in osteoarthritis in order to develop new analgesics.
  • TLR7 Toll-like receptor 7 in the cell.
  • DRG small-diameter dorsal root ganglion neurons (sensory nerves) that are involved in sensory functions such as pain, and is known to be involved in pain control.
  • Patent Document 1 (US 9267139 B2) exemplifies osteoarthritis as an example of a musculoskeletal disease, and discloses a method for reducing or controlling miR-29b-1 expression by focusing on miRNA as a treatment method.
  • BMSCs bone marrow-derived mesenchymal stem cells
  • Patent Document 2 discloses a method for treating cartilage degenerative diseases such as osteoarthritis and intervertebral disc degeneration by controlling the expression of miR-140. It is not a therapeutic drug for osteoarthritis pain, but treats osteoarthritis by controlling cartilage differentiation. There are many reports that cartilage degeneration does not correlate with pain, which is fundamentally different from the present invention.
  • Patent Document 3 Japanese Patent Publication No. 2012-504651 relates to a method for treating an inflammatory condition by suppressing the activity of TLR3, and osteoarthritis is listed as one aspect thereof. They are just listed.
  • Patent Document 4 Japanese Patent Publication No. 2011-502170 uses a TLR4 antagonist for the treatment or prevention of osteoarthritis disease.
  • TLR4 When TLR4 is activated, the release of inflammatory cytokines increases, and this is an invention that is linked to the onset of osteoarthritis disease by an inflammatory reaction, but TLR7, 8, and 9 are not focused.
  • Patent Document 5 Japanese Patent Publication No. 2016-512511 is an invention that focuses on TLR4 antagonists to reduce pain. Osteoarthritis is mentioned as the target disease, but TLR7, 8, and 9 are not focused. In addition, tests are conducted for lower back pain, vulva pain, trigeminal neuralgia, migraine, etc., but no test is performed for osteoarthritis.
  • the TLR antagonist is completely different if the type of receptor and the site of action are different.
  • the purpose of the present invention is to clarify the cause of pain in osteoarthritis and to contribute to the development of a therapeutic agent that relieves pain in osteoarthritis.
  • microRNA which is considered to be related to pain
  • microRNA-21 (miR-21) was found to be involved in osteoarthritis pain.
  • the present invention is as follows. [1] A pain relieving drug for osteoarthritis containing an miR-21 inhibitor as an active ingredient. [2] The pain relieving drug for osteoarthritis according to [1], wherein the miR-21 inhibitor binds to miR-21 in joint fluid and inhibits its function. [3] The drug for alleviating pain caused by osteoarthritis according to [1] or [2], wherein the miR-21 inhibitor is a nucleic acid that binds to miR-21 and inhibits its function. [4] Any of [1] to [3], wherein the miR-21 inhibitor is a nucleic acid comprising a sequence that is complementary to the entire base sequence represented by SEQ ID NO: 1 or a partial sequence that continues.
  • a pain relieving drug for osteoarthritis containing a TLR antagonist as an active ingredient.
  • a pain relieving drug for osteoarthritis containing the miR-21 inhibitor of any one of [1] to [4] as an active ingredient and a TLR antagonist of any of [5] to [9] A pain relieving drug for osteoarthritis in combination with a pain relieving drug for osteoarthritis, which is included as a component.
  • FIG. 6A shows the result of PAM
  • FIG. 6B shows the result of von Frey test.
  • FIG. 7A shows the result of PAM
  • FIG. 7B shows the result of von Frey test.
  • FIG. 8A shows the result of PAM
  • FIG. 8A shows the result of PAM
  • FIG. 8B shows the result of von Frey test. It is a figure which shows the progress (A) of PAM and the progress (B) of von Frey test until 56 days after ACLT operation in an osteoarthritis animal model. It is a figure which shows the expression of miR-21 by an osteoarthritis animal model.
  • FIG. 10A shows the expression level in the synovium
  • FIG. 10B shows the expression level in the synovial fluid. It is a figure which shows the result of confirmation of a miR-21 discharge
  • the present invention is a pain relieving drug for osteoarthritis, comprising an miR-21 inhibitor or a TLR 7, 8 and 9 antagonist as an active ingredient.
  • Osteoarthritis is a joint disease with chronic arthritis, causing synovitis and causing pain and swelling in the joint. Osteoarthritis develops in knee joints, hip joints, ankle joints, toe joints, shoulder joints, elbow joints, wrist joints, finger joints, spinal intervertebral joints, and the like.
  • miR-21 is increased in synovial membrane and synovial fluid in osteoarthritis model animals. miR-21 is thought to be upregulated in synovial cells, and the expressed miR-21 is released to the outside of the cell and acts on other cells, which may contribute to osteoarthritis pain. Therefore, in osteoarthritis pain patients, the pain can be alleviated by inhibiting the activity of miR-21.
  • TLR7 is expressed in small-diameter dorsal root ganglion (DRG) neurons (sensory nerves) that are involved in sensory functions such as pain, and is known to be involved in pain control.
  • DRG small-diameter dorsal root ganglion
  • MicroRNA released outside the cell may bind to intracellular TLR7 (Toll-like receptor-7) and induce pain, and TLR7 antagonists can relieve pain in pain patients .
  • the present invention is a pain relieving drug for osteoarthritis containing a miR-21 inhibitor as an active ingredient, and a pain relieving drug for osteoarthritis containing a TLR7, 8, and 9 antagonist as an active ingredient It is.
  • the present invention is a pain relieving drug for osteoarthritis comprising a combination of a miR-21 inhibitor and a TLR7, 8, or 9 antagonist.
  • miR-21 inhibitors include those that inhibit miR-21 expression and those that inhibit miR-21 silencing function. It can inhibit the activity and function of miR-21. miR-21 inhibitors are also referred to as miR-21 activity inhibitors.
  • MiR-21 (has-miR-21-5p) consists of a 22-base sequence represented by UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 1).
  • a nucleic acid consisting of a sequence containing a sequence complementary to the sequence represented by SEQ ID NO: 1 can bind to miR-21 and inhibit the activity of miR-21.
  • a nucleic acid consisting of a sequence containing a sequence complementary to the sequence represented by SEQ ID NO: 1 has Micro RNA Binding Site (MBS), and the MBS portion binds to miR-21. That is, the miR-21 inhibitor includes a nucleic acid consisting of a sequence comprising a sequence complementary to the entire base sequence represented by SEQ ID NO: 1 or a partial sequence that is continuous.
  • MBS Micro RNA Binding Site
  • the nucleic acid is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 97% of the sequence represented by SEQ ID NO: 1. It has a complementarity of 98%, more preferably at least 99%, particularly preferably 100%.
  • the base length of the nucleic acid complementary to the miRNA sequence is 5 to 50 bases, preferably 6 to 24 bases, more preferably 8 to 22 bases.
  • a sequence that is 100% complementary to the sequence represented by SEQ ID NO: 1 is represented by UCAACAUCAGUCUGAUAAGCUA (SEQ ID NO: 2).
  • the nucleic acid may be DNA or RNA.
  • the nucleotides constituting the nucleic acid may be natural nucleotides, modified nucleotides, artificial nucleotides, or a combination thereof.
  • the miR-21 inhibitor is preferably modified to improve the thermal stability of the duplex complex of nucleic acid and miR-21 that is the inhibitor.
  • Modified examples include nucleic acids in which some or all of the bases are modified, for example, at least one may be modified, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the bases may be modified.
  • Modified bases include 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, or 5-iodocytosine; 5-fluorouracil, 5-bromouracil, 5-iodouracil, or 5-hydroxyuracil; 2-thiothymine N6-methyladenine or 8-bromoadenine; and N2-methylguanine or 8-bromoguanine.
  • modified substance examples include a nucleic acid in which a part or all of the sugar moiety is modified, for example, at least one of which may be modified, and is at least 10%, at least 20%, at least 30%, at least 40%. %, At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the bases may be modified.
  • modified sugars include, for example, bicyclic sugars, 5′-vinyl, 5′-methyl, 4′-S, 2′-F, 2′-OCH 3 (2′-methoxy or 2′-methoxy). O-methyl group), and 2′-O (CH 2 ) 2 OCH 3 substituents.
  • Nucleic acids containing bicyclic sugar moieties are referred to as bridged nucleotides (BNA), and as such nucleic acids, sugars with a methyleneoxy (4'-CH 2 -O-2 ') bridge (LNA TM ), 2 ', 4'-BNA ), ethyleneoxy (4' - (CH 2) 2 -O-2 ') a sugar having a crosslinked (ENA), 4'-CH ( CH 3) -O-2' bridge Sugar (cEt, constrained ethyl), sugar having 4′-CH (CH 2 OCH 3 ) -O-2 ′ bridge (cMOE, constrained MOE), sugar having amide bridge (AmNA, Amido-bridged nucleic acid) Etc.
  • BNA bridged nucleotides
  • some or all of the phosphodiester bonds of the nucleic acid may be modified, for example, at least one may be modified, at least 10%, at least 20%, at least 30%, at least 40% , At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the bonds may be modified.
  • Such bond modifications include phosphorothioate bonds, phosphorodithioate bonds, phosphorodiamidate bonds, phosphoramidate bonds, and the like. These modified bonds are more nuclease resistant than naturally occurring bonds.
  • the nucleic acid may include a nucleotide mimic such as a peptide nucleic acid or a morpholino nucleic acid.
  • the nucleic acid acting as a miR-21 inhibitor may contain at least one modified nucleotide as described above. For example, it may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% modified nucleotides.
  • Modified nucleotides can include modified phosphodiester bonds, modified sugar moieties, and / or modified nucleobases.
  • At least one of the phosphodiester bonds of the nucleic acid that acts as an miR-21 inhibitor may be modified and is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% , At least 70%, at least 80%, at least 90%, or 100% of the bonds may be modified.
  • At least one sugar moiety of the nucleic acid that acts as an miR-21 inhibitor may be modified. For example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% sugar moieties may be modified Good.
  • At least one of the nucleic acid bases acting as miR-21 inhibitors may be a modified nucleobase. For example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the bases may be modified. .
  • the nucleic acid that acts as an miR-21 inhibitor may have a single-stranded structure or a double-stranded structure.
  • nucleic acids that act as miR-21 inhibitors include S-TuD (Stud) (Synthetic Tough Decoy), IDT (registered trademark) miRNA inhibitor.
  • S-TuD is a double-stranded RNA composed mainly of 2'-O-Methyl RNA.
  • Micro-RNA Binding Site MMS specifically binds to the mature miRNA complexed with RISC and miRNA activity Strongly inhibits even at low concentrations.
  • S-TuD is described in WO2017 / 047097, WO2010 / 047216, and the like. S-Tud can be obtained from Gene Design Co., Ltd.
  • IDT (registered trademark) miRNA inhibitor is an oligonucleotide having a special modification of 2-O-Met and ZEN, and is described in Lennox KA, et al., Mol Ther Nucleic Acids. 2, e117 (2013) .
  • IDT (registered trademark) miRNA inhibitor can be obtained from Integrated DNA Technologies.
  • the above-described oligonucleotide miR-21 activity inhibitor can be synthesized by a known chemical synthesis method or enzymatic transcription method.
  • the chemical synthesis method include a phosphorothioate method, a phosphoramidite method, and a phosphotriester method.
  • the enzymatic transcription method include a method using RNA polymerase.
  • TLR antagonists act on TLR7, TLR8, or TLR9 and inhibit these effects. Therefore, as a TLR antagonist, a compound known to inhibit the action of any of TLR7, TLR8 and TLR9 can be used.
  • TLR7 and TLR8 use low molecular weight compounds such as imidazoquinoline and single-stranded RNA as ligands, and TLR9 uses unmethylated CpGDNA as a ligand. Inhibits binding and acts as a TLR antagonist.
  • a TLR antagonist is also called a TLR antagonist or TLR inhibitor.
  • TLR8 antagonists are preferable.
  • single-stranded RNA of TLR is recognized by TLR7 in rodents, but is recognized by TLR8 in humans.
  • TLR8 There are many reports that support this for miR-21 among single-stranded RNAs (Heil F et al. Science. 2004; 303 (5663), 1526-1529: M. Fabbri et al., RNA Biology 10: 2, 169 -174; February 2013: X. Chen et al., RNA (2013), 19: 737-739: M. Fabbri et al., PNAS, E2110-E2116, published online July 2, 2012). Therefore, it is clear that TLR8 antagonists have a pain relieving action in humans.
  • TLR antagonists are, for example, Barrat FJ et al., J Exp Med. 2005; 202 (8), 1131-1139: Hennessy EJ et al., Nat Rev Drug Discov. 2010; 9 (4), 293-307. : Wu YW et al., Acta Pharmacol Sin. 2015; 36 (12): 1395-1407 .: Ferreira TP et al., Front Immunol. 2016; 7,95.
  • TLR antagonists examples include IRS-954, DV1179, IMO-3100, IMO-8400, INH-ODN-24888, VTX-763, TMX-302, IMO-92003, hydroxychloroquine, CPG-523644, and the like.
  • polynucleotides that bind to TLR and reduce TLR signaling examples include the following polynucleotides.
  • TLR binds to DNA having a CpG motif to form a complex and promotes signaling.
  • a polynucleotide that reduces TLR signaling binds to TLR more strongly than DNA having a CpG motif and inhibits activation of TLR.
  • These polynucleotides are sometimes referred to as inhibitory oligodeoxynucleotides (iODN).
  • the nucleotide in the above polynucleotide may be a natural nucleotide, a modified nucleotide, an artificial nucleotide, or a combination thereof.
  • Modified nucleotides include those described above.
  • part or all of the phosphodiester bond of the nucleic acid may be modified. Examples of such modifications include phosphorothioate bond, phosphorodithioate bond, phosphorodiamidate bond, and phosphoramidate bond. Is mentioned.
  • low molecular weight compounds such as chloroquine (4-aminoquinoline), hydroxychloroquine, quinacrine (9-aminoacridine), quinazoline derivatives, cyclohexene derivatives, and the like can be given.
  • Examples of such compounds include DV1179, IMO-3100, IMO-8400, VTX-763, TMX-302, IMO-92003, hydroxychloroquine, and compounds below CPG-523644.
  • a miR-21 inhibitor and a TLR antagonist will be used in combination to alleviate pain due to osteoarthritis, and to produce a synergistic effect.
  • at least one miR-21 inhibitor and at least one TLR antagonist may be used in combination.
  • the present invention includes a combination preparation or a combination kit for alleviating pain due to osteoarthritis, which combines a miR-21 inhibitor and a TLR antagonist.
  • the present invention includes a pain relieving drug for osteoarthritis comprising a miR-21 inhibitor used in combination with a TLR antagonist, and a degenerative joint comprising a TLR antagonist used in combination with a miR-21 inhibitor Includes palliative pain relief drugs.
  • the miR-21 inhibitor and the TLR antagonist may be administered simultaneously or separately. Moreover, you may administer sequentially, and the order of administration may be the miR-21 inhibitor first and the TLR antagonist first.
  • the route of administration of the pain relieving drug for osteoarthritis including the miR-21 activity inhibitor or TLR antagonist of the present invention is not limited, and oral, inhalation, injection (intravenous injection) as long as the relieving drug can reach the joint , Subcutaneous injection, intradermal injection, intramuscular injection, intraarticular injection), transdermal (external use), nasal, transmucosal, intranasal, intratracheal and the like. Among these, administration by injection into the joints of osteoarthritis patients is preferable. Intra-articular administration refers to administration into the joint cavity.
  • the dosage form of the pain relieving drug for osteoarthritis including the miR-21 activity inhibitor or TLR antagonist of the present invention is not limited, but is preferably a solution, emulsion, suspension, solution, injection, tablet Granules, sprays, capsules, syrups, suppositories, ointments, tapes and the like.
  • the pain relieving agent for osteoarthritis of the present invention is a pharmacologically acceptable carrier, diluent, excipient, binder, disintegrant, lubricant, flavoring agent, ordinarily used in the pharmaceutical field.
  • Coloring agents sweetening agents, flavoring agents, suspending agents, wetting agents, emulsifying agents, dispersing agents, auxiliary agents, preservatives, buffering agents, stabilizers, coating agents and the like may be included.
  • the carrier and excipient include lactose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate, gum arabic and the like
  • binders include polyvinyl alcohol, polyvinyl ether, Examples include ethyl cellulose, gum arabic, shellac, and sucrose.
  • aqueous solution for injection isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants. You may use together.
  • suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants. You may use together.
  • oily liquid sesame oil, soybean oil and the like are used, and as a solubilizing agent, benzyl benzoate, benzyl alcohol and the like may be used in combination.
  • the content of miR-21 activity inhibitor or TLR antagonist, which is an active ingredient in a pain relieving drug for osteoarthritis including a carrier, etc. depends on the dosage form, but is usually about 1 to 50% by weight. is there.
  • the therapeutic agent for pain relief due to osteoarthritis including the miR-21 activity inhibitor or TLR antagonist of the present invention may be administered in a therapeutically effective amount.
  • the therapeutically effective amount can be appropriately changed depending on the severity of the patient's disease state, sex, age, weight, and the like.
  • a dose of about 1 ⁇ g to about 2000 mg, more preferably about 10 ⁇ g to about 1000 mg, more preferably about 100 ⁇ g to about 500 mg per kg body weight of a patient can be administered in a single administration.
  • TLR antagonists about 0.1 ⁇ g to about 2000 mg, preferably about 1 ⁇ g to about 1000 mg, more preferably about 10 ⁇ g to about 500 mg per kg body weight of the patient may be administered in a single administration.
  • the administration can be divided into 1 to several times a day, for example, 4 to 6 times, and may be intermittently administered once every several days or weeks.
  • the pain relieving drug for osteoarthritis of the present invention can be used for alleviating pain due to osteoarthritis in any animal.
  • it can be suitably used for alleviating pain caused by osteoarthritis in mammals such as humans, monkeys, cows, sheep, horses, dogs, and cats.
  • microRNAs are positively released outside the cell and affect nearby cells in addition to the conventionally known intracellular translational inhibitory effect. Therefore, it was hypothesized that a part of the microRNA was released into the joint cavity and acted on other cells, thereby contributing to osteoarthritis pain.
  • Targeting extracellular microRNA in the joint cavity not only avoids systemic effects by local administration of nucleic acid drugs, but also does not require intracellular translocation of nucleic acids, so it is effective from the viewpoint of drug delivery.
  • the possibility of verification is high. Therefore, development as a next-generation drug is also expected.
  • microRNA-21 increased in the quantitative polymerase chain reaction (quantitative PCR) of synovial membrane and synovial fluid. Confirmed that.
  • TLR7 is expressed in small-diameter dorsal root ganglion (DRG) neurons (sensory nerves) that are involved in sensory functions such as pain, and is known to be involved in pain control.
  • DRG dorsal root ganglion
  • Example 1 it was confirmed that this hyperalgesia was improved by simultaneously injecting a TLR antagonist together with miR-21.
  • TLR antagonists that can be used as pain relieving drugs.
  • the nucleic acid prepared as a TLR antagonist acts on both TLR7, 8, and 9, so if it is a substance that inhibits the functions of TLR7, 8, 9, it is expected to show the same effect .
  • IDT miRNA Inhibitor also known as a miR-21 inhibitor in Example 3 described later
  • the pain generated in ACLT model rats was similarly relieved for a long time.
  • miR-21 inhibitors There are various types of miR-21 inhibitors, but generally known ones can be used.
  • PAM Pressure Press Application Measurement
  • FIG. 1A shows the progress of PAM after ACLT
  • FIG. 1B shows the progress of von Frey test after ACLT.
  • the threshold of pain sensation was significantly decreased from 14 to 14 days after ACLT in PAM, and from 7 to 28 days after ACLT in von Frey test. I understood.
  • the miR-140 shown in International Publication No. 2013/151058 is unknown to either miR-140-3p or miR-140-5p, but both were elevated in the synovial membrane. According to WO 2013/151058, a decrease in miR-140 in cartilage causes osteoarthritis of the knee, which is a contradictory result. In addition, the expression level of any microRNA is extremely small compared to miR-21. From the above, it is unclear whether miR-29b-1 and miR-140 shown in US Pat. No. 9,267,139 and International Publication No. 2013/151058 are involved in pain in osteoarthritis.
  • FIG. 3 shows the expression level of miR-21 in synovial membrane (A) and synovial fluid (B).
  • miR-21 expression increases in synovial membrane and synovial fluid of osteoarthritis model rats. Therefore, if miR-21 expression can be suppressed, pain is expected to be alleviated.
  • TLR7, 8, and 9 are at least part of the mechanism of arthralgia caused by miR-21, and TLR7, 8, and 9 antagonists can suppress hyperalgesia caused by miR-21. It was.
  • Example 2 Administration experiment of miR-21 inhibitor to ACLT model rats (1) 14 days after ACLT surgery ACLT model rats were injected with S-TuD (obtained from Gene Design Co., Ltd.), which inhibits miR-21 function, into the knee joint cavity and evaluated for pain sensation. On the 7th, 14th, and 21st days after, in the von Frey test, the pain threshold on the 7th and 14th days after administration was significantly reduced.
  • FIG. 6A shows the PAM result
  • FIG. 6B shows the von Frey test result. After the onset of pain, it can be seen that once miR-21 inhibitor is injected into the joint once, it has a pain relieving effect continuously.
  • Example 3 Administration experiment of miR-21 inhibitor to ACLT model rats (2) IDT (registered trademark) miRNA Inhibitor (obtained from Integrated DNA Technologies (IDT)), a miR-21 inhibitor, was injected into the knee joint space of ACLT model rats on the 14th day after ACLT surgery, and PAM, von Frey test The pain sensation was evaluated. As with S-TuD, it can be seen that the pain threshold was reduced after administration.
  • FIG. 7A shows the PAM result
  • FIG. 7B shows the von Frey test result. Accordingly, it is predicted that the pain threshold value is mitigated for miR-21 inhibitors.
  • Example 4 Administration experiment of TLR antagonists to ACLT model rats TLR7, 8, 9 antagonists were administered by injection into the knee joint space of ACLT model rats 14 days after ACLT surgery to evaluate pain sensation As a result, the pain threshold was significantly reduced on days 7, 14, and 21 after administration in PAM, and on days 7 and 14 after administration in von Frey test.
  • FIG. 8A shows the PAM result
  • FIG. 8B shows the von Frey test result.
  • FIG. 9A shows the results of PAM after ACLT
  • FIG. 9B shows the progress of von Frey test after ACLT.
  • Reference Example 1 was data up to 28 days after ACLT, but further investigation was conducted until 56 days after ACLT.
  • PAM the threshold of pain was significantly reduced in the ACLT group from the 14th day to the 56th day after surgery.
  • the threshold value tended to be low in the ACLT group from the 35th day to the 56th day after surgery.
  • FIG. 13 (A) shows staining with DiI
  • FIG. 13 (B) shows immunochemical staining for TLR7
  • FIG. 13 (C) shows a stained image obtained by superimposing two of (A) and (B). Approximately 26.7% of the cells labeled with DiI expressed TLR7.
  • FIG. 14 is a fluorescent immunostained image of CD11b and TLR7, which are markers of macrophage-like synoviocytes (synovial A cells), and it was found that TLR7 is expressed in macrophage-like synoviocytes.
  • FIG. 15 is a fluorescent immunostained image of Thy-1 and TLR7, which are markers of fibroblast-like synoviocytes (synovial B cells), and that TLR7 is also expressed in fibroblast-like synoviocytes. all right. It was possible to show that the site of action of miR-21 is in the joint.
  • the VAS will mark the patient's pain level (related to cancer pain pharmacotherapy). Guidelines (2010 edition).
  • group (3) was more painful than the other groups.
  • the expression of miR-21 in the synovial fluid is increased in the (3) group compared to the (1) group and (2) group, and the (3) group is approximately about the (1) group. 50-fold expression was observed.
  • the expression of miR-21 in the synovium was increased about 1.5 times in the (3) group compared to the (2) group. In other words, miR-21 expression was increased in osteoarthritis with strong pain in both synovial fluid and synovium.
  • Example 5 Effect of TLR7, 8, 9 antagonist (evaluation by PAM)
  • PAM evaluation by PAM
  • the threshold of pain was significantly higher 1 hour after administration compared to the group administered miR-21 and the control drug.
  • Example 6 Effects of TLR7-specific antagonists Experiments similar to those in Examples 1 and 5 using TLR7, 8, and 9 antagonists were conducted using TLR7-specific antagonists. In the group administered with TLR7-specific antagonist at the same time as miR-21, the pain threshold was significantly higher in both the PAM and von Frey tests at 1 hour after administration than in the group administered miR-21 and the control drug. it was high.
  • FIG. 18 shows the result of PAM
  • FIG. 19 shows the result of von Frey test.
  • TLR7-specific antagonist As clarified in Heil F et al. Science. 2004; 303 (5663), 1526-1529, single-stranded RNA of TLR is recognized by TLR7 in rodents, but is recognized by TLR8 in humans. Therefore, from the results of the present invention, it is considered that a TLR8 antagonist has a pain relieving action in humans.
  • the present invention can be used to relieve pain due to osteoarthritis.

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Abstract

Un objectif de la présente invention est d'élucider la cause de la douleur de l'arthrose et de contribuer au développement d'un agent thérapeutique pour soulager la douleur de l'arthrose. L'invention concerne également un agent pour soulager la douleur de l'arthrose, l'agent comprenant un inhibiteur de MiR-21 en tant que principe actif, ou un agent pour soulager la douleur de l'arthrose, l'agent comprenant un antagoniste de TLR en tant que principe actif.
PCT/JP2019/009867 2018-03-12 2019-03-12 Soulagement de la douleur de l'arthrose par ciblage de micro-arn -21 WO2019176901A1 (fr)

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Citations (3)

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JP2011502170A (ja) * 2007-10-31 2011-01-20 セントコア・オーソ・バイオテツク・インコーポレーテツド 変形性関節症疾患の治療又は予防のためのトール様受容体４拮抗物質の使用
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TANAKA, SHIHO ET AL.: "Comparison of miRNA expressions in osteoarthritis and rheumatoid arthritis - Examination in synovial tissues", PROGRAMS AND ABSTRACTS OF THE 29TH MEETING OF THE JAPANESE SOCIETY OF CARTILAGE METABOLISM, vol. 29, 2016, pages 105 *
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