Oligonucleotide for inhibiting TRPV1 expression, composition and application

Abstract

The invention discloses oligonucleotide for inhibiting TRPV1 expression, a composition and application. In particular, the oligonucleotides and compositions disclosed herein can be used to treat, prevent and / or ameliorate TRPV1-related diseases, including pain, cardiovascular diseases, mental diseases, diabetes, and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to oligonucleotides and compositions for inhibiting TRPV1 expression, as well as their use in treating, preventing and / or improving TRPV1-related diseases, such as pain, cardiovascular disease, mental illness, diabetes, and neuroprotection. Background Art

[0002] Antisense oligonucleotide (ASO) drugs are a class of artificially synthesized single-stranded oligonucleotides with a length of 14-30 nt. They inhibit the expression of target genes by binding to the target gene mRNA in a sequence-specific manner, and are molecular drugs that regulate at the gene level. With the continuous advancement of modification technology and delivery systems, ASO drugs are expected to cover a wider range of indications and replace some existing therapies, and the potential market size is very broad.

[0003] Transient Receptor Potential Vanilloid 1 (TRPV1), also known as transient receptor potential vanilloid subtype 1 or vanilloid receptor-1 (VR1), is the first member of the transient receptor potential (TRP) superfamily found to exist in mammals. It is mainly expressed in the terminals of small and medium-sized sensory neurons in the central and peripheral regions, and is also found in different brain regions such as the cerebellum, hypothalamus, cerebral cortex, olfactory bulb, midbrain, hippocampus, etc. Studies have shown that TRPV1 is related to the pathogenesis of many diseases, such as pain, inflammation, cardiovascular disease, cough, mental illness, diabetes, and neuroprotection.

[0004] In the case of tissue inflammation and nerve damage, TRPV1 on the nociceptors is sensitized by various pro-inflammatory and pro-pain factors, increasing the chance of channel opening and triggering pain hypersensitivity. This neurogenic inflammation is associated with a variety of acute or chronic pain conditions, such as arthritis, irritable bowel syndrome, pancreatitis, migraine, and cancer pain. TRPV1 is also involved in some itch pathways and works together with itch receptors. Summary of the invention

[0005] The present invention provides a compound comprising an antisense oligonucleotide that can specifically and effectively inhibit the expression of transient receptor potential vanilloid 1 (TRPV1), which can be used to treat, prevent and / or improve TRPV1-related diseases, such as pain, eye disease, chronic pain, cardiovascular disease, mental illness, diabetes and neuroprotection by knocking down TRPV1 mRNA levels and thereby reducing the expression of TRPV1 protein.

[0006] The antisense oligonucleotide of the invention can inhibit the expression of TRPV1 mRNA, and the inhibition level thereof varies from 1% to 99%, and the antisense oligonucleotide of the invention has no significant cytotoxicity.

[0007] In a first aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, comprising a single-stranded antisense oligonucleotide complementary to a TRPV1 nucleic acid or a TRPV1 homolog nucleic acid. In some embodiments, the compound consists of a single-stranded antisense oligonucleotide complementary to a TRPV1 nucleic acid or a TRPV1 homolog nucleic acid.

[0008] In some embodiments, the antisense oligonucleotide includes a portion complementary to a target nucleic acid. In some embodiments, the target nucleic acid is TRPV1 mRNA. In some embodiments, the target nucleic acid is TRPV1 pre-mRNA. In some embodiments, the target nucleic acid includes introns, exons, or untranslated regions. In some embodiments, the target nucleic acid is, for example, TRPV1 mRNA shown in NCBI accession numbers NM_080705.4, NM_080704.4, NM_080706.3, NM_018727.5.

[0009] In some embodiments, in any of the above-mentioned compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide can specifically hybridize to human TRPV1 nucleic acid.

[0010] In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to an equal length portion of human TRPV1 nucleic acid.

[0011] In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide is complementary to any of the following in human TRPV1 nucleic acid: the 15th exon of NM_080705.4, the 16th exon of NM_080704.4, the 14th exon of NM_080706.3, the 15th exon of NM_018727.5, the 16th exon of NM_080706.3, the 16th exon of NM_080707.4, the 16th exon of NM_080707.5 ... The 8th exon of NM_080705.4, the 9th exon of NM_080704.4, the 7th exon of NM_080706.3, the 8th exon of NM_018727.5, the 6th exon of NM_080705.4, the 7th exon of NM_080704.4, the 5th exon of NM_080706.3, and the 6th exon of NM_018727.5.

[0012] In some embodiments, in any of the above-mentioned compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide consists of 20 linked nucleosides, for example, the single-stranded antisense oligonucleotide consists of the nucleobase sequence shown in SEQ ID NO: 1-92.

[0013] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleobases and / or one or more modified internucleoside linkages.

[0014] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, the modified sugar is independently selected from 2'-MOE substituted ribose or 2'-deoxyribose.

[0015] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, each sugar in the single-stranded antisense oligonucleotide is a 2'-MOE-modified sugar or a 2'-deoxy sugar.

[0016] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, the modified nucleobase is 5-methylcytosine.

[0017] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, each cytosine in the single-stranded antisense oligonucleotide is 5-methylcytosine.

[0018] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, the internucleoside linkage is phosphorothioate.

[0019] In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, each internucleoside linkage of the single-stranded antisense oligonucleotide is a phosphorothioate.

[0020] In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide comprises, consists of or consists essentially of:

[0021] A gap segment consisting of linked 2'-deoxynucleosides;

[0022] a 5' wing segment consisting of linked nucleosides; and

[0023] a 3' wing segment consisting of linked nucleosides;

[0024] The 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, and the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment; the gap segment consists of 7 to 12 connected deoxynucleosides, and the 5' wing segment and the 3' wing segment consist of 2 to 7 connected ribonucleosides, respectively; and the nucleosides of the 5' wing segment and the 3' wing segment are both 2'-MOE nucleosides, all cytosines on the single-stranded antisense oligonucleotide are 5-methylcytosine, and all internucleoside linkages on the single-stranded antisense oligonucleotide are phosphorothioates.

[0025] In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide comprises, consists of or consists essentially of:

[0026] A gap segment consisting of 10 linked 2'-deoxynucleosides;

[0027] a 5' wing segment consisting of 5 linked nucleosides; and

[0028] A 3' wing segment consisting of 5 linked nucleosides;

[0029] The 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, and the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment; and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-MOE nucleosides, all cytosines on the single-stranded antisense oligonucleotide are 5-methylcytosine, and all internucleoside linkages on the single-stranded antisense oligonucleotide are phosphorothioates.

[0030] In some embodiments, the modified oligonucleotide comprises one or more ligands of N-acetylgalactosamine (GalNAc) derivatives. Preferably, the ligands of N-acetylgalactosamine (GalNAc) derivatives are attached to the oligonucleotide via a linker. Preferably, the linker is a monovalent, divalent or trivalent branched linker.

[0031] In some embodiments, in any of the above-mentioned compounds or pharmaceutically acceptable salts thereof, the single-stranded antisense oligonucleotide is SG20-1, SG20-2, SG20-3, SG20-4, SG20-5, SG20-6, SG20-7, SG20-8, SG20-9, SG20-10, SG20-16, SG20-17, SG20-18, SG20-19, SG20-20, SG20-21, SG20-22, SG20-23, SG20-24, SG 20-25, SG20-26, SG20-27, SG20-28, SG20-29, SG20-30, SG20-31, SG20-32, SG20-33, SG20-34, SG20-35, SG20-36, SG2 0-37, SG20-38, SG20-39, SG20-40, SG20-41, SG20-42, SG20-43, SG20-44, SG20-45, SG20-46, SG20-47, SG20-48, SG20 -49, SG20-50, SG20-51, SG20-52, SG20-53, SG20-54, SG20-55, SG20-56, SG20-57, SG20-58, SG20-59, SG20-60, SG20- 61. SG20-62, SG20-63, SG20-64, SG20-65, SG20-66, SG20-67, SG20-68, SG20-69, SG20-70, SG20-71, SG20-72, SG20-7 3. SG20-74, SG20-75, SG20-76, SG20-77, SG20-78, SG20-79, SG20-80, SG20-81, SG20-82, SG20-83, SG20-84, SG20-85 , SG20-86, SG20-87, SG20-88, SG20-89, SG20-90, SG20-91, SG20-92, SG20-93, SG20-94, SG20-95, SG20-96, SG20-97;

[0032] Preferably, it is SG20-7, SG20-9, SG20-18, SG20-28, SG20-35, SG20-37, SG20-38, SG20-39, SG20-40, SG20-41, SG20-42, SG20-43, SG20-44, SG20-45, SG20-46, SG20-47, SG20-48, SG20-49, SG20-50, SG20-51, SG2 0-52, SG20-53, SG20-54, SG20-56, SG20-58, SG20-59, SG20-63, SG20-64, SG20-67, SG20-69, SG20-70, S G20-71, SG20-73, SG20-75, SG20-76, SG20-77, SG20-78, SG20-87, SG20-89, SG20-90, SG20-92, SG20-93.

[0033] In some embodiments, in any of the above-described compounds or pharmaceutically acceptable salts thereof, the compound comprises a prodrug moiety.

[0034] In some embodiments, in any of the above-mentioned compounds or pharmaceutically acceptable salts thereof, the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

[0035] In a second aspect, the present invention provides any one of the above compounds or a pharmaceutically acceptable salt thereof for use as a medicament.

[0036] The compounds disclosed in the present invention or their pharmaceutically acceptable salts can be incorporated into a composition. Therefore, in a third aspect, the present invention also provides a composition comprising any of the above-described compounds or their pharmaceutically acceptable salts, and a pharmaceutically acceptable diluent, carrier and / or excipient. In some embodiments, the composition is a pharmaceutical composition. The pharmaceutically acceptable diluent, carrier, excipient can be any suitable diluent, carrier, excipient conventionally used in the art.

[0037] In a fourth aspect, the present invention provides a method for inhibiting TRPV1 gene expression in cells in vivo or in vitro, the method comprising introducing into the cells an effective amount of any of the above-described compounds or pharmaceutically acceptable salts thereof or any of the above-described compositions.

[0038] In some embodiments, in the above methods, the cell is in a subject.

[0039] In some embodiments, in the above methods, the subject is a human.

[0040] In some embodiments, in any of the methods described above, the TRPV1 gene expression is inhibited by at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0041] In a fifth aspect, the present invention provides use of any of the above-mentioned compounds or pharmaceutically acceptable salts thereof or any of the above-mentioned compositions in the preparation of drugs for treating, preventing and / or ameliorating TRPV1-related diseases.

[0042] In some embodiments, in the above use, the TRPV1-related disease is selected from one or more of the following: pain, eye disease, chronic pain, cardiovascular disease, mental illness, diabetes. Preferably, the pain is eye pain.

[0043] In a sixth aspect, the present invention provides a method for treating, preventing and / or ameliorating TRPV1-related diseases, comprising administering a therapeutically effective amount of any of the above-mentioned compounds or pharmaceutically acceptable salts thereof or any of the above-mentioned compositions to a patient in need thereof.

[0044] In some embodiments, in the above methods, the patient is a human.

[0045] In some embodiments, in any of the above methods, the TRPV1-related disease is selected from one or more of the following: pain, eye disease, chronic pain, cardiovascular disease, mental illness, diabetes. Preferably, the pain is eye pain.

[0046] In some embodiments, in any of the methods described above, the compound or a pharmaceutically acceptable salt thereof or composition is administered to the patient by subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and / or cerebrospinal administration.

[0047] Administration may be systemic or local.The compositions of the present invention may be provided in the form of solutions, suspensions, emulsions, tablets, pills, multiparticulates, capsules (including capsules containing liquids), powders, suppositories, aerosols or in any other form.

[0048] In a seventh aspect, the present invention also provides use of any of the above-mentioned compounds or pharmaceutically acceptable salts thereof or any of the above-mentioned compositions in the preparation of neuroprotective drugs.

[0049] Beneficial Effects

[0050] The antisense oligonucleotide provided by the present invention can effectively inhibit the expression of TRPV1 at the transcription level, and thus can be used to treat, prevent and / or improve TRPV1-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The effects of antisense oligonucleotides with different sequences on knocking down TRPV1 gene expression in human retinal pigment epithelial cells (ARPE19) were evaluated by real-time fluorescence quantitative PCR at the end of the test.

[0052] Figure 2 It is a cytotoxicity test of candidate antisense oligonucleotides in HepG2 cells, which is evaluated by detecting the fluorescent signal by microplate reader at the end of the test.

[0053] Figure 3 The analgesic effect of antisense oligonucleotides in a mouse model of ocular pain.

[0054] Figure 4 The analgesic effect of antisense oligonucleotides in the New Zealand rabbit model of ocular pain. DETAILED DESCRIPTION

[0055] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0056] The term "nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides lacking a nucleobase.

[0057] The term "2'-deoxynucleoside" refers to a nucleoside comprising a 2'-H(H)furanosyl sugar moiety as found in naturally occurring deoxyribonucleotides (DNA). A "2'-deoxynucleoside" may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0058] "Oligonucleotide" means a polymer of linked nucleosides, each of which may be modified or unmodified, independently of one another. Unless otherwise indicated, an oligonucleotide consists of 8-80 linked nucleosides. "Modified oligonucleotide" means an oligonucleotide in which at least one sugar, nucleobase, or internucleoside linkage is modified. "Unmodified oligonucleotide" means an oligonucleotide that does not contain any sugar, nucleobase, or internucleoside linkage modification.

[0059] The term "TRPV1 nucleic acid" refers to any nucleic acid encoding TRPV1. For example, TRPV1 nucleic acid includes a DNA sequence encoding TRPV1, a pre-mRNA sequence transcribed from a DNA encoding TRPV1 (including genomic DNA with introns and exons), and an mRNA sequence encoding TRPV1. "TRPV1 mRNA" refers to an mRNA encoding a TRPV1 protein.

[0060] The sequence of human TRPV1 mRNA includes, but is not limited to, the TRPV1 mRNAs shown in the National Center for Biotechnology Information (NCBI) accession numbers NM_080705.4, NM_080704.4, NM_080706.3, and NM_018727.5. Additional examples of TRPV1 mRNA sequences can be readily obtained using publicly available databases such as GenBank, UniProt, and OMIM.

[0061] As used herein, "target segment" refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during transcription of the TRPV1 gene, including the mRNA of the product after RNA treatment as the initial transcription product. In one example, the target portion of the sequence is at least long enough to serve as a substrate for RNase H cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the TRPV1 gene. In one example, the target sequence is in the exon region of the TRPV1 pre-mRNA. In another example, the target sequence is in the intron region of the TRPV1 pre-mRNA.

[0062] The terms "gapmer", "gap-mer", "gap polymer" or "chimeric antisense oligonucleotide" all refer to a form of antisense oligonucleotide containing a central block of deoxynucleotide monomers long enough to induce RNase H cleavage, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external region. The internal region may be referred to as a "gap" or "spacer", and the external region may be referred to as a "wing". In certain embodiments, the antisense oligonucleotide is a gapmer.

[0063] The term "antisense oligonucleotide" refers to a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.

[0064] The term "directly connected" means that there are no intervening elements between the connected elements.

[0065] The term "modified internucleoside linkage" refers to a substitution or any change relative to a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

[0066] The term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymine and uracil.

[0067] The term "modified sugar" refers to modified deoxyribose or modified ribose. The modification includes, but is not limited to, replacing the 2'-OH group of the ribose ring with "2'-O-methoxyethyl" (also known as 2'-MOE).

[0068] The term "modified nucleosides" refers to nucleosides independently having a modified sugar moiety or a modified nucleobase.

[0069] The term "modified nucleotide" refers to nucleotides independently having a modified sugar moiety, a modified internucleoside linkage or a modified nucleobase.

[0070] "Complementary" with respect to an oligonucleotide means that the nucleobase sequence of such an oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof, when the two nucleobase sequences are aligned in opposite directions. As described herein, unless otherwise indicated, nucleobase matching or complementary nucleobases are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (m C) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not necessarily have nucleobase complementarity at every nucleoside and may include one or more nucleobase mismatches. In contrast, with respect to oligonucleotides, "fully complementary" or "100% complementary" means that such oligonucleotides have nucleobase matches at every nucleoside without any nucleobase mismatches.

[0071] The term "pharmaceutically acceptable salt" refers to a physiologically or pharmaceutically acceptable salt, i.e., a salt that retains the desired biological activity of the parent oligonucleotide and does not impart an undesirable toxicological effect thereto, including but not limited to metal complexes and salts of inorganic and organic acids. Pharmaceutically acceptable salts also include metal salts, such as aluminum salts, calcium salts, iron salts, magnesium salts, manganese salts, sodium salts, potassium salts and complex salts. In addition, pharmaceutically acceptable salts include, but are not limited to, acidic salts such as acetate, aspartate, alkylsulfonate, arylsulfonate, benzenesulfonate, benzoate, bicarbonate, butyrate, lactate, lactobionate, maleate, malate, malonate, mandelate, methanesulfonate, methylnitrate, methylsulfate, nitrate, oxalate, p-nitromethanesulfonate, pantothenate, phosphate, hydrogenphosphate, dihydrogenphosphate, phthalate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfamate, p-aminobenzenesulfonate, sulfonate, sulfate, tannate, and tartrate.

[0072] The term "phosphorothioate bond" refers to an internucleoside bond in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate bond (P=S) is a modified internucleoside bond.

[0073] The term "prodrug" refers to a therapeutic agent that is prepared in an inactive form and is converted to an active form within the body or its cells by the action of endogenous enzymes or other chemicals or conditions.

[0074] The term "single-stranded oligonucleotide" refers to an oligonucleotide that is not hybridized to a complementary strand.

[0075] The term "TRPV1-related disease" refers to any disease associated with TRPV1 nucleic acid or its expression product. Such diseases include pain, eye disease, chronic pain, cardiovascular disease, mental illness, diabetes. Preferably, the pain is eye pain.

[0076] cEt nucleoside refers to a nucleoside that includes a bicyclic sugar portion containing a 4'-CH(CH3)-O-2' bridge. The structural formula of cEt nucleoside is as follows:

[0077]

[0078] Wherein, B represents the base part.

[0079] The term "complementary" refers to the ability of polynucleotides to pair with each other to form base pairs. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can be base paired in a Watson-Crick manner (e.g., A and T, A and U, C and G), or in any other manner that allows the formation of a duplex, such as the Hoogsteen base pairing method, the wobble base pairing method.

[0080] As used herein, the phrase "inhibiting TRPV1 gene expression" encompasses inhibiting the expression of any TRPV1 gene (such as mouse TRPV1 gene, rat TRPV1 gene, monkey TRPV1 gene or human TRPV1 gene) and variants or mutants of the TRPV1 gene encoding the TRPV1 protein.

[0081] Inhibition of TRPV1 gene expression comprises any level of TRPV1 gene inhibition, such as at least partial inhibition of TRPV1 gene expression, such as at least about 20% inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0082] TRPV1 gene expression can be assessed based on the level of any variable related to TRPV1 gene expression, such as TRPV1 mRNA level or TRPV1 protein level. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from a similar untreated or control (e.g., buffer-only control or inert agent control) treated subject, cell or sample.

[0083] Subjects include any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0084] The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to the amount of the antisense oligonucleotide of the present invention or its pharmaceutically acceptable salt or composition, when administered to a cell, tissue or subject alone or in combination with other therapeutic drugs, that is effective in preventing or ameliorating the symptoms of one or more diseases or conditions or the development of the disease or condition. The effective amount for a specific subject may vary depending on a variety of factors, such as the disease to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. When an active ingredient is administered to an individual alone, the therapeutically effective amount refers to the individual ingredient. When a combination is administered, the therapeutically effective amount refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether it is administered in combination, continuously or simultaneously. The therapeutically effective amount will alleviate symptoms usually by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40% and most preferably by at least 50%.

[0085] The term "treating" includes amelioration or cessation of a disorder or symptom thereof. Treatment includes inhibition, e.g., reducing the overall frequency of onset of a disorder or symptom thereof.

[0086] The term "improvement" includes an improvement or alleviation of at least one indicator, sign or symptom of a disease or its symptoms; for example, improvement includes a delay or slowing of the progression or severity of one or more indicators of a disease or its symptoms. The progression or severity of an indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0087] The term "preventing" includes avoiding the initiation of a disorder or its symptoms.

[0088] Antisense compounds

[0089] Antisense compounds refer to oligomeric compounds that can hybridize with target nucleic acids through hydrogen bonds. Antisense compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense oligonucleotides, siRNA and other oligonucleotides that hybridize with target nucleic acids and regulate their expression.

[0090] In certain embodiments, the antisense compound is 20 subunits in length. In certain embodiments, the antisense compound is an antisense oligonucleotide and the linked subunits are modified or unmodified ribonucleotides or deoxyribonucleotides.

[0091] Complementarity

[0092] When a sufficient number of nucleobases in the antisense oligonucleotide can form hydrogen bonds with corresponding nucleobases in the target nucleic acid, the antisense oligonucleotide and the target nucleic acid are complementary to each other, whereby antisense inhibition of the target nucleic acid occurs.

[0093] The antisense oligonucleotide may have nucleobases that are not complementary to the TRPV1 nucleic acid, as long as the antisense oligonucleotide remains capable of specific hybridization to the TRPV1 nucleic acid.

[0094] In certain embodiments, the antisense oligonucleotides herein or a designated portion thereof are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a TRPV1 nucleic acid, a target segment or a designated portion thereof. Complementarity can be routinely calculated using BLAST programs (Basic Local Alignment Search Tool) and the like known in the art.

[0095] The non-complementary nucleobase can be located at the 5' end or the 3' end of the antisense oligonucleotide. Alternatively, the non-complementary one or more nucleobases are located at a position inside the antisense oligonucleotide. In certain embodiments, the non-complementary nucleobase is located in the wing segment of the antisense oligonucleotide.

[0096] Modification

[0097] The antisense oligonucleotides of the present invention comprise at least one modification selected from the group consisting of a modified internucleoside linkage, a modified sugar and a modified nucleobase.

[0098] Modified internucleoside linkages

[0099] The naturally occurring internucleoside linkage of RNA and DNA is 3' to 5' phosphodiester bond. In certain embodiments, due to the desired characteristics, such as enhanced cellular uptake, enhanced affinity to target nucleic acid and increased stability in the presence of nuclease, the antisense oligonucleotides described herein with one or more modified (that is, non-naturally occurring) internucleoside linkages are generally selected, rather than the antisense oligonucleotides with naturally occurring internucleoside linkages. In certain embodiments, the modified internucleoside linkage is phosphorothioate.

[0100] Modified sugar moiety

[0101] The modified sugar moiety can confer nuclease stability, binding affinity or some other beneficial biological properties to the antisense oligonucleotide. The furanose ring of the nucleoside can be modified by a variety of methods, including but not limited to: adding a substituent, especially at the 2' position; bridging the atoms of two asymmetric rings to form a bicyclic nucleic acid, etc. In certain embodiments, the modified sugar is 2'-MOE substituted ribose or 2'-deoxyribose.

[0102] Modified Nucleobases

[0103] Nucleobase modifications can confer antisense oligonucleotides with improved nuclease stability, binding affinity, or some other beneficial biological properties. Modified nucleobases include synthetic and natural nucleobases, such as 5-methylcytosine. Certain nucleobase substitutes, including 5-methylcytosine substitutes, are particularly effective for improving the affinity of antisense compounds to target nucleic acids.

[0104] In contrast, "unmodified" or "natural" nucleobases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0105] In certain embodiments, the antisense oligonucleotide targeting TRPV1 nucleic acid comprises one or more modified sugars, one or more modified nucleobases and / or one or more modified internucleoside linkages. In certain embodiments, the modified sugar is independently selected from ribose or 2'-deoxyribose substituted by 2'-MOE. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, the modified internucleoside linkage is phosphorothioate. In certain embodiments, each sugar in a single-stranded antisense oligonucleotide is a 2'-MOE modified sugar or a 2'-deoxy sugar. In certain embodiments, each cytosine in a single-stranded antisense oligonucleotide is a 5-methylcytosine. In certain embodiments, each internucleoside linkage of the single-stranded antisense oligonucleotide is a phosphorothioate. In certain embodiments, each sugar in a single-stranded antisense oligonucleotide is a 2'-MOE modified sugar or a 2'-deoxy sugar, each cytosine is a 5-methylcytosine, and each internucleoside linkage is a phosphorothioate.

[0106] Antisense Oligonucleotides

[0107] Provided herein are antisense oligonucleotides for inhibiting TRPV1 gene expression.

[0108] In certain embodiments, the antisense oligonucleotide targeted to the TRPV1 gene consists of 20 linked nucleosides.

[0109] RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, greatly enhancing the efficiency of oligonucleotides in inhibiting gene expression.

[0110] In chimeric antisense oligonucleotides, the gap segment is generally used as a substrate for RNase H endonuclease cleavage, and the wing segment comprises a modified nucleoside. In certain embodiments, these regions of chimeric antisense oligonucleotides are distinguished by the type of sugar moieties constituting each different region, and are distinguished herein by the nucleoside modified with 2'-MOE. "Wing-gap-wing" motif is often described as "XYZ", wherein "X" represents the length of 5' wing region, "Y" represents the length of gap region, and "Z" represents the length of 3' wing region, wherein 5' wing region is directly connected to gap region, gap region and 3' wing region, i.e., there is no insertion nucleotide. X, Y and Z can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 arbitrary values. Chimeric antisense oligonucleotides described herein include but are not limited to: 5-10-5, 2-13-5, 4-12-4, 5-9-6, 6-8-6.

[0111] In certain embodiments, the antisense oligonucleotide targeting a TRPV1 nucleic acid has a 5-10-5 gapmer motif. In certain embodiments, the antisense oligonucleotide targeting a TRPV1 nucleic acid consists of the nucleobase sequence shown in SEQ ID NO: 1-92.

[0112] In certain embodiments, antisense oligonucleotides targeted to a TRPV1 nucleic acid may be targeted to TRPV1 or any portion thereof. In certain embodiments, the 15th exon of human TRPV1 mRNA NM_080705.4, the 16th exon of NM_080704.4, the 14th exon of NM_080706.3, the 15th exon of NM_018727.5, the 8th exon of NM_080705.4, the 9th exon of NM_080704.4, the 7th exon of NM_080706.3, the 8th exon of NM_018727.5, the 6th exon of NM_080705.4, the 7th exon of NM_080704.4, the 5th exon of NM_080706.3, and the 6th exon of NM_018727.5 are targeted.

[0113] In some embodiments, the antisense oligonucleotides of the present invention contain one or more modified sugars, one or more modified nucleobases and / or one or more modified internucleoside linkages, and these modifications do not cause the antisense oligonucleotides to significantly weaken or lose their function of regulating target gene expression. On the other hand, these modifications may allow the antisense oligonucleotides of the present invention to resist endogenous nuclease degradation, increase the stability of the oligonucleotide chain in plasma, tissues and cells, and make the antisense oligonucleotides sufficiently stable to reach and enter the target cells expressing the TRPV1 gene, contact the target nucleic acid, and act therewith, so that the antisense oligonucleotides maintain a high target gene inhibitory activity. These modifications can also increase the affinity of the oligonucleotide chain to the target nucleic acid, making the two more strongly bound, further improving the stability of the hybrid double chain, and achieving a higher antisense activity.

[0114] The antisense oligonucleotides prepared in this article are shown in Table 1. These antisense oligonucleotides consist of: a gap segment consisting of 10 connected 2'-deoxynucleosides, a 5' wing segment consisting of 5 connected nucleosides, and a 3' wing segment consisting of 5 connected nucleosides; the 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, and the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment; and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-MOE nucleosides, all cytosines on the antisense oligonucleotides are 5-methylcytosine, and all internucleoside linkages on the antisense oligonucleotides are phosphorothioates.

[0115] "2'-deoxynucleoside" means a nucleoside comprising a 2'-H(H)furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0116] "2'-O-methoxyethyl" (also known as 2'-MOE) refers to 2'-O(CH2)2-OCH3 in place of the 2'-OH group of the ribosyl ring.

[0117] "2'-MOE nucleoside" refers to a nucleoside comprising a 2'-MOE modified sugar moiety. The 2'-MOE modification may have enhanced stability and targeting, and generally has lower toxicity.

[0118] Phosphorothioate (P=S) refers to replacing the non-bridging oxygen atom in the phosphodiester bond with a sulfur atom, replacing the phosphodiester bond with a thiophosphate diester bond, that is, the connection between the two nucleosides is a thiophosphate-based connection, and its structural formula is shown below:

[0119]

[0120] Antisense oligonucleotides containing thiophosphate linkages have good water solubility and stability, are easy to synthesize in large quantities, and have a longer half-life, thus meeting the needs of clinical treatment.

[0121] "5-methylcytosine" refers to cytosine having a methyl group attached to the 5' position.

[0122] Those skilled in the art will appreciate that the antisense oligonucleotides described herein can be obtained by conventional methods for preparing antisense oligonucleotides in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial custom services. The modified nucleotides can be introduced into the antisense oligonucleotides described herein by using nucleotide monomers with corresponding modifications, and methods for preparing nucleotide monomers with corresponding modifications and methods for introducing modified nucleotides into antisense oligonucleotides are also well known to those skilled in the art.

[0123] Composition

[0124] The present invention provides a composition comprising the antisense oligonucleotide disclosed herein or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable diluent, carrier and / or excipient (e.g., PBS buffer, physiological saline, water). The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0125] The pharmaceutically acceptable diluents, carriers and / or excipients used in the present invention include any suitable pharmaceutically acceptable diluents, carriers and / or excipients known in the art.

[0126] Compositions comprising antisense oligonucleotides encompass any pharmaceutically acceptable salt, ester, or salt of such ester that can provide its biologically active metabolite when administered. Therefore, the disclosure also relates to pharmaceutically acceptable salts, prodrugs, and pharmaceutically acceptable salts of such prodrugs of antisense oligonucleotides. Suitable salts can be sodium salts, potassium salts.

[0127] Prodrugs may include additional nucleosides incorporated at one or both ends of the antisense oligonucleotide that are degraded by enzymes in vivo to form active antisense oligonucleotides.

[0128] The antisense oligonucleotides or pharmaceutically acceptable salts thereof of the present invention can be used alone or in combination with any compound for treating, preventing and / or ameliorating TRPV1-related diseases in a pharmaceutical composition, which is expected to enhance the beneficial therapeutic effects of the antisense oligonucleotides or pharmaceutically acceptable salts thereof of the present invention.

[0129] The composition of the present disclosure may also be used in combination with one or more other drugs. Such combined use is expected to have a synergistic effect in treating, preventing and / or improving TRPV1-related diseases.

[0130] application

[0131] Conventional methods in the art can be used to detect the inhibition level of the antisense oligonucleotides disclosed herein on TRPV1 nucleic acid. In some embodiments, real-time fluorescence quantitative PCR is used to detect the inhibition of the expression of TRPV1 mRNA by antisense oligonucleotides using ACTB as an internal reference gene. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 1% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 10% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 20% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 30% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 40% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 50% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 60% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit more than 70% of TRPV1 mRNA expression. In some embodiments, the antisense oligonucleotides of the present invention can inhibit the expression of TRPV1 mRNA by more than 80%. In some embodiments, the antisense oligonucleotides of the present invention can inhibit the expression of TRPV1 mRNA by more than 90%.

[0132] The present invention provides antisense oligonucleotides or pharmaceutically acceptable salts thereof or compositions that can be used to treat, prevent and / or improve TRPV1-related diseases; in certain embodiments, the TRPV1-related diseases are selected from one or more of the following: pain, eye diseases (e.g., dry eye), chronic pain, cardiovascular diseases, mental illnesses, and diabetes. Preferably, the pain is eye pain.

[0133] Administration to a patient may be by any suitable route known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal.

[0134] The present invention confirms that the antisense oligonucleotide disclosed in the present invention can effectively knock down the expression level of TRPV1 mRNA without significant cytotoxicity.

[0135] Example 1: Preparation of chimeric antisense oligonucleotides by solid phase technology

[0136] The antisense oligonucleotides shown in Table 1 were synthesized using solid phase technology against human TRPV1 mRNA. Unless otherwise stated, all reagents and solutions used to synthesize these oligomeric compounds were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and m C residue. Phosphoramidite solutions of all monomers (β-D-2'-deoxyribonucleosides and β-D-2'-(MOE) ribonucleosides) used were 0.06 M solutions in anhydrous acetonitrile.

[0137] 500nmol synthesis column made of Universal CPG solid support was filled on LK-48E synthesizer, and the specified sequence was synthesized using phosphoramidite coupling method. For the coupling step, phosphoramidite monomer was delivered with an amount exceeding 4 times of the loading on the solid support and phosphoramidite condensation was continued for 10min. All other steps were in accordance with the standard protocol supplied by the manufacturer. A solution of 3vol% trichloroacetic acid in dichloromethane was used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl of nucleotides. 5-benzylthiotetrazole (5-BTT) (0.35M, containing 0.5vol% N-methylimidazole (NMI)) in anhydrous acetonitrile was used as an activator during the coupling step. The phosphorothioate linkage was introduced by sulfurizing a 0.2M solution of diphenylacetyl disulfide (PADS) in a volume ratio of 1:1 pyridine / acetonitrile for 3 minutes.

[0138] After the synthesis of the specified sequence, the solid support-bound specified sequence was suspended in ammonia water (25wt%-30wt%) and heated at 85°C for 2h. The solid support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high pressure liquid chromatography to prepare the chimeric antisense oligonucleotides shown in Table 1.

[0139] The chimeric antisense oligonucleotides in Table 1 are all 20 nucleosides in length and are designed as 5-10-5 spacers; wherein the gap segment consists of 10 connected 2'-deoxynucleosides, the 5' wing segment and the 3' wing segment each consist of 5 connected nucleosides, the 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment, and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-MOE nucleosides, all cytosines in the entire spacer are 5-methylcytosine, and all internucleoside bonds in the entire spacer are phosphorothioate (P=S) bonds.

[0140] Table 1 Antisense oligonucleotides

[0141]

[0142]

[0143]

[0144] In Table 1, A, T, C, and G each represent the base composition of each nucleotide.

[0145] Example 2: Real-time fluorescence quantitative PCR detection of the effect of antisense oligonucleotides of different sequences on reducing TRPV1 gene expression

[0146] The cells used in this experiment were human retinal pigment epithelial cells (ARPE19). These cells were inoculated in DMEM / F12 + 10% FBS medium in a 24-well plate, with 1.5 × 10 5 Cells were transfected with antisense oligonucleotides of different sequences shown in Table 1 (powdered antisense oligonucleotides were first dissolved in ddH2O to a storage concentration of 100 μM, and then diluted to 20 μM with ddH2O for experiments) into the corresponding wells 12 hours later using Lipofectamine RNAiMax (Thermo Fisher) transfection reagent. The final concentration of antisense oligonucleotides was 100 nM, and DEPC water was used for transfection in the blank control group. After 24 hours of continuous culture at 37°C and 5% CO2, cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template to synthesize oligo (dT) (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3', SEQ ID NO: 93) as a reverse transcription primer, and mmlv reverse transcriptase (Promega) was used for reverse transcription to obtain cDNA. Afterwards, the expression of TRPV1 gene was detected by real-time fluorescence quantitative PCR using cDNA as a template and ACTB as an internal reference gene, and the data were analyzed using the 2^-ΔΔCT method. Compared with the cell group treated with DEPC water (i.e., blank control group), the knockdown efficiency of antisense oligonucleotide was calculated. Figure 1 The knockdown efficiency is shown in Table 3.

[0147] Table 2 Sequences of detection primers

[0148]

[0149] Table 3 Knockdown efficiency

[0150]

[0151]

[0152]

[0153] The results of real-time fluorescence quantitative PCR showed that among the 92 antisense oligonucleotides tested, except for SG20-5, SG20-6, SG20-8, SG20-21, SG20-26, SG20-33, SG20-61, SG20-85, SG20-94 and SG20-96, the remaining antisense oligonucleotides significantly inhibited TRPV1 mRNA expression, and their knockdown efficiency ranged from 10% to 99%.

[0154] Example 3: Caspase-Glo 3 / 7 detection of cytotoxicity of antisense oligonucleotides of different sequences

[0155] Antisense oligonucleotides that can effectively knock down TRPV1 pre-mRNA / mRNA expression were transfected into HepG2 cells, and the cytotoxicity was reflected by the content of Caspase 3 / 7.

[0156] HepG2 cells in DMEM + 10% FBS (fetal bovine serum) serum medium were seeded in a white opaque 96-well plate, with 3×10 cells per well. 4 12 hours later, different antisense oligonucleotides were transfected into the corresponding wells using Lipofectamine RNAiMax (Thermo Fisher) transfection reagent. The final concentration of oligonucleotides was 0.1 μM and 1 μM. The positive control group was transfected with cEt modified sequence 200 (sequence 5'-CTGGTGCTGCCTGTAG-3' (SEQ ID NO:98), the antisense oligonucleotide has a length of 16 nucleosides and is designed as a 3-10-3 spacer; wherein the gap segment consists of 10 connected 2'-deoxynucleosides, the 5' wing segment and the 3' wing segment each consist of 3 connected nucleosides, the 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment, and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-cEt nucleosides, all cytosines in the entire spacer are 5-methylcytosine, and all internucleoside bonds in the entire spacer are phosphorothioate (P=S) bonds), and the wells transfected with DEPC water are recorded as 0 μM. After culturing for 24 hours at 37°C and 5% CO2, fresh medium was replaced and 50 μL of fresh medium was added to each well. Caspase-Glo 3 / 7 reagent (Promega, catalog number G8091) with an equal volume to the medium was added to each well. After incubation at room temperature for 1 hour, the fluorescence signal value was detected by an ELISA reader and compared with the fluorescence value of the corresponding wells transfected with DEPC water (control group) to measure the cytotoxicity of antisense oligonucleotides with different sequences. The results are shown in the figure. Figure 2 As shown in Table 4. The control group in Table 4 is the DEPC water group, which is a negative control group; sequence 200 is a positive control group.

[0157] Table 4 Cytotoxicity

[0158]

[0159] The results showed that the positive control had strong cytotoxicity, and all the antisense oligonucleotides tested had no significant cytotoxicity. Compared with the DEPC water group, except for SG20-48 which had weak cytotoxicity, there was no statistical difference in other ASO groups.

[0160] Example 4: Analgesic effect of antisense oligonucleotides in an eye pain mouse model

[0161] C57BL / 6 mice were used, male, 6-8 weeks old. The experiment was designed to administer drugs to the left and right eyes respectively, with the left eye given an invalid sequence SG-089 (i.e., scramble, sequence 5'-CCTTCCCTGAAGGTTCCTCC-3' (SEQ ID NO: 99)) that does not target any gene, and the right eye given candidate ASO sequences: SG20-42, SG20-43 and SG20-44, with 10 mice in each group. In addition, a positive control group was set up, with a total of 8 mice, SG-089 was given to the left eye, and the positive control siRNA SYL-1001 (sequence sense chain: 5'-AAGCGCAUCUUCUACUUCA-3' (SEQ ID NO: 100); antisense chain: 5'-UGAAGUAGAAGAUGCGCUU-3' (SEQ ID NO: 101)) was given to the right eye. The mice were given drugs for 5 consecutive days before the experiment. On the first day, 100 μg / mouse was injected subconjunctivally. Then, 250 μg / mouse was given by eye drops once a day for 4 days. The solvent was Gibco PBS. Six hours after the drug administration on the fifth day, 10 μl of 0.1% capsaicin was used for treatment.

[0162] Preparation method of 0.1% capsaicin: First, add 0.01g capsaicin to 150μl 100% ethanol to make a capsaicin solution. Take 850μL of Tween-20 and 9.0ml of artificial tears respectively, and then slowly add the capsaicin solution to the Tween-20 / artificial tears mixture while vortexing to finally obtain 0.1% (3.3mM) capsaicin.

[0163] Each group of mice was further divided into two groups, each with 5 mice. One group was treated with capsaicin in the left eye, and the other group was treated with capsaicin in the right eye. After capsaicin treatment, the number of times the mice scratched their eyes within 30 seconds was counted. After 15 minutes of capsaicin treatment, photos of the capsaicin-treated eyes were taken, and the eyelid opening and closing of the eye was calculated (the eyelid opening and closing calculation formula is: eyelid opening and closing = palpebral fissure height (H) / palpebral fissure width (W)). The experimental results are shown in Table 5 and Figure 3 shown.

[0164] Table 5 Analgesic effect of antisense oligonucleotides in the eye pain mouse model

[0165]

[0166] The results showed that compared with the negative control SG-089, the positive control, SG20-42, SG20-43 and SG20-44 could significantly increase the eyelid opening and closing degree of mice and reduce the number of scratching times, and SG20-42, SG20-43 and SG20-44 had better analgesic effects than the positive control.

[0167] Example 5: Analgesic effect of antisense oligonucleotides in the New Zealand rabbit model of eye pain

[0168] New Zealand white rabbits were used, male, 2-2.5 kg. The experiment was designed to administer drugs to the left and right eyes respectively, with the left eye given the invalid sequence SG-089 (i.e. scramble) that does not target any gene, and the right eye given the candidate ASO sequences: SG20-42, SG20-43 and SG20-44, of which 4 New Zealand white rabbits were in the SG20-42 and SG20-43 groups, and 3 New Zealand white rabbits were in the SG20-44 group. Continuous administration was performed for 5 days before the start of the experiment, with 500 μg / rabbit injected subconjunctivally on the first day, and then eye drops were administered twice a day for 4 days, 2 mg / rabbit each time, and the solvent was Gibco PBS. Four hours after administration on the fifth day, 30 μl of 1% capsaicin was used for treatment.

[0169] Preparation method of 1% capsaicin: 0.1g capsaicin + 150μl 15% ethanol (1.5ml anhydrous ethanol + 8.5ml distilled water) + 850μl 8.5% Tween-80 (0.85ml Tween-80 + 9.15ml distilled water) to dissolve, and then dilute with 9.0ml of normal saline to a final concentration of 33mM.

[0170] Each group of New Zealand white rabbits was further divided into two groups, each with 2 rabbits. One group was treated with capsaicin in the left eye, and the other group was treated with capsaicin in the right eye (SG20-44 left eye group had 1 rabbit). After capsaicin treatment, the number of times the rabbit scratched its eye within 120 seconds was counted. Five minutes after capsaicin treatment, photos of the capsaicin-treated eye were taken, and the eyelid opening and closing degree of the eye was calculated. The experimental results are shown in Table 6 and Figure 4 shown.

[0171] Table 6 Analgesic effect of antisense oligonucleotides in the New Zealand rabbit model of eye pain

[0172]

[0173] The results showed that compared with the negative control SG-089, SG20-42 could significantly increase the opening and closing degree of rabbit eyelids, and SG20-42 and SG20-44 could significantly reduce the number of scratching times.

[0174] SG20-42 showed significant analgesic effects using different measurement indicators in different animal models.

[0175] The specific implementation modes of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details in the above implementation modes. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0176] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0177] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, comprising a single-stranded antisense oligonucleotide complementary to TRPV1 nucleic acid, wherein the single-stranded antisense oligonucleotide consists of 20 linked nucleosides.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide consists of the nucleobase sequence shown in SEQ ID NO: 1-92.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleobases and / or one or more modified internucleoside linkages. 4 . The compound according to claim 3 , or a pharmaceutically acceptable salt thereof, wherein each of the modified sugars is independently selected from 2′-MOE substituted ribose or 2′-deoxyribose. 5 . The compound according to claim 4 , or a pharmaceutically acceptable salt thereof, wherein each sugar in the single-stranded antisense oligonucleotide is 2′-MOE-substituted ribose or 2′-deoxyribose. 6 . The compound according to claim 3 , or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is 5-methylcytosine. 7 . The compound or pharmaceutically acceptable salt thereof according to claim 6 , wherein each cytosine in the single-stranded antisense oligonucleotide is 5-methylcytosine.

8. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate.

9. The compound according to claim 8, or a pharmaceutically acceptable salt thereof, wherein each internucleoside linkage of the single-stranded antisense oligonucleotide is phosphorothioate.

10. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide comprises, consists of, or consists essentially of: A gap segment consisting of linked 2'-deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides; The 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, and the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment; the gap segment consists of 7 to 12 connected deoxynucleosides, and the 5' wing segment and the 3' wing segment consist of 2 to 7 connected ribonucleosides, respectively; and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-MOE nucleosides, all cytosines on the single-stranded antisense oligonucleotide are 5-methylcytosine, and all internucleoside linkages on the single-stranded antisense oligonucleotide are phosphorothioates.

11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide comprises, consists of, or consists essentially of: A gap segment consisting of 10 linked 2'-deoxynucleosides; A 5' wing segment consisting of five linked nucleosides; and A 3' wing segment consisting of 5 linked nucleosides; The 5' end of the gap segment is directly connected to the 3' end of the 5' wing segment, and the 3' end of the gap segment is directly connected to the 5' end of the 3' wing segment; and the nucleosides of the 5' wing segment and the 3' wing segment are all 2'-MOE nucleosides, all cytosines on the single-stranded antisense oligonucleotide are 5-methylcytosine, and all internucleoside linkages on the single-stranded antisense oligonucleotide are phosphorothioates.

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide is SG20-1, SG20-2, SG20-3, SG20-4, SG20-5, SG20-6, SG20-7, SG20-8, SG20-9, SG20-10, SG20-16, SG20-17, SG20-18, SG20-19, SG20-20, SG20-21, SG20-22, SG20-23, SG20-24 in Table 1 herein , SG20-25, SG20-26, SG20-27, SG20-28, SG20-29, SG20-30, SG20-31, SG20-32, SG20-33, SG20-34, SG20-35, SG20-36, SG20-37, SG20-38, SG20-39, SG20-40, SG20-41, SG20-42, SG20-43, SG20-44, SG20-45, SG20-46, SG20-47, SG20-48, SG 20-49, SG20-50, SG20-51, SG20-52, SG20-53, SG20-54, SG20-55, SG20-56, SG20-57, SG20-58, SG20-59, SG20-60, SG2 0-61, SG20-62, SG20-63, SG20-64, SG20-65, SG20-66, SG20-67, SG20-68, SG20-69, SG20-70, SG20-71, SG20-72, SG20- 73. SG20-74, SG20-75, SG20-76, SG20-77, SG20-78, SG20-79, SG20-80, SG20-81, SG20-82, SG20-83, SG20-84, SG20-8 5. SG20-86, SG20-87, SG20-88, SG20-89, SG20-90, SG20-91, SG20-92, SG20-93, SG20-94, SG20-95, SG20-96, SG20-97; Preferably, it is SG20-7, SG20-9, SG20-18, SG20-28, SG20-35, SG20-37, SG20-38, SG20-39, SG20-40, SG20-41, SG20-42, SG20-43, SG20-44, SG20-45, SG20-46, SG20-47, SG20-48, SG20-49, SG20-50, SG20-51, SG2 0-52, SG20-53, SG20-54, SG20-56, SG20-58, SG20-59, SG20-63, SG20-64, SG20-67, SG20-69, SG20-70, S G20-71, SG20-73, SG20-75, SG20-76, SG20-77, SG20-78, SG20-87, SG20-89, SG20-90, SG20-92, SG20-93.

13. A composition comprising the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier and / or excipient.

14. Use of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof or the composition according to claim 13 in the preparation of a medicament for treating, preventing and / or ameliorating TRPV1-related diseases.

15. Use of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof or the composition according to claim 13 in the preparation of a neuroprotective drug.

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