Methods for treating neurological disorders

Abstract

A method for treating neurological disorders, including epilepsy, using modified oligonucleotides that inhibit AMPAR is provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 453,814, filed on 22 March 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] This specification provides methods for treating neurological disorders, including epilepsy. Background of the Invention

[0003] Epilepsy is a common neurological disorder affecting approximately 50 million people worldwide, with about 0.5–1% of the general population experiencing it long-term. Furthermore, about 5% of the population will experience at least one epileptic seizure at some point in their lives. This is described in Wahab, A., “Difficulties in Treatment and Management of Epilepsy and Challenges in New Drug Development,” Pharmaceuticals, Vol.3(7), July 2010.

[0004] Epilepsy is a serious illness, and the mortality rate for people with epilepsy is two to three times higher than that of the general population. People with epilepsy may also suffer from a variety of social, emotional, psychological, and medical challenges, including social isolation, unemployment, psychological problems, a reduced quality of life, and medical comorbidities due to medication and seizures.

[0005] Currently, there is no known cure for epilepsy. Treatment aims to alleviate symptoms and prevent seizures. For most patients, antiepileptic drugs are prescribed as lifelong treatment. However, the long-term use of current treatments is limited by side effects, withdrawal symptoms, interactions with other drugs, and the financial burden.

[0006] Furthermore, it is reported that approximately 30% of all epilepsy patients show resistance to pharmaceutical therapy, and approximately 15 - 35% of all epilepsy patients cannot achieve long-term remission. See Wahab.

[0007] Epilepsy disorder has been treated for many years, and over the decades, a great deal of funding has been invested in the development of improved anti-epileptic drugs and considerable research has been done. However, the cell-based mechanisms of the disease state still remain largely mysterious. Recently, evidence has emerged that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor is involved in epilepsy and epileptogenesis. Hanada, T., “The AMPA Receptor as a Therapeutic Target in Epilepsy: Preclinical and Clinical Evidence,” Dovepress, Vol.7 (39 - 50), Sept. 18, 2014.

[0008] Therefore, improved pharmaceutical agents for treating epilepsy and other nervous system disorders are still needed.

Summary of the Invention

[0009] The present disclosure relates to a method of treating a nervous system disorder of a subject, optionally a method of treating epilepsy, comprising administering to the subject a therapeutically effective amount of a compound comprising a modified oligonucleotide that inhibits AMPAR.

[0010] Embodiment 1. A method of treating a nervous system disorder of a subject, comprising administering to the subject a therapeutically effective amount of a modified oligonucleotide consisting of 4 - 15 linked nucleosides, wherein the last 4 nucleosides at the 3'-end of the modified oligonucleotide have the nucleobase sequence UUUG, and at least 2 of the last 4 nucleosides at the 3'-end of the modified oligonucleotide are bicyclic nucleosides.

[0011] Embodiment 2: The method according to Embodiment 1, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleosides of the modified oligonucleotide are bicyclic nucleosides.

[0012] Embodiment 3: The method according to Embodiment 1 or Embodiment 2, wherein each bicyclic nucleoside is independently selected from S-cEt nucleoside, LNA nucleoside, and ENA nucleoside.

[0013] Embodiment 4: The method according to Embodiment 3, wherein each bicyclic nucleoside is an S-cEt nucleoside.

[0014] Embodiment 5: The method according to any one of the prior embodiments, wherein at least one nucleoside bond of the modified oligonucleotide is a modified nucleoside bond.

[0015] Embodiment 6: The method according to Embodiment 5, wherein each nucleoside bond in the modified oligonucleotide is a modified nucleoside bond.

[0016] Embodiment 7: The method according to Embodiment 5 or Embodiment 6, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0017] Embodiment 8 The method according to any one of Embodiments 1 to 3, wherein the modified oligonucleotide consists of 4-9, 4-10, 4-11, 4-12, 4-13, or 4-14 linked nucleosides.

[0018] Embodiment 9 The modified oligonucleotide is a method according to any one of the prior embodiments, wherein the modified oligonucleotide consists of four linked nucleosides.

[0019] Embodiment 10 The modified oligonucleotide is U F U M U S G S or U SU S U S G S where the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, the method according to Embodiment 9.

[0020] Embodiment 11. The modified oligonucleotide consists of 9 linked nucleosides, the method according to any one of Embodiments 1 to 8.

[0021] Embodiment 12. The modified oligonucleotide contains the nucleic acid base sequence 5'-AGCACUUUG-3', and each cytosine is independently selected from non-methylated cytosine and 5-methylcytosine, the method according to any one of Embodiments 1 to 8 and 11.

[0022] Embodiment 13. The modified oligonucleotide has the following nucleoside pattern in the 5' to 3' direction N S N S N M N F N F N F N M N S N S and the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, the method according to Embodiment 11 or Embodiment 12.

[0023] Embodiment 14. The modified oligonucleotide is selected from the modified oligonucleotides in Table 1, the method according to any one of the preceding embodiments.

[0024] Embodiment 15: The method according to any one of the prior embodiments, wherein a pharmaceutically acceptable salt of the modified oligonucleotide is administered to the subject.

[0025] Embodiment 16: The method according to Embodiment 15, wherein the pharmaceutically acceptable salt is a sodium salt.

[0026] Embodiment 17 A method for treating a nervous system disorder of a target, wherein the target has a structure [ka] The method comprising administering a therapeutically effective amount of a modified oligonucleotide having, or a pharmaceutically acceptable salt thereof.

[0027] Embodiment 18: The method of Embodiment 17, comprising administering a pharmaceutically acceptable salt of the modified oligonucleotide to the subject.

[0028] Embodiment 19: The method according to Embodiment 18, comprising administering the sodium salt of the modified oligonucleotide to the subject.

[0029] Embodiment 20 A method for treating a neurological disorder of a target, wherein the target has a structure [ka] The method comprising administering a therapeutically effective amount of a modified oligonucleotide having [a specific characteristic].

[0030] Embodiment 21 The modified oligonucleotide is formulated into a pharmaceutical composition for administration to the subject, the pharmaceutical composition comprising the modified oligonucleotide in an aqueous solution, according to any one of the prior embodiments.

[0031] Embodiment 22: The method according to Embodiment 21, wherein the aqueous solution is physiological saline.

[0032] Embodiment 23 The method according to any one of the prior embodiments, wherein the neurological disorder is hearing loss, motor disorder, amyotrophic lateral sclerosis (ALS), pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke, or glioblastoma.

[0033] Embodiment 24 The method according to any one of the prior embodiments, wherein the neurological disorder is epilepsy.

[0034] Embodiment 25 A method according to any one of the prior embodiments, comprising administering at least one additional therapy to the subject.

[0035] Embodiment 26: The method according to Embodiment 25, wherein the additional therapy is an antiepileptic drug. [Brief explanation of the drawing]

[0036] [Figure 1] Figures A and B are graphs showing the latency (seconds) to observed seizures (clonic and tonic, respectively) in mice administered 60 mpk, 65 mpk, and 70 mpk of pentylenetetrazole (PTZ) in the dose pilot study of Example 3. [Figure 2] This graph shows the time course of the Racine score after administering 60 mpk, 65 mpk, and 70 mpk of PTZ to mice in the dose pilot study of Example 3. [Figure 3] This bar graph shows the survival data 30 minutes after administering 60 mpk, 65 mpk, and 70 mpk of PTZ to mice in the dose pilot study of Example 3. [Figure 4] This graph shows the latency data to tonic seizures obtained from the experiment in Example 4, which compares the CNS effects of RG4326, a negative control compound (NCC), and perampanel on PTZ-induced seizures in mice. [Figure 5] This graph shows the latency data to cardiac arrest (e.g., death) obtained from the study in Example 4, which compares the CNS effects of RG4326, NCC, and perampanel on PTZ-induced seizures in mice. [Figure 6] This graph shows racine score data obtained at 5-minute intervals up to 30 minutes after PTZ administration, from the study in Example 4, which compares the CNS effects of RG4326, NCC, and perampanel on PTZ-induced seizures in mice. [Figure 7] This graph shows survival data up to 30 minutes after PTZ administration in the study of Example 4, comparing the CNS effects of RG4326, NCC, and perampanel on PTZ-induced seizures in mice. [Figure 8] This document presents data from the 6Hz-induced seizure model in Example 6, reporting the percentage of animals in each group that exhibited forelimb clonus seizures after 6Hz current administration. [Figure 9] This document shows data obtained from the 6Hz-induced seizure model in Example 6, and reports the percentage of animals in each group that exhibited tail-raising seizures after 6Hz current administration. [Figure 10] This document shows data obtained from the 6Hz-induced seizure model in Example 7, and reports the number of mice in each group that exhibited tail-raising seizures after 6Hz current administration. [Figure 11] A and B represent the ATP content of cells cultured using the control substance and glutamic acid, as described in Example 8. [Figure 12] A to H represent the ATP content of cells cultured for 24 hours using the test compounds in Table 18 before the addition of glutamic acid, as described in Example 8. [Figure 13] A and B represent the ATP content of cells cultured with the control substance and AMPA, as described in Example 8. [Figure 14] A to H represent the ATP content of cells cultured for 24 hours using the test compounds in Table 18 before the addition of AMPA, as described in Example 8. [Figure 15-1] A and B show calcium release from cells cultured with the control substance and various concentrations of AMPA, as described in Example 8. [Figure 15-2] C to G show calcium release from cells cultured with the control substance and various concentrations of AMPA, as described in Example 8. [Figure 15-3] H~L indicates calcium release from cells cultured with the control substance and various concentrations of AMPA, as described in Example 8. [Figure 16] As described in Example 8, calcium release from cells cultured with the control substance and glutamic acid is shown. [Figure 17] A-H show calcium release from cells cultured for 24 hours using the test compounds in Table 18 before the addition of glutamic acid, as described in Example 8. [Figure 18] As described in Example 8, calcium release from cells cultured for 24 hours with a control substance before the addition of AMPA is shown. [Figure 19] As described in Example 8, calcium release from cells cultured for 24 hours using the test compounds in Table 18 before the addition of APMA is shown. [Modes for carrying out the invention]

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. If there are multiple definitions for a term herein, the definition in this section shall prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and for the treatment of patients. Specific such techniques and procedures can be found, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994, and “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 18th edition, 1990, which are incorporated herein by reference for any purpose. Where permitted, all patents, patent applications, published applications and gazettes, GENBANK sequences, websites, and other published materials referenced throughout the disclosure herein are incorporated by reference in their entirety unless otherwise specified. Where URLs or other such identifiers or addresses are referenced, it is understood that such identifiers may change, and certain information on the Internet may change, but equivalent information can be found by searching the Internet. References to them demonstrate the availability and public dissemination of such information.

[0038] Before disclosing and describing the compositions and methods of the present invention, it should be understood that the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural nouns unless otherwise explicitly indicated by the context.

[0039] definition "AMPA" (or "AMPA-R") stands for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor.

[0040] "Quality of life" refers to the extent to which an individual's physical, psychological, and social functions are impaired by the disease and / or treatment of the disease. In individuals with paroxysmal disorders, the quality of life may be reduced.

[0041] "To slow down the deterioration of ~" and "gradual deterioration" mean to reduce the rate at which a medical condition progresses to an advanced stage.

[0042] "Improving average life expectancy" means extending the lifespan of a subject by treating one or more symptoms of a disease.

[0043] "Subject" means a human or non-human animal selected for treatment or therapy.

[0044] "Those who need it" means those who have been identified as needing treatment or therapy.

[0045] "Subjects suspected of having a disease" refers to subjects who exhibit one or more clinical indicators of a disease.

[0046] "AMPAR-related diseases" refers to diseases or conditions that are regulated by AMPAR activity.

[0047] "Administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0048] "Pareral administration" refers to administration via infusion or injection. Parenteral administration methods include, but are not limited to, subcutaneous, intravenous, and intramuscular administration.

[0049] "Subcutaneous administration" means administration directly beneath the skin.

[0050] "Intravenous administration" means administering the drug intravenously.

[0051] "Simultaneous administration" refers to the co-administration of two drugs in any manner in which their pharmacological effects appear simultaneously in the patient. Simultaneous administration does not require that both drugs be administered in a single pharmaceutical composition, in the same dosage form, or via the same route of administration. The effects of both drugs do not need to appear simultaneously. The effects only need to overlap for a certain period and do not need to have the same extent.

[0052] "Period" refers to the duration during which an activity or event persists. In certain embodiments, the treatment period is the duration during which a certain dose of a pharmaceutical agent or pharmaceutical composition is administered.

[0053] "Therapy" refers to a method of treating a disease. In certain embodiments, therapies may include, but are not limited to, chemotherapy, radiation therapy, or the administration of pharmaceutical agents.

[0054] "Treatment" means applying one or more specific procedures used to improve at least one indicator of a disease. In certain embodiments, a specific procedure is the administration of one or more medicinal agents.

[0055] "Improvement" means reducing the severity of at least one indicator of a condition or disease. In certain embodiments, improvement includes delaying or slowing the progression of one or more indicators of a condition or disease. The severity of an indicator may be determined by subjective or objective measures known to those skilled in the art.

[0056] "At risk of developing" means that the subject has a predisposition to developing a condition or disease. In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but not enough symptoms to warrant a diagnosis of the condition or disease. In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a low degree to which a diagnosis of the condition or disease is necessary.

[0057] "Preventing onset" means preventing the onset of a condition or disease in subjects at risk of developing the condition or disease. In certain embodiments, subjects at risk of developing the condition or disease receive treatment similar to that received by subjects who already have the condition or disease.

[0058] "Delaying onset" means delaying the onset of a condition or disease in a subject at risk of developing the condition or disease. In certain embodiments, subjects at risk of developing the condition or disease receive treatment similar to that received by subjects who already have the condition or disease.

[0059] "Dose" refers to a specific amount of a pharmaceutical agent provided in a single dose. In certain embodiments, the dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, if subcutaneous administration is desired, the desired dose requires a volume that cannot be readily addressed by a single injection. In such embodiments, the desired dose may be achieved using two or more injections. In certain embodiments, the dose may be administered in two or more injections to minimize injection site reactions in the individual. In certain embodiments, the dose is administered as a slow infusion.

[0060] "Dosage unit" refers to the form in which the pharmaceutical agent is provided. In certain embodiments, the dosage unit is a vial containing lyophilized oligonucleotides. In certain embodiments, the dosage unit is a vial containing reconstituted oligonucleotides.

[0061] "Therapeutic dose" refers to the amount of pharmaceutical agent that provides a therapeutic benefit to an animal.

[0062] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, and includes pharmaceutical agents. For example, a pharmaceutical composition may include a sterile aqueous solution.

[0063] A "pharmaceutical agent" refers to a substance that produces a therapeutic effect when administered to a target.

[0064] "Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that produces the desired effect.

[0065] "Pharmacologically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound provided herein, i.e., a salt that retains the desired biological activity of the compound and does not have undesirable toxicological effects when administered to a subject. Non-limiting exemplary pharmaceutically acceptable salts of the compounds provided herein include sodium and potassium salt forms. As used herein, the terms "compound," "oligonucleotide," and "modified oligonucleotide" include their pharmaceutically acceptable salts unless otherwise specified.

[0066] "Physiological saline solution" refers to an aqueous solution of sodium chloride.

[0067] An "acceptable safety profile" refers to a pattern of side effects that falls within a clinically acceptable range.

[0068] "Side effects" refer to physiological responses resulting from treatment other than the desired effects. In certain embodiments, side effects may include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. Such side effects may be detected directly or indirectly. For example, elevated serum aminotransferase levels may indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin may indicate hepatotoxicity or abnormal liver function.

[0069] "Blood" refers to whole blood as well as blood fractions such as serum and plasma.

[0070] "Anti-miR" refers to an oligonucleotide having a nucleic acid base sequence complementary to microRNA. In certain embodiments, the anti-miR is a modified oligonucleotide.

[0071] "Anti-miR-17" refers to a modified oligonucleotide having a nucleic acid base sequence that is at least 80%, at least 85%, at least 90%, or at least 95% complementary to miR-17.

[0072] "miR-17" refers to a mature miRNA with the nucleic acid base sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (Sequence ID 1).

[0073] "Target nucleic acids" refer to nucleic acids that are designed to hybridize with oligomeric compounds.

[0074] "Targeting" refers to the process of designing and selecting nucleic acid base sequences that hybridize to a target nucleic acid.

[0075] "Targeting" means having a nucleic acid base sequence that allows for hybridization to the target nucleic acid.

[0076] "Modulation" means a change in function, quantity, or activity. In certain embodiments, modulation means an increase in function, quantity, or activity. In certain embodiments, modulation means a decrease in function, quantity, or activity.

[0077] "Expression" refers to any function or step in which the encoded information of a gene is converted into a structure that exists within and operates within a cell.

[0078] "Nucleic acid base sequence" refers to the order of adjacent nucleic acid bases in an oligomeric compound or nucleic acid, and is typically enumerated in 5' to 3' orientation, regardless of any sugars, ligations, and / or modifications of nucleic acid bases.

[0079] "Adjacent nucleic acid bases" refers to nucleic acid bases that are directly adjacent to each other within a nucleic acid.

[0080] "Nucleic acid base complementarity" refers to the ability of two nucleic acid bases to pair non-covalently via hydrogen bonds.

[0081] "Complementary" means that one nucleic acid can hybridize with another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide that can hybridize with a target nucleic acid.

[0082] "Fully complementary" means that each nucleic acid base of an oligonucleotide can pair with a nucleic acid base at its corresponding position in the target nucleic acid. In certain embodiments, an oligonucleotide is fully complementary to a microRNA (also called 100% complementary). That is, each nucleic acid base of an oligonucleotide is complementary to a nucleic acid base at its corresponding position in the microRNA. A modified oligonucleotide may be fully complementary to a microRNA and have multiple linked nucleosides shorter than the length of the microRNA. For example, an oligonucleotide having 16 linked nucleosides, where each nucleic acid base of the oligonucleotide is complementary to a nucleic acid base at its corresponding position in the microRNA, is fully complementary to the microRNA. In certain embodiments, an oligonucleotide in which each nucleic acid base is complementary to a nucleic acid base within a region of the microRNA stem-loop sequence is fully complementary to the microRNA stem-loop sequence.

[0083] "Percent complementarity" refers to the proportion of nucleic acid bases in oligonucleotides that are complementary to the isolength portion of the target nucleic acid. Percent complementarity is calculated by dividing the number of nucleic acid bases in oligonucleotides that are complementary to the nucleic acid base at the corresponding position in the target nucleic acid by the total number of nucleic acid bases in the oligonucleotide.

[0084] "Percent identity" means the number of nucleic acid bases in the first nucleic acid that are identical to the nucleic acid bases in the corresponding positions in the second nucleic acid, divided by the total number of nucleic acid bases in the first nucleic acid. In certain embodiments, the first nucleic acid is a microRNA, and the second nucleic acid is a microRNA. In certain embodiments, the first nucleic acid is an oligonucleotide, and the second nucleic acid is an oligonucleotide.

[0085] "Hybridizing" refers to the annealing of complementary nucleic acids that occurs due to the complementarity of nucleic acid bases.

[0086] A "mismatch" refers to a nucleic acid base of the first nucleic acid that cannot form a Watson-Crick pair with the corresponding nucleic acid base of the second nucleic acid at its corresponding position.

[0087] In the context of nucleic acid base sequences, "identical" means having the same nucleic acid base sequence, regardless of sugars, ligations, and / or nucleic acid base modifications, and regardless of the methylation status of any pyrimidine present.

[0088] "MicroRNA" refers to endogenous non-coding RNA with a length of 18-25 nucleic acid bases, and is the product of cleavage of premicroRNA by an enzyme dicer. Examples of mature microRNA can be found in the microRNA database known as miRBase (http: / / microrna.sanger.ac.uk / ). In certain embodiments, microRNA is abbreviated as "miR".

[0089] An "oligomeric compound" refers to a compound containing multiple linked monomer subunits. Oligomer compounds include oligonucleotides.

[0090] An "oligonucleotide" refers to a compound containing multiple linked nucleosides, each of which may be modified or unmodified independently of the others.

[0091] "Naturally occurring internucleoside bonds" refers to phosphodiester linkages between nucleosides from 3' to 5'.

[0092] "Natural sugars" refers to sugars found in DNA (2'-H) or RNA (2'-OH).

[0093] "Nucleoside bond" refers to a covalent bond between adjacent nucleosides.

[0094] "Linked nucleosides" refers to nucleosides that are linked together by covalent bonds.

[0095] A "nucleic acid base" refers to a heterocyclic portion that can pair with another nucleic acid base in a non-covalent bond.

[0096] "Nucleoside" refers to a nucleic acid base linked to a sugar portion.

[0097] A "nucleotide" refers to a nucleoside that has a phosphate group covalently bonded to the sugar portion of the nucleoside.

[0098] A compound containing a modified oligonucleotide consisting of multiple linked nucleosides means a compound containing a modified oligonucleotide having a specific number of linked nucleosides. Therefore, the compound may contain additional substituents or conjugates. Unless otherwise specified, the compound does not contain any additional nucleosides beyond those of the modified oligonucleotide.

[0099] A "modified oligonucleotide" refers to a single-stranded oligonucleotide that has one or more modifications compared to naturally occurring terminal, sugar, nucleic acid base, and / or nucleoside-to-nucleoside bonds. Modified oligonucleotides may contain unmodified nucleosides.

[0100] A "modified nucleoside" refers to a nucleoside that has undergone any modification from a naturally occurring nucleoside. Modified nucleosides may have modified sugars and unmodified nucleic acid bases. Modified nucleosides may have modified sugars and modified nucleic acid bases. Modified nucleosides may have natural sugars and modified nucleic acid bases. In certain embodiments, the modified nucleoside is a bicyclic nucleoside. In certain embodiments, the modified nucleoside is a non-bicyclic nucleoside.

[0101] "Modified nucleoside bonds" refer to any changes from naturally occurring nucleoside bonds.

[0102] "Phosphothioate nucleoside bond" refers to a bond between nucleosides where one of the non-bridged atoms is a sulfur atom.

[0103] The term "modified sugar portion" refers to substitution and / or any alteration from natural sugars.

[0104] "Unmodified nucleic acid bases" refer to naturally occurring heterocyclic bases of RNA or DNA. Purines are based on adenine (A) and guanine (G), while pyrimidines are based on thymine (T), cytosine (C) (such as 5-methylcytosine), and uracil (U).

[0105] "5-methylcytosine" refers to cytosine containing a methyl group attached to the 5th position of the cytosine ring.

[0106] "Non-methylated cytosine" refers to cytosine that does not have a methyl group attached to the 5th position of the cytosine ring.

[0107] "Modified nucleic acid base" refers to any nucleic acid base that is not an unmodified nucleic acid base.

[0108] The term "sugar portion" refers to naturally occurring furanosyl or modified sugar portions.

[0109] "Modified sugar portion" refers to the substituted sugar portion or sugar substitute.

[0110] "2'-O-methyl sugar" or "2'-OMe sugar" refers to a sugar that has an O-methyl modification at the 2' position.

[0111] "2'-O-methoxyethyl sugar" or "2'-MOE sugar" refers to a sugar that has an O-methoxyethyl modification at the 2' position.

[0112] "2'-Fluoro" or "2'-F" refers to a sugar that has a fluoro modification at the 2' position.

[0113] A “bicyclic sugar moiety” refers to a modified sugar moiety containing a 4- to 7-membered ring (including, but not limited to, a furanosyl) that includes a bridge that combines two atoms of a 4- to 7-membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4- to 7-membered ring is a sugar ring. In certain embodiments, the 4- to 7-membered ring is a furanosyl. In certain such embodiments, the bridge combines the 2'-carbon and 4'-carbon of the furanosyl. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

[0114] The term "locked nucleic acid (LNA) sugar moiety" refers to a substituted sugar moiety that contains a (CH2)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0115] The term "ENA sugar portion" refers to the substituted sugar portion that contains a (CH2)2-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0116] The term "restricted ethyl (cEt) sugar moiety" refers to a substituted sugar moiety containing a CH(CH3)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. In certain embodiments, the CH(CH3)-O bridge is restricted to S orientation. In certain embodiments, the CH(CH3)-O bridge is restricted to R orientation.

[0117] The term "S-cEt sugar moiety" refers to a substituted sugar moiety that contains an S-restricted CH(CH3)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0118] The term "R-cEt sugar moiety" refers to a substituted sugar moiety that includes an R-constrained CH(CH3)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0119] "2'-O-methyl nucleoside" refers to a 2'-modified nucleoside that has a 2'-O-methyl sugar modification.

[0120] "2'-O-methoxyethyl nucleoside" refers to a 2'-modified nucleoside having a 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleosides may contain modified or unmodified nucleic acid bases.

[0121] "2'-Fluoronucleoside" refers to a 2'-modified nucleoside that has a 2'-fluorosaccharide modification. 2'-Fluoronucleosides may contain modified or unmodified nucleic acid bases.

[0122] A "bicyclic nucleoside" refers to a 2'-modified nucleoside that has a bicyclic sugar moiety. Bicyclic nucleosides can have modified or unmodified nucleic acid bases.

[0123] "cEt nucleoside" refers to a nucleoside containing a cEt sugar moiety. cEt nucleosides may contain modified or unmodified nucleic acid bases.

[0124] "S-cEt nucleoside" refers to a nucleoside that contains an S-cEt sugar moiety.

[0125] "R-cEt nucleoside" refers to a nucleoside that contains the R-cEt sugar moiety.

[0126] "β-D-deoxyribonucleoside" refers to a naturally occurring DNA nucleoside.

[0127] "β-D-ribonucleoside" refers to a naturally occurring RNA nucleoside.

[0128] "LNA nucleoside" refers to a nucleoside that contains the LNA sugar portion.

[0129] "ENA nucleoside" refers to a nucleoside that contains the ENA sugar portion.

[0130] A "hydrogen bond receptor" refers to a hydrogen bond component that does not supply a shared hydrogen atom.

[0131] A "hydrogen bond donor" refers to a bond or molecule that supplies hydrogen atoms for a hydrogen bond.

[0132] overview Anti-miR-17 compounds have previously been found to be useful in the treatment of polycystic kidney disease (PKD). Screening for optimal pharmaceutically acceptable properties of a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides identified compound RGLS4326 (or RG4326) as a candidate for clinical development for the treatment of human PKD. Compound RG4326, its manufacturing method, in vitro and in vivo data demonstrating its efficacy in PKD models, and its metabolic stability, pharmacokinetic, and safety profiles are described in WO2018 / 106566A1, which is incorporated herein by reference in its entirety.

[0133] Following the pursuit of clinical development and consultations with the FDA, it was shown that the dose / duration limit for RG4326 could be far below 1 mg / kg for long-term chronic administration. However, after clinical trials to study RG4326 in humans for use in the treatment of PKD were initiated, CNS-related findings were observed with high doses of RG4326 in a non-clinical toxic mouse model. RG4326 was found to be an antagonist of AMPAR, a glutamate receptor and ion channel at excitatory synapses in the central nervous system (CNS). AMPAR mediates rapid excitatory neurotransmission and is a crucial component of all neural networks.

[0134] Surprisingly, the AMPAR antagonist activity of the compounds provided herein, including the compounds in Table 1, which had previously been considered undesirable candidates for the advancement of PKD drug development, has now been found to be beneficial in advancing the clinical development of these compounds and other compounds for use in the treatment of neurological disorders, including epilepsy.

[0135] The compounds provided herein, including those listed in Table 1, are competitive inhibitors of AMPAR and have therefore been found to be useful in the treatment of neurological disorders. compound

[0136] This specification provides compounds comprising modified oligonucleotides consisting of 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, or 4-15 linked nucleosides, or pharmaceutically acceptable salts thereof. In certain embodiments, the modified oligonucleotide consists of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides.

[0137] In certain embodiments, the last four nucleosides at the 3' end of the modified oligonucleotide have the nucleic acid base sequence UUUG.

[0138] In certain embodiments, at least two of the last four nucleosides at the 3' end of the modified oligonucleotide are bicyclic nucleosides. In certain embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleosides of the modified oligonucleotide are bicyclic nucleosides. In certain embodiments, each bicyclic nucleoside is independently selected from S-cEt nucleosides, LNA nucleosides, and ENA nucleosides.

[0139] In certain embodiments, at least one nucleoside bond of the modified oligonucleotide is a modified nucleoside bond. In certain embodiments, the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0140] In certain embodiments, the modified oligonucleotide consists of four linked nucleosides. In certain embodiments, the modified oligonucleotide is UUUG. In some embodiments, the modified oligonucleotide is U F U M U S G Sor U S U S U S G S Therefore, the nucleoside following the subscript "M" is the 2'-O-methyl nucleoside, the nucleoside following the subscript "F" is the 2'-fluoro nucleoside, and the nucleoside following the subscript "S" is the S-cEt nucleoside.

[0141] In certain embodiments, the modified oligonucleotide consists of nine linked nucleosides. In certain embodiments, the modified oligonucleotide contains the nucleic acid sequence 5'-AGCACUUUG-3', and each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine.

[0142] In certain embodiments, the modified oligonucleotide has the following nucleoside pattern oriented from 5' to 3'. N S N S N M N F N F N F N M N S N S It has, Nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, and nucleosides followed by the subscript "S" are S-cEt nucleosides.

[0143] In certain embodiments, the compound consists of a modified oligonucleotide.

[0144] In certain embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0145] In a particular embodiment, the modified oligonucleotide is RG4326, and has the following sequence and chemical modification pattern: A S G S C M A F C F UF U M U S G S It has the following characteristics. However, nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, nucleosides followed by the subscript "S" are S-cEt nucleosides, each cytosine is unmethylated cytosine, and all bonds are phosphorothioate bonds.

[0146] RG4326 can be further described as having the following structure: [ka]

[0147] This specification provides modified oligonucleotides having the following structure. [ka]

[0148] In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide has fewer cationic counterions (Na) per molecule than phosphorothioate and / or phosphodiester bonds. + (e.g., some phosphorothioate and / or phosphodiester bonds are protonated). In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide contains fewer than eight cationic counterions (Na) per molecule of the modified oligonucleotide. + This includes, for example, pharmaceutically acceptable salts of modified oligonucleotides may, on average, contain 1, 2, 3, 4, 5, 6, or 7 cationic counterions per molecule of the modified oligonucleotide, with the remaining phosphorothioate and / or phosphodiester bonds being protonated.

[0149] Table 1 below shows some specific, non-limiting, exemplary compounds provided herein. TIFF2026510893000006.tif147170

[0150] How to use A method is provided for treating a target neurological disorder, the method comprising administering a therapeutically effective amount of a compound provided herein (including the compounds in Table 1), which includes a modified oligonucleotide that binds to and inhibits AMPA, or a pharmaceutically acceptable salt thereof, to the target. In some embodiments, the neurological disorder is epilepsy.

[0151] In some embodiments, the neurological disorder is drug-resistant epilepsy. In other embodiments, the neurological disorder is non-drug-resistant epilepsy. In some embodiments, the compounds provided herein are used as a first-line treatment.

[0152] In some embodiments, the compounds provided herein are used to treat central or peripheral nervous system disorders in a subject. In some embodiments, the nervous system disorders include hearing loss, motor disorders, amyotrophic lateral sclerosis (ALS), pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke, or glioblastoma.

[0153] In one embodiment, the compounds provided herein are formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable diluents or excipients. In some embodiments, the compounds provided herein are formulated into physiological saline. In some embodiments, the pharmaceutical composition is a lyophilized composition.

[0154] In some embodiments, the compounds provided herein are any one of the compounds listed in Table 1.

[0155] In any of the embodiments provided herein, the subject is a human subject. In certain embodiments, the human subject is an adult. In certain embodiments, an adult is at least 18 years of age. In certain embodiments, the human subject is a child subject, i.e., the subject is under 18 years of age. The child population may be defined by a regulatory body. In certain embodiments, the human subject is an adolescent. In certain embodiments, an adolescent is at least 12 years of age and under 18 years of age. In certain embodiments, the human subject is a child. In certain embodiments, a child is at least 2 years of age and under 12 years of age. In certain embodiments, the human subject is an infant. In certain embodiments, an infant is at least 1 month of age and under 2 years of age. In certain embodiments, the subject is a newborn. In certain embodiments, a newborn is under 1 month of age.

[0156] Any of the compounds provided herein may be used for therapeutic purposes.

[0157] Any of the compounds provided herein may be used for the preparation of pharmaceuticals. Any of the compounds provided herein may be used for the preparation of pharmaceuticals for the treatment of neurological disorders, preferably epilepsy.

[0158] Any of the modified oligonucleotides provided herein may be used for the preparation of pharmaceuticals. Any of the modified oligonucleotides provided herein may be used for the preparation of pharmaceuticals for the treatment of neurological disorders.

[0159] Any of the pharmaceutical compositions provided herein may be used for the treatment of neurological disorders.

[0160] Specific additional therapies Treatment of any neurological disorder or any of the conditions enumerated herein may comprise two or more therapies. Accordingly, in certain embodiments, methods for treating subjects having or suspected of having a neurological disorder are provided herein, which comprises administering at least one therapy in addition to administering an AMPAR-mediated compound provided herein.

[0161] In certain embodiments, at least one additional therapy comprises a pharmaceutical agent. In certain embodiments, the additional pharmaceutical agent is an antiepileptic drug (AED). In certain embodiments, the AED is selected from carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, everolimus, gabapentin, lacosamide, lamotrigine, levetiracetam, oxycarbazepine, perampanel, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, sodium valproate, stiripentol, thiagabine, topiramate, vigabatrin, and zonisamide.

[0162] Specific pharmaceutical compositions Pharmaceutical compositions comprising compounds or modified oligonucleotides provided herein and pharmaceutically acceptable diluents are provided herein. In certain embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In certain embodiments, the aqueous solution is physiological saline. As used herein, the pharmaceutically acceptable diluent is understood to be a sterile diluent. Preferred routes of administration include, but are not limited to, intravenous and subcutaneous administration. In certain embodiments, administration is intravenous. In certain embodiments, administration is subcutaneous. In certain embodiments, administration is oral.

[0163] In certain embodiments, the pharmaceutical composition is administered in the form of a drug unit (e.g., a tablet, capsule, bolus, etc.). For example, in certain embodiments, the drug unit is in the form of a tablet, capsule, or bolus injection.

[0164] In certain embodiments, the pharmaceutical preparation is a modified oligonucleotide, which is prepared with a suitable diluent, adjusted to pH 7.0–9.0 with an acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified oligonucleotide is then reconstituted with a suitable diluent, such as an aqueous solution like water, or a physiologically compatible buffer such as saline, Hanks' solution, or Ringer's solution. The reconstituted product is administered by subcutaneous injection or intravenous infusion. The lyophilized pharmaceutical preparation may be packaged in a 2 mL clear glass vial (ammonium sulfate treated), stoppered with a bromobutyl rubber closure, and sealed with an aluminum overseal.

[0165] In certain embodiments, the pharmaceutical compositions provided herein may further contain other auxiliary components conventionally found in pharmaceutical compositions at their established levels of use in the art. Thus, for example, a composition may contain additional suitable pharmaceutically active materials, such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents.

[0166] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. Such additional materials also include, but are not limited to, excipients such as alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In various embodiments, if such materials are added, they should not excessively interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if necessary, mixed with adjuvants that do not adversely interact with the oligonucleotide(s) of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, fragrances, and / or aromatic substances. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent, enabling the preparation of highly concentrated solutions.

[0167] Lipid moieties are used in nucleic acid therapy in various ways. In one method, nucleic acids are introduced into pre-formed liposomes or lipoplexes made from a mixture of cationic and neutral lipids. In another method, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to muscle tissue.

[0168] In certain embodiments, the pharmaceutical compositions provided herein include a lipid moiety complexed with a polyamine compound or nucleic acid. In certain embodiments, such preparations include one or more compounds having individually defined structures by formula (Z) or pharmaceutically acceptable salts thereof. [ka] In the formula, each a and X b C is independent of each occurrence. 1~6 It is an alkylene, where n is 0, 1, 2, 3, 4, or 5, each R independently is H, and at least about 80% of the R portions of the compound of formula (Z) in the preparation, at least n+2 are not H, m is 1, 2, 3, or 4, and Y is O, NR 2 , or S, R 1 is alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents, R 2is H, alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents, except that if n is 0, at least n+3 R moieties are not H. Such preparations are described in PCT Publication WO / 2008 / 042973, which is incorporated herein by reference in its entirety for the disclosure of lipid preparations. Certain additional preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (May 1, 2008), which is incorporated herein by reference in its entirety for the disclosure of lipid preparations.

[0169] In certain embodiments, the pharmaceutical compositions provided herein are prepared using known techniques including, but not limited to, mixing, dissolving, granulation, herb preparation, granulation, granulation, granulation, emulsification, encapsulation, embedding, or tableting processes.

[0170] In certain embodiments, the pharmaceutical compositions provided herein are solid (e.g., powders, tablets, and / or capsules). In certain embodiments of such embodiments, the solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using components known in the art, but are not limited to starches, sugars, diluents, granulators, lubricants, binders, and disintegrants.

[0171] In certain embodiments, the pharmaceutical compositions provided herein are formulated as depot formulations. Certain such depot formulations typically exhibit a longer-acting effect than non-depot formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, the compounds of the present invention can be formulated as suitable polymers or hydrophobic materials (e.g., emulsions in acceptable oils) or ion-exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0172] In certain embodiments, the pharmaceutical compositions provided herein include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions that include a hydrophobic compound. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0173] In certain embodiments, the pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the present invention to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0174] In certain embodiments, the pharmaceutical compositions provided herein include sustained-release systems. Non-limiting examples of such sustained-release systems are semipermeable matrices of solid hydrophobic polymers. In certain embodiments, depending on their chemical properties, sustained-release systems may release the pharmaceutical agent for hours, days, weeks, or months.

[0175] Certain pharmaceutical compositions for injection are provided in unit dosage forms, for example, in ampoules or in multi-dose containers.

[0176] In certain embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of modified oligonucleotide. In certain embodiments, a therapeutically effective amount is sufficient to prevent, alleviate, or improve the symptoms of a disease, or to extend the survival time of the subject being treated.

[0177] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to a more biologically, pharmacokinetically, or therapeutically active form of the oligonucleotide. In certain embodiments, the prodrug is useful because it is easier to administer than the corresponding active form. For example, in certain cases, the prodrug may be more biologically available (e.g., by oral administration) than the corresponding active form. In certain cases, the prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrug is less water-soluble than the corresponding active form. In certain cases, such a prodrug has excellent permeability across cell membranes where water solubility impairs mobility. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In certain cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, the prodrug comprises a short peptide (polyamino acid) bonded to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0178] In certain embodiments, prodrugs are produced by modifying a pharmaceutically active compound so that the active compound is regenerated upon in vivo administration. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, mask side effects or toxicity, improve the flavor of a drug, or alter other properties or characteristics of a drug. With knowledge of pharmacodynamic processes and in vivo drug metabolism, those skilled in the art can design prodrugs of a compound if the pharmaceutically active compound is known (see, for example, Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pages 388–392).

[0179] Preferred routes of administration include, but are not limited to, oral administration, rectal administration, transmucosal administration, intestinal administration, enteral administration, topical administration, suppository administration, inhalation administration, intrathecal administration, intraventricular administration, intraperitoneal administration, intranasal administration, intraocular administration, intratumoral administration, and parenteral administration (e.g., intravenous, intramuscular, intrathecal, and subcutaneous). In certain embodiments, a pharmaceutical intrathecal agent is administered to achieve local exposure rather than systemic exposure. For example, a pharmaceutical composition may be injected directly into the desired site of action (e.g., the kidney).

[0180] Specific kit The present invention also provides kits. In some embodiments, the kit comprises one or more compounds, including modified oligonucleotides disclosed herein. In some embodiments, the kit may be used to administer the compounds to a target.

[0181] In certain embodiments, the kit includes a pharmaceutical composition ready for administration. In some embodiments, the pharmaceutical composition is contained in vials. Multiple vials (e.g., 10) may be contained in, for example, a dispensing pack. In some embodiments, the vials are manufactured for use with syringes. The kit may also include instructions for using the compound.

[0182] In some embodiments, the kit includes a pharmaceutical composition in a pre-filled syringe (e.g., a single-dose syringe with a 27-gauge, 1 / 2-inch needle and needle guard) rather than in a vial. Multiple (e.g., 10) pre-filled syringes may be present, for example, in a dispensing pack. The kit may also include instructions for administering compounds containing the modified oligonucleotides disclosed herein.

[0183] In some embodiments, the kit contained the modified oligonucleotides provided herein as a lyophilized drug product and a pharmaceutically acceptable diluent. In preparation for administration to a subject, the lyophilized drug product is reconstituted in the pharmaceutically acceptable diluent.

[0184] In some embodiments, in addition to the compounds containing the modified oligonucleotides disclosed herein, the kit may further include one or more of the following: a syringe, an alcohol swab, a cotton ball, and / or a gauze pad. [Examples]

[0185] The following embodiments are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the present invention.

[0186] Those skilled in the art will readily adopt the principles underlying this discovery to design various compounds without departing from the spirit of the present invention.

[0187] Abbreviation In this specification, the following abbreviations may be useful when considering the following examples. TIFF2026510893000008.tif170170

[0188] Example 1 Example 1A: AMPAR coupling The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutically appropriate properties. RGLS4326 preferentially distributes to renal and vascular cysts, displaces miR-17 from translationally active polysomes, and desuppresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in a human in vitro ADPKD model and multiple PKD mouse models after subcutaneous administration. A Phase 1 single-dose elevation (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a Phase 1 multiple-dose elevation (MAD) clinical trial in healthy volunteers in May 2018. A Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) commenced in October 2020.

[0189] Nonclinical toxicity studies following the initiation of the Phase 1 MAD clinical trial revealed central nervous system (CNS) related findings, including abnormal gait, decreased motor activity, and / or debilitation, with high doses of RGLS4326. To identify potential off-target pharmacology candidates, a panel of 174 targets, including G protein-coupled receptors, transporters, ion channels, nuclear receptors, and cytokine receptors, was evaluated in vitro for possible interactions with RGLS4326. RGLS4326 had a 50% inhibitory concentration (IC50) of 4.6 uM (14.2 ug / mL) based on ligand binding, and a functional IC50 of 300–600 nM (0.9–1.8 ug / mL) based on patch-clamp activity. 50 It was found to be an antagonist of the AMPA glutamate receptor. The AMPA receptor is an ion channel on excitatory synapses in the CNS, mediating rapid excitatory neurotransmission and therefore a crucial component of all neural networks. Such interaction with the AMPA receptor can explain the CNS-mediated findings observed with high doses of RGLS4326 in nonclinical toxicity models.

[0190] Example 1B: Compound having AMPAR antagonist activity The activity of anti-miR-17 compounds was increased in the presence of higher concentrations of anti-miR-17 compounds to AMPAR present on rat brain synaptic membranes. 3 The binding of the [H]AMPA ligand was evaluated by a radioligand binding assay. Anti-miR-17 compounds with affinity for AMPAR were evaluated by [ 3 It binds to the [H]AMPA ligand and also competes for the binding of this ligand.

[0191] The assay was performed according to previously published methods (Honore et al., J Neurochem., 1982, 38(1):173-178; Olsen et al., Brain Res., 1987, 402(2):243-254). 5.0 nM ligand [ 3 [H]AMPA, 1.0 mM nonspecific ligand L-glutamic acid, and uM concentration anti-miR compounds were incubated with synaptic membranes prepared from Wistar rat cerebral cortex for 90 minutes. The compounds shown in Table 2 were tested in three experiments. Anti-miR compounds targeting microRNAs other than miR-17 were used as control compounds (RG5124 targeting miR-33a; RG5365 targeting let-7a; RG8093 targeting miR-214). RGLS4326 and RG-NG-1001 were also tested in each experiment because they were demonstrated to bind to AMPAR and inhibit its activity. 3 The amount of [H]AMPA ligand was quantified by radioactive ligand binding and is shown in Tables 3, 4, and 5. As the data shows, the compounds differ in their ability to inhibit the binding of the radiolabeled ligand to AMPAR. TIFF2026510893000009.tif72170TIFF2026510893000010.tif80170TIFF2026510893000011.tif62170

[0192] As shown in Tables 3-5 above, AMPAR binding was further profiled using a series of shortened-length sequences (i.e., 4-nucleotide "tetramers") based on data obtained using anti-miR-17 compounds in radioligand binding assays. In these studies, RGLS4326 was included as a positive control for effective AMPAR-R binding. RG-NH-1026 was included as a negative control for the absence of effective AMPAR binding. As shown in Table 6, RG8524 and RG8525 demonstrated the ability to inhibit the binding of radiolabeled ligands to AMPAR, and RG8525 met the significance criteria of the assay (≥50% inhibition; NC = not calculated (IC)). 50 Because it does not meet the standards. TIFF2026510893000012.tif82170

[0193] To evaluate the functional antagonistic effects of anti-miR-17 oligonucleotides against AMPAR, specific oligonucleotides were tested using manual whole-cell patch-clamp technique, which records membrane current as a measure of AMPAR activity.

[0194] Manual whole-cell patch-clamp studies were performed by Metrion Biosciences (Cambridge, UK). Whole-cell voltage clamp experiments were conducted at room temperature (18–21°C) using an EPC10 patch-clamp amplifier with Patchmaster software (HEKA Elektronik). Glass patch pipettes were prepared from borosilicate glass capillaries (Harvard Apparatus) to resistances of 1.4–2.5 MΩ. Membrane currents were recorded using the whole-cell patch-clamp technique. ChanTest® GluA1 / GluA4 EZCells were clamped at a holding potential of -80 mV, and membrane currents were induced by 10 μM(S)-AMPA delivered using a VC38 perfusion system (ALA Scientific Instruments). The minimum current amplitude was measured after each application of 10 μM(S)-AMPA. The fractional changes in current amplitude generated by each compound concentration were calculated and compared to the control current (prior compound), and expressed as the percentage change (% inhibition) for each cell. The compounds tested are shown in Table 7. TIFF2026510893000013.tif62170

[0195] RG4326 is transmitted from the rat cerebral cortex to the synaptic membrane. 3 We discovered that it inhibits the binding of [H]AMPA ligand and inhibits whole-cell patch-clamp studies of HEK293 cells that overexpress rGluA1 and rGluA2 in response to 3 mM glutamate.

[0196] Therefore, RGLS4326 has a 50% inhibitory concentration (IC) of 4.6 μM (14.2 μg / mL) based on ligand binding. 50 ), and based on patch-clamp activity, functional IC50 with a concentration of 300-600 nM (0.9-1.8 ug / mL) 50 We found that it possesses AMPAR's antagonist properties.

[0197] Example 2 Maximum Tolerable Dose (MTD) Pilot Study and Comparative Dose Evaluation The compounds were evaluated using the maximum tolerated dose (MTD) and pentylenetetrazole (PTZ) seizure models. The experiment was conducted in two parts. Part 1 (Example 2A) involved pilot studies of RG4047 and RG4326 at four dose levels each (32 mice) at the maximum tolerated dose (MTD), and included PTZ dose determination (16 mice). Part 2 (Example 2B) involved PTZ studies of RG4047 and RG4326 at two dose levels each (90 mice). Negative controls (non-AMPAR-bound compounds) were included in both parts of the study, and perampanel was used as the reference group.

[0198] Each study used 6-7 week old C57Bl / 6J male mice (Jackson Laboratories). Mice were randomly assigned to the treatment group, and the studies were blinded. The animals were allowed to acclimate for at least 5 days and housed in a 12-hour light / dark cycle (lights on at 7am). No more than 4 mice were housed in each cage (i.e., ventilated cage rack system). Standard rodent feed and water were provided freely.

[0199] Example 2A - MTD Pilot Test The following parameters were used in this study. 1. Route of administration (multiple routes possible): ICV administration of RG4047 and RG4326; IP administration of PTZ. 2. Dosage volume (multiple options allowed): For RG compounds, 4 µl, 5 ml / kg (PTZ) 3. Preparations (multiple preparations are possible): Vehicle, Ca 2+ and Mg 2+ Free dPBS 4. Dose levels: PTZ was tested at 60, 65, and 75 mg / kg. 5. Number of doses: 1 6. Exam period: 8 days 7. Number of groups: 14 8. Number of animals per group: Groups 1-12 (4 animals each), Groups 13-14 (8 animals each). 9. Total number of animals: 64

[0200] For ICV administration, mice were anesthetized and positioned for injection. An incision was made in the skin above the skull, and a microdrill was used to create a small hole in the skull above the target. Stereotactic coordinates for injection into both the right and left ventricles were -0.4 mm anterior-posterior (AP), + / - 1.0–1.5 mm medial-lateral (ML), and -3.0 mm dorsoventricular (DV) from the bregma (Hironaka et al, 2015). In the animals, 4 μl was injected unilaterally into the right ventricle. The compound was injected over 1–2 minutes, and the needle was left in place for 0.5–1 minute before removal. The incision was closed with sutures, wound clips, or VetBond.

[0201] The design of this study is further illustrated in Tables 8A and 8B below. TIFF2026510893000014.tif207170TIFF2026510893000015.tif131170

[0202] For groups 1-12, animals were monitored for 7 days after ICV treatment (day 0), and daily health checks, weight, and mortality rates were recorded. On day 7, the brain and kidneys were collected, fixed (in 10% formalin), and stored while awaiting histological examination.

[0203] For groups 13-14, the vehicle was administered to the right ventricular ventricle of the mice on day 0. 24 hours after ICV administration, PTZ was injected at a rate of 5 ml / kg as the final dose. Immediately after PTZ administration, the animals were observed for 30 minutes. In this case, the time to the initial myoclonus response and the tonic hind limb extensor response was measured. 30 minutes. The maximum latency to seizure duration was imposed.

[0204] Racine Scale: Clinical behavior was scored using the following adaptive Racine scale: Score 0, no seizures observed; Score 1, paralysis (freezing, staring, glaring), spasms of the trichomes, clonus of the mouth or face. Score 2, sudden jump, tail lift, legs spread, head nod Score 3, forelimb clonus, forelimb extension, multiple sudden movements or pulls Score 4, standing and bilateral forelimb clonus, running and running / bouncing Score 5, total limb clonus, standing and falling (falling backward) Score 6: Ankylous hind limb extension, cardiac arrest

[0205] Behavior was scored by blinded observers. The highest score reached was recorded every 5 minutes for 30 minutes after PTZ administration.

[0206] The data were analyzed using one-way or two-way ANOVA followed by Dunnett's test or Fisher LSD post-hoc test to determine the difference between treatments. Data are expressed as mean ± SEM. Comparisons between the Peramp panel reference group and the vehicle group were analyzed using t-tests. A p-value of less than 0.05 was used to indicate statistical significance.

[0207] The results of the MTS studies for groups 1-12 are shown in Table 9 below. The negative control compound showed good tolerability at high doses (consistent with previous ICV studies). In the pilot study, the MTD for RG4326 was predicted to be approximately 2.5 < 5.0 μg ICV. Mortality was observed at high doses of RG4326 and RG4047 (e.g., 600, 300, 100 ug), so the MTD dosage parameters were modified accordingly. The MTD was not achieved for RG4047 and was therefore likely less than 2.5 ug. All animal deaths were reported to occur within the first 5-8 hours after ICV infusion. Mice injected with 2.5 ug of RG4326 were reported to show some immediate signs of respiratory distress, and a heating pad was provided. All animals were reported to have fully recovered by day 2 of observation. The results of previous studies support the good tolerability of ICV doses of the negative control compound. Based on this data, this model selected 2.0 ug as the maximum dose of RG4326. TIFF2026510893000016.tif86170

[0208] Example 3 PTZ optimal dose pilot study To determine the optimal dose of PTZ for use in this experiment, pilot studies were conducted considering PTZ (IP) at doses of 60, 65, and 70 mg / kg. In these pilot studies, animals were administered PTZ three times and then observed at 5-minute intervals for 30 minutes using the Racine scale. Behavior was scored by blinded observers. The highest score achieved at 5-minute intervals during the 30-minute observation period was recorded. Immediately after PTZ injection (time 0), the time from latency to the onset of clonic seizures was assessed, and signs of tonic seizures in the animals were monitored from there. Clinical behavior was recorded at 5-minute intervals for 30 minutes.

[0209] Figures 1A and 1B show the latency to seizure data (clonic and tonic seizures, respectively) for three doses: 60 mpk, 65 mpk, and 70 mpk. As shown in Figures 1A and 1B, PTZ induced the rapid onset of clonic seizures (Figure 1A) and tonic seizures (Figure 1B) at all doses. However, mortality (shown in Figure 1B) showed an increasing trend with higher doses (70 mpk). In comparison, no deaths were reported at the lowest test dose of 60 mpk.

[0210] Figure 2 shows the time course of the Racine score after PTZ administration in this experiment. As shown in Figure 2, the lowest dose of PTZ, 60 mpk, was able to induce clonic / tonic seizures, but the seizure intensity rapidly subsided 5 minutes after the first PTZ dose. Conversely, PTZ of 70 mpk induced a higher mean Racine score, which was consistent with the desired result of an increased incidence of tonic seizures in the model.

[0211] Figure 3 is a bar graph showing the survival data in this experiment. The final survival data comparing the effects of 60mpk, 65mpk, and 70mpk doses 30 minutes after PTZ treatment revealed that the survival rate decreased with 70mpk, with 4 out of 6 injected mice surviving, compared to 6 out of 6 mice with 60mpk and 4 out of 5 mice with 65mpk.

[0212] Based on this data, a 70 mpk PTZ dose was selected as the appropriate effective concentration for inducing a sufficient seizure profile and increasing the mortality risk in the model. Higher doses of PTZ (e.g., 80 mpk, 100 mpk) have been profiled in previous studies and found to have an excessively strong impact on seizure latency, racine scoring, and, most importantly, 30-minute survival.

[0213] Example 4 PTZ test of RG4326 compared to Perampanel The effects of the test compound RG4326 on the CNS were compared with those of a negative control compound, a vehicle, and perampanel. Seizures were induced in test mice by administering PTZ at a dose of 70 mpk as determined in Example 3. The mouse groups were pre-treated with RG4326, NCC, and perampanel (or untreated in the vehicle group) before PTZ administration, and data were then collected on the effect of the test drug (compared to the vehicle) on the CNS effects from PTZ-induced seizures. The results of this PTZ study demonstrated that RG4326 exhibited significant anti-seizure effects across all major study endpoints, including seizure and cardiac arrest latency, Racine scale scoring, and overall survival.

[0214] The effect of RG4326 on the CNS by Racine scoring, compared to perampanel or a negative control compound (NCC), was evaluated using the following parameters with the PTZ dose of 70 mpk determined in Example 3. 1. Route of administration (multiple routes possible): ICV RG4326 and NCC; PTZ (IP); Perampanel (PO) 2. Dosage volume (multiple options allowed): 10 ml / kg (perampanel), test substance 4 uL, 5 ml / kg (PTZ) 3. Preparation(s): dPBS; PER vehicle physiological saline 4. Dosage levels: Perampanel, 2 mg / kg; PTZ, 70 mg / kg 5. Number of doses: 1 6. Exam period: 2 days 7. Number of animals per group: 15 8. Number of groups: 8 9. Total number of animals: 120

[0215] The design of this study is further illustrated in Table 10 below. As shown in Table 10, the test drugs RG4326 and NCC were administered to all mice in groups 2-7 by ICV administration 24 hours prior to PTZ administration, at the doses shown in Table 10. Group 1 received only sterile vehicle PBS without any pretreatment. Group 8 received approximately 2 mg / kg of perampanel as a reference compound 30 minutes prior to oral (PO) administration of PTZ. ICV administration of the test drugs was carried out as described in Example 2A above.

[0216] To evaluate the effects on the central nervous system (CNS), mice were first acclimated to the treatment room for at least 30 minutes. Pentylenetetrazole (PTZ; Sigma Aldrich) was formulated in water at a concentration of 16 mg / ml. To induce seizures, PTZ was administered by injection at a volume of 5 ml / kg, with a final dose of 70 mg / kg (IP). Immediately after PTZ administration, the animals were observed for 30 minutes, and the latency of clonic and tonic hindlimb extension responses, as well as Racine scale scoring, were recorded to compare the effects of RG4326, NCC, and perampanel on PTZ-induced seizures. TIFF2026510893000017.tif93170

[0217] Latency to tonic seizure. Comparative latency data to tonic seizure obtained from this study are reported in Figure 4. Figure 4 shows the mean duration (seconds) of the period from latency to the onset of tonic seizure in all groups. As shown in Figure 4, animals administered perampanel (reference compound) at a concentration of 2 mg / kg showed a significant increase in the duration from latency to tonic seizure compared to animals treated with a vehicle (****P<0.0005, 1-week ANOVA, Dunnett). The RG4326 treatment group showed significant differences (*P<0.05) at 0.25 and 0.5 ug compared to the vehicle. Despite the tendency for latency to increase with additional treatment conditions, no significant difference was observed in the time span from latency to the onset of tonic seizure in any of the other groups compared to animals treated with a vehicle.

[0218] Latency to cardiac arrest. Comparative data on latency to cardiac arrest (e.g., death) obtained from this study are shown in Figure 5. Figure 5 reports the data from this study regarding latency comparisons to cardiac arrest (e.g., death). Figure 5 reports the average vertical bar graph of the time (seconds) from latency to tonic cardiac arrest for all groups. As shown in Figure 5, animals administered perampanel (reference compound) at a concentration of 2 mg / kg showed a significant increase in the time span from latency to tonic cardiac arrest compared to animals treated with a vehicle (***P<0.005, 1-week ANOVA, Dunnett). RG4326 at 0.5 ug showed significance (*P<0.05). Other treatment groups showed a tendency for an increase in the time span from latency to cardiac arrest (similar to the time from latency to tonic attack), but no significant difference was shown in the time from latency to cardiac arrest compared to animals treated with a vehicle.

[0219] Racine scoring. Racine scores for all groups obtained at 5-minute intervals up to 30 minutes after PTZ administration are reported in the line graph of Figure 6. When collecting the Racine scores in Figure 6, all animals with the endpoint of cardiac arrest were scored with the maximum score (6) for all remaining time points within the 30-minute observation interval. Animals administered perampanel (reference compound) at a concentration of 2 mg / kg showed a statistically significant decrease in Racine score at all time points after PTZ administration compared to animals treated with a vehicle (*P<0.05, 2-week ANOVA, Dunnett). Animals treated with RG4326 showed a significant decrease in Racine score in the 0.5 ug treatment group. Compared to animals treated with a vehicle, the other groups did not show any significant difference at any time point.

[0220] Time course of mortality after PTZ administration. Deaths occurring in all groups were recorded for each 5-minute observation period up to 30 minutes after PTZ administration. Survival data are reported in the line graph in Figure 7. Tables 11 and 12 below further report the total number of deaths at each interval and over time (Table 11) and the mortality rate based on the starting sample size for each cohort (Table 12). As can be seen from this data, survival rates after PTZ were clearly significantly improved with 2 mpk perampanel (reference compound) (Group 8 in Tables 11 and 12, and the line graph) and 0.5 ug of RG4326 (Group 4 in Tables 11 and 12, and the graph) (Group 8) (**P<0.005, log-rank, Mantel-Cox test). TIFF2026510893000018.tif113170TIFF2026510893000019.tif145170

[0221] In summary, the study in Example 4 tested RG4326's ability to provide protection against PTZ-induced seizures when administered by ICV injection 24 hours prior to PTZ administration. Based on pilot data aimed at establishing the MTD of RG4326 in this model, doses of 2.0 ug, 1.0 ug, 0.5 ug, and 0.25 ug were selected. Perampanel was selected as the reference compound and tested at 2 mg / kg (PO) based on published data on optimized efficacy in the PTZ model. Hanada et al, Epilepsia (2011) 52(7):1331-40. The results of this PTZ study demonstrated that RG4326 (0.5 ug ICV treatment) exhibited significant anti-seizure effects, impacting all key study endpoints, including seizure and cardiac arrest latency, Racine scale scoring, and overall survival.

[0222] Example 5 MTD trial for evaluation of RG4326 in a 6Hz seizure model Two MTD dose studies of RG4326 were conducted using two different mouse models to determine the dose to use when evaluating the compound in 6Hz-induced seizure experiments (Examples 6 and 7). Specifically, Study 5A used C57Bl / 6J male mice (Jackson Laboratories), 6-7 weeks old (same as Example 2), while Study 5B used Swiss:Rjorl male mice, 5 weeks old (Janvier).

[0223] In these studies (5A / 5B), mice were placed under isoflurane anesthesia (5% for induction, 2% for maintenance, <100% O2) and administered 5 mg / kg of sc-carprofen (Rimadyl®). They were then placed in a stereotactic frame. A midline sagittal incision was made in the scalp, and a hole was made in the skull above the left ventricle. A stainless steel cannula (outer diameter 0.51 mm) was stereotactically fixed into the left ventricle at the following coordinates: +0.5 posterior to bregma, L ± 0.7 mm, V = -2.7 mm. After a 2-minute delay to allow brain tissue to slide along the cannula, 4 μL of a solution containing 0.625 mg / mL of RG4326 was slowly infused over 2 minutes. After infusion, the cannula was left in place for an additional 5 minutes to prevent backflow of the solution along the cannula track.

[0224] Mice were administered 5 mg / g of sc-carprofen (Limadil®) 24 and 48 hours after surgery.

[0225] Mice were monitored 3 to 7 days after surgery (starting 24 hours after ICV administration), and their body weight was taken daily to check their health status. For mice monitored for 7 days, body weight was taken on day 1 and day 7 post-surgery to check their health status.

[0226] RG4326 was evaluated at four different doses in four distinct groups, as shown in Table 13 below. TIFF2026510893000020.tif57170

[0227] The combined results of both studies 5A and 5B demonstrated that RG4326 was generally well-tolerated in subjects. In study 5A, six mice were injected with 4 μL of a 0.625 mg / mL solution (total 2.5 μg per ICV). At the end of anesthesia, the mice remained lying on one side. The mice were quiet for the first few hours post-operatively, although they occasionally scratched. No toxic effects were observed in the six administered mice at 24, 48, or 72 hours. In study 5B, four mice were injected with four different doses of RG4326 (0.75, 1.0, 1.25, and 1.875 mg / mL, 4 μL). One mouse that received the highest dose (1.875 mg / mL, i.e., 7.5 μg / mouse) was found dead around 24 hours after ICV injection. All other mice remained in good health until the end of the pilot study (7 days post-administration). Table 14 summarizes the MTD data for RGLS4326 in the Study 5A and 5B mouse models. Based on these results, a 4ug MTD was selected for the 6Hz tests in Examples 6 and 7. TIFF2026510893000021.tif113170

[0228] Example 6 RG4326 in 6Hz seizure model #1 Examples 6 (and 7) tested RG4326 for its anticonvulsant effect in a 6Hz psychomotor seizure test in mice. The 6Hz psychomotor test method used in these examples followed the method described by Brown et al. (J. Pharmacol. Exp. Ther. 107, 273-283, 1953). A few drops of tetracaine solution (1%) were applied to each eye of the mouse for local anesthesia before transcorneal stimulation. After 1 to 10 minutes, the mice were administered a rectangular current (44mA, rectangular pulse: pulse width 0.2ms, duration 3 seconds, (6Hz)) via a corneal electrode connected to a constant current shock generator (Ugo Basile: type 7801).

[0229] In this example, six groups were used, with 15 mice in each group. Before the 6 Hz-induced seizures, as described in the previous paragraph, the mice were pretreated with three doses of RG4326 (24 hours before the test), with vehicle as a control and perampanel as a reference (60 minutes before the test). The design of this study is further described in Table 15 below. TIFF2026510893000022.tif76170

[0230] The mice were placed under isoflurane anesthesia (5% for induction, 2% for maintenance, under 100% O2), and 5 mg / kg of carprofen (Rimadyl®) was administered s.c. Then they were placed in a stereotaxic frame. A mid-sagittal incision was made in the scalp, and a hole was drilled in the skull above the left ventricle. A unilateral cannula (Plastics One, Phymep) was steadily placed in the left lateral ventricle using the following coordinates: 0.5 mm posterior to bregma, L ± 0.7 mm, V = -2.7 mm, and fixed on the skull using dental cement. After surgery, the transplanted mice were maintained in individual macrolon cages and allowed to recover for at least 5 - 7 days. The mice were administered 5 mg / kg of s.c. carprofen (Rimadyl®) 24 hours and 48 hours after surgery. In this Example 6, a total of 64 animals were transplanted over 2 days to obtain at least 15 mice per group.

[0231] Around 5 - 7 days after surgery, RG4326 was administered ICV to the mice in groups 4 - 6. These substances were slowly ICV injected over 2 - 4 minutes (1 - 2 μL / min). 24 hours after the RG4326 administration, a 6 Hz-induced seizure test was performed. For the mice in group 2 (perampanel), the reference compound (4 mg / kg) was administered orally 60 minutes before the 6 Hz seizure test.

[0232] The results for the number of seizures reflected by forelimb clonus and / or tail elevation were recorded immediately after the current administration. Seizures were recorded as either none (0 = no seizure) or present (1 = seizure - forelimb clonus or tail elevation).

[0233] Data from the tests of this Example 6 are reported in FIGS. 8 and 9. They report the percentage of animals in each group that showed forelimb clonus (FIG. 8) and tail - elevation (FIG. 9) seizures after 6 - Hz current administration. For the p.o. - administered neutral vehicle control (0.2% HPMC in distilled water) given 60 minutes before the test, the mean forelimb seizure score was 0.9 ± 0.1. For the vehicle control (PBS) administered ICV 24 hours before the test, the mean forelimb seizure score was 13 out of 15 mice, and for tail - elevation, 14 out of 15 mice. When RG4326 (0.0625, 0.125, and 0.5 mg / mL, i.e., 0.25, 0.5, and 2 μg / mouse) was administered ICV 24 hours before the test, a significant modification of the seizure score was seen at the highest dose of 2.0 μg compared to the vehicle control (Fisher's direct test, p < 0.05). Perampanel (4 mg / kg) administered p.o. 60 minutes before the test completely suppressed forelimb clonus and tail - elevation compared to the neutral vehicle control (p < 0.001). These results suggest that RG4326 has an antiseizure effect against 6 Hz, and the dose range tested was 0.0625 - 0.5 mg / mL (0.25, 0.5, and 2 μg / mouse) ICV, with the strongest effect shown at 2.0 μg.

[0234] Example 7 RG4326 in the 6 - Hz Model #2 The 6 - Hz study of Example 7 was repeated for different doses and pretreatment times for RG4326 administration and for different doses for perampanel. In this example, the effect of RG4326 on 6 - Hz - induced CNS effects was evaluated by administering RG4326 3 - 4 hours and 24 hours before 6 - Hz administration (in contrast to only 24 hours used in Example 6). In Example 7, perampanel (2 mg / kg p.o.) was administered p.o. at a dose of 2 mg / kg (compared to 4 mg / kg in Example 6) 60 minutes before the test. In this example, transplantation was performed on a total of 75 animals over 4 days to obtain at least 15 mice per group. The design of this study is further described in Table 16 below. TIFF2026510893000023.tif124170

[0235] Quantitative data (scores) from the test substances were analyzed by comparing the treatment group to the vehicle control using Fisher's direct test. These results are reported in Figure 10 and Table 17 below.

[0236] In the neutral vehicle control group (0.2% HPMC in distilled water administered po 60 minutes prior to the test), the mean forelimb seizure score was 0.9 ± 0.1, and all mice (15 / 15) exhibited tail elevation. In the vehicle control group (PBS administered via ICV 3–4 hours prior to the test), the mean forelimb seizure score was 0.7 ± 0.2, and all mice (15 / 15) exhibited tail elevation. ICV administration of RG4326 (0.5 mg / mL, i.e., 2 μg / mouse) 3–4 hours prior to the test significantly reduced the number of mice exhibiting tail elevation compared to the vehicle control group (-33%, p<0.05). Interestingly, at this dose, RG4326 did not affect the forelimb seizure score (compared to the data in Figure 8 showing 6 Hz 24 hours after ICV). No effect on these parameters was observed at 0.125 and 1 mg / mL, i.e., 0.5 and 4 μg / mouse. In the group treated with 1 mg / mL, three mice showed significant sedation and respiratory reduction. ICV administration of RG4326 (1 mg / mL, i.e., 4 μg / mouse) 24 hours prior to the study significantly reduced the number of mice exhibiting tail-lifting compared to vehicle controls (-53%, p<0.01). Perampanel (2 mg / kg) administered po 60 minutes prior to the study significantly reduced mean forelimb clonus compared to neutral vehicle controls (-67%, p<0.01), but did not affect the number of mice exhibiting tail-lifting compared to RG4326.

[0237] In summary, these results demonstrate the anticonvulsant effect of 0.5 mg / mL RG4326 administered via ICV 3-4 hours prior to the 6 Hz test in mice. The anticonvulsant effect was also observed with 1 mg / mL RG4326 administered via ICV 24 hours prior to the test. Significant sedation and respiratory reduction were observed in three mice with 1 mg / mL RG4326.

[0238] In summary, RG4326 was profiled in two tests, including a 6 Hz psychomotor test, in Examples 6 and 7 reported herein. In the Example 6 test, RG4326 was found to reduce seizure incidence (forelimb clonus and tail elevation) after 2.0 ug of RG4326 was administered by ICV 24 hours prior to 6 Hz. In the Example 7 test, the effects of administering a higher dose of RG4326 24 hours prior to the 6 Hz current and the effects of administering RG4326 by ICV closer to the 6 Hz current (i.e., 3-4 hours) were evaluated. The results of the Example 7 test showed improved seizure suppression with 4.0 ug of RG4326 administered 24 hours prior (tail elevation), in addition to the seizure suppression with 2.0 ug of RG4326 administered 3-4 hours prior to 6 Hz. TIFF2026510893000024.tif112170

[0239] Example 8 Evaluation of AMPA-R compounds for glutamate and AMPA-induced excitotoxicity in rat primary cortical neurons. The compounds shown in Table 18 were evaluated for glutamate and AMPA-induced excitotoxicity in primary cortical neurons of rats. TIFF2026510893000025.tif79170

[0240] Testing system Primary rat cortical neurons were obtained from E18-19 rat embryos (Sprague-Dawley rats).

[0241] cell culture The cortex was harvested from E18-19 rat embryos and dissociated enzymatically and mechanically. Dissociated cells (10,000 cells / well) were seeded in 70 μL of nerve growth medium (Neurobasal + 2% SM1 neurotrophic agent + L-glutamine + HEPES) in a poly-D-lysine coated imaging plate (384 wells). The cells were incubated at 37°C and 5% CO2.

[0242] Half of the culture medium was replaced twice a week. A total of five plates were prepared for the experiment.

[0243] Evaluation of glutamate or AMPA-induced excitotoxicity After 9 days in vitro, half of the culture medium was removed, and 35 μL of the test item was added to the culture medium at a 2-fold concentration.

[0244] After 24 hours, AMPA or glutamic acid was added to the wells, and the wells were treated as follows: - Wells were incubated with the test item: 17.5 μL was removed and 17.5 μL of 4X glutamate or AMPA was added. - Wells were incubated with reference material: 35 μL was taken out, 17.5 μL of 4X reference material (MK-801, Perampanel, or CNQX) was added to each well, followed by 17.5 μL of 4X glutamate or AMPA.

[0245] After 72 hours, half of the supernatant was discarded, and an intracellular ATP content assay was performed using an ATP kit (CellTiter-Glo® 3D cell survival assay).

[0246] Each experimental condition was tested in four wells, as shown below. TIFF2026510893000026.tif115170TIFF2026510893000027.tif115170

[0247] Glutamate significantly reduced ATP content compared to the untreated (NT) control. See Figure 11A. MK-81 and perampanel each significantly increased ATP content in the presence of glutamate. See Figures 11A and 11B.

[0248] RG8431, RG4326, RG4047, RG6224, RG8534, and RG8535 significantly increased the ATP content in the presence of glutamate. RG8525 showed a slight but significant increase in the ATP content at 1 μM and no significant effect was observed on RG8524 at high doses. See Figures 12A - 12H.

[0249] AMPA significantly decreased the ATP content compared to the untreated (NT) control. See Figure 13A. CNQX and perampanel each significantly increased the ATP content in the presence of glutamate. See Figures 13A and 13B.

[0250] RG4326, RG4047, RG6224, RG8534, and RG8535 significantly increased the ATP content in the presence of AMPA. RG8431 did not change the ATP content, but RG8524 and RG8525 significantly increased the ATP content in the presence of high doses of AMPA. See Figures 14A - 14H.

[0251] Calcium measurements Pilot experiment to determine the dose of AMPA

[0252] After 8 days in vitro, the growth medium was discarded and replaced with 30 μL of calcium probe in physiological saline (containing 1.5 mM calcium) at 37°C / 5% CO2 for 60 minutes.

[0253] For calcium measurements, AMPA, perampanel, and CNQX were prepared at 6-fold concentration (6 μL added to 30 μL). The final vehicle concentration was adjusted for all conditions. Before automatically adding the compound or control during recording, the basal calcium level was measured for 1 minute. The intracellular calcium signal was recorded for an additional 5 - 10 minutes. The sampling rate was approximately 1 point per second.

[0254] Each experimental condition was tested in quadruplicate wells as shown below. TIFF2026510893000028.tif156170

[0255] AMPA significantly increased calcium release in a dose-dependent manner compared to the untreated condition at all doses (Figure 15A). Furthermore, AMPA administered with 0.1% DMSO also significantly increased calcium release in a dose-dependent manner compared to 0.1% DMSO at all doses (Figure 15B).

[0256] Paranpanel (1, 3, 10, and 30 μM) nearly completely suppressed calcium release in the presence of 3, 10, 30, and 100 μM AMPA, and dose-dependently reduced calcium release in the presence of 300 μM AMPA (Figures 15C-15G).

[0257] CNQX (1, ​​10, and 25 μM) significantly and dose-dependently reduced calcium release in the presence of 3 μM and 10 μM AMPA (no clear effect was observed at 0.1 μM). CNQX (10 and 25 μM) significantly and dose-dependently reduced calcium release in the presence of 30 μM and 100 μM AMPA. In this experiment, CNQX did not reduce calcium release in the presence of 300 μM AMPA (Figures 15H-15L).

[0258] At the end of the pilot study, it was decided to use a 100 μM dose of AMPA to induce a clear calcium response. 10 μM perampanel and 25 μM CNQX were selected as reference materials for the main experiment.

[0259] Main experiment After 9 days in vitro, half of the culture medium was removed, and 35 μL of the test substance was added to the culture medium at a 2-fold concentration.

[0260] After 24 hours, the growth medium was discarded and replaced with 30 μL of calcium probe in physiological saline (containing 1.5 mM calcium) at 37°C / 5% CO2 for 60 minutes.

[0261] For calcium measurement, glutamate and MK-801 or AMPA and perampanel / CNQX were prepared in 6-fold concentrations (6 μL added to 30 μL). Final vehicle concentrations were adjusted under all conditions. Basal calcium levels were measured for 1 minute before auto-addition of the compound or control during recording. Intracellular calcium signals were recorded for a further 5–10 minutes. The sampling rate was approximately 1 point per second.

[0262] Each experimental condition was tested in a four-well system as shown below. TIFF2026510893000029.tif109170TIFF2026510893000030.tif115170

[0263] As shown in Figure 16, glutamate increased calcium release compared to the untreated (NT) control, while MK-801 significantly decreased calcium release in the presence of glutamate.

[0264] RG8431 (1 and 5 μM), RG4326 (1 and 50 μM), RG4047 (1, 5, and 50 μM), RG8524 (0.25 and 10 μM), RG8525 (0.25 and 10 μM), and RG6224 (10, 25, and 50 μM) slightly but significantly increased calcium release when incubated for 24 hours before glutamate addition. RG8534 (50 μM) significantly increased calcium release, while RG8535 had no significant effect on calcium release. See Figures 17A-17H.

[0265] As shown in Figure 18, AMPA increased calcium release compared to the untreated (NT) control, while CNQX and perampanel significantly reduced calcium release in the presence of AMPA.

[0266] RG8431 (1, 5, 10, 25, and 50 μM), RG4047 (1, 5, 10, 25, and 50 μM), and RG8524 (5 μM) significantly reduced calcium release in the presence of AMPA. RG4326 (1, 5, 10, 25, and 50 μM) and RG8535 (1, 5, 10, 25, and 50 μM) significantly reduced calcium release in a dose-dependent manner, while RG8525, RG6224, and RG8534 did not significantly alter calcium release. See Figures 19A-19H.

[0267] These results indicate that most of the test compounds exhibit neuroprotective effects when incubated 24 hours prior to glutamate or AMPA.

Claims

1. A method for treating a target neurological disorder, comprising administering to the target a therapeutically effective amount of a modified oligonucleotide consisting of 4 to 15 linked nucleosides, wherein the last four nucleosides at the 3' end of the modified oligonucleotide have the nucleic acid base sequence UUUG, and at least two of the last four nucleosides at the 3' end of the modified oligonucleotide are bicyclic nucleosides.

2. The method according to claim 1, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleoside of the modified oligonucleotide is a bicyclic nucleoside.

3. The method according to claim 1 or claim 2, wherein each bicyclic nucleoside is independently selected from S-cEt nucleosides, LNA nucleosides, and ENA nucleosides.

4. The method according to claim 3, wherein each bicyclic nucleoside is an S-cEt nucleoside.

5. The method according to any one of the prior claims, wherein at least one nucleoside bond of the modified oligonucleotide is a modified nucleoside bond.

6. The method according to claim 5, wherein each nucleoside bond in the modified oligonucleotide is a modified nucleoside bond.

7. The method according to claim 5 or claim 6, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

8. The method according to any one of claims 1 to 3, wherein the modified oligonucleotide consists of 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, or 4 to 14 linked nucleosides.

9. The method according to any one of the prior claims, wherein the modified oligonucleotide consists of four linked nucleosides.

10. The modified oligonucleotide is U Ｆ U Ｍ U Ｓ G Ｓ or U Ｓ U Ｓ U Ｓ G Ｓ The method according to claim 9, wherein the nucleoside following the subscript "M" is 2'-O-methylnucleoside, the nucleoside following the subscript "F" is 2'-fluoronucleoside, and the nucleoside following the subscript "S" is S-cEt nucleoside.

11. The method according to any one of claims 1 to 8, wherein the modified oligonucleotide consists of nine linked nucleosides.

12. The method according to any one of claims 1 to 8 and 11, wherein the modified oligonucleotide comprises the nucleic acid base sequence 5'-AGCACUUUG-3', and each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine.

13. The modified oligonucleotide has the following nucleoside pattern oriented from 5' to 3'. N Ｓ N Ｓ N Ｍ N Ｆ N Ｆ N Ｆ N Ｍ N Ｓ N Ｓ It has, The method according to claim 11 or claim 12, wherein the nucleoside following the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside following the subscript "F" is a 2'-fluoro nucleoside, and the nucleoside following the subscript "S" is an S-cEt nucleoside.

14. The modified oligonucleotide is selected from the modified oligonucleotides in Table 1, according to the method of any one of the prior claims.

15. The method according to any one of the prior claims, wherein a pharmaceutically acceptable salt of the modified oligonucleotide is administered to the subject.

16. The method according to claim 15, wherein the pharmaceutically acceptable salt is a sodium salt.

17. A method for treating a target neurological disorder, wherein the target has a structure 【Chemistry 1】 The method comprising administering a therapeutically effective amount of a modified oligonucleotide having, or a pharmaceutically acceptable salt thereof.

18. The method according to claim 17, comprising administering a pharmaceutically acceptable salt of the modified oligonucleotide to the subject.

19. The method according to claim 18, comprising administering the sodium salt of the modified oligonucleotide to the subject.

20. A method for treating a target neurological disorder, wherein the target has a structure 【Chemistry 2】 The method comprising administering a therapeutically effective amount of a modified oligonucleotide having [a specific characteristic].

21. The method according to any one of the prior claims, wherein the modified oligonucleotide is formulated into a pharmaceutical composition for administration to the subject, and the pharmaceutical composition comprises the modified oligonucleotide in an aqueous solution.

22. The method according to claim 21, wherein the aqueous solution is physiological saline.

23. The method according to any one of the prior claims, wherein the neurological disorder is hearing loss, motor disorder, amyotrophic lateral sclerosis (ALS), pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke, or glioblastoma.

24. The method according to any one of the prior claims, wherein the neurological disorder is epilepsy.

25. The method according to any one of the prior claims, comprising administering at least one additional therapy to the subject.

26. The method according to claim 25, wherein the additional therapy is an antiepileptic drug.

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