Compounds for inhibiting il-1β expression

Abstract

This invention discloses a nicked oligonucleotide (ASO) and a pharmaceutical composition comprising the nicked oligonucleotide, wherein the nicked oligonucleotide compound specifically inhibits IL-1β eRNA transcription and wherein the nicked oligonucleotide comprises about 14 to about 25 nucleotide bases, having a (3' to 5') 3' wing region containing 3 to 7 chemically modified RNA bases, an interstitial region containing at least 8 DNA bases, and a 5' wing region containing 3 to 7 chemically modified RNA bases, and wherein the nicked oligonucleotide is substantially complementary to the 14-25 base region on the IL-1β eRNA (SEQ ID NO. 1). Preferably, each nicked oligonucleotide is linked by a phosphate thioester (P=S) nucleotide internucleotide bond that runs through the nicked oligonucleotide; and wherein the modified nucleotide bases are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof.

Description

[0001] Prior related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 542514, filed on October 4, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence declaration

[0004] This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated herein by reference in its entirety. The XML copy was created on October 3, 2024, and is named J1742-00103_SL.xml, with a size of 568,719 bytes. Technical Field

[0005] This disclosure provides a gapmer-type ASO (antisense oligonucleotide) and a pharmaceutical composition comprising the gapmer, wherein the gapmer compound specifically inhibits IL-1β-eRNA transcription and wherein the gapmer comprises about 14 to about 25 nucleotide bases, the gapmer having a (3' to 5') 3' wing region containing 3 to 7 chemically modified RNA bases, an interstitial region containing at least 8 DNA bases, and a 5' wing region containing 3 to 7 chemically modified RNA bases, and wherein the gapmer is substantially complementary to the 14-25 base region on IL-1β eRNA (SEQ ID NO. 1). Preferably, the gapmer nucleotides are each linked by a phosphate thioester (P=S) nucleotide internucleotide bond that runs through the gapmer; and wherein the modified nucleotide bases are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof. This disclosure further provides a method for treating sepsis, comprising administering to a sepsis-stricken individual an effective amount of a pharmaceutical composition containing a nick body, wherein the nick body compound specifically inhibits IL-1β eRNA transcription and wherein the nick body comprises about 14 to about 25 nucleotide bases, the nick body having a (3' to 5') 3' wing region containing 3 to 7 chemically modified RNA bases, a gap region containing at least 8 DNA bases, and a 5' wing region having 3 to 7 chemically modified RNA bases, and wherein the nick body is substantially complementary to the 14-25 base region on IL-1β eRNA (SEQ ID NO. 1). Background Technology

[0006] The following includes information that may help in understanding the invention. This document does not acknowledge that any information, publications, or documents specifically or implicitly referenced herein are prior art or material to the invention described and claimed herein. All publications, patents, related applications, and other written or electronic materials mentioned or identified herein are incorporated herein by reference in their entirety. The information contained herein is part of the application as it is repeated in the application and should be considered part of the text and content of the application.

[0007] Long non-coding RNAs (lncRNAs) are non-coding transcripts that typically exceed 200 nucleotides (200 nt). LncRNAs derived from enhancers are often referred to as enhancer RNAs (eRNAs). One therapeutic target is IL-1β-eRNA, an eRNA that regulates IL-1β transcriptional activation. Therefore, transcriptional inhibitors or regulators of IL-1β eRNA are needed to address acute and chronic inflammation. (Mattick, JS, et al.) Nat Rev Mol Cell Biol 24, 430–447 (2023)). Long non-coding transcripts have been found in many species. DNA (cDNA) sequencing projects, such as FANTOM, have determined the complexity of these transcripts in humans (Carninci P, et al. (September 2005), Science. 309 (5740): 1559–1563).

[0008] Interleukin-1β (IL-1β) is a major cytokine at the apex of inflammation and a key endogenous mediator of immune training. It induces potent pro-inflammatory functions, which are essential for inducing immune training and providing immune protection (Arts, RJW et al. BCG vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity. Cell Host Microbe 23, 89–100. e5 (2018)). Transcription of IL-1β eRNA (enhancer RNA) is required to trigger a strong IL-1β-mediated inflammatory response. However, excessive production of IL-1β eRNA can be detrimental and contribute to many hyperinflammatory and autoimmune diseases, such as sepsis, rheumatoid arthritis, gout, and other autoinflammatory syndromes (Dinarello, CA. A clinical perspective of IL-1β as the gatekeeper of inflammation. Eur. J. Immunol. 41, 1203–1217 (2011)). Therefore, transcriptional inhibitors or regulators of IL-1β-eRNA are needed to address acute and chronic inflammation.

[0009] LncRNAs (long non-coding RNAs) derived from enhancers are often referred to as enhancer RNAs (eRNAs). One therapeutic target is IL-1β-eRNA, an eRNA that regulates IL-1β transcriptional activation (Ilott, NE et al. Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes. Nat. Commun. 5, 3979 (2014)). Therefore, transcriptional inhibitors or regulators of IL-1β eRNA are needed to address acute and chronic inflammation. Summary of the Invention

[0010] The inventions described and claimed herein have many attributes and embodiments, including but not limited to those set forth, described, or referenced in this brief overview. It is not intended to be all-encompassing, and the inventions described and claimed herein are not limited to or restricted by the features or embodiments identified in this description. This description is for illustrative purposes only and not for limitation.

[0011] This disclosure provides a nicked oligonucleotide (ASO) and a pharmaceutical composition comprising the nicked oligonucleotide, wherein the nicked oligonucleotide compound specifically inhibits IL-1β eRNA transcription and wherein the nicked oligonucleotide comprises about 14 to about 25 nucleotide bases, has a (3' to 5') 3' wing region containing 3 to 7 chemically modified RNA bases, an interstitial region containing at least 8 DNA bases, and a 5' wing region containing 3 to 7 chemically modified RNA bases, and wherein the nicked oligonucleotide is substantially complementary to the 14-25 base region on the IL-1β eRNA (SEQ ID NO. 1). Preferably, each nicked oligonucleotide is linked by a phosphate thioester (P=S) nucleotide internucleotide bond that runs through the nicked oligonucleotide; and wherein the modified nucleotide base is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof.

[0012] This disclosure provides a nicked compound that is complementary to region A (bases 58 to 80 of SEQ ID NO. 1) of IL-1β eRNA and inhibits the transcription of various acute inflammatory genes regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotide bases; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein each nicked nucleotide is linked by a phosphate-thionucleotide inter-linked bond, a phosphate-thioester inter-linked chain, or a combination thereof spanning the nicked nucleotide; and wherein the modified nucleotide bases are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. Preferably, region A of the IL-1β eRNA is bases 65-80 of SEQ ID NO. 1. Most preferably, the notched compound complementary to region A is selected from the group consisting of SEQ ID NO. 64, 65, 68, 69 and combinations thereof.

[0013] This disclosure provides a nicked compound that is complementary to region B (SEQ ID NO. 1, bases 1153 to 1172) of IL-1β eRNA and inhibits the transcription of various acute inflammatory genes regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotide bases; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein each nicked nucleotide is linked by a phosphate-thionucleotide inter-linked bond, a phosphate-thioester inter-linked chain, or a combination thereof; and wherein the modified nucleotide bases are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. Preferably, the nicked compound complementary to region B is selected from the group consisting of SEQ ID NO. 50, 174, and combinations thereof.

[0014] This disclosure provides a nicked compound that is complementary to region C (SEQ ID NO. 1 bases 1245 to 1297) of IL-1β eRNA and inhibits the transcription of various acute inflammatory genes regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotide bases; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein each nicked nucleotide is linked by a phosphate-thionucleotide inter-linked bond, a phosphate-thioester inter-linked chain, or a combination thereof spanning the nicked nucleotide; and wherein the modified nucleotide bases are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. Preferably, region C of the IL-1β eRNA is SEQ ID NO. 1 bases 1246-1261. More preferably, the notched compound complementary to region B is selected from the group consisting of: SEQ ID NO. 25, 51, 121, 122 and combinations thereof. Detailed Implementation

[0015] This invention relates to IL-1β eRNA transcription regulators and compositions comprising the same for regulating IL-1β eRNA transcription. In some embodiments, the IL-1β eRNA transcription regulator is an IL-1β eRNA transcription inhibitor.

[0016] In some implementations, the IL-1β eRNA transcription inhibitor contains an antisense molecule. See the examples below.

[0017] The first series of nick body compounds disclosed herein are designed to target different regions of the human IL-1 long non-coding RNA IL-1βeRNA (FANTOM CAT number: FTMT20800006485.1) (SEQ ID NO. 1).

[0018] Human IL-1β-eRNA sequence:

[0019] The corresponding mouse IL1β eRNA, or IL-1β eRNA sequence (SEQ ID NO. 2): .

[0020] definition

[0021] The following terms have the following meanings: "2′-substituted nucleosides" refer to nucleosides containing a 2′-substituted sugar moiety. When referring to the sugar moiety, "2′-substituted" means a sugar moiety containing at least one 2′-substituent other than H or OH.

[0022] "2′-Deoxynucleoside" refers to a nucleoside containing a 2′-H furanose moiety that is naturally found in deoxyribonucleosides (DNA). 2′-Deoxynucleosides can contain modified nucleosides or RNA nucleosides (e.g., uracil).

[0023] "2′-O-methoxyethyl" (also known as 2′-MOE and 2′-O(CH2)2-OCH3) refers to the O-methoxy-ethyl modification at the 2′ position of the furanyl ring. Sugars modified with 2′-O-methoxyethyl ester are modified sugars.

[0024] "2′-O-methoxyethyl nucleotide" refers to a nucleotide containing a sugar moiety modified with 2′-O-methoxyethyl ester.

[0025] "5-Methylcytosine" refers to cytosine modified with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0026] "Approximately" refers to a value provided that is plus or minus 7%.

[0027] "Active agent" refers to one or more substances in a pharmaceutical composition that provide therapeutic benefit when administered to an individual. For example, in some embodiments, an antisense oligonucleotide targeting IL-1β-eRNA is an active agent.

[0028] "Active target region" or "target region" refers to the region targeted by one or more active antisense compounds. "Active antisense compound" refers to an antisense compound that reduces the transcription of a target gene or the resulting protein level.

[0029] "Administration" refers to the provision of a medicine to an individual, including but not limited to administration by a medical professional and self-administration.

[0030] As used herein, the term “IL-1β-eRNA transcription regulator” nick body ASO compound (sometimes also referred to as an IL-1β-eRNA transcription “inhibitor” nick body ASO compound) is a compound that prevents, inhibits, and / or reduces IL-1β-eRNA transcription.

[0031] “Inhibition” or “regulation” should not be interpreted as meaning that the function, activity, expression, transport and / or assembly of IL-1β-eRNA is completely inhibited, inactivated or completely regulated, although this may be preferred, but should be understood to include any reduction in the function, activity or expression of IL-1β-eRNA.

[0032] "Improvement" refers to the reduction of at least one indicator, sign, or symptom of a related disease, disorder, or condition. The severity of the indicator can be determined by subjective or objective measures known to a person skilled in the art.

[0033] "Antisense activity" refers to any detectable and / or measurable change resulting from the hybridization of an antisense compound with its target nucleic acid. Antisense activity is defined as a reduction in the amount or expression of the target nucleic acid or the protein encoded by it compared to the levels of the target nucleic acid or protein in the absence of the antisense compound.

[0034] "Antisense compound" refers to an oligomeric compound that can achieve at least one antisense activity.

[0035] "Alkyl" refers to a saturated aliphatic hydrocarbon group containing 1 to 8 (e.g., 1 to 6 or 1 to 4) carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl. Alkyl groups can be substituted with one or more substituents (i.e., optionally substituted), such as halogens; alicyclic groups [e.g., cycloalkyl or cycloalkenyl]; heterocyclocyclic groups [e.g., heterocycloalkyl or heterocycloalkenyl]; aryl; heteroaryl; alkoxy; aryl; heteroaryl; acyl [e.g., (aliphatic)carbonyl, (alicyclo)carbonyl, or (heterocyclocyclic)carbonyl]; nitro; cyano; amide [e.g., (cycloalkyl)carbonylamino, arylcarbonylamino, arylalkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkyl)carbonylamino, heteroarylcarbonyl] [Amino, heteroarylalkylcarbonylamino, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl or heteroarylaminocarbonyl]; amino [e.g. aliphatic amino, alicyclic amino or heterocyclocyclic amino]; sulfonyl [e.g. aliphatic -S(O)2-]; sulfinyl; thioalkyl; thiooxy; ureo; thiourea; aminosulfonyl; sulfonamide; oxo; carboxyl; carbamoyl; alicyclic epoxy; heterocyclic epoxy; aryloxy; heteroaryloxy; arylalkoxy; heteroarylalkoxy; alkoxycarbonyl; alkylcarbonyloxy; or hydroxyl. Some examples of substituted alkyl groups include, but are not limited to, carboxylalkyl groups (such as HOOCalkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl); cyanalkyl; hydroxyalkyl; alkoxyalkyl; acylalkyl; aralkyl; (alkoxyaryl)alkyl; (sulfonylamino)alkyl (such as alkyl-S(O)2-aminoalkyl); aminoalkyl; amamidealkyl; (alicycloalkyl); or haloalkyl.

[0036] "alkylene" refers to a difunctional alkyl group.

[0037] A "bifunctional" part refers to a chemical group attached to the main chemical structure in two places, such as a linker part. A bifunctional part can be attached to the main chemical structure at any two chemically feasible substitutable points. Unless otherwise stated, a bifunctional part can be attached in either direction; for example, the bifunctional part "NO" can be attached in either the -NO- or -ON- direction.

[0038] A “chemically distinct region” refers to a region in an antisense compound that is chemically different from another region in the same antisense compound. For example, a region containing a 2′-O-methoxyethyl nucleotide is chemically different from a region containing a nucleotide without the 2′-O-methoxyethyl modification.

[0039] "Chimeric antisense compounds" are antisense compounds that have at least two chemically distinct regions.

[0040] "Combined administration" refers to the administration of two or more drugs to an individual. The two or more drugs may be in a single drug composition or in a separate drug composition. Each of the two or more drugs may be administered via the same or different routes of administration. Combined administration includes parallel or sequential administration.

[0041] "Complementarity" refers to the ability of the nucleobases of the first and second nucleic acids to pair.

[0042] "Continuous nucleobases" refers to nucleobases that are adjacent to each other. "Diluent" refers to a component in the composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, the diluent in an injectable composition can be a liquid, such as a saline solution.

[0043] "Dosage" refers to a single administration or a specified quantity of a drug administered over a specified period of time. In some embodiments, the dosage may be administered in the form of one, two, or more pills, tablets, or injections. For example, in some embodiments requiring subcutaneous administration, the desired dosage requires a volume that is not easily accommodated by a single injection; therefore, two or more injections may be used to achieve the desired dosage. In some embodiments, the drug is administered via prolonged or continuous infusion. Dosage may be expressed as an hourly, daily, weekly, or monthly dose.

[0044] "Effective dose" refers to the amount of active pharmaceutical ingredient sufficient to achieve the desired physiological outcome in an individual who requires the drug. Effective doses can vary from person to person, depending on the individual's health and physical condition, the individual's taxonomic group, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0045] "Completely complementary" or "100% complementary" means that every nucleobase of the first nucleic acid has a complementary nucleobase in the second nucleic acid. In some embodiments, the first nucleic acid is an antisense compound and the target nucleic acid is the second nucleic acid. "Basically complementary" means that the oligonucleotide has at most two mismatched bases that are not complementary to the second nucleic acid. For example, for a decameric oligonucleotide, a substantially complementary sequence can have 8, 9, or 10 bases complementary to the second nucleic acid. "Basically complementary" includes "completely complementary" sequences (where the oligonucleotide sequence has no bases that do not match the second nucleic acid).

[0046] When referring to RNA or DNA bases, “chemical modification” has the meaning understood in the art, including nucleoside bases selected from the group consisting of: 2′-substituted nucleosides, ′-O-methoxyethyl (also known as 2′-MOE and 2′-O(CH2)2-OCH3), 2′-deoxynucleosides, 2′-O-methoxyethyl nucleotides, 5-methylcytosine, monocyclic nucleosides, bicyclic nucleosides, 4′-2′ bicyclic nucleosides, 4′ to 2′ bicyclic nucleosides, locked nucleic acids, and nucleoside mimics, all of which are as defined herein.

[0047] "Cutable portion" refers to a bond or group of atoms that can be cut under physiological conditions, such as in a cell or animal body.

[0048] When referring to oligonucleotides, "complementarity" means that when the nucleobase sequences of an oligonucleotide and another nucleic acid are aligned in opposite directions, at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof can form hydrogen bonds with the nucleobases of another nucleic acid or one or more regions thereof. Complementary nucleobases are nucleobases that can form hydrogen bonds with each other.

[0049] Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (B) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine. Complementary oligonucleotides and / or nucleic acids do not need to have nucleobase complementarity on every nucleoside. Instead, some mismatches are tolerable. "Complete complementarity" or "100% complementarity" means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid on every nucleoside of the oligonucleotide.

[0050] In the context of oligonucleotides, "continuous" refers to nucleosides, nucleobases, sugar moieties, or internucleotide bonds that are adjacent to each other. For example, "continuous nucleobases" refers to nucleobases that are adjacent to each other in the sequence.

[0051] A “gap body” refers to a modified oligonucleotide containing an internal “gap” region with multiple DNA nucleotides located between an outer region containing one or more nucleotides. The nucleotides containing the internal region are chemically distinct from those containing the outer region. The internal region is typically referred to as the “gap,” while the outer region is typically referred to as the “wing.” Unless otherwise stated, a “gap body” refers to a glycosylation motif. Unless otherwise stated, the sugar portion of the nucleotide in the central region of the gap body is an unmodified 2′-deoxyribosome. Therefore, the term “MOE gap body” refers to a gap body with a 2′-MOE nucleotide glycosylation motif on both wings and a 2′-deoxyribosome gap. Unless otherwise stated, an MOE gap body may contain one or more modified nucleotide internucleotide bonds and / or modified nucleobases, and such modifications do not necessarily follow the glycosylation pattern of a gap body.

[0052] "Hybridization" refers to the pairing or annealing of complementary oligonucleotides and / or nucleic acids. While not limited to specific mechanisms, the most common hybridization mechanisms involve hydrogen bonds between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds.

[0053] "Closest" means that there are no intermediate elements between the adjacent elements.

[0054] In this article, the terms “individual” or “subject” may be used interchangeably to refer to a human or non-human animal selected for treatment or therapy.

[0055] "Modified nucleotide bases" and "modified nucleosides" refer to deoxyribonucleotides or ribonucleotides that have been modified to have one or more chemical moieties not found in natural nucleic acids. Examples of modified nucleotide bases and "modified nucleosides" are compounds of formula Ia, Ib, IIa, or IIb described herein.

[0056] "Non-bicyclic modified sugar moieties" refers to the sugar moieties of modified nucleotide bases, as described in this article, where the chemical modification does not involve the conversion of the sugar moieties into bicyclic or polycyclic systems.

[0057] "Monocyclic nucleoside" refers to a nucleoside containing a modified sugar moiety that is not a bicyclic sugar moiety. In some embodiments, the sugar moiety of the nucleoside or a sugar moiety analogue may be modified or substituted at any position.

[0058] "2′-modified sugar" refers to a furanose modified at the 2′ position. Such modifications include the substituents described herein.

[0059] “Bicyclic nucleoside” (BNA) refers to a modified nucleoside containing a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides containing a bridge between the 4′ and 2′ ribose ring atoms. The synthesis of bicyclic nucleosides has been disclosed, for example, in 7399845, WO / 2009 / 006478, WO / 2008 / 150729, US2004-0171570, US Patent 7427672, Chattopadhyaya et al. J. Org. Chem. 2009, 74, 118-134, WO 99 / 14226, and WO 2008 / 154401. The synthesis and preparation of methyleneoxy (4′-CH2-O-2′)BNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation have also been described in WO98 / 39352 and WO 99 / 14226. Analogs of methyleneoxy (4′-CH2-O-2′)BNA and 2′-thio-BNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (WO 99 / 14226). Furthermore, the synthesis of 2′-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog, has been described in this field (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino and 2′-methylamino-BNA have been prepared, and their thermostability with complementary RNA and DNA duplexes has been reported. A carbocyclic bicyclic nucleotide with a 4′-(CH2)3-2′ bridge and an alkenyl analog bridge 4′-CH=CH-CH2-2′ has been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies are also described (Srivastava et al., J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

[0060] "4′-2′ bicyclic nucleoside" or "4′ to 2′ bicyclic nucleoside" is a bicyclic nucleoside containing a furanose ring. The furanose ring contains a bridge connecting the two carbon atoms of the furanose ring, which connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

[0061] Locked nucleic acid (LNA) is a modified nucleotide base in which the chemical modification converts the sugar moiety into a bicyclic or polycyclic system. Two specific examples of locked nucleic acid compounds are β-D-methyleneoxynucleotide, or "restricted methyl" (cMe) nucleotide; and β-D-ethoxynucleotide, or "restricted ethyl" (cEt) nucleotide.

[0062] "Mismatch" or "non-complementarity" means that when the first and second oligonucleotides are matched, the nucleobases of the first oligonucleotide are not complementary to the corresponding nucleobases of the second oligonucleotide or the target nucleic acid.

[0063] "Motif" refers to the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or nucleoside bonds in an oligonucleotide.

[0064] "Nucleoside" refers to an unmodified nucleobase or a modified nucleobase. Unmodified nucleobases are adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). Modified nucleobases are a group of atoms other than the unmodified A, T, C, U, or G that can pair with at least one unmodified nucleobase. "5-Methylcytosine" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any of the five unmodified nucleobases. A "nucleoside sequence" refers to a continuous sequence of nucleobases in a nucleic acid or oligonucleotide, unaffected by any sugar or nucleoside internucleotide modifications.

[0065] A nucleoside is a compound consisting of a nucleobase and a sugar moiety. The nucleobase and sugar moiety are either unmodified or modified independently. A modified nucleoside is a nucleoside containing a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abase-free nucleosides lacking a nucleobase. A linked nucleoside is a nucleoside linked by a continuous sequence (i.e., no additional nucleosides between the linked nucleosides).

[0066] "Nucleoside mimics" include those structures used to replace bonds at one or more positions in a sugar or sugar and base, not necessarily in an oligomer, such as nucleoside mimics having, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranoyl, bicyclic, or tricyclic sugar mimics (e.g., non-furanose units). Nucleotide mimics include those structures used to replace bonds at one or more positions in a nucleoside and oligomer, such as, for example, peptide nucleic acids or morpholino (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Sugar substitutes overlap with the slightly broader term nucleoside mimics, but are used only to indicate the substitution of a sugar unit (furanose ring). The tetrahydropyranoyl ring provided herein illustrates an example of a sugar substitute in which the furanose group has been replaced by a tetrahydropyranoyl ring system.

[0067] "Parenteral administration" refers to administration by injection (e.g., bolus) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intraventricular administration.

[0068] "Pharmaceutically acceptable carriers" refer to physiologically and pharmaceutically acceptable carriers for compounds. Pharmaceutically acceptable carriers retain the desired biological activity of the parent compound without imparting undesirable toxicological effects.

[0069] "Pharmaceutical composition" refers to a mixture of substances suitable for administration to animals. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution. In some embodiments, the pharmaceutical composition exhibits activity in free uptake assays in certain cell lines.

[0070] "Pharmaceutically acceptable derivatives" include pharmaceutically acceptable carriers, conjugates, prodrugs, or isomers of the compounds described herein.

[0071] "Thiophosphate bond" refers to the bond between nucleosides, where the phosphodiester bond is modified by replacing a non-bridging oxygen atom with a sulfur atom.

[0072] "Partial" refers to a defined number of consecutive (i.e., linked) nucleobases in a nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the target nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the antisense compound.

[0073] "Single-stranded oligonucleotides" refer to oligonucleotides that have not hybridized with their complementary strands.

[0074] "Specific hybridization" means that the antisense compound has sufficient complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under conditions requiring specific binding, i.e., physiological conditions for in vivo assay and treatment.

[0075] “Glycan moiety” refers to either an unmodified or modified sugar moiety. As used herein, “unmodified sugar moiety” refers to the OH(H) ribosyl moiety present in RNA (“unmodified RNA sugar moiety”) or the 2-H(H) deoxyribosyl moiety present in DNA (i.e., “unmodified DNA sugar moiety”). An unmodified sugar moiety has one hydrogen atom at positions 1′, 3′, and 4′, one oxygen atom at position 3′, and two hydrogen atoms at position 5′. As used herein, “modified sugar moiety” or “modified sugar” refers to a modified furanose moiety or a sugar substitute.

[0076] "Sugar substitutes" refer to sugar moieties modified other than the furanyl group, which can link a nucleobase to another group, such as a nucleoside internucleotide, conjugated group, or terminal group in an oligonucleotide. Nucleosides modified with sugar substitutes can be incorporated into one or more positions within an oligonucleotide, and such oligonucleotides can hybridize with complementary oligomers or target nucleic acids.

[0077] “Targeting” refers to the process of designing and selecting antisense compounds that will specifically hybridize with target nucleic acids and induce the desired effect.

[0078] "Target fragment" refers to the nucleotide sequence of the target nucleic acid targeted by the antisense compound. "5' target site" refers to the nucleotide closest to the 5' end of the target fragment. "3' target site" refers to the nucleotide closest to the 3' end of the target fragment.

[0079] "Target nucleic acid" and "target RNA" refer to the nucleic acid that an antisense compound is intended to affect, such as IL-1β-eRNA.

[0080] "Target region" refers to a portion of the target nucleic acid that the oligomeric compound is designed for hybridization.

[0081] "Therapeutic effective dose" refers to the amount of medication that provides therapeutic benefit to an individual.

[0082] "Treatment" refers to the administration of a pharmaceutical composition to achieve alteration or improvement of a disease, disorder, or symptom.

[0083] "Unmodified nucleotides" refer to nucleotides composed of naturally occurring nucleobases, sugar moieties, and nucleoside bonds. Unmodified nucleotides are RNA nucleotides (i.e., β-D-ribonucleoside) or DNA nucleotides (i.e., α-D-deoxyribonucleoside).

[0084] Antisense ASO compound motif

[0085] This invention provides a nick-body antisense oligonucleotide (ASO) compound (“nick-body compound”) that is substantially complementary to a region of IL-1β eRNA (including 100% complementarity over the entire length of the nick-body compound) and inhibits the transcription of multiple acute inflammatory genes regulated by IL-1β-eRNA. “Substantially complementary” means no more than two mismatches over the entire length of the nick-body. In some embodiments, there is one mismatch between the nick-body compound and the target region on IL-1β eRNA. In some embodiments, there are two mismatches between the nick-body compound and the target region on IL-1β-eRNA. In some embodiments, the nick-body compound comprises a modified oligonucleotide of 12 to 29 linked nucleosides. The nick-body compound is substantially complementary to a region of IL-1β eRNA (SEQ ID NO. 1) (e.g., no more than one nucleotide mismatch over the entire length of the nick-body compound) and inhibits the transcription of multiple acute inflammatory genes regulated by IL-1β-eRNA.

[0086] The nick body comprises an internal region of a nucleoside having multiple nucleotides or linked nucleotides, situated between an external region of a nucleoside having multiple nucleotides or linked nucleotides that are chemically different from the internal region. In the case of antisense oligonucleotides with a nick body motif, the interstitial fragment is typically used as a substrate for endonuclease cleavage, while the wing fragment contains a modified nucleoside. The regions of the nick body (5' wing, interstitial sequence, and 3' wing) are distinguished by the type of sugar moiety comprising each distinct region. The types of sugar moiety used to distinguish the nick body regions may include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O-CH3, etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include nucleosides with 4′-(CH2)nO-2′ bridges, where n=1 or n=2). Preferably, each distinct region contains a uniform sugar moiety. The wing-space-wing motif is “XYZ”, where “X” represents the length of the 5' wing region, “Y” represents the length of the space region, and “Z” represents the length of the 3' wing region. Generally, the notch form referred to as “XYZ” has a configuration in which the space segment is immediately adjacent to both the 5' and 3' wing segments. Therefore, there is no intermediate nucleotide between the 5' wing segment and the space segment, or between the space segment and the 3' wing segment. Typically, X and Z are nucleotides with the same chemical modification, but they can also be nucleotides with different chemical modifications. Preferably, Y is between 8 and 15 nucleotides. X or Z can be any one of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Therefore, notched bodies include, but are not limited to, for example, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, or 5-8-5.

[0087] In one embodiment, the nick body has ten 2′-deoxyribonucleotide spacer segments adjacent to and between five chemically modified nucleoside wing segments. In some embodiments, the chemical modification in the wings comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification. Preferably, the space-widened antisense oligonucleotide has eight 2′-deoxyribonucleotide spacer segments adjacent to and between five chemically modified nucleoside wing segments. Alternatively, the chemical modification comprises a 2′-sugar modification. Alternatively, the chemical modification comprises a 2′-MOE sugar modification.

[0088] The nick body has an interstitial segment consisting of eight 2′-deoxyribonucleotides, adjacent to and between five to six chemically modified nucleosides. The chemical modifications include 2′-sugar modifications, such as 2′-MOE sugar modifications. The nick body compound has a nucleotide sequence substantially complementary to the nucleotide sequence of SEQ ID NO: 1 throughout its length, for example, complementary to one of the regions AC described herein, including the entire length of region B of SEQ ID NO: 1 (IL-1β eRNA nucleotides 1153-1172), region A of IL-1β eRNA nucleotides 58-83, and region C of SEQ ID NO: 1 (IL-1β eRNA nucleotides 1245-1297).

[0089] Chemical modifications to antisense oligonucleotides can enhance their resistance to nucleases and potentially their ability to enter cells. For example, phosphate-thionucleotide oligonucleotides can be used. Other deoxynucleotide analogs include methylphosphonates, aminophosphates, dithiophosphates, N3'P5'-aminophosphates, and oligonucleotide phosphate-thiophosphates and their 2'-O-alkyl analogs, as well as 2'-O-methylribonucleotide methylphosphonates. Alternatively, mixed backbone oligonucleotides (“MBOs”) can be used. An MBO contains a phosphate-thionucleotide oligodeoxynucleotide fragment and appropriately placed modified oligodeoxynucleotide or oligoribonucleotide fragments. MBOs, with their phosphate-thionucleotide bond fragments and other modified oligonucleotide fragments such as methylphosphonates, are nonionic and highly resistant to nucleases or 2'-O-alkyl oligoribonucleotides.

[0090] In some embodiments, the oligonucleotide sugar moiety is a modified sugar moiety. In some embodiments, the modified sugar moiety may be the sugar moiety of a conformationally restricted sugar. In some embodiments, the conformationally restricted sugar may be a locked nucleotide (locked nucleic acid or LNA). In some embodiments, the locked nucleotide may be selected from one of the following types: 2′-O-CH2-4′ (oxo-LNA), 2′-CH2-CH2-4′ (methylene-LNA), 2′-NH-CH2-4′ (amino-LNA), 2′-N(CH3)-CH2-4′ (methylamino-LNA), 2′-S-CH2-4′ (thio-LNA), and 2′-Se-CH2-4′ (seleno-LNA). In some embodiments, the conformationally restricted sugar may be a bridging nucleic acid (BNA). Some conformationally restricted sugars may be locked nucleic acids, such as Formulas III and IV of U.S. Patent No. 10,465,188, which are incorporated herein by reference.

[0091] It can be used to synthesize antisense oligonucleotides. See, for example, Stein CA and Krieg AM (eds), Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss).

[0092] Hybridization

[0093] Hybridization occurs between the nick body and the target nucleic acid (SEQ ID NO.1). The most common mechanism of hybridization involves hydrogen bonds between complementary nucleobases of the nucleic acid molecules (e.g., Watson-Crick hydrogen bonds, Hugustine hydrogen bonds, or reverse Hugustine hydrogen bonds). Hybridization can occur under various conditions. The stringent conditions are sequence-dependent and determined by the properties and composition of the nucleic acid molecule to be hybridized.

[0094] Modified sugar portion

[0095] Antisense compounds may optionally contain one or more nucleosides in which the glycosyl group has been modified. Such sugar-modified nucleosides can confer antisense compounds enhanced nuclease stability, increased binding affinity, or other beneficial biological properties. The nucleosides comprise a chemically modified furanose ring moiety. Examples of chemically modified furanose rings include, but are not limited to, the addition of substituents (including 5′ and 2′ substituents, bridging non-homogeneous ring atoms to form bicyclic nucleic acids (BNAs), and the use of S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C). 12 Alkyl or protecting groups) replace the ribose epoxide atom and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleosides (WO2008 / 101157, for other disclosed 5′,2′-disubstituted nucleosides) or ribose epoxide atoms replaced with S, further substituted at the 2′-position (US Patent Application 2005 / 0130923), or 5′-substituted BNA (WO2007 / 134181, wherein LNA is substituted, for example, with 5′-methyl or 5′-vinyl).

[0096] Antisense oligonucleotides may be part of a composition that may contain oligonucleotides comprising multiple regions of IL-1β eRNA (SEQ ID NO: 1).

[0097] When this document refers to a particular protein, derivatives, variants, and fragments may be considered and included. Protein derivatives and variants are well understood by those skilled in the art and may involve amino acid sequence variants with insertions, substitutions, or deletions known in the art.

[0098] Modified nucleotide bases

[0099] The modified nucleotide bases include those of formula Ia, Ib, IIa, or IIb:

[0100] in

[0101] Each X is independently O or S, where 0, 1, or 2 instances of X are S; Each W is independently H, OH, halogen, or -O-C1-6 alkyl, wherein the alkyl group may be optionally substituted by up to three C1-4 alkyl groups, C1-4 alkoxy groups, halogens, amino groups, CN, NO2, or OH; each Qa is independently a bifunctional C1-6 alkylene group, which may be optionally substituted by up to two C1-4 alkyl groups, C1-4 alkoxy groups, halogens, or OH; and each Qb is independently a bond or bifunctional moiety selected from -O-, -S-, -NO-, -N(R)-, -C(O)-, -C(O)O-, and -C(O)N(R)-, wherein R is an unsubstituted C1-4 alkyl group.

[0102] In one implementation, each X is O. In another implementation, an instance of X is S.

[0103] In one embodiment, the nick body comprises one or more nucleotides of formula Ia or Ib, wherein W is a halogen. In another embodiment, W is fluorine. In a further embodiment, the nick body comprises one or more nucleotides of formula Ia. In a further embodiment, the nick body comprises one or more nucleotides of formula Ib.

[0104] In one embodiment, the nick body comprises one or more nucleotides of formula Ia or Ib, wherein W is -OC. 1-6 Alkyl groups, wherein the alkyl group may be selected from at most three carbon atoms. 1-4 Alkyl, C 1-4 Alkyl, halogen, amino, or OH substitutions. In a further embodiment, W is -OC. 1-6 Alkyl, wherein the alkyl group may be C 1-4 Alkyl substitution. In a further embodiment, W is unsubstituted -OC. 1-6 Alkyl group. In another further embodiment, W is -OC 1-6 Alkyl, wherein the alkyl group is C 1-4 Alkyl substitution. In a further embodiment, W is selected from methoxy and -O-CH2CH2-OCH3. In one embodiment, the nicked body comprises one or more nucleotides of formula Ia. In one embodiment, the nicked body comprises one or more nucleotides of formula Ib.

[0105] In one embodiment, the nick body comprises one or more β-D nucleotides of formula IIa or α-L nucleotides of formula IIb, wherein Q a It is an unsubstituted bifunctional C 1-6 Alkylene and Q b It is a bond or bifunctional part selected from -O-, -S-, -NO- and -N(R)-.

[0106] In another implementation, Q a Selected from -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2(CH3)- and Q b It is a bond or bifunctional moiety selected from -O-, -S-, -N(R)-O-, and -N(R)-, where R is H or C. 1-6 alkyl.

[0107] In one embodiment of formula IIa or IIb, Q a It is -CH2- and Q b Yes -O-. In another embodiment of formula IIa or IIb, Q a It is -CH2-CH2- and Q b Yes -O-. In another embodiment of formula IIa or IIb, Q a It is -CH2- and Q b It is -N(R)-O-, where R is H or C. 1-6 Alkyl group. In another embodiment of formula IIa or IIb, Q a It is -CH(CH3)- and Q b Yes -O-. In another embodiment of formula IIa or IIb, Q a It is -CH2- and Q b Yes -S-. In another embodiment of formula IIa or IIb, Q a It is -CH2- and Qb is -N(R)-, where R is H or C. 1-6 Alkyl group. In another embodiment of formula IIa or IIb, Q a It is -CH2-CH(CH3)- and Q b It is a key.

[0108] In some embodiments, the nick body comprises one or more nucleotides selected from nucleotides modified as follows:

[0109] Many other bicyclic and tricyclic sugar substitution ring systems can be used to modify nucleosides to incorporate into antisense compounds (see, for example, review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).

[0110] application

[0111] The notched bodies described herein can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be local, pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via a nebulizer); intratracheal, intranasal, epidermal and percutaneous, oral, or parenteral. The compounds and compositions described herein can be delivered in a manner targeting specific tissues, such as bone marrow or brain. The compounds and compositions described herein are administered via parenteral administration. “Parenteral administration” means administration by injection or infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, or intracranial administration, such as intracerebellar, intrathecal, intraventricular, intraventricular, intravascular, intracerebral, or intraventricular administration. Administration can be continuous, chronic, transient, or intermittent.

[0112] Parenteral administration is also performed via infusion. Infusion can be chronic, continuous, brief, or intermittent, and can be done by pump or injection. In some implementations, parenteral administration is subcutaneous.

[0113] preparation

[0114] The pharmaceutical compositions of this invention include, for example, compositions comprising, a nickbody compound containing an IL-1β eRNA transcription inhibitor. The pharmaceutical formulations of this invention may further comprise one or more pharmaceutically acceptable excipients.

[0115] The pharmaceutical formulation of this invention may further comprise one or more pharmaceutically acceptable excipients.

[0116] The IL-1β eRNA transcription inhibitor nick body compound may be present in the formulation in a substantially isolated form. It is understood that the product may be mixed with a carrier or diluent that does not interfere with the intended purpose of the product, but is still considered substantially isolated. The product of the present invention may also be in a substantially purified form, in which case it typically contains about 80%, 85%, or 90%, such as at least about 88%, at least about 90%, 95%, or 98%, or at least about 99% of the dry matter of the oligonucleotide or formulation.

[0117] Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for both veterinary and human pharmaceutical use. For example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, delay agents, polymers and lipids, emulsions, etc. As further examples, suitable liquid carriers, particularly for injectable solutions, include water, saline solutions, glucose solutions, etc., and carriers particularly suitable for administering pharmaceutical agents, such as liposomes.

[0118] Suitable carriers and diluents include buffered aqueous solutions, saline solutions, glucose, glycerol, isotonic saline solutions such as phosphate-buffered saline, isotonic water, and combinations thereof. In some embodiments, the carrier may include propylene glycol, dimethyl isosorbide, and water, and more specifically, phosphate-buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols. In some embodiments, pharmaceutically acceptable carriers or diluents may be or contain thermosetting poloxamer (which may be a liquid or gel depending on temperature), carboxycellulose (e.g., carboxymethyl cellulose), collagen (e.g., type I collagen), collagen materials containing procollagen, hyaluronic acid or derived hyaluronic acid, and / or oils (e.g., emu oil). Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers.

[0119] The composition may be in any standard known dosage form, including tablets, pills, capsules, semi-solid dosage forms, powders, sustained-release dosage forms, solutions, suspensions, elixirs, aerosols, injections, gels, creams, transdermal delivery devices (e.g., transdermal patches), implants (such as ocular implants), or any other suitable composition.

[0120] Preferably, the IL-1β eRNA transcription inhibitor nick body compound is combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical composition.

[0121] Pharmaceutically acceptable salts may also be present, such as inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, such as citrate, acetate, propionate, malonate, benzoate, etc.

[0122] In addition, if desired, wetting agents or emulsifiers, stabilizers or pH buffers or preservatives may also be present. In some embodiments, the pharmaceutical composition of the invention will contain suitable acceptable buffers, such as acetate buffers, citrate buffers, phosphate buffers, borate buffers and mixtures thereof. In some embodiments, buffers that can be used in the invention include boric acid, sodium borate, sodium phosphate (including monobasic, dibasic, and tribasic phosphates, such as sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate), and mixtures thereof. In some embodiments, the preservative may be stable chlorine dioxide, cationic polymers, or quaternary ammonium compounds. In some embodiments, the pharmaceutical composition may also contain wetting agents, nutrients, thickeners, antioxidants, etc., such as disodium ethylenediaminetetraacetate, alkali metal hexametaphosphate, citric acid, sodium citrate, sodium metabisulfite, sodium thiosulfate, N-acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose and mixtures thereof. In some embodiments, the pharmaceutical formulation of this invention will not include preservatives. In some embodiments, the IL-1βeRNA transcription inhibitor nickbody composition or formulation comprises disodium hydrogen phosphate heptahydrate or potassium phosphate, sodium dihydrogen phosphate, or both.

[0123] Nucleic acid uptake in mammalian cells can be enhanced using known transfection techniques, including the use of transfection agents. These techniques can be employed with certain IL-1β eRNA transcription inhibitor nicksome compounds. Examples of useful transfection agents include cationic agents (such as calcium phosphate and DEAE dextran), liposomes (such as lipofectam™ and transfectam™), and surfactants.

[0124] The composition can be formulated according to standard techniques known in the art, including those found, for example, in standard references such as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins, 2000.

[0125] Any container suitable for storing and / or administering the pharmaceutical composition may be used in the combination products of this invention. Those skilled in the art will understand suitable containers. For example, such containers include vials and syringes. Containers may be appropriately sterilized and sealed.

[0126] Such compositions contain pharmaceutically acceptable solvents, such as water or saline, diluents, carriers, or adjuvants. The pharmaceutical compositions can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be local (including ocular and mucous membrane delivery, including vaginal and rectal delivery), pulmonary (by inhalation or blowing of powders or aerosols, including via nebulizers); intratracheal, intranasal, epidermal, and percutaneous, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (intrathecal or intraventricular administration).

[0127] Compounds can also be mixed, coupled, or otherwise combined with other molecules, molecular structures, or mixtures of compounds, such as liposomes, receptor-targeting molecules, or other formulations, to aid in uptake, distribution, and / or absorption.

[0128] The term "pharmaceutically acceptable carrier" refers to a carrier that is physiologically and pharmaceutically acceptable to a compound, i.e., a carrier that retains the desired biological activity of the parent compound without imparting undesirable toxicological effects. Preferred examples of pharmaceutically acceptable carriers and their uses for oligonucleotides are further described in U.S. Patent 6,287,860, which is incorporated herein by reference. Sodium carriers have proven to be a suitable form for oligonucleotide drugs.

[0129] Formulations include liposome formulations. The term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayer. Liposomes are single-compartment or multi-compartment vesicles with a membrane formed of lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes and are believed to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to encapsulate DNA rather than complex with it. Both cationic and non-cationic liposomes are used to deliver DNA into cells.

[0130] Liposomes also include "sterically stable" liposomes, which are liposomes containing one or more specific lipids that, when incorporated into liposomes, can extend cycle life compared to liposomes lacking such specific lipids. Liposomes and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein by reference.

[0131] Preferred formulations for topical application include mixtures of oligonucleotides with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Preferred lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearate phosphatidylcholine), anion-positive (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

[0132] lipid nanoparticles

[0133] LNPs are multi-component systems, typically composed of ionizable amino lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipids. All components contribute to the efficient delivery of nucleic acid drugs and particle stability (Schroeder et al., J. Intern. Med. (2010;267:9-21). Cationic lipids electrostatically aggregate negatively charged RNA into nanoparticles, and the use of positively charged ionizable lipids at acidic pH is thought to enhance endosome escape. Clinical and non-clinical delivery formulations are primarily based on cationic lipids, such as DLin-MC3-DMA (MC3). (Kanasty et al.) Nat. Mater. 2013;12:967–977; and Xue et al. Curr. Pharm. Des. 2015;21:3140–3147).

[0134] Further LNPs include nanoemulsions having a perfluorocarbon component (a), an emulsifying component (b), and an endocytosis-enhancing component (c). The perfluorocarbon component (a) consists of at least one perfluorocarbon compound, the emulsifying component (b) includes phospholipids and optionally accessory lipids, and the endocytosis-enhancing component (c) contains at least one compound that induces cellular uptake of the nanoemulsion. The perfluorocarbon compound of component (a) is preferably selected from compounds having a C-structure. m F 2m+1 X, XC m F 2m X, XC n F 2n OC o F 2o X, N(C) o F 2o X)3 and N(C o F 2o Compounds of type +1)3, where m is an integer from 3 to 10, n and o are integers from 1 to 5, and X is independent of any further occurrences selected from Cl, Br, and I. Examples of perfluorocarbon compounds are perfluorooctyl bromide and perfluorotributylamine.

[0135] Examples of emulsifiers include phospholipids, such as phospholipid compounds represented by Formula I: (I), Among them, R 1 and R 2 Independently selected from H and C 16-24 Acyl residues, which can be saturated or unsaturated and can carry 1 to 3 R residues. 3And one or more of the C atoms can be O or NR 4 Substitution, X is selected from H, -(CH2). p -N(R 4 )3 + -(CH2) p -CH(N(R 4 )3 + )-COO - -(CH2) p -CH(OH)-CH2OH and -CH2(CHOH) p -CH2OH (where p is an integer from 1 to 5); R 3 Independently selected from H, lower alkyl groups, F, Cl, CN, and OH; and R 4 It is independently selected from H, CH3 and CH2CH3, or their pharmacologically acceptable carriers.

[0136] Following subcutaneous administration, it is expected that most of LNP and its mRNA vector will remain at the injection site, resulting in high local concentrations. Since LNP is known to be pro-inflammatory, this is primarily attributed to the ionizable lipids present in LNP (Sabnis et al.). Mol. Ther. (2018;26:1509–1519), therefore it is expected that the mRNA formulated in subcutaneous LNP injection will be associated with dose-limiting inflammatory responses. Combined administration of dexamethasone and LNP can reduce the immune inflammatory response following intravenous injection (Abrams et al.). Mol. Ther. 2010;18:171-180). Chen et al. ( J. Control. Release. (2018;286:46–54.) showed that systemic administration of LNP-containing nucleic acids and lipophilic dexamethasone prodrugs reduced immune stimulation.

[0137] Dosage

[0138] The optimal dosing regimen is calculated by measuring drug accumulation in the patient's body. The optimal dose depends on the relative potency of the individual oligonucleotides and is typically estimated based on the EC50, which has been found to be effective in in vitro and in vivo animal models. Generally, the dose is 0.01 μg to 100 g per kg body weight and can be administered once or more daily, weekly, monthly, or annually, or at desired intervals. After successful treatment, maintenance therapy may be necessary to prevent relapse of the disease state, in which oligonucleotides are administered at a maintenance dose ranging from 0.01 μg to 100 g per kg body weight, once or more daily.

[0139] Therapeutic effective doses include, but are not limited to, the doses described herein. These doses and other therapeutically effective doses are administered according to one or more therapeutically effective dose regimens described herein.

[0140] Products / Reagent Kits

[0141] In another embodiment of the invention, an article of manufacture or "kit" containing material that can be used to inhibit IL-1β eRNA is provided. The kit comprises a container holding a composition containing one or more modulators, such as IL-1β eRNA modulators, such as IL-1β eRNA transcription inhibitor nickbody compounds described herein. Suitable containers include, for example, bottles, vials, etc. The container can be formed from various materials, such as glass or plastic. The kit may also contain a pharmaceutically acceptable carrier. In some embodiments, the kit may also include components for administering the composition, such as syringes, needles, microneedles, etc.

[0142] manufacture

[0143] The oligonucleotides of the present invention can be manufactured using solid-phase chemistry for synthesizing oligonucleotides, chemistry known in the art for synthesizing and preparing peptides and peptide-like compounds, and chemistry known in the art for synthesizing organic compounds. In one aspect, formulations of the invention will comprise a salt of the oligonucleotide of the invention, such as a sodium salt of the oligonucleotide of the invention. The kit may also comprise a pharmaceutically acceptable carrier.

[0144] In some embodiments, the formulation of the invention is substantially pure. Substantially pure means that the formulation contains less than about 10%, 5%, or 1%, preferably less than about 0.1%, of any nucleotide or non-nucleotide impurities. In some embodiments, total impurities, including metabolites of the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound, will not exceed 15%. In some embodiments, total impurities, including metabolites of the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound, will not exceed 12%. In some embodiments, total impurities, including metabolites of the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound, will not exceed 11%. In other embodiments, total impurities, including metabolites of the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound, will not exceed 10%.

[0145] A sterile composition comprising the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound of the present invention is prepared by aseptic processing by dissolving the IL-1β eRNA transcription inhibitor nick-somatic ASO (antisense oligonucleotide) compound in a formulation carrier. In one embodiment, the formulation may also be sterilized by filtration. The excipients used to manufacture the formulation of the present invention are widely used in pharmaceuticals and are published in accordance with pharmacopoeia standards.

[0146] Example

[0147] Example 1

[0148] This embodiment provides a screening system for in vitro detection of candidate nick bodies that inhibit gene transcription regulated by IL-1β eRNA. The effects of candidate nick body compounds on target nucleic acid expression (e.g., messenger RNA) are screened using real-time polymerase chain reaction (RT-PCR).

[0149] The THP-1 human mononuclear cell line (derived from acute leukemia patients) was purchased from InvivoGen. THP-1 cells were maintained in complete culture medium consisting of RPMI 1640, 1% (2 mM) GlutaMAX L-glutamine supplement, 25 mM HEPES, 10% FBS, 100 µg / ml Normocin, penicillin-streptomycin (100 U / ml), blastomycin (10 µg / ml), and Zeocin (100 µg / ml). Prior to screening with vaccine, the THP-1 mononuclear cell culture was divided 50% to allow the cells to re-enter the exponential growth phase. 250,000 cells were seeded per well in four replicates in 96-well plates, with each well containing 180 µL of complete culture medium. The nick body compound for each test was added to the THP-1 cells at a final concentration of 10 µM and gently mixed. The plates were incubated at 37°C and 5% CO2 for 24 hours. Then, LPS (10 ng / mL) was added to each well, and the plate was incubated at 37°C and 5% CO2 for another 24 hours.

[0150] Antisense regulation of specific gene expression by IL-1β eRNA was detected by real-time PCR (RT-PCR). RNA analysis was performed on total cellular RNA or poly(A)+ mRNA. RNA was isolated and prepared using TRIZOL® reagents (Thermo Fisher Scientific) and the Direct-zol RNA mini-extraction kit (ZymoResearch) according to the manufacturer's recommended protocol.

[0151] Target RNA levels were quantified using the CFX Real-Time qPCR Detection System (Bio Rad) via quantitative real-time PCR. Prior to real-time PCR, isolated RNA underwent a reverse transcriptase (RT) reaction to produce complementary DNA (cDNA), which was then used as the substrate for real-time PCR amplification. RT reaction reagents and real-time PCR reagents were purchased from Thermo Fisher Scientific, and their usage protocols were provided by the manufacturer. The gene (or RNA) target levels obtained by real-time PCR were normalized using the expression levels of stably expressed housekeeping genes, such as HPRT or RPL37A. Total RNA was quantified using a Qubit fluorometer (Invitrogen / Scientific) and the Qubit RNA HS Detection Kit (Thermo Fisher Scientific catalog number Q32852), according to the manufacturer's protocol. The Qubit fluorometer was calibrated to standard.

[0152] A series of cleavage compounds are shown in Table 1. These cleavages are designed to target different regions of human IL-1β eRNA (SEQ ID NO. 1). The cleavage compounds in Table 1 are chimeric oligonucleotides (“cleavages”) with different conformations. For example, a cleavage with a conformation of 20 (5-10-5) nucleotides consists of a central “gap” region of 10 2′-deoxynucleotides flanked by 5 nucleotide “wings” on both sides (5′ and 3′ directions). These wings are composed of nucleosides modified with 2′-methoxyethyl (2′-MOE) sugars. The internucleotide (backbone) bonds throughout the oligonucleotide sequence are phosphate thiophosphates. The cytidine residues are 5-methylcytidine, and unless otherwise specified, they are cytidine residues in this case. A cleavage with a conformation of 16 (3-10-3) nucleotides consists of a central “gap” region of 10 2′-deoxynucleotides flanked by 3 nucleotide “wings” on both sides (5′ and 3′ directions). In some embodiments, the wings are composed of nucleosides modified with locked nucleic acids (LNAs) using cMe locked nucleic acid modification. The internucleotide (backbone) bonds throughout the oligonucleotide sequence are phosphate thiophosphates. The cytidine residues are 5-methylcytidine, and unless otherwise stated, they are cytidine residues in this case.

[0153] The notched compounds used are shown in Table 1. In Table 1, the abbreviations for nucleoside modifications are: M = 2'-methoxyethyl (2'-MOE) modified nucleoside; L = locked nucleic acid (LNA) modified nucleoside (cMe); d = 2′-deoxynucleoside.

[0154]

[0155] Table 1.

[0156] Since IL-1β-eRNA is a lncRNA that regulates IL-1β transcription, the effect of the nick body compounds on IL-1β transcription was analyzed by quantitative real-time PCR. Similarly, the effects of the nick body compounds on cytotoxicity and Toll-like receptor (TLR) signaling activation were analyzed by detecting the gene transcription of TNFRSF10B and secreted embryonic alkaline phosphatase (SEAP), respectively. Data are the average of four replicates of THP1 cells treated with the nick body compounds listed in Table 1.

[0157] Table 2 shows the fold changes in the expression of IL1β, TNFRSF10B, and SEAP genes in THP1 cells in the presence of the nick body compounds listed in Table 1. "ND" indicates "no data" if present. Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to negative controls (SEQ ID NO. 131 was used as a negative control for the MOE nick body and SEQ ID NO. 132 as a negative control for the LNA nick body). An expression value <1.0 indicates that transcription of the gene is repressed, and an expression value >1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0158]

[0159] Table 2.

[0160] The data in Table 2 show that, in this experiment, the notched bodies SEQ ID NO. 48, 50, 51, 64, 65 and 122 exhibited at least 50% inhibition of human IL1β expression.

[0161] Based on the screening results in Tables 1 and 2, a group of chimeric phosphate-thiolated nick-form compounds that effectively target human IL-1β eRNA (SEQ ID NO. 1) were synthesized. Table 3 provides the configuration, chemical modifications, and sequences of the newly synthesized chimeric phosphate-thiolated nick-form compounds. Abbreviations for nucleoside modifications in Table 3: M = 2'-methoxyethyl (2'-MOE) modified nucleoside; L = locked nucleic acid (LNA) modified nucleoside (cMe); d = 2'-deoxynucleoside; 2'Md = 2'OMe modified deoxynucleoside.

[0162]

[0163] Table 3.

[0164] The effects of the chimeric phosphate-thiocyanate nick compounds listed in Table 3 on the transcription of IL1β, TNFRSF10B, and SEAP in THP1 cells were analyzed by quantitative real-time PCR. Table 4 shows the gene expression of IL1B, TNFRSF10B, and SEAP by the chimeric phosphate-thiocyanate nick compounds SEQ ID NO. 50, 51, 69, and 84 targeting the human IL-1β eRNA sequence (SEQ ID NO. 1). Data were normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to negative controls (SEQ ID NO. 131 was used as a negative control for the MOE nick compound and SEQ ID NO. 132 was used as a negative control for the LNA nick compound). An expression value <1.0 indicates that the transcription of the gene was repressed, and an expression value >1.0 indicates that the gene was induced to be transcribed. For example, a value of 0.25 means that gene transcription was repressed by 75%.

[0165]

[0166] Table 4.

[0167] Based on the screening data in Tables 1-4, three nick regions (SEQ ID NO: 64, 65, 68, 69, 50, 25, 51, 121, 122, and 131) were identified in the target IL-1β-eRNA sequence (SEQ ID NO. 1). Table 5 provides the locations of the IL-1β eRNA target regions (A, B, and C) in the human IL-1β eRNA sequence (SEQ ID NO. 1) and the average inhibition of IL1β gene expression by nick regions targeting individual regions.

[0168]

[0169] Table 5.

[0170] All nicks targeting positions 58-80, 1153-1172, and 1245-1297 of IL-1β eRNA regions A, B, and C, respectively, showed an inhibitory effect of more than 40% on IL-1β gene expression.

[0171] Example 2: Inhibition of IL-1β eRNA in LPS-stimulated CD14-positive monocytes

[0172] Percoll mononuclear cells from healthy donors (n=3) were seeded at 1 x 10⁶ cells / well in 24-well (Sarstedt) serum-free RPMI 1640 containing GlutaMAX (Gibco), 10% FBS (Gibco), and 1% penicillin-streptomycin (Gibco) and incubated at 37°C, 5% CO₂ for 1 hour. The plated cells were washed with serum-free RPMI and treated with RPMI + 10% mixed human serum containing the nicked ASO compound SEQ ID NO. 69 or SEQ ID NO. 132, and incubated overnight at 37°C, 5% CO₂. Each treatment was performed twice. The plated cells were then treated with 10 ng / ml ultrapure LPS (Invivogen) and incubated overnight at 37°C, 5% CO₂. Cells treated with nick bodies were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was quantified using the NanoDrop® (Thermo Fisher Scientific) manufacturer's recommended protocol. Total RNA was subjected to reverse transcriptase (RT) reaction using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol to generate complementary DNA, which was used as a substrate for quantitative real-time polymerase chain reaction (RT-qPCR). Target RNA levels were quantified by RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad) with SsoAdvanced Universal SYBR Green according to the manufacturer's recommended protocol. The target amount obtained by RT-qPCR was normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0173] Table 6 presents the fold change in IL1B gene expression in LPS-treated monocytes (n=3 donors) in the presence of the nick body compound (SEQ ID NO. 69). Data are normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to LPS-treated monocytes with the control nick body compound (SEQ ID NO. 132). An expression value <1.0 indicates that transcription of the gene is repressed, and an expression value >1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0174]

[0175] Table 6.

[0176] Example 3: Inhibitory effect of IL-1β eRNA on prostate adenocarcinoma cell line PC-3

[0177] PC-3 cells (ATCC) were seeded at 6 x 10⁴ cells per well in 24-well Sarstedt RPMI 1640 plates containing GlutaMAX (Gibco), 10% FBS (Gibco), 1% penicillin-streptomycin (Gibco), and the cut-body compound SEQ ID NO. 50. The cut-body compound was added at 5 μM (low) or 30 μM (high) doses. Each treatment was performed in quadruplicates. The plated cells were incubated at 37°C and 5% CO₂ for 24 hours. The cut-body treated cells were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA miRVana Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was quantified using NanoDrop® (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Following the manufacturer's recommended reagents and protocols, total RNA was subjected to reverse transcriptase (RT) reactions using the iScript cDNA Synthesis Kit (Bio-Rad) to generate complementary DNA, which was used as a substrate for quantitative real-time polymerase chain reaction (RT-qPCR). Target RNA levels were quantified by RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad) with SsoAdvanced Universal SYBR Green, following the manufacturer's recommended protocols. The target levels obtained by RT-qPCR were normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0178] Table 7 shows the fold change in IL1B gene expression in PC-3 cells in the presence of the nick body compound. Data were normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to untreated PC-3 cells. An expression value <1.0 indicates that transcription of the gene is repressed, and an expression value >1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0179]

[0180] Table 7.

[0181] Example 4: Inhibitory effect of IL-1β eRNA in PBMCs treated with LPS and R848

[0182] Peripheral blood mononuclear cells (PBMCs) from three healthy donors with a mean age of 56.3 ± 16.8 years were purchased from CTL Europe and cryopreserved. The cryopreserved PBMCs were thawed in pre-warmed wash medium (RPMI (Dutch modified) + 20% FBS + 2 mM GlutaMAX + 1 mM sodium pyruvate + 1% penicillin / streptomycin). The PBMCs were then washed three times in pre-warmed wash medium and centrifuged at 500 rpm for 10 min at room temperature. Centrifugation was repeated twice before resuspending the cells in pre-warmed medium (RPMI (Dutch modified) + 10% FBS + 2 mM GlutaMAX + 1 mM sodium pyruvate + 1% penicillin / streptomycin). Next, cells were counted and seeded at approximately 1 x 10⁵ cells per well in a 96-well U-bottom tissue culture plate (CellSTAR) containing a TLR mixture with 100 ng / mL ultrapure LPS (Invivogen) and 500 ng / mL Resiquimod (R848, Invivogan), with or without the nick body compound (SEQ ID50). The nick body compound was added to a final concentration of 5 μM. Each treatment was performed in duplicate, and the plated cells were incubated at 37°C and 5% CO₂ for 48 hours. The plated cells were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was then quantified using NanoDrop® (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Finally, total RNA was subjected to reverse transcriptase (RT) using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol to produce complementary DNA. Following the manufacturer's recommended protocol, SsoAdvanced Universal SYBRGreen was used, with complementary DNA as the substrate for quantitative real-time polymerase chain reaction (RT-qPCR). Target RNA levels were quantified via RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad). The target levels obtained by RT-qPCR were normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0183] Table 8 shows the fold change in IL1B gene expression in TLR-stimulated PBMCs in the presence of the gap-body compound (SEQ ID NO. 50). Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to PBMCs treated with a TLR mixture without the gap-body compound. An expression value <1.0 indicates that transcription of the gene is repressed, and an expression value >1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0184]

[0185] Table 8.

[0186] Example 5: IL-1β eRNA inhibits LPS-stimulated mouse macrophage cell line, RAW264.7

[0187] This embodiment provides cross-reactivity of clefsome compounds from different species (human and mouse) for in vivo testing in mouse models. Homology regions of the target sequences of human (SEQ ID NO. 1) and mouse (SEQ ID NO. 2) IL-1β eRNA were compared, but no sequences longer than 20 nucleotides were found. Three clefsome antisense sequences complementary to human and mouse IL-1β eRNA were designed with no more than one mismatch with mouse IL-1β eRNA. These clefsomes were designed to function in both in vitro human cell models and in vivo mouse models. However, the relative antisense potency of the two forms may not be equal due to imperfect homology with IL-1β eRNA or another.

[0188]

[0189] Table 9A.

[0190] RAW 264.7 cells (ATCC) were seeded at 2.5 x 10⁵ cells per well in 24-well (Sarstedt) DMEM (Gibco) containing 10% FBS (FisherScientific) and 1% penicillin / streptomycin (Gibco). The plated cells were incubated at 37°C and 5% CO₂ for 24 hours. Cells were then treated with 10 ng / ml ultrapure LPS (Invitrogen) and incubated at 37°C and 5% CO₂ for 24 hours. Each treatment was performed in quadruplicates. Cells were then treated with 5 μmol (low), 10 μmol (medium), and 30 μmol (high) of a nick body compound (SEQ ID NO. 106).

[0191] Table 9B shows the fold change in IL1β gene expression in LPS-stimulated RAW264.7 cells in the presence of the nick body compound (SEQ ID NO. 106). Data are normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to RAW 264.7 cells treated with an LPS mixture without the nick body compound. An expression value <1.0 indicates that transcription of the gene is repressed, and an expression value >1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0192]

[0193] Table 9B.

[0194] All patents, publications, scientific articles, websites, and other documents and materials cited or referenced herein demonstrate the skill level of a person skilled in the art to which this invention pertains, and each such cited document and material is incorporated herein by reference to the same extent as if it were individually incorporated in its entirety or fully set forth herein. The applicant reserves the right to incorporate any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents into this specification. No application, patent, or publication mentioned in this specification is, and should not be construed as, an admission or in any way implying that they constitute valid prior art or are part of common general knowledge in any country of the world.

[0195] The specific methods and compositions described herein represent preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon consideration of this specification, and these objects, aspects, and embodiments are contained within the spirit of the invention as defined by the scope of the claims. It will be readily understood by those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described herein illustratively can be suitably practiced without any one or more elements or limitations that are not specifically disclosed herein as necessary. Thus, for example, in each case herein, in embodiments or examples of the invention, any one of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced by any of the other two terms in the specification. Furthermore, the terms “comprising,” “including,” “containing,” etc., are used extensively and without limitation. The methods and processes described herein illustratively can be suitably practiced in different sequences of steps, and they are not necessarily limited to the sequence of steps indicated herein or in the claims. Furthermore, as used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “a (a), an” and “the” include plural references. Under no circumstances should this patent be construed as being limited to the specific embodiments, implementations, or methods specifically disclosed herein. Under no circumstances should this patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless the applicant expressly and unreservedly adopts such statement in their responsive written statement. Furthermore, the provision of headings, subheadings, etc., is for the purpose of enhancing the reader's understanding of this document and should not be construed as limiting the scope of the invention. Any embodiment of the aspects, implementations, or components of the invention described herein should be considered non-limiting.

[0196] The terms and expressions used are descriptive rather than limiting, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof. However, it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined in the appended claims.

[0197] This invention provides a broad and general description of the invention. Each narrower group of species and subgenera falling within the scope of the genus's disclosure also constitutes part of this invention. This includes the genus description of the invention, but with the conditional or negative limitation that any subject matter is removed from that genus, regardless of whether the excluded material is specifically cited herein.

[0198] Other embodiments are within the scope of the following claims. Furthermore, in the context of the description of features or aspects of the invention according to the Markush group, those skilled in the art will recognize that the invention is therefore also described according to any single member or subgroup of members of the Markush group.

Claims

1. A nick body compound for inhibiting IL-1β eRNA transcription, comprising about 14 to about 25 nucleotide bases, said nick body compound having a (3' to 5') 3' wing region comprising 3 to 7 chemically modified RNA bases, an interstitial region comprising at least 8 DNA bases; and a 5' wing region comprising 3 to 7 chemically modified RNA bases; and wherein said nick body is substantially complementary to the 14-25 base region on IL-1β eRNA (SEQ ID NO. 1).

2. The nick body compound for inhibiting IL-1β transcription according to claim 1, wherein each of the nick body nucleotides is linked by a phosphate thioester (P=S) nucleotide internucleotide bond through the nick body; and wherein the modified nucleotide base modification is selected from the group consisting of: 2'-methoxyethyl (MOE) modification, locked nucleic acid (LNA) modification, 2'F-ANA modification, 2'-O-methoxyethyl (2'OMe) modification, and combinations thereof.

3. A nick body compound that inhibits transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotide bases; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein the nick body sequence is complementary to region A of bases 58 to 80 of the IL-1β eRNA (SEQ ID NO. 1).

4. The nick body compound according to claim 3 that inhibits transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, wherein the modified nucleotide base modification is selected from the group consisting of: 2'-methoxyethyl (MOE) modification, locked nucleic acid (LNA) modification, 2'F-ANA modification, 2'-O-methoxyethyl (2'OMe) modification, and combinations thereof.

5. The nick body compound of claim 3 that inhibits transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, wherein the nick body compound complementary to region A is selected from the group consisting of: SEQ ID NO. 64, 65, 68, 69 and combinations thereof.

6. A nick body compound for inhibiting transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotide bases; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein the nick body sequence is complementary to region B of IL-1β eRNA (SEQ ID NO. 1) bases 1153 to 1172.

7. The nick body compound of claim 6 that inhibits transcription of the acute inflammatory IL-1 gene regulated by IL-1β eRNA, wherein the modified nucleotide base is selected from the group consisting of: 2'-methoxyethyl (MOE) modification, locked nucleic acid (LNA) modification, 2'F-ANA modification, 2'-O-methoxyethyl (2'OMe) modification, and combinations thereof.

8. The nick body compound for inhibiting acute inflammatory IL-1β gene transcription regulated by IL-1β eRNA according to claim 6, wherein the nick body compound complementary to region B is SEQ ID NO. 50, 174 and combinations thereof.

9. A nick body compound for inhibiting transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, comprising: (a) a 5' wing sequence having about 3 to about 7 wing-modified nucleotide bases; (b) a central interstitial region sequence having about 8 to about 15 2' deoxynucleotides; and (c) a 3' wing sequence having about 3 to about 7 wing-modified nucleotide bases; wherein the nick body sequence is complementary to region C of IL-1β eRNA (SEQ ID NO. 1) from base 1245 to base 1297.

10. The nick body compound of claim 9 that inhibits transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, wherein the modified nucleotide base is selected from the group consisting of: 2'-methoxyethyl (MOE) modification, locked nucleic acid (LNA) modification, 2'F-ANA modification, 2'-O-methoxyethyl (2'OMe) modification, and combinations thereof.

11. The nick body compound of claim 9 that inhibits transcription of the acute inflammatory IL-1β gene regulated by IL-1β eRNA, wherein the nick body compound complementary to region C is selected from the group consisting of: SEQ ID NO. 25, 51, 121, 122 and combinations thereof.

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