Nucleic acid molecule conjugate with an extended in vivo half-life

Abstract

The present invention relates to the field of biomedicine. In particular, the present invention relates to a nucleic acid molecule conjugate with extended in vivo half-life, the nucleic acid molecule being conjugated to a fatty acid and a coumarin derivative. More particularly, the nucleic acid molecule is an aptamer, in particular an aptamer against sclerostin.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. In particular, the present invention relates to a nucleic acid molecule conjugate having an extended in vivo half-life, wherein the nucleic acid molecule is conjugated to a fatty acid and a coumarin derivative. More specifically, the nucleic acid molecule is an aptamer, particularly an aptamer against sclerostin.

Background Art

[0002] Aptamers are short single-stranded oligonucleotides that bind to their targets through conformational complementarity (Ellington and Szostak 1990, Tuerk and Gold 1990). Aptamers can be selected and adapted for positive and negative targets. Compared to therapeutic antibodies, aptamers have similar affinity and specificity but have several important advantages. Regarding immunogenicity, aptamers are not recognized as foreign by the immune system due to their small size and do not stimulate a negative immune response (Keefe, Pai et al., 2010). Regarding production and cost, aptamers are identified in vitro under various selection conditions and can be easily synthesized by chemical methods, so production is less costly and less risky (Banerjee 2010). Regarding stability, aptamers are temperature-resistant, so they have an indefinite shelf life, allow transportation without any special requirements for cooling, and eliminate the need for a continuous cold chain (Jayasena 1999). Pegaptanib, an aptamer against vascular endothelial growth factor (VEGF) for the treatment of age-related macular degeneration, is well used clinically (Jellinek, Green et al., 1994, Ruckman, Green et al., 1998, Ng and Adamis 2006, Que-Gewirth and Sullenger 2007).

[0003] However, unmodified aptamers are susceptible to degradation by enzymes widely present in the body. More importantly, since the molecular weight of unmodified aptamers is not large enough, unmodified aptamers are easily filtered and excreted by the kidneys. This shortens the half-life of unmodified aptamers very significantly and limits the therapeutic effect when used clinically. Clinically, nucleic acid aptamers are often covalently modified with PEG having a large molecular weight (20 kDa, 40 kDa) to make their overall molecular weight larger than the glomerular filtration molecular weight cut-off (30 - 50 kDa), thereby increasing the circulating half-life of the molecules in the body. However, the large molecular weight of the PEG component makes the proportion of the aptamer in the active ingredient very small. At the same time, due to problems with solubility and solution fluidity, the presence of PEG limits the maximum dose and restricts the clinical application of many aptamer drugs.

[0004] Therefore, improving the enzyme stability of the aptamer itself while extending the circulating half-life of the aptamer through chemical modification with small molecules has become an important research direction in the development of aptamer drugs. Therefore, there remains a need in the art for modified aptamers with an extended in vivo half-life and enhanced in vivo efficacy.

[0005] Osteoporosis is a disease with reduced bone mass and bone strength, leading to an increased risk of fractures (Hamersma, Gardner et al., 2003). Various drugs for osteoporosis treatment are mainly bone resorption inhibitors, which inhibit bone resorption to prevent further bone mass loss (Russell, Watts et al., 2008, Pennypacker, Duong et al., 2011). Parathyroid hormone (PTH) peptide is the only available anabolic agent that stimulates bone formation to reverse established osteoporosis (Compston 2007, Greenspan, Bone et al., 2007). Unfortunately, long-term treatment with PTH brings about the risk of osteosarcoma (Whitfield 2001, Orwoll, Scheele et al., 2003). Therefore, alternative anabolic agents that can promote bone formation without any adverse effects are highly needed.

[0006] Sclerostin is a promising target for developing therapeutic agents in established osteoporosis (Rey and Ellies 2010). Humanized monoclonal antibodies against human sclerostin have been reported to have good tolerance in clinical trials, promote bone formation, and increase bone mass. However, for therapeutic antibodies, there are several major concerns, including high immunogenicity (Padhi, Jang et al., 2011, Padhi, Allison et al., 2014), high cost and labor-intensive production (Baker 2015, Bradbury and Pluckthun 2015, Groff, Brown et al., 2015), and instability requiring a continuous cold chain for transportation and storage (Jayasena 1999). Therefore, alternative anti-sclerostin agents that are non-immunogenic, easy to produce, have low cost, and high stability are desirable for bone anabolic therapy.

Summary of the Invention

[0007] The present invention provides at least the following embodiments. Embodiment 1. A nucleic acid molecule conjugate with an extended in vivo half-life, wherein the nucleic acid molecule is conjugated to a fatty acid and / or a coumarin derivative.

[0008] Embodiment 2. The nucleic acid molecule conjugate according to Embodiment 1, wherein the fatty acid is selected from dodecanedioic acid, palmitic acid (PA), tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA), and preferably, the fatty acid is dodecanedioic acid.

[0009] Embodiment 3. The nucleic acid molecule conjugate according to Embodiment 1 or 2, wherein the coumarin derivative is selected from 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin)oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, and 6-methoxy-2-oxo-2H-chromene-3-carboxylic acid, and preferably, the coumarin derivative is 4-hydroxycoumarin.

[0010] Embodiment 4. The nucleic acid molecule conjugate according to any one of Embodiments 1 to 3, wherein the fatty acid, such as dodecanedioic acid, is conjugated to the 5'-end of the nucleic acid molecule, or the coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the 5'-end of the nucleic acid molecule, or the fatty acid, such as dodecanedioic acid, and the coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the 5'-end of the nucleic acid molecule.

[0011] Embodiment 5. The nucleic acid conjugate according to any one of Embodiments 1 to 4, wherein a fatty acid, such as dodecanedioic acid, is conjugated to the nucleic acid molecule through a linker, or a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the nucleic acid molecule via a linker, or a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the nucleic acid molecule through a linker.

[0012] Embodiment 6. The nucleic acid conjugate according to any one of Embodiments 1 to 5, wherein the nucleic acid molecule is a DNA molecule, an RNA molecule, or a DNA / RNA hybrid molecule.

[0013] Embodiment 7. The nucleic acid conjugate according to any one of Embodiments 1 to 6, wherein the nucleic acid molecule is double-stranded, or the nucleic acid molecule is single-stranded, or the nucleic acid molecule contains single-stranded and double-stranded portions.

[0014] Embodiment 8. The nucleic acid conjugate according to any one of Embodiments 1 to 7, wherein the nucleic acid molecule has a length of about 1 to about 250 bp / nt, about 1 to about 200 bp / nt, about 1 to about 150 bp / nt, about 1 to about 100 bp / nt, about 1 to about 50 bp / nt, about 1 to about 30 bp / nt, about 1 to about 20 bp / nt, about 1 to about 15 bp / nt, about 1 to about 10 bp / nt.

[0015] Embodiment 9. The nucleic acid conjugate according to any one of Embodiments 1 to 8, wherein the nucleic acid molecule is an aptamer, siRNA, antisense RNA, or shRNA, and preferably, the nucleic acid molecule is an aptamer.

[0016] Embodiment 10. The nucleic acid conjugate according to any one of Embodiments 1 to 9, wherein the nucleic acid molecule contains one or more modifications that confer enhanced nuclease resistance to the nucleic acid molecule.

[0017] Embodiment 11. The nucleic acid conjugate according to Embodiment 10, wherein the modification includes a 3'-inverted deoxythymidine (3'idT) modification.

[0018] Embodiment 12. The nucleic acid molecule conjugate according to Embodiment 10, wherein the modification comprises replacing one or more naturally occurring nucleotides with a modified nucleotide selected from the group consisting of 2'-fluoro, 2'-methoxyethyl, 2'-methoxy or 2'-allyloxy modified nucleotides, preferably a 2'-methoxy modified nucleotide.

[0019] Embodiment 13. The nucleic acid molecule conjugate according to Embodiment 10, wherein the modification comprises an internucleotide modification such as an internucleotide phosphorothioate bond modification.

[0020] Embodiment 14. The nucleic acid molecule conjugate according to Embodiment 10, wherein the aptamer comprises a 2'-methoxy (2'-OMe) modification and / or a 3'-inverted deoxythymidine (3'idT) modification.

[0021] Embodiment 15. The in vivo half-life of the nucleic acid molecule conjugate is at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, or longer compared to the corresponding nucleic acid molecule not containing the conjugated fatty acid and / or coumarin derivative, according to any one of Embodiments 1 to 14.

[0022] Embodiment 16. An aptamer conjugate against sclerostin, comprising an aptamer against sclerostin conjugated to a fatty acid and / or a coumarin derivative, wherein the aptamer is i) a nucleotide sequence having at least about 90% identity, at least about 91% identity, at least about 90% identity, about 92% identity, at least about 93% identity, at least about 94% identity, or at least about 95% identity to any one of SEQ ID NOs: 1 to 17, or ii) at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides among any one of SEQ ID NOs: 1 to 17 comprising an aptamer that specifically binds to sclerostin an aptamer conjugate.

[0023] Embodiment 17. The aptamer conjugate according to Embodiment 16, wherein the aptamer comprises any one nucleotide sequence of SEQ ID NOs: 1 to 17 and 19 to 25.

[0024] Embodiment 18. The aptamer conjugate according to Embodiment 16 or 17, wherein the aptamer is conjugated to a fatty acid and a coumarin derivative.

[0025] Embodiment 19. The fatty acid is selected from dodecanedioic acid, palmitic acid (PA), tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA), and preferably, the fatty acid is dodecanedioic acid. The aptamer conjugate according to any one of Embodiments 16 to 18.

[0026] Embodiment 20. The coumarin derivative is selected from 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin)oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, and 6-methoxy-2-oxo-2H-chromene-3-carboxylic acid, and preferably, the coumarin derivative is 4-hydroxycoumarin. The aptamer conjugate according to any one of Embodiments 16 to 19.

[0027] Embodiment 21. The aptamer conjugate according to any one of Embodiments 16 to 20, wherein a fatty acid, such as dodecanedioic acid, is conjugated to the 5'-end of the aptamer, or a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the 5'-end of the aptamer, or a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the 5'-end of the aptamer.

[0028] Embodiment 22. The aptamer conjugate according to any one of Embodiments 16 to 21, wherein a fatty acid, such as dodecanedioic acid, is conjugated to the aptamer through a linker, or a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the aptamer via a linker, or a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the aptamer through a linker.

[0029] Embodiment 23. The aptamer conjugate according to any one of Embodiments 16 to 22, wherein the aptamer has a K for sclerosis tin of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM or lower. d Embodiment 24. The aptamer conjugate according to any one of Embodiments 16 to 23, wherein the aptamer is capable of inhibiting the biological activity of sclerosis tin.

[0030] Embodiment 25. The aptamer conjugate according to any one of Embodiments 16 to 24, wherein the aptamer is capable of blocking the antagonistic effect of sclerosis tin in a cell-based Wnt signaling assay.

[0031] Embodiment 25. The aptamer conjugate according to any one of Embodiments 16 to 24, wherein the aptamer is capable of blocking the antagonistic effect of sclerosis tin in a cell-based Wnt signaling assay.

[0032] Embodiment 26. The aptamer conjugate according to any one of Embodiments 16 to 25, wherein the aptamer inhibits the biological activity of sclerostin with an EC50 value of less than 100 μg / ml, preferably less than 50 μg / ml, preferably less than 40 μg / ml, preferably less than 30 μg / ml, preferably less than 20 μg / ml, preferably less than 10 μg / ml or lower, for example, inhibits the antagonistic effect of sclerostin in the Wnt signaling pathway.

[0033] Embodiment 27. The aptamer conjugate according to any one of Embodiments 16 to 26, wherein the aptamer comprises one or more modifications that confer enhanced nuclease resistance to the nucleic acid molecule.

[0034] Embodiment 28. The aptamer conjugate according to Embodiment 27, wherein the modification comprises an inverted deoxythymidine (3'idT) modification.

[0035] Embodiment 29. The aptamer conjugate according to Embodiment 27, wherein the modification comprises substituting one or more naturally occurring nucleotides with a modified nucleotide selected from the group consisting of 2'-fluoro, 2'-methoxyethyl, 2'-methoxy or 2'-allyloxy modified nucleotides, preferably a 2'-methoxy modified nucleotide.

[0036] Embodiment 30. The aptamer conjugate according to Embodiment 27, wherein the modification comprises an internucleotide modification such as an internucleotide phosphorothioate bond modification.

[0037] Embodiment 31. The aptamer conjugate according to Embodiment 27, wherein the aptamer comprises a 2'-methoxy (2'-OMe) modification and / or an inverted deoxythymidine (3'idT) modification.

[0038] Embodiment 32. A method for treating a sclerostin-related disease, comprising the step of administering a therapeutically effective amount of an aptamer conjugate against sclerostin according to any one of Embodiments 16 to 31 to a subject in need thereof, for example, the method wherein the subject is a human.

[0039] Embodiment 33. The method according to Embodiment 32, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, fractures, osteoarthritis and myeloma, hypophosphatemic rickets, and triple-negative breast cancer.

[0040] Embodiment 34. A pharmaceutical composition comprising at least one aptamer conjugate against sclerostin according to any one of Embodiments 16 to 31 and a pharmaceutically acceptable carrier or excipient.

[0041] Embodiment 35. Use of an aptamer conjugate against sclerostin according to any one of Embodiments 16 to 31 or the pharmaceutical composition according to Embodiment 34 in the preparation of a medicament for treating a sclerostin-related disease.

[0042] Embodiment 36. The use according to Embodiment 35, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, fractures, osteoarthritis and myeloma, hypophosphatemic rickets, and triple-negative breast cancer.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0044] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art that are apparent to those skilled in the art. Standard handbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin, "Genes IV"; and Roitt et al., "Immunology" (8th Edition) and the general background art cited herein are, for example, referred to. Further, unless otherwise indicated, all methods, steps, techniques, and operations not specifically described in detail may be practiced and are practiced in a manner known per se and apparent to those skilled in the art. For example, standard handbooks, the general background art referred to above, and further references cited therein are referred to again.

[0045] Definition As used herein, the term "nucleotide" refers to ribonucleotides or deoxyribonucleotides or modified forms and analogs thereof. Chemical species including purines (e.g., adenine, hypoxanthine, guanine and their derivatives and analogs) and pyrimidines (e.g., cytosine, uracil, thymine and their derivatives and analogs) are included as nucleotides.

[0046] As used herein, the terms "nucleic acid", "oligonucleotide", and "polynucleotide" are used interchangeably to refer to polymers of nucleotides, including DNA, RNA, DNA / RNA hybrids, and modifications of these types of nucleic acids, oligonucleotides, and polynucleotides, and including the attachment of various entities or components to the nucleotide units at any position. The terms "polynucleotide", "oligonucleotide", and "nucleic acid" include double-stranded or single-stranded molecules. Nucleic acids, oligonucleotides, and polynucleotides are superordinate terms to the term aptamer, and thus the terms nucleic acid, oligonucleotide, and polynucleotide include, but are not limited to, aptamers.

[0047] As used herein, the term "aptamer" refers to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. Desired effects include, but are not limited to, binding to a target, catalytically changing a target, reacting with a target in a manner that modifies or alters the target or the target's functional activity, covalently binding to a target, and facilitating a reaction between a target and another molecule. In one embodiment, the effect is a specific binding affinity for a target molecule (such as sclerostin), where such target molecule is a three-dimensional chemical structure other than a polynucleotide that binds to a nucleic acid ligand through a mechanism independent of Watson / Crick base pairing or triple helix formation, and where the aptamer is not a nucleic acid having a known physiological function that is bound by the target molecule. In this context, the "specific binding affinity" of an aptamer for its target (such as sclerostin) means that the aptamer binds to its target with a generally higher degree of affinity than it binds to other, non-target constituents in a mixture or sample.

[0048] The term "identity" of a sequence has the meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds., M Stockton Press, New York, 1991). There are numerous methods for measuring identity between two polynucleotides or polypeptides, but the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)). An example of an algorithm suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter "BLAST") as described in, e.g., Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res., 15:3389-3402, 1997. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI").The default parameters used in determining sequence identity using software available from NCBI, such as BLASTN (for nucleotide sequences), are described in McGinnis et al., Nucleic Acids Res., 32:W20-W25, 2004.

[0049] Nucleic acid molecule conjugate with extended in vivo half-life Fatty acid modification (conjugation) is another means of extending the in vivo half-life of drug molecules. However, fatty acid modification has generally been applied only to proteins or peptides, and there are no reports of the application of fatty acid modification to nucleic acid molecules. Furthermore, while fatty acids are lipophilic, nucleic acid molecules are water-soluble, and thus, those skilled in the art would expect it to be difficult to conjugate nucleic acid molecules. In contrast, the inventors have surprisingly found that the in vivo half-life of nucleic acid molecules, such as aptamers, can be significantly extended by conjugating the nucleic acid molecules, such as aptamers, to fatty acids, thereby improving their clinical utility.

[0050] Furthermore, the inventors have surprisingly found that coumarin derivatives, such as 4-hydroxycoumarin, have a high affinity for HSA, and their combination with fatty acids, such as dodecanedioic acid, can synergistically extend the in vivo half-life of the nucleic acid molecules conjugated thereto.

[0051] Accordingly, in one aspect, the present invention provides a nucleic acid molecule conjugate having an extended in vivo half-life, wherein the nucleic acid molecule is conjugated to a fatty acid and / or a coumarin derivative.

[0052] In another aspect, the present invention provides a method of extending the in vivo half-life of a nucleic acid molecule, the method comprising conjugating the nucleic acid molecule to a fatty acid and / or a coumarin derivative.

[0053] In some embodiments, fatty acids include, but are not limited to, palmitic acid (PA), dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), and the like. In some preferred embodiments, the fatty acid is palmitic acid. In some other preferred embodiments, the fatty acid is octadecanedioic acid. In some more preferred embodiments, the fatty acid is dodecanedioic acid.

[0054] In some embodiments, coumarin derivatives include, but are not limited to, 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin)oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, 6-methoxy-2-oxo-2H-chromene-3-carboxylic acid. In some preferred embodiments, the coumarin derivative is 4-hydroxycoumarin.

[0055] In some embodiments, a fatty acid, such as dodecanedioic acid, is conjugated to the 5'-end of a nucleic acid molecule. In some embodiments, a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the 5'-end of a nucleic acid molecule. In some embodiments, a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the 5'-end of a nucleic acid molecule.

[0056] In some embodiments, a fatty acid, such as dodecanedioic acid, is conjugated to a nucleic acid molecule through a linker. In some embodiments, a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to a nucleic acid molecule via a linker. In some embodiments, a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to a nucleic acid molecule through a linker.

[0057] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is a DNA / RNA hybrid molecule. In some embodiments, the nucleic acid molecule is double-stranded. In some embodiments, the nucleic acid molecule is single-stranded. In some embodiments, the nucleic acid contains single-stranded and double-stranded portions. In some embodiments, the nucleic acid molecule is an oligonucleotide.

[0058] In some embodiments, the nucleic acid molecule is about 1 to about 500 bp / nt in length. In some embodiments, the nucleic acid molecule is about 1 to about 250 bp / nt, about 1 to about 200 bp / nt, about 1 to about 150 bp / nt, about 1 to about 100 bp / nt, about 1 to about 50 bp / nt, about 1 to about 30 bp / nt, about 1 to about 20 bp / nt, about 1 to about 15 bp / nt, about 1 to about 10 bp / nt in length. In some embodiments, the nucleic acid molecule is about 500 bp / nt, about 250 bp / nt, about 200 bp / nt, about 150 bp / nt, about 100 bp / nt, about 90 bp / nt, about 80 bp / nt, about 70 bp / nt, about 60 bp / nt, about 50 bp / nt, about 40 bp / nt, about 30 bp / nt, about 20 bp / nt, about 10 bp / nt in length.

[0059] In some embodiments, the nucleic acid molecule can be an aptamer, siRNA, antisense RNA, shRNA, etc. In some preferred embodiments, the nucleic acid molecule is an aptamer.

[0060] In some embodiments, the nucleic acid molecule of the present invention may also include one or more modifications. For example, the modification is a modification that confers enhanced nuclease resistance to the nucleic acid molecule.

[0061] Examples of modifications include 3' and 5' modifications such as 3' and 5' capping. In some embodiments, the nucleic acid molecule is capped with an inverted deoxythymidine at the 3' end, i.e., a 3' inverted deoxythymidine (3'idT) modification.

[0062] The modification can also include substitution with one or more modified nucleotides of naturally occurring nucleotides. For example, modified nucleotides include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and / or 2'-allyloxy modified nucleotides (i.e., the 2'-hydroxy group of ribose is substituted by fluorine, methoxyethyl, methoxy or allyloxy, etc.). Modified nucleotides can also include C5-modified pyrimidines. The term "C5-modified pyrimidine" refers to a pyrimidine having a modification at the C5 position. C5-modified pyrimidines can enhance the nuclease resistance of oligonucleotides and are known in the art. See, for example, PCT application WO 2011 / 130195 and the references cited therein. In some preferred embodiments, the modification is a 2'-methoxy (2'-OMe) modification. In some embodiments, the modification such as 2'-methoxy (2'-OMe) modification is performed on one or more nucleotides, such as 4 nucleotides, at the 5' and / or 3' ends of the nucleic acid molecule.

[0063] The modification also includes internucleotide modifications having uncharged bonds (such as methylphosphonate, phosphotriester, phosphoamine ester, carbamate, etc.), internucleotide modifications having charged bonds (such as phosphorothioate, dithiophosphate, etc.), internucleotide modifications having intercalating agents (such as acridine, psoralen, etc.), internucleotide modifications containing chelating agents (such as metals, radioactive metals, boron, oxidizing metals, etc.), internucleotide modifications containing alkylating agents, and internucleotide modifications containing modified linkages (such as alpha-anomeric nucleic acids, etc.).

[0064] In some embodiments, the nucleic acid molecule may include a combination of the modifications described above. For example, the aptamer may include a 2'-methoxy (2'-OMe) modification and / or a 3'-inverted deoxythymidine (3'idT) modification.

[0065] In some embodiments, the in vivo half-life of the nucleic acid molecule conjugates of the present invention is at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, or longer compared to the corresponding nucleic acid molecules that do not contain the conjugated fatty acid and / or coumarin derivative.

[0066] Aptamer conjugate with extended in vivo half-life Based on the protein-selection technology, the inventors used sclerostin as the target protein for positive screening and irrelevant proteins for negative screening, and finally selected aptamers that have high affinity and specifically bind to sclerostin. The sclerostin described herein is preferably human sclerostin, for example, sclerostin whose amino acid sequence is shown in SEQ ID NO: 18.

[0067] Exemplary human sclerostin amino acid sequence: QGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY (SEQ ID NO: 18).

[0068] In one aspect, the present invention is an aptamer conjugate against sclerostin, comprising an aptamer against sclerostin conjugated to a fatty acid and / or coumarin derivative. The aptamer comprises a nucleotide sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity to any one of SEQ ID NOs: 1 to 17, or the aptamer comprises at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides among any one of SEQ ID NOs: 1 to 17. Provided is an aptamer conjugate against sclerostin. In some embodiments, the aptamer provides an aptamer that specifically binds to sclerostin. In some preferred embodiments, the aptamer comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 17 and 19 to 25, more preferably the aptamer comprises a nucleotide sequence of any one of SEQ ID NOs: 1, 3, 10, 19 to 23 or 25.

[0069] In one aspect, the present invention is an aptamer conjugate against Dickkopf-1 (DKK1), comprising an aptamer against DKK1 conjugated to a fatty acid and / or a coumarin derivative. The aptamer comprises a nucleotide sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity or at least about 99% identity to any one of SEQ ID NOs: 26, or the aptamer comprises at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides among any one of SEQ ID NOs: 26. Provided is an aptamer conjugate against DKK1. In some embodiments, the aptamer specifically binds to DKK1. In some preferred embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 26.

[0070] In some embodiments, examples of the fatty acid include, but are not limited to, palmitic acid (PA), dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), and the like. In some preferred embodiments, the fatty acid is palmitic acid. In some other preferred embodiments, the fatty acid is octadecanedioic acid. In some more preferred embodiments, the fatty acid is dodecanedioic acid.

[0071] In some embodiments, examples of the coumarin derivative include, but are not limited to, 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin)oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, 6-methoxy-2-oxo-2H-chromene-3-carboxylic acid. In some preferred embodiments, the coumarin derivative is 4-hydroxycoumarin.

[0072] In some embodiments, a fatty acid, such as dodecanedioic acid, is conjugated to the 5'-end of the aptamer. In some embodiments, a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the 5'-end of the aptamer. In some embodiments, a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the 5'-end of the aptamer.

[0073] In some embodiments, a fatty acid, such as dodecanedioic acid, is conjugated to the aptamer through a linker. In some embodiments, a coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the aptamer via a linker. In some embodiments, a fatty acid, such as dodecanedioic acid, and a coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the aptamer through a linker.

[0074] In some embodiments, the aptamer or aptamer conjugate against sclerostin of the present invention has a Kd (dissociation constant) for sclerostin of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM or lower. The Kd is measured, for example, by enzyme-linked oligonucleotide assay (ELONA).

[0075] In some embodiments, the aptamer or aptamer conjugate against sclerostin of the present invention inhibits the biological activity of sclerostin. "Inhibits" means that the biological activity of sclerostin is reduced in the presence of the aptamer or aptamer conjugate compared to the absence of the aptamer, for example, by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, and even up to at least about 90%.

[0076] As used herein, the term "biological activity" refers to an effect on one or more intracellular or extracellular processes that may affect physiological or pathophysiological processes. The biological activities of sclerostin include, but are not limited to, antagonizing the Wnt signaling pathway.

[0077] In some embodiments, the aptamer or aptamer conjugate of the present invention against sclerostin can inhibit the antagonistic effect of sclerostin in the Wnt signaling pathway. For example, the aptamer or aptamer conjugate of the present invention against sclerostin can block the antagonistic effect of sclerostin in the cell-based Wnt signaling pathway.

[0078] In some embodiments, the aptamer or aptamer conjugate of the present invention against sclerostin inhibits the biological activity of sclerostin, such as inhibiting the antagonistic effect of sclerostin in the Wnt signaling pathway, with an EC50 value of less than 100 μg / ml, preferably less than 50 μg / ml, preferably less than 40 μg / ml, preferably less than 30 μg / ml, preferably less than 20 μg / ml, preferably less than 10 μg / ml or lower. In some embodiments, the EC50 value is determined in vitro by a TOP-Wnt-induced luciferase reporter gene assay in osteoblasts.

[0079] In some embodiments, the aptamer of the present invention may also include one or more modifications. For example, the modification is a modification that imparts enhanced nuclease resistance to the aptamer and / or enhances the in vivo half-life of the aptamer.

[0080] Examples of the modification include 3' and 5' modifications such as 3' and 5' capping. In some embodiments, the aptamer is capped using inverted deoxythymidine at the 3' end, i.e., 3'inverted deoxythymidine (3'idT) modification.

[0081] The modification can also include substitution with one or more modified nucleotides of naturally occurring nucleotides. For example, modified nucleotides include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and / or 2'-allyloxy modified nucleotides (i.e., the 2'-hydroxy group of ribose is substituted by fluorine, methoxyethyl, methoxy or allyloxy, etc.). Modified nucleotides can include C5-modified pyrimidines. The term "C5-modified pyrimidine" refers to a pyrimidine having a modification at the C5 position. C5-modified pyrimidines can enhance the nuclease resistance of oligonucleotides and are known in the art. See, for example, PCT application WO 2011 / 130195 and the references cited therein. In some preferred embodiments, the modification is a 2'-methoxy (2'-OMe) modification. In some embodiments, the modification such as 2'-methoxy (2'-OMe) modification is performed on one or more nucleotides, such as 4 nucleotides, at the 5' and / or 3' ends of the aptamer.

[0082] The modification also includes internucleotide modifications having uncharged bonds (such as methylphosphonate, phosphotriester, phosphoamine ester, carbamate, etc.), internucleotide modifications having charged bonds (such as phosphorothioate, dithiophosphate, etc.), internucleotide modifications having intercalating agents (such as acridine, psoralen, etc.), internucleotide modifications containing chelating agents (such as metals, radioactive metals, boron, oxidizing metals, etc.), internucleotide modifications containing alkylating agents, and internucleotide modifications containing modified linkages (such as alpha-anomer nucleic acids, etc.).

[0083] In some embodiments, the aptamer may include a combination of various modifications described above. For example, the aptamer may include 2'-methoxy (2'-OMe) modification and / or 3'-inverted deoxythymidine (3'idT) modification.

[0084] In some specific embodiments, the aptamer conjugate comprises the following sequence structure: FA-linker-C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT (where FA represents a fatty acid, (OMe) represents a 2'-methoxy (2'-OMe) modification of the corresponding nucleotide, and idT represents a 3'-reverse deoxythymidine modification). In some specific embodiments, the fatty acid is palmitic acid (PA). In some specific embodiments, the fatty acid is octadecanedioic acid. In some preferred embodiments, the fatty acid is dodecanedioic acid (DA).

[0085] In some specific embodiments, the aptamer conjugate comprises the following sequence structure: fatty acid / coumarin derivative-linker-C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT (where (OMe) represents a 2'-methoxy (2'-OMe) modification of the corresponding nucleotide, and idT represents a 3'-reverse deoxythymidine modification). In some preferred embodiments, the fatty acid is dodecanedioic acid (DA). In some preferred embodiments, the coumarin derivative is 4-hydroxycoumarin.

[0086] Method for preparing nucleic acid molecule conjugate In one aspect, the present invention provides a method for preparing the nucleic acid molecule conjugate of the present invention, wherein the nucleic acid molecule is conjugated to a fatty acid. The method includes activating the carboxyl group of the fatty acid to form an active ester structure, and then reacting with a nucleic acid molecule structure selected from the following S1-S5

[0087]

Chemical formula

[0088] Nucleic acid molecules, such as aptamers, and fatty acids are as defined above. In the nucleic acid molecule structures of S1 to S5, the functional group at the 5'-end is inserted by using commercially available amino monomers under conventional nucleic acid solid-phase synthesis conditions.

[0089] In one aspect, the present invention provides a method for preparing a nucleic acid molecule conjugate of the present invention, wherein the nucleic acid molecule is conjugated to a fatty acid and a coumarin derivative, reacting a fatty acid or a chemical intermediate thereof suitable for the reaction with the amino group of the nucleic acid molecule structure of S6, and reacting a coumarin derivative or a chemical intermediate thereof suitable for the reaction with the sulfhydryl group of the nucleic acid molecule structure of S6.

[0090]

Chemical formula

[0091] Nucleic acid molecules, such as aptamers, fatty acids, and coumarin derivatives are as defined above.

[0092] Treatment of disease In another aspect, the present invention provides a method for treating a disease with an aptamer conjugate against sclerostin of the present invention, which comprises administering a therapeutically effective amount of the aptamer conjugate against sclerostin of the present invention to a subject in need thereof.

[0093] The diseases treated by the aptamer conjugate against sclerostin of the present invention are sclerostin-related diseases such as sclerostin-mediated diseases.

[0094] As used herein, "sclerostin-related diseases" include diseases in which bone mineral density (BMD) is abnormally and / or pathologically low compared to healthy subjects. Diseases characterized by low BMD and / or bone fragility include, but are not limited to, primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (osteonecrosis), fractures and implant healing (dental implants and hip implants), and bone loss due to other diseases (e.g., associated with HIV infection, cancer or arthritis). Other "sclerostin-related diseases" include, but are not limited to, hypophosphatemic rickets, rheumatoid arthritis, osteoarthritis, arthritis and osteolytic lesions.

[0095] As used herein, "sclerostin-related diseases" include sclerostin-related cancers such as myeloma (e.g., multiple myeloma with osteolytic lesions), breast cancer (such as triple-negative breast cancer), colon cancer, melanoma, hepatocellular carcinoma, epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer or pancreatic cancer and any metastases thereof.

[0096] "Sclerostin-related diseases" may also include, at least, the states of the kidneys and cardiovascular system due to the expression of sclerostin in the kidneys and cardiovascular structure (cardiovasculature). Examples of said diseases include, but are not limited to, kidney disorders such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, systemic lupus erythematosus, Goodpasture syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasms, sickle cell disease, and glomerular lesions associated with systemic diseases such as chronic inflammatory diseases), tubule diseases (e.g., acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary sponge kidney, medullary cystic kidney disease, renal glycosuria, and tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug- and toxin-induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy), acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, gout, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarction), or tumors (e.g., renal cell carcinoma and nephroblastoma).

[0097] Examples of said sclerostin-related diseases include, but are not limited to, cardiovascular disorders such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, cor pulmonale, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., obstructive lesions of valves and blood vessels, atrial or ventricular septal defects, and patent ductus arteriosus), or myocardial diseases (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

[0098] In another aspect, the present invention provides a method for treating a disease with an aptamer conjugate against DKK1 of the present invention, the method comprising administering a therapeutically effective amount of the aptamer conjugate against DKK1 of the present invention to a subject in need thereof.

[0099] The diseases treated by the aptamer conjugate against DKK1 of the present invention are DKK1-related diseases such as, for example, DKK1-mediated diseases.

[0100] As used herein, "DKK1-related diseases" include myeloma (e.g., multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma, multiple myeloma), breast cancer, colon cancer, melanoma, hepatocellular carcinoma, epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and sclerostin-related cancers such as any metastases thereof.

[0101] In some embodiments, the DKK1-related disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, fractures, osteoarthritis and myeloma.

[0102] The subject may be any animal including, but not limited to, cats, dogs, horses, pigs and cows (domestic, breeding or wild animals), with human subjects being preferred. As used herein, the terms patient, individual and subject may be used interchangeably.

[0103] The subject can be male or female. Preferably, the human subject is at risk of fracture, more preferably, the human subject is at risk of or suffering from osteoporosis. The human subject is preferably female, more preferably a female at risk of or suffering from postmenopausal osteoporosis. It is expected that the method of the present invention can be beneficial to the subject at any stage of osteoporosis.

[0104] As used herein, "treating" an individual suffering from a disease or disease state means that the symptoms of the individual are partially or completely alleviated, or remain unchanged, after treatment. Thus, treatment includes prevention, treatment and / or cure. Prevention refers to the prevention of the possibility of a disease and / or the prevention of the worsening of symptoms or the progression of the disease.

[0105] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material or composition that contains a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, recover from, arrest or partially arrest the symptoms of a disease or condition. As used herein, "therapeutic effect" means an effect resulting from the treatment of an individual that modifies, generally restores or alleviates, or cures the symptoms of a disease or disease state.

[0106] The dosing regimen of the aptamer conjugate is selected according to various factors including, for example, the patient's type, race, age, weight, gender and medical condition; the severity of the condition being treated; the route of administration; the patient's renal and hepatic function; and the specific aptamer conjugate or its salt being used. A normally skilled physician can readily determine and specify the effective amount of the composition necessary to prevent, combat or arrest the progression of the condition.

[0107] Typically, the dosing regimen of the aptamer conjugate is from about 1 μg / kg body weight to about 100 mg / kg body weight per day.

[0108] Exemplary treatment regimens are once a day, once every two days, once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months or once every three to six months, or involve administration with an initially short dosing interval (such as once a week to once every three weeks) followed by an extended interval (such as once a month to once every three to six months). The frequency and interval of administration can be determined by one of ordinary skill in the art according to the pharmacokinetic parameters of the aptamer conjugate.

[0109] Pharmaceutical composition In another aspect, the present invention also provides a pharmaceutical composition comprising an aptamer conjugate against at least one sclerostin of the present invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is used, for example, to treat sclerostin-related diseases.

[0110] In another aspect, the present invention also provides a pharmaceutical composition comprising at least one aptamer conjugate against DKK1 of the present invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is used, for example, for treating DKK1-related diseases.

[0111] The aptamer conjugates described herein can be utilized in any pharmaceutically acceptable dosage form including, but not limited to, injectable dosage forms, liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release formulations, sustained release formulations, pulsatile release formulations, immediate release formulations, and mixtures of immediate release and controlled release formulations. Specifically, the aptamers described herein can be formulated for: (a) administration selected from any of oral, pulmonary, intravenous, intraarterial, intrathecal, intraarticular, rectal, ophthalmic, colonic, parenteral, intravesical, intravaginal, intraperitoneal, local, buccal, nasal, and topical administration; (b) a dosage form selected from any of liquid dispersions, gels, aerosols, ointments, creams, tablets, sachets, and capsules; (c) a dosage form selected from any of lyophilized formulations, dry powders, fast melt formulations, controlled release agents, delayed release formulations, sustained release formulations, pulsatile release formulations, and mixtures of immediate release and controlled release formulations; or (d) any combination thereof.

[0112] Solutions or suspensions for parenteral, intradermal, or subcutaneous application may contain one or more of the following components: (1) a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; (2) an antibacterial agent such as benzyl alcohol or methylparaben; (3) an antioxidant such as ascorbic acid or sodium bisulfite; (4) a chelating agent such as ethylenediaminetetraacetic acid; (5) a buffer such as acetate, citrate, or phosphate; and (6) an agent for adjusting the osmotic pressure such as sodium chloride or glucose. The pH may be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0113] A pharmaceutical composition suitable for injectable use may contain a sterile aqueous solution (if water-soluble) or a dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include saline, bacteriostatic water, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The pharmaceutical composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. As used herein, the term "stable" means remaining in a condition or state suitable for administration to a patient.

[0114] The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents, such as sugars, polyalcohols such as mannitol or sorbitol, and inorganic salts such as sodium chloride. Sustained absorption of the injectable composition can be brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0115] A sterile injectable solution can be prepared by filter sterilization following incorporation of the required amount of the active reagent (such as an aptamer conjugate) into a suitable solvent containing one or a combination of the ingredients listed above. Generally, a dispersant is prepared by incorporating at least one aptamer conjugate into a sterile vehicle containing a basic dispersion medium and any other necessary ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods include vacuum drying and lyophilization, both of which result in a powder of any additional desirable ingredients derived from its pre-sterilized solution in addition to the aptamer conjugate.

[0116] Oral compositions generally contain an inert diluent or an edible carrier. They can be encapsulated, for example, in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the aptamer conjugate may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, rinsed, spat out, or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition.

[0117] For administration by inhalation, the compound is delivered in the form of an aerosol spray, a nebulized solution, or a dry powder from a suitable device, such as a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide. For transmucosal or transdermal administration, a suitable penetrant for the permeated barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved through the use of nasal drops or suppositories. For transdermal administration, the active agent is generally formulated in an ointment, salve, gel, or cream known in the art. The agent can also be prepared in the form of a suppository (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or a retention enema for rectal delivery.

[0118] In one embodiment, the aptamer conjugate is formulated for topical administration. As used herein, "topical administration" refers to the delivery of an aptamer conjugate to an animal by contacting a formulation containing the aptamer conjugate directly or otherwise with all or a part of the skin (epidermis) of the animal. The term encompasses several routes of administration including, but not limited to, topical and transdermal. The general requirement for these modes of administration is efficient delivery to the target tissue or layer. In one aspect, topical administration is used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the aptamer. In another aspect, topical administration is used as a means to selectively deliver the aptamer conjugate to the epidermis or dermis of an animal or to specific layers thereof.

[0119] For topical administration, the aptamer conjugate may be formulated into pharmaceutically acceptable ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings and aerosols, medicinal powders, medicated adhesives, foams, for example, ointments, gels and creams may contain suitable conventional additives and excipients including preservatives or solvents to assist in the penetration and softening of the drug. Such topical formulations may also contain compatible conventional carriers such as ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more generally, such carriers will constitute up to about 80% by weight of the formulation. Specific formulations for topical delivery of aptamers are described in the art.

[0120] In one embodiment, the aptamer conjugate is prepared using a carrier that protects against rapid elimination from the body. For example, controlled release formulations can be used, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can also be used. Methods for the preparation of such formulations will be apparent to those skilled in the art.

[0121] Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art.

[0122] In addition, suspensions of the aptamer conjugate can also be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers that are not lipids can also be used for delivery. Optionally, the suspension can also contain suitable stabilizers or agents to increase the solubility of the compound and to allow the preparation of highly concentrated solutions.

[0123] In some cases, for ease of administration and uniformity of dosage, it may be particularly advantageous to formulate the aptamer conjugate in a dosage unit form in an oral or parenteral composition. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of the aptamer conjugate calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier. The specifications for the dosage unit forms of the aptamer conjugates described herein are directly dependent on, and determined by, the unique characteristics of the particular aptamer conjugate, and the limitations inherent in the art of compounding such as the detailed therapeutic effect to be achieved and the active agents for the treatment of individuals.

[0124] A pharmaceutical composition comprising at least one aptamer conjugate may contain one or more pharmaceutical excipients. Examples of such excipients include, but are not limited to, binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Exemplary excipients include: (1) binders including various celluloses and cross-linked polyvinylpyrrolidone, crystalline celluloses such as Avicel PH101 and Avicel PH102, silicified crystalline cellulose (ProSolv SMCC (trademark)), tragacanth gum, and gelatin; (2) fillers including various starches, lactose, lactose monohydrate, and anhydrous lactose; (3) disintegrants including alginic acid, Primogel, corn starch, lightly cross-linked polyvinylpyrrolidone, potato starch, corn starch, and modified starch, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof; (4) lubricants including agents that act on the fluidity of compressed powders, including magnesium stearate, colloidal silicon dioxide such as Aerosil 200, talc, stearic acid, calcium stearate, and silica gel; (5) glidants such as colloidal silicon dioxide; (6) preservatives including potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of p-hydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride; (7) diluents such as pharmaceutically acceptable inert fillers including crystalline cellulose such as Avicel PH101 and Avicel PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose DCL21; dibasic calcium phosphate such as Emcompress; mannitol; starch; sorbitol; sucrose; and glucose;Sweeteners containing any natural or artificial sweeteners such as (8) sucrose, saccharin, sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; flavoring agents such as (9) peppermint, methyl salicylate, orange flavoring agent, Magnasweet (trademark of MAFCO), bubble gum flavor, fruit flavor, etc.; and (10) foaming agents containing an effervescent couple such as an organic acid and a carbonate or bicarbonate.

Example

[0125] The present invention is further described by the following examples, but the scope of the present invention is not limited to the described examples.

[0126] Example 1. Concentration and Selection of High-Affinity Aptamers for Sclerostin The ssDNA library consists of an 18-nt conserved region at each end and a random region in the middle. Two ssDNA libraries containing randomized sequences of different lengths were used in this project. The long ssDNA library contains a 40-nt random region (5’-CGTACGGTCGACGCTAGC-(N) 40 -CACGTGGAGCTCGGATCC-3’), and the short ssDNA contains a 25-nt random region (5’-CGTACGGTCGACGCTAGC-(N) 25 -CACGTGGAGCTCGGATCC-3’). A forward primer (FP: 5’-CGTACGGTCGACGCTAGC-3’) and a biotinylated reverse primer (Bio-RP: 5’-biotin-GGATCCGAGCTCCACGTG-3’) were synthesized for the amplification of ssDNA during selection. All oligos were purified by HPLC after synthesis.

[0127] The protein-selection method was implemented to identify high-affinity aptamers (Ellington and Szostak 1990, Tuerk and Gold 1990). 100 - 30 pmole of His6-sclerostin protein was immobilized on NTA magnetic beads at 4°C for 1 hour (Murphy, Fuller et al., 2003). 1 nmole of the ssDNA library was denatured at 95°C for 5 minutes, rapidly cooled to 4°C, and then incubated with the immobilized sclerostin protein at R.T. for 0.5 - 1 hour. Unbound sequences were removed using wash buffer. After washing, the bound DNA-protein-NTA was recovered, resuspended in H2O / Tween 20, and applied to PCR amplification. PCR was performed using an unmodified forward primer and a biotinylated reverse primer (Step 1: 95°C for 1 minute for initial denaturation; Step 2: 95°C for 30 seconds for denaturation, 56°C for 30 seconds for annealing, 72°C for 30 seconds for extension, repeated for 12 cycles; and Step 3: 72°C for 5 minutes for final extension). The PCR product was applied to streptavidin magnetic beads through biotin-streptavidin binding. The single-stranded sequences were regenerated by treatment with 0.2 M NaOH. Negative selection was performed against other His6-tagged irrelevant proteins immobilized on NTA magnetic beads. A total of 20 rounds of selection were performed for each selection. The DNA pool from the final round was subjected to high-throughput next-generation sequencing (NGS).

[0128] Figure 1 shows that the affinity of the DNA pool for sclerostin increases after the 10th and 20th selections, indicating that high-affinity sclerostin aptamers are enriched by selection.

[0129] Example 2. Characterization of the Specificity of Candidate Sclerostin Aptamers Based on the NGS results, representative aptamers with high occurrence rates were synthesized for specificity assays. The detailed sequences of these aptamer candidates are listed in Table 1.

[0130] To determine the specificity of aptamer candidates to sclerostin, representative aptamer candidates and random sequences (RS) (negative controls) were synthesized using N-terminal biotinylation modification, and 1 μM of each aptamer / RS was used to determine specificity to sclerostin using enzyme-linked oligonucleotide assay (ELONA). 160 ng of purified recombinant human sclerostin was coated in a 96-well microtiter plate in 100 μl PBS at 4 °C overnight. The plate was then blocked with blocking buffer (PBS, 0.1% Tween 20 and 1% BSA) for 1 h at room temperature and washed 4 times with SelectB&W buffer (PBS, 1 mM MgCl2, 0.1% Tween 20 and 0.1% BSA). Aptamer candidates were denatured at 95 °C for 10 min and rapidly cooled on ice for 10 min before use. 1 μM biotinylated aptamer was added to each well, then SelectB&W buffer was added up to 100 μl, and the mixture was incubated for 45 min at room temperature with continuous gentle shaking. After binding, the plate was washed 4 times with SelectB&W buffer to remove non-specific and very weak binding, and then washed 4 times with PBST + 0.1% BSA. 100 μl of streptavidin-HRP / goat anti-human IgG Fc-HRP (diluted 1:10,000 in PBST + 0.1% BSA) was added to each well, incubated for 30 / 60 min, and washed 4 times with PBST + 0.1% BSA. 50 μl of TMB was added to each well and incubated for 20 min. The reaction was stopped by adding 50 μl of 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader (Stoltenburg, Krafcikova et al., 2016). To determine the binding ability of aptamer candidates to hepatocytes / PBMC, the characterization step was the same as ELONA. 300,000 cells were incubated with each aptamer candidate, and centrifugation was used for washing and separation purposes.

[0131] Aptamer candidates identified from both long and short ssDNA libraries showed high selectivity for human sclerostin when binding to hepatocytes and PBMCs was compared (Figure 2). Aptamer candidates, aptscl 6, 9, 15, 27, 34, 36, 46, 51, 56, 132, and 140 identified from the long ssDNA library, and aptscl 1, 2, 3, 5, 8, 12, 16, 22, 29, and 32 identified from the short ssDNA library, showed high binding specificity for sclerostin and were selected for subsequent affinity characterization.

[0132] Example 3. Binding Affinity Characterization of Candidate Sclerostin Aptamers Enzyme-linked oligonucleotide assay (ELONA) was performed to determine the binding affinity of aptamer candidates to sclerostin (Drolet, Moon-McDermott et al., 1996). Similarly, enzyme-linked immunosorbent assay (ELISA) was performed to determine the binding affinity of anti-sclerostin antibodies to human sclerostin (Engvall and Perlmann 1971). 160 ng of purified recombinant human sclerostin was coated by incubating overnight at 4 °C in 100 μl of PBS in a 96-well microtiter plate. The plate was then blocked for 1 hour at room temperature with blocking buffer (PBS, 0.1% Tween 20 and 1% BSA) and washed 4 times with Select-B&W buffer (PBS, 1 mM MgCl2, 0.1% Tween 20 and 0.1% BSA). Aptamer candidates were denatured at 95 °C for 10 minutes and rapidly cooled on ice for 10 minutes before use. Appropriate concentrations of biotinylated aptamer / antibody were added to each well, followed by addition of Select-B&W buffer to 100 μl, and the plate was incubated for 45 minutes at room temperature with continuous gentle shaking. After binding, the plate was washed 4 times with Select-B&W buffer to remove non-specific and very weak binding, and then washed 4 times with PBST + 0.1% BSA. 100 μl of streptavidin-HRP / goat anti-human IgG Fc-HRP (diluted 1:10000 in PBST + 0.1% BSA) was added to each well, incubated for 30 / 60 minutes, and washed 4 times with PBST + 0.1% BSA. 50 μl of TMB was added to each well and incubated for 20 minutes. The reaction was stopped by adding 50 μl of 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader (Stoltenburg, Krafcikova et al., 2016). Data were analyzed using Origin software (OriginLab, Northampton, MA). The non-linear curve fitting model Hyperbl was used to plot the binding curve. The equation of the Hyperbl model is y = P1×x / (P2 + x), where P2 is the Kd value.

[0133] For the aptamer candidates identified from the ssDNA library containing a 40-nt random region, aptscl 6, 9, 15, 46, 56, and 132 had dissociation constant (Kd) values in the nanomolar level (the Kd values were 4.2, 3.4, 45.6, 43.1, and 42.2 nM, respectively) and showed high affinity for sclerostin (Figure 3). On the other hand, aptscl 36, 140, 136, and the random sequence (RS) did not fit. For the aptamer candidates identified from the ssDNA library containing a 25-nt random region, aptscl 32, 29, 22, 16, 3, 2, and 1 had Kd values of 0.18, 0.28, 0.76, 0.02, 0.04, 0.006, and 0.02 nM, respectively, and showed higher binding affinity for sclerostin (Figures 4-1 and 4-2). The random sequence showed low binding ability to sclerostin and did not fit. In comparison, the Kd value of the anti-sclerostin antibody to sclerostin was 3.55 nM.

[0134] Example 4. In vitro evaluation of the inhibitory ability of candidate sclerostin aptamers on the activity of osteosclerosis To study the inhibitory intensity of the antagonistic effect of the aptamer against sclerostin in Wnt signaling, the TOP-Wnt-induced luciferase reporter assay was used in osteoblast MC3T3-E1 cells (van Bezooijen, Svensson et al., 2007, Shum, Chan et al., 2011).

[0135] MC3T3-E1 cells were seeded in 24-well plates and transfected with the corresponding reporter plasmid (100 ng), Wnt3a plasmid (800 ng), and sclerostin plasmid (800 ng), and the next day, FuGENE HD transfection reagent (Promega) was used if necessary. Ten hours after transfection, the culture medium was replaced with fresh medium, and the cells were treated with aptamer / antibody. Twenty-four hours after treatment, each well of the cells was lysed with 100 μl of passive lysis buffer, and 20 μl was taken for analysis. Luciferase Assay Reagent II and Stop&Glo reagent were prepared and automatically added by a SpectraMax i3x Multi-Mode Detection Platform (Molecular Device) according to the manufacturer's protocol (Promega), and the data were analyzed thereby (Grentzmann, Ingram et al., 1998, McNabb, Reed et al., 2005).

[0136] As shown in Figure 5, aptscl 56, aptscl 6, aptscl 3, and the anti-sclerostin antibody could effectively inhibit the antagonistic effect of sclerostin in Wnt signaling and release Wnt-induced luciferase activity. The inhibition of sclerostin was dose-dependent, and the response was stable when the concentrations of aptscl 56 and 6 reached 25 and 47.4 μg / ml, respectively. During treatment with the antibody, the response was not yet stable even when the concentration increased to 20 mg / ml. Furthermore, the inhibition strengths of aptscl 56, aptscl 6, and aptscl 3 were analyzed using non-linear curve fitting. The EC50s for aptscl56, aptscl6, and aptscl 3 were 19.7 μg / ml, 36.8 μg / ml, and 18.2 μg / ml, respectively.

[0137]

Table 1

[0138] Example 5. Cleavage and Characterization of Aptscl3 Aptscl3, which showed high affinity and inhibitory strength for sclerosis, was cleaved (Table 2). The binding affinity and in vitro inhibitory strength were carried out using the same protocol as in the previous study. Aptscl 3-1, -2, -3, -4, and -5 had Kd values of 0.86, 0.52, 0.2, and 0.22 nM, respectively, leaving high binding affinity for sclerosis. On the other hand, aptscl 3-6 could not fit the binding curve in this concentration range and showed low binding ability to sclerosis (Figure 6-1). Furthermore, aptscl3-5 retained high inhibitory strength against the antagonistic effect of sclerosis in Wnt signaling (EC50 = 28.4 μg / ml) (Figure 6-2).

[0139] [Table 2]

[0140] Example 6. Evaluation of Serum Stability of Chemically Modified Aptamer Candidates The inventors selected DNA aptamers against sclerostin and ultimately developed two cleavage aptamers called aptscl 56 and aptscl 3-5, which have dissociation constants in the low nanomolar range and bind specifically and tightly to sclerostin. The bulky 2'-O-methyl (2'-OMe) modification of nucleic acid aptamers has been previously used as a post-selection modification because of the enhanced nuclease resistance and increased duplex melting temperature observed in clinical cases (Fine, Martin et al., 2005; Gupta, Hirota et al., 2014). 3'-end capping with inverted dT is also a common strategy for aptamers in disease treatment in ongoing or completed clinical trials (Padilla, Sousa et al., 1999; Ruckman, Green et al., 1998). Together, this example evaluates whether the serum stability of aptscl 56 and aptscl 3-5 can be improved by 2'-OMe and 3'-end inverted dT (3'-idT) modifications.

[0141] Experimental design: Modified nucleotides were introduced during synthesis. The serum metabolic stability of modified and unmodified aptamers was evaluated in freshly prepared mouse serum. All aptamer samples were incubated at 37°C for 0, 2, 4, 8, 12, 24, 36, 48, and 72 hours using 10% and 100% mouse serum, respectively. At the designated times, the aptamer samples were snap-frozen in a dry ice bath and then stored at -80°C until all samples were collected for evaluation. The stability of all aptamer samples was represented by the band density of the untreated aptamer remaining after incubation, which could be determined by agarose gel electrophoresis.

[0142] DNA synthesis protocol Modified and unmodified DNA sequences were synthesized on a K&A H8 standard DNA / RNA synthesizer at the 1 μmol scale using commercially available 5'-O-DMT-2'-deoxynucleoside (ABz, CAc, GiBu, and T) phosphoramidite monomers, 5'-O-DMT-2'-O-methyl nucleoside (ABz, CAc, GiBu, and T) phosphoramidite monomers, and / or 5'-O-DMT-2'-F-nucleoside (ABz, CAc, GiBu, and T) phosphoramidite monomers (Beaucage and Caruthers 2001). The modified sequence of aptscl56 is CGGG G TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CT GCCC -idT, and the underlined nucleotides are 2'-OMe modified. The modified sequence of aptscl 3-5 is GCTA G CTGTT GTACA TCGCC TTACG CA CGT G -idT, and the underlined nucleotides are 2'-OMe modified.

[0143] Evaluation protocol: The band densities of all aptamer samples were assayed by a molecular imager (Bio-Rad) (Klussmann, Nolte et al., 1996, Siller-Matula, Merhi et al., 2012). Binding affinity and in vitro inhibition strength were performed using the same protocol as in previous studies.

[0144] Results: For aptscl 56, the unmodified aptamer was completely degraded after 48 hours in 10% serum and remained after only 8 hours in 100% serum. The 2'-OMe and 3'idT modified aptscl 56 remained after 72 hours in 10% mouse serum and were degraded after 12 hours. After 72 hours, a small amount of the modified aptamer still remained (Figure 7).

[0145] For aptscl 3-5, the unmodified aptamer was degraded after 24 hours in 10% mouse serum. The 2'-OMe and 3'idT modified aptscl 3-5 may survive for 48 hours in 10% mouse serum. In 100% serum, the unmodified aptscl 3-5 was rapidly and completely degraded after 8 hours, while the modified aptscl 3-5 could remain intact after 72 hours (Figure 8).

[0146] The chemically modified aptscl 56 and aptscl 3-5 had Kd values of 6.55 and 0.54 nM respectively, showing high binding affinity to sclerostin. Furthermore, the chemically modified aptscl 56 and aptscl 3-5 could effectively inhibit the antagonism of sclerostin in Wnt signaling in cells, with inhibition strengths of 14 and 11 μg / ml respectively (Figure 9).

[0147] Conclusion: Modification with 2'-OMe and 3'idT can further promote the development of aptscl 56 and aptscl 3-5 towards therapeutic nuclease-resistant aptamers. Example 7. Preparation of fatty acid-modified aptamer candidates and their in vivo half-life The nucleic acid molecule aptamer aptscl56 that specifically targets sclerostin was identified in the above example. Its specific sequence is 5'-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3' (SEQ ID NO: 1) is.

[0148] The obtained sequence was modified to sequence APC001 (the 2'-position of the sugar ring of 4 nucleotides at each of the 5'-end and 3'-end was modified with methoxy, and the 3'-end of the nucleic acid strand was modified with idT): C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT.

[0149] The nucleotide sequence is CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCCUGCC (SEQ ID NO: 25) is as follows.

[0150] In this example, APC001 was conjugated and modified with a fatty acid (FA) at the 5'-end to obtain FA-C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT .

[0151] The effects of conjugating palmitic acid (PA) and octadecanedioic acid (OA) separately on the in vivo half-life of the aptamer were tested.

[0152] Conjugation method In the present invention, a chemical modification technique was used to perform fatty acid coupling at the 5'-end of the adapter nucleic acid strand. Due to the structural similarity of the nucleic acid strand itself, it is not easy to couple directly on the nucleobase. It is necessary to extend an active amino group at the end of the nucleic acid strand. In the present invention, different amino phosphoramidite monomers were used to construct a linking arm to perform amino modification at the end of the nucleic acid strand. These phosphoramidite monomers can be specifically incorporated directly into the nucleic acid solid-phase synthesis cycle to perform a coupling reaction to obtain a target compound as follows.

[0153] 1) 5'-amino modifier 5 2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite

[0154]

Chemical formula

[0155] The following structure S1 can be obtained for coupling with the carboxyl group of the fatty acid.

[0156]

Chem.

[0157] 2) 5'-Amino modifier C6 6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite

[0158]

Chem.

[0159] The following structure S2 can be obtained for coupling with the carboxyl group of a fatty acid.

[0160]

Chem.

[0161] 3) 5'-Amino modifier C12 12-(4-Monomethoxytritylamino)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0162]

Chem.

[0163] The following structure S3 can be obtained for coupling with the carboxyl group of a fatty acid.

[0164]

Chem.

[0165] 4) 5'-Amino modifier TEG CE-phosphoramidite 10-(O-Trifluoroacetylamide-N-ethyl)-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

[0166]

Chem.

[0167] The following structure S4 can be obtained for coupling with the carboxyl group of fatty acids.

[0168]

Chem.

[0169] 5) 5'-Amino-modified C3-TFA 3-(Trifluoroacetylamino)propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite

[0170]

Chem.

[0171] The following structure S5 can be obtained for coupling with the carboxyl group of fatty acids.

[0172]

Chem.

[0173] Specifically, using the S2 structure of APC001, coupling reactions with palmitic acid and octadecanedioic acid were respectively carried out to obtain APC001PA and APC001OA.

[0174] In vivo half-life analysis Pharmacokinetic studies of APC001, APC001PA, and APC001OA were conducted in 6-month-old female naive Sprague-Dawley rats maintained under controlled conditions (12-hour light cycle, 20 °C) with free access to standard laboratory chow. The rats were treated by single subcutaneous injection with APC001, APC001PA, and APC001OA. Blood samples were collected from the rats in each group at various time points, and plasma was separated. Samples were extracted using the Sangon UNIQ-10 column universal DNA extraction kit, and the concentrations of APC001, APC001PA, and APC001OA in plasma at various time points were measured by HPLC. Then, the in vivo half-life was calculated using the pharmacokinetic software DS.

[0175] The in vivo half-lives of APC001, APC001PA, and APC001OA are shown in the following table.

[0176] [Table 3]

[0177] By conjugating aptamers with fatty acids, their half-lives in vivo can be significantly extended.

[0178] Example 8. Preparation of Aptamer Candidates Modified with Fatty Acids and Coumarin Derivatives and Their In Vivo Half-Lives 1. Virtual Screening of Coumarin Derivatives: Warfarin (WA) has a strong affinity for serum albumin (HSA), but due to its anticoagulant effect, it is not suitable as a small molecule coupling reagent. By virtual high-throughput screening method, the present invention identified 4-hydroxycoumarin which has a high affinity for HSA and no anticoagulant function (Table 3). Virtual high-throughput screening was carried out using Schrodinger_Suites_2021-2 software, which is software capable of simulating small molecules and macromolecular systems and was used to perform docking simulations of the selected compounds with full-length HSA and FABP respectively.

[0179]

Table 4

[0180] 2. Verification of the affinity between 4-hydroxycoumarin and HSA and verification of the anticoagulant function: The affinity of warfarin and 4-hydroxycoumarin for HSA was determined by surface plasmon resonance (SPR) technology. 4-Hydroxycoumarin was found to have a slightly stronger affinity for HSA than warfarin. And by thrombin time (TT) measurement test, it was found that 4-hydroxycoumarin has no anticoagulant function. The results are shown in Figure 10.

[0181] 3. Virtual screening of fatty acid derivatives: Octadecanedioic acid (OA) has a strong affinity for serum albumin (HSA), but since OA can also bind to fatty acid-binding protein (FABP), it causes off-target effects and is not suitable as a small molecule coupling reagent. By virtual high-throughput screening, the present invention obtained dodecanedioic acid (DA) having a high affinity for HSA and a low affinity for FABP (Table 4). Virtual high-throughput screening was carried out using Schrodinger_Suites_2021-2 software, which is software capable of simulating small and macromolecular systems and was used to perform docking simulations of the selected compounds with full-length HSA and FABP, respectively.

[0182]

Table 5

[0183] 4. Verification of the affinity between dodecanedioic acid and HSA: The affinities of dodecanedioic acid and octadecanedioic acid for HSA and FABP were determined by surface plasmon resonance (SPR) technology, respectively. Dodecanedioic acid was found to have a slightly stronger affinity for HSA than octadecanedioic acid and a weaker affinity for FABP than octadecanedioic acid. The results are shown in Figure 11.

[0184] 5. Coupling method In the present invention, chemical modification technology was used to couple a fatty acid and a coumarin derivative to the 5'-end of the aptamer nucleic acid strand. Due to the structural similarity of the nucleic acid strand itself, it is not easy to couple directly on the nucleobase, and an active group is required to extend at the end of the nucleic acid strand. In the present invention, the following 5'-amino-modified C7-TFA monomer was used to construct a connecting arm to modify the end of the nucleic acid strand.

[0185] 5'-amino-modified C7-TFA

Chemical formula

[0186] The following structure S6 was obtained. [Chemical formula]

[0187] Next, 4-hydroxycoumarin was connected to the sulfhydryl group of S6, and dodecanedioic acid was connected to the amino group of S6.

[0188] 6. Detection of the in vivo half-life of APC001 conjugated with 4-hydroxycoumarin and dodecanedioic acid APC001 conjugated with 4-hydroxycoumarin and dodecanedioic acid was named APC001OC, and its structure is as follows: OC-C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT (Here, OC represents conjugated 4-hydroxycoumarin and dodecanedioic acid). The pharmacokinetic research method is the same as that in the previous example.

[0189] The results of the in vivo half-life of the 4-hydroxycoumarin and dodecanedioic acid double-modified sclerostin nucleic acid aptamer and its derivatives disclosed in the present invention, compared with APC001 without 4-hydroxycoumarin and dodecanedioic acid double modification, are shown as follows:

[0190] [Table 6]

[0191] Here, OA represents octadecanedioic acid modification, HC represents 4-hydroxycoumarin modification, DA represents dodecanedioic acid modification, and OC represents 4-hydroxycoumarin and dodecanedioic acid modification.

[0192] Detection of the in vivo half-life of an aptamer against DKK1 conjugated with 7.4-hydroxycoumarin and dodecanedioic acid The sequence of the aptamer against DKK1 (APTDKK1) is as follows: CGTACGGTCGACGCTAGCTGGTGGTTGGGGTGGGTGGT (SEQ ID NO: 26)

[0193] APTDKK1 conjugated with 4-hydroxycoumarin and dodecanedioic acid was named APTDKK1OC, and its structure is as follows: OC-CGTACGGTCGACGCTAGCTGGTGGTTGGGGTGGGTGGT

[0194] Here, OC represents conjugated 4-hydroxycoumarin and dodecanedioic acid. The pharmacokinetic research method is the same as that of the previous example.

[0195] The results of the in vivo half-life of the 4-hydroxycoumarin and dodecanedioic acid double-modified DKK1 nucleic acid aptamer and its derivatives disclosed in the present invention, compared with APTDKK1 without 4-hydroxycoumarin and dodecanedioic acid double modification, are shown as follows:

[0196]

Table 7

[0197] Here, OA represents octadecanedioic acid modification, HC represents 4-hydroxycoumarin modification, DA represents dodecanedioic acid modification, and OC represents 4-hydroxycoumarin and dodecanedioic acid modification.

Claims

1. An aptamer conjugate against sclerostin, comprising an aptamer against sclerostin conjugated to a fatty acid, wherein the aptamer comprises any one nucleotide sequence of SEQ ID NOs: 1 to 17 and 19 to 25, wherein the fatty acid is octadecanedioic acid, and the aptamer is an aptamer conjugate that specifically binds to sclerostin.

2. The aptamer conjugate according to claim 1, wherein the aptamer is conjugated to a fatty acid and a coumarin derivative.

3. The aptamer conjugate according to claim 2, wherein the coumarin derivative is selected from 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin)oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, and 6-methoxy-2-oxo-2H-chromene-3-carboxylic acid.

4. The aptamer conjugate according to any one of claims 1 to 3, wherein the fatty acid is conjugated to the 5'-end of the aptamer.

5. The aptamer conjugate according to any one of claims 1 to 4, wherein the fatty acid is conjugated to the aptamer through a linker.

6. The aptamer has a K for sclerosis tin of less than 100 nM ｄ The aptamer conjugate according to any one of claims 1 to 5, which has

7. The aptamer conjugate according to any one of claims 1 to 6, wherein the aptamer can inhibit the biological activity of sclerostin.

8. The aptamer conjugate according to any one of claims 1 to 7, wherein the aptamer can block the antagonistic effect of sclerostin in a cell-based Wnt signaling assay.

9. The aptamer conjugate according to any one of claims 1 to 8, wherein the aptamer inhibits the biological activity of sclerostin with an EC50 value of less than 100 μg / ml.

10. The aptamer conjugate according to any one of claims 1 to 9, wherein the aptamer comprises one or more modifications that confer enhanced nuclease resistance to the nucleic acid molecule.

11. The aptamer conjugate according to claim 10, wherein the modification comprises a 3'-inverted deoxythymidine (3'idT) modification.

12. The aptamer conjugate according to claim 10, wherein the modification comprises replacing one or more naturally occurring nucleotides with a modified nucleotide selected from the group consisting of 2'-fluoro, 2'-methoxyethyl, 2'-methoxy or 2'-allyloxy modified nucleotides.

13. The aptamer conjugate according to claim 10, wherein the modification comprises an internucleotide modification.

14. The aptamer conjugate according to claim 10, wherein the aptamer comprises a 2'-methoxy (2'-OMe) modification and / or a 3'-inverted deoxythymidine (3'idT) modification.

15. A pharmaceutical composition comprising the aptamer conjugate according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier or excipient.

16. Use of the pharmaceutical composition according to claim 15 or the aptamer conjugate according to any one of claims 1 to 14 in the preparation of a medicament for treating a sclerostin-related disease.

17. The use according to claim 16, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, fracture, osteoarthritis and myeloma, hypophosphatemic rickets, and triple negative breast cancer.

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