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WO2019154410A1 - 针对骨硬化蛋白的适体及其用途 - Google Patents

针对骨硬化蛋白的适体及其用途 Download PDF

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Publication number
WO2019154410A1
WO2019154410A1 PCT/CN2019/074764 CN2019074764W WO2019154410A1 WO 2019154410 A1 WO2019154410 A1 WO 2019154410A1 CN 2019074764 W CN2019074764 W CN 2019074764W WO 2019154410 A1 WO2019154410 A1 WO 2019154410A1
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Prior art keywords
aptamer
osteosclerosis
modification
preferably less
aptscl56
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French (fr)
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张戈
于媛媛
倪帅健
何伊欣
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Eagle Biotechnology Ltd
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Eagle Biotechnology Ltd
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Priority to JP2020565005A priority Critical patent/JP7120531B2/ja
Priority to CN201980012952.6A priority patent/CN111712573B/zh
Priority to US16/969,314 priority patent/US12338441B2/en
Priority to EP19751167.8A priority patent/EP3754021A4/en
Publication of WO2019154410A1 publication Critical patent/WO2019154410A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end

Definitions

  • the invention relates to the field of biomedicine.
  • the invention relates to aptamers for sclerostin and their use, in particular for their use in the treatment of osteosclerosis-associated diseases such as osteoporosis.
  • Osteoporosis is a disease of reduced bone mass and reduced bone strength that leads to an increased risk of fracture (Hamersma, Gardner et al., 2003).
  • the various drugs used to treat osteoporosis are primarily anti-resorptive agents that inhibit bone resorption to prevent further bone loss (Russell, Watts et al., 2008; Pennypacker, Duong et al., 2011).
  • Parathyroid hormone (PTH) is the only anabolic agent that can be used to stimulate bone formation to reverse established osteoporosis (Compston 2007; Greenspan, Bone et al., 2007).
  • long-term treatment with PTH has a risk of causing osteosarcoma (Whitfield 2001; Orwoll, Scheele et al., 2003). Therefore, there is an urgent need for alternative anabolic drugs that promote bone formation without side effects.
  • Osteosclerosis is a promising target for the development of osteoporosis drugs (Rey and Ellies, 2010).
  • Humanized monoclonal antibodies against human osteosclerosis have been reported to be well tolerated in clinical trials to promote bone formation and increase bone mass.
  • therapeutic antibodies have several major problems, including high immunogenicity (Padhi, Jang et al., 2011; Padhi, Allison et al., 2014), with high production costs (Baker, 2015; Bradbury and Pluckthun 2015; Groff , Brown et al., 2015), unstable, requiring continuous cold chain transport and storage (Jayasena, 1999). Therefore, for bone anabolic therapy, alternative anti-osteosclerosis agents that are non-immunogenic, easy to produce, low cost, and highly stable are desired.
  • Aptamers are short single-stranded oligonucleotides that bind to their targets by conformational complementation (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Aptamers have similar affinities and specificities compared to therapeutic antibodies, but have some important advantages. For immunogenicity, aptamers are not recognized by the immune system as foreign and do not stimulate negative immune responses because of low molecular weight (Keefe, Pai et al., 2010). In terms of production and cost, aptamers can be identified in vitro under a variety of selection conditions and can be readily synthesized by chemical methods, resulting in lower production costs and lower risks (Banerjee, 2010).
  • aptamers In terms of stability, aptamers have an indefinite shelf life because they are temperature insensitive and do not have any special cooling requirements during transport, so continuous cold chains are not required (Jayasena, 1999).
  • Pegaptanib an aptamer specific for vascular endothelial growth factor (VEGF)
  • VEGF vascular endothelial growth factor
  • the invention provides an aptamer for osteosclerosis, said aptamer
  • Ii) comprising at least 30, at least 35, at least 40, at least 45, at least 50 or more contiguous nucleotides of any one of SEQ ID NOS: 1-17,
  • the aptamer comprises the sequence of any one of SEQ ID NOs: 1-17 or 19-24, wherein the aptamer specifically binds to osteosclerin.
  • the aptamer of less than 100nM osteosclerosis protein preferably less than 50 nM, preferably less than 40nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10nM or less K d.
  • the aptamer is capable of inhibiting the biological activity of osteosclerin. In some embodiments, the aptamer is capable of blocking the antagonism of osteosclerin in a cell-based Wnt signaling assay. In some embodiments, the aptamer has an EC50 of less than 100 ⁇ g/ml, preferably less than 50 ⁇ g/ml, preferably less than 40 ⁇ g/ml, preferably less than 30 ⁇ g/ml, preferably less than 20 ⁇ g/ml, preferably less than 10 ⁇ g/ml or less. The value inhibits the biological activity of osteosclerosis, for example, inhibits the antagonism of osteosclerosis to the Wnt signaling pathway.
  • the aptamer further comprises one or more modifications that confer enhanced nuclease resistance to the aptamer and/or enhance the in vivo half-life of the aptamer.
  • the modification comprises a 3' reverse deoxythymidine (3' idT) modification.
  • the modification comprises substituting one or more naturally occurring nucleotides with a modified nucleotide selected from the group consisting of 2'-fluoro, 2'-methoxy B. A nucleotide modified with a 2'-methoxy or 2' propyleneoxy group, preferably a 2'-methoxy modified nucleotide.
  • the modification comprises a modification between nucleotides, such as a phosphorothioate linkage modification between nucleotides.
  • the modification comprises a PEG modification.
  • the aptamer comprises a 2'-methoxy (2'-OMe) modification, a 3' reverse deoxythymidine (3' idT) modification, and/or a PEG modification.
  • the invention provides a method of treating a bone sclerostin-related disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an aptamer of the invention against osteosclerosis, such as a human.
  • the osteosclerosis-associated disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), ischemic osteonecrosis, rheumatoid arthritis, fracture, osteoarthritis And myeloma.
  • the invention provides a pharmaceutical composition comprising at least one aptamer of the invention for osteosclerosis, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides the use of an aptamer for osteosclerosis or a pharmaceutical composition according to the present invention for the preparation of a medicament for use in the treatment of a bone sclerostin-related disease.
  • the osteosclerosis-associated disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), ischemic osteonecrosis, rheumatoid arthritis, fracture, osteoarthritis And myeloma.
  • Figure 1 shows the enrichment of osteosclerin high affinity aptamers by SELEX.
  • A Binding affinity of enriched ssDNA and unselected libraries for sclerostin.
  • B Binding affinities of the enriched ssDNA library and the unselected library to the control protein.
  • Figure 2 shows the specific characterization of aptamer candidates. Aptamer candidates showed high selectivity for human osteosclerosis compared to binding to hepatocytes and PBMC.
  • Figure 3 shows the affinity of aptamer candidates identified from ssDNA libraries with a 40 nt random region for recombinant human osteosclerosis proteins.
  • the dissociation constant (Kd) of aptamer candidates and antibodies against osteosclerin was calculated by nonlinear curve fitting analysis.
  • the Kd values of aptamer candidates for osteosclerosis were: utscl 6 of 4.2 nM, aptscl 9 of 3.4 nM, aptscl 15 of 45.6 nM, aptscl 46 of 43.1 nM, aptscl 56 of 43.1 nM and aptscl 132 of 42.2 nM.
  • the Kd value of the anti-osteosclin antibody was 3.55 nM.
  • Figure 4 shows the binding affinity of aptamer candidates identified from ssDNA libraries with 25 nt random regions to recombinant human osteosclerosis proteins.
  • the dissociation constant (Kd) of each candidate was calculated by nonlinear curve fitting analysis.
  • the Kd values of each aptamer candidate and antibody were: aptscl 32 was 0.18 nM, aptscl 29 was 0.28 nM, aptscl 22 was 0.76 nM, aptscl 16 was 0.22, aptscl 3 was 0.04 nM, aptscl 2 was 0.006 nM, aptscl 1 It is 0.02 nM.
  • the Kd value of the anti-osteosclin antibody was 3.55 nM.
  • Figure 5 shows the assessment of the ability of aptamer candidates and antibodies to inhibit using a TOP-Wnt induced luciferase reporter assay.
  • A Wnt signaling-mediated luciferase activity in MC3T3-E1 cells treated with aptamer candidates compared to antibodies.
  • Aptscl 56, aptscl 6, aptscl 3 and anti-osteoclast antibodies can effectively inhibit the antagonism of osteosclerosis to Wnt signaling and release Wnt-induced luciferase activity.
  • concentrations of aptscl 56 and aptscl 6 reached 25 ⁇ g/ml and 47.4 ⁇ g/ml, respectively, the response was stable.
  • Figure 6 shows the characterization of truncated aptscl3 binding to osteosclerin.
  • aptscl3-1, -2, -3, -4, and -5 maintain high affinity for osteosclerosis proteins, while aptscl3-6 shows low binding ability to sclerostin and cannot fit the affinity analysis curve;
  • Figure 7 shows the serum stability assessment of modified aptscl56 compared to unmodified aptscl56. All aptamers were treated with 10% and 100% mouse serum for 0 to 72 hours. Almost all unmodified aptscl56 degraded after 48 hours of incubation in 10% mouse serum. 2'-OMe and 3'-idT modified aptscl56 were maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl56 rapidly degraded completely after 8 hours; at 72 hours, a small amount of modified aptamer remained.
  • Figure 8 shows the serum stability assessment of modified aptscl3-5 compared to unmodified aptscl3-5. All aptamers were treated with 10% and 100% mouse serum for 0 to 72 hours. After incubation for 24 hours in 10% mouse serum, Aptscl3-5 degraded. The 2'-OMe and 3'idT modified aptscl3-5 were maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl3-5 rapidly degraded completely after 8 hours, while modified aptscl3-5 remained intact after 72 hours.
  • Figure 9 shows the affinity and inhibitory potency of chemically modified aptscl56 and aptscl3-5. Both aptscl56 and aptscl3-5 maintain high affinity and in vitro inhibitory potency against osteosclerin after chemical modification.
  • Figure 10 shows the pharmacokinetics of a single subcutaneous injection of Aptscl56 in 6 rats: pharmacokinetic profile fitted by software DAS (left), actual pharmacokinetic profile (right).
  • Figure 11 shows the pharmacokinetics of a single subcutaneous injection of PEG40K-aptscl56 in 6 rats: pharmacokinetic profile fitted by software DAS (left), actual pharmacokinetic profile (right).
  • Figure 12 shows the pharmacokinetics of a single subcutaneous injection of Aptscl56 (blue) and PEG40K-aptscl56 (orange) in rats, respectively.
  • Figure 13 shows the evaluation of the efficacy of PEG40K-aptscl56/aptscl3 in bone metabolism in ovariectomized rats induced by osteoporosis.
  • (a) A schematic showing the experimental design of the study.
  • (b) Representative 3D microarchitecture images and micro-CT parameters for each group of distal femoral trabeculae.
  • (c) Representative 3D microarchitecture images and micro-CT parameters for each group of proximal humerus trabeculae.
  • BMD bone density
  • Tb.BV/TV relative bone mass of trabecular bone
  • Tb.vBMD trabecular volume mineral density
  • Tb.Th trabecular thickness
  • Tb.N trabecular number
  • Tb.Sp trabecular spacing
  • Tb.conn.D trabecular connection density
  • Tb. SMI trabecular structure model index
  • MAR mineral deposition rate
  • BFR/BS bone formation rate
  • PINP intact N-terminus of procollagen I
  • OPG osteoprotective effect.
  • SHAM-BS SHAM baseline; OVX-BS: pre-treatment OVS baseline; SHAM+Veh: vehicle-treated SHAM rats; OVX+Veh: vehicle-treated OVX rats; OVX+aptscl56: treated with PEG40K-aptscl56 OVX rats; OVX+RS: OVX rats treated with PEG40K-random sequence.
  • nucleotide refers to a ribonucleotide or deoxyribonucleotide, or a modified form thereof, and analogs thereof. Nucleotides include species including purines (eg, adenine, hypoxanthine, guanine and their derivatives and analogs) and pyrimidines (eg, cytosine, uracil, thymine, and their derivatives and the like) ()).
  • purines eg, adenine, hypoxanthine, guanine and their derivatives and analogs
  • pyrimidines eg, cytosine, uracil, thymine, and their derivatives and the like
  • nucleic acid As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides, and include DNA, RNA, DNA/RNA hybrids, and nucleic acids of these kinds, Modifications of oligonucleotides and polynucleotides, including the addition of various entities or moieties at any position in the nucleotide unit.
  • polynucleotide “oligonucleotide” and “nucleic acid” include double or single stranded molecules.
  • nucleic acids, oligonucleotides and polynucleotides are terms broader than the term aptamer, and thus the terms nucleic acid, oligonucleotide and polynucleotide include aptamers but are not limited to aptamers.
  • aptamer refers to a non-naturally occurring nucleic acid having the desired effect on a target molecule. Desirable effects include, but are not limited to, binding to the target, catalyzing a change in the target, modifying or altering the functional activity of the target or the target, reacting with the target, covalently attaching the target, and promoting the The reaction between the target and other molecules.
  • the effect is directed to a specific binding affinity of a target molecule (eg, osteosclerin), such a target molecule being a three dimensional chemical structure rather than a polynucleotide that is independent of Watson/Crick base pairing Or a triple helix forming mechanism to bind the aptamer, wherein the aptamer is not a nucleic acid having a known physiological function that is bound by the target molecule.
  • a target molecule eg, osteosclerin
  • Sequence identity has art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule.
  • BLAST basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • the present inventors used the osteosclerin as a target protein for positive screening, and negatively screened with an unrelated protein, and finally selected an aptamer that specifically binds to osteosclerin with high affinity.
  • the osteosclerin described herein is preferably a human osteosclerosis protein, for example, an osteosclerin having the amino acid sequence set forth in SEQ ID NO: 18.
  • the invention provides an aptamer for osteosclerosis comprising at least about 90% identity, at least about 91% identity, at least any of SEQ ID NOS: 1-17, at least 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 a nucleotide sequence of 99% identity, or the aptamer comprises at least 30, at least 35, at least 40, at least 45, at least 50 of or any one of SEQ ID NOs: 1-17 or More consecutive nucleotides.
  • the aptamer specifically binds to osteosclerin.
  • the aptamer comprises the nucleotide sequence of any one of SEQ ID NOS: 1-17 and 19-24, and more preferably, the aptamer comprises SEQ ID NOS: 1, 3, A nucleotide sequence of one of 10 or 19-23.
  • the aptamer of the invention has a K d (dissociation constant) for osteosclerosis 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 less. ).
  • the K d is determined, for example, by an enzyme-linked oligonucleotide assay (ELONA).
  • the aptamer of the invention inhibits the biological activity of osteosclerosis proteins.
  • “Inhibiting” means that the biological activity of osteosclerin is reduced in the presence of the aptamer, for example by at least about 10%, at least about 20%, at least about 30%, at least in comparison to the absence of the aptamer. About 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or even at least about 90%.
  • biological activity refers to the action on one or more cells or extracellular processes that can affect a physiological or physiologic process.
  • the biological activities of osteosclerosis include, but are not limited to, antagonizing the Wnt signaling pathway.
  • the aptamer of the invention is capable of inhibiting the antagonism of osteosclerin to the Wnt signaling pathway.
  • the aptamer of the invention is capable of blocking the antagonism of osteosclerin in a cell-based Wnt signaling assay.
  • the aptamer of the invention is less than 100 ⁇ g/ml, preferably less than 50 ⁇ g/ml, preferably less than 40 ⁇ g/ml, preferably less than 30 ⁇ g/ml, preferably less than 20 ⁇ g/ml, preferably less than 10 ⁇ g/ml or more.
  • a small EC50 value inhibits the biological activity of osteosclerosis, for example, inhibits the antagonism of osteosclerosis to the Wnt signaling pathway.
  • the EC50 value is determined in vitro by a TOP-Wnt-induced luciferase reporter assay in osteoblasts.
  • the aptamer of the invention may also comprise one or more modifications.
  • the modification is a modification that confers enhanced nuclease resistance to the aptamer and/or enhances the in vivo half-life of the aptamer.
  • modifications include, for example, 3' and 5' modifications, such as 3' and 5' capping.
  • the aptamer is modified at the 3' end with a reverse deoxythymidine cap, i.e., 3' reverse deoxythymidine (3' idT).
  • the modification may also include replacing one or more naturally occurring nucleotides with a modified nucleotide.
  • the modified nucleotides include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy, and/or 2' propyleneoxy modified nucleotides (ie, ribose The 2' position hydroxyl group is substituted by fluorine, methoxyethyl, methoxy or propyleneoxy or the like).
  • the modified nucleotide may also include a C-5 modified pyrimidine.
  • C-5 modified pyrimidine refers to a modified pyrimidine at the C-5 position.
  • C-5 modified pyrimidines are capable of enhancing the nuclease resistance of oligonucleotides and are known in the art, for example, see International Patent Application WO 2011/130195 and the literature cited therein.
  • the modification is a 2'-methoxy (2'-OMe) modification.
  • the modification e.g., 2'-methoxy (2'-OMe) modification, is carried out at one or more, e.g., 4 nucleotides, 5' and/or 3' ends of the aptamer.
  • the modifications also include internucleotide modifications, such as internucleotide modifications with uncharged bonds (eg, methylphosphonates, phosphotriesters, urethanes, carbamates, etc.) and Internucleotide modification of a charge bond (eg, phosphorothioate, phosphorodithioate, etc.), internucleotide modification of an intercalating agent (eg, acridine, psoralen, etc.), containing a chelating agent (eg, Internucleotide modification of metals, radioactive metals, boron, oxidizing metals, etc., including internucleotide modifications of alkylating agents and internucleotide modifications of modified linkages (eg, alpha anomeric nucleic acids, etc.).
  • internucleotide modifications such as internucleotide modifications with uncharged bonds (eg, methylphosphonates, phosphotriesters, urethanes, carba
  • the modification may also include a pegylation modification (PEG modification).
  • PEG modification pegylation modification
  • the half-life of the aptamer can be extended by conjugation to PEG.
  • the PEG has a molecular weight of from about 1 kDa to about 100 kDa, such as from about 10 kDa to about 80 kDa, from about 20 kDa to about 60 kDa, from about 30 kDa to about 50 kDa, and about 40 kDa.
  • the PEG can be conjugated to the 5' end of the aptamer.
  • the PEG can be conjugated to the 3' end of the aptamer.
  • the aptamer can comprise a combination of the various modifications described above.
  • the aptamer may comprise a 2'-methoxy (2'-OMe) modification, a 3' reverse deoxythymidine (3' idT) modification, and/or a PEG modification.
  • the PEG has a molecular weight of about 40 kDa.
  • the invention provides a method of treating a disease by an aptamer of osteosclerin of the invention, the method comprising administering to a subject in need thereof a therapeutically effective amount of an aptamer of the invention against osteosclerin.
  • the disease treated by the aptamer of the osteosclerin of the present invention is, for example, an osteosclerosis-related disease such as an osteosclerin-mediated disease.
  • osteosclerin-associated disease includes disorders in which bone mineral density (BMD) is abnormal and/or pathologically low relative to a healthy subject.
  • Diseases characterized by low BMD and/or bone fragility include, but are not limited to, primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), ischemic necrosis (bone Necrosis), fracture and implant healing (dental implants and hip implants), bone loss due to other conditions (eg, associated with HIV infection, cancer, and arthritis).
  • Other "osteosclerosis-associated diseases” include, but are not limited to, rheumatoid arthritis, osteoarthritis, arthritis, and osteolytic lesions.
  • osteosclerin-associated diseases include osteosclerosis-associated cancers, such as myeloma (eg, multiple myeloma with osteolytic lesions), breast cancer, colon cancer, melanoma, hepatocellular carcinoma, Epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and any metastases thereof.
  • the "osteosclerosis-associated disease” may also include nephropathy and cardiovascular disease caused by at least osteosclerin expression in the kidney and in the cardiovascular.
  • kidney diseases such as glomerular diseases (eg, acute and chronic glomerulonephritis, progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, and Systemic lupus erythematosus, Goodpasch's syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasia, sickle cell disease, and chronic inflammation associated with systemic disease, glomerular damage), renal tubules Diseases (eg, acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial disease (eg, pyelonephritis)
  • glomerular diseases
  • the diseases also include, but are not limited to, cardiovascular diseases such as ischemic heart disease (eg, angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (eg, , rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse and aortic stenosis, congenital heart disease (eg, occlusion of the valve and blood vessels, septal or ventricular septal defect, and long-term survival) Arterial catheter), or cardiomyopathy (eg, myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).
  • ischemic heart disease eg, angina pectoris, myocardial infarction, and chronic ischemic heart disease
  • hypertensive heart disease eg., pulmonary heart disease, valvular heart disease (eg, , rheumatic fever and rheumatic heart disease, end
  • the subject can be any animal (domestic, domestic or wild) including, but not limited to, cats, dogs, horses, pigs and cattle, and preferably human subjects.
  • the terms patient, subject, and subject are used interchangeably.
  • the subject can be male or female.
  • the human subject is at risk of fracture, more preferably the human subject is at risk of osteoporosis or has osteoporosis.
  • the human subject is preferably a female, and more preferably a female at risk of osteoporosis after amenorrhea or suffering from postmenopausal osteoporosis. It is contemplated that the methods of the invention may be beneficial to subjects at any stage of osteoporosis.
  • treating means that the symptoms of the subject are partially or completely relieved or remain unchanged after treatment.
  • treatment includes prevention, treatment, and/or cure.
  • Prevention refers to prevention of underlying disease and/or prevention of worsening symptoms or disease progression.
  • therapeutically effective amount refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition.
  • therapeutic effect refers to an effect caused by the treatment of a subject that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures a disease or condition.
  • a dosage regimen utilizing the aptamer for osteosclerin is selected according to a variety of factors including, for example, the type, type, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated The route of administration; the function of the kidney and liver of the patient; and the specific aptamer or salt thereof for the osteosclerin used.
  • a generally skilled physician can readily determine and specify an effective amount of the composition required to prevent, combat or inhibit the progression of the condition.
  • the dosage regimen for the aptamer of osteosclerin is from about 1 [mu]g/kg body weight to about 100 mg/kg body weight per day.
  • An exemplary treatment regimen requires daily dosing, once every two days, once weekly, twice weekly, once every two weeks, once every three weeks, once every four weeks, once a month, every 3 months once, every 3-6 months, or the initial dosing interval is slightly shorter (such as once a week to once every three weeks).
  • the post-dosing interval is longer (eg once a month to once every 3-6 months) ).
  • the frequency and interval of administration can be determined by one skilled in the art based on the pharmacokinetic parameters of the aptamer.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one aptamer of the invention for osteosclerosis, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is used, for example, for the treatment of osteosclerosis-associated diseases.
  • aptamers described herein can be used in any pharmaceutically acceptable dosage form including, but not limited to, injectable dosage forms, liquid dispersions, gels, sprays, ointments, creams, lyophilized preparations, dry powders, tablets, Capsules, controlled release formulations, quick melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, and the like.
  • the aptamers described herein can be formulated as: (a) selected from the group consisting of oral, pulmonary, intravenous, intraarterial, intrathecal, internal joint, rectum, eye, colon, parenteral, intracranial, intravaginal , administered intraperitoneally, topically, buccally, nasally, and topically; (b) selected from the group consisting of liquid dispersions, gels, sprays, ointments, creams, tablets, sachets, and capsules.
  • Dosage form (c) a dosage form selected from the group consisting of lyophilized preparations, dry powders, quick-melt preparations, controlled release preparations, delayed release preparations, extended release preparations, pulsatile release preparations, and mixed immediate release and controlled release preparations Or (d) any combination of them.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may contain one or more of the following components: (1) sterile diluents, for example, water for injection, saline solution, fixed oil, polyethylene a diol, glycerol, propylene glycol or other synthetic solvent; (2) an antibacterial agent such as benzyl alcohol or p-hydroxybenzyl ester; (3) an antioxidant such as ascorbic acid or sodium sulfite; (4) a chelating agent such as ethylenediaminetetra Acetic acid; (5) a buffer such as acetate, citrate or phosphate; and (5) a substance for regulating tension, such as sodium chloride or glucose.
  • the pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide.
  • Formulations for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions adapted for injectable use may include sterile aqueous solutions (where are water-soluble) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition should be sterile and should be fluid to the extent that it is easy to inject.
  • the pharmaceutical composition should be stable under the conditions of manufacture and storage and should be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • stable as used herein means to maintain a state or condition suitable for administration to a patient.
  • the carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of 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, for example, p-hydroxybenzyl ester, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, polyols (e.g., mannitol or sorbitol), and inorganic salts (e.g., sodium chloride) in the compositions.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions, such as, such,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
  • Sterility can be prepared by incorporating the active agent (eg, an aptamer for osteosclerosis) in a desired amount with one or a combination of the above-listed ingredients (if desired) followed by filter sterilization.
  • Injectable solution e.g., dispersions are prepared by incorporating at least one aptamer to osteosclerin to a sterile carrier containing a base dispersion medium and any other desired ingredients.
  • exemplary methods of preparation include vacuum drying and freeze drying, both of which obtain the powder of the aptamer for osteosclerosis as well as from previous no The bacteria filter any additional desired components of the solution.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • they can be filled into gelatin capsules or compressed into tablets.
  • the aptamer for osteosclerin may be incorporated into an excipient and used in the form of a tablet, lozenge or capsule.
  • a pharmaceutically compatible binding agent and/or adjuvant material can be included as part of the composition.
  • a suitable propellant eg, a gas (eg, carbon dioxide), a nebulized liquid, or a dry powder from a suitable device
  • a suitable penetrant for the barrier to be permeated is used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be achieved by the use of nasal sprays or suppositories.
  • the active agents are formulated into ointments, salves, gels or creams as are known in the art.
  • the agents may also be prepared in the form of a suppository (for example, a conventional suppository base such as cocoa butter and other glycerides) or a retention enemas for rectal delivery.
  • the aptamer for osteosclerin is formulated for topical administration.
  • topical administration refers to the delivery of an osteosclerin suitable by contacting (directly or otherwise) a formulation comprising the aptamer for osteosclerin with all or part of the skin (epidermal) of an animal. Body to the animal. The term encompasses several routes of administration including, but not limited to, topical administration and transdermal administration. A general need for these modes of administration is effective delivery to the target tissue or layer.
  • topical application is used as a means of penetrating the epidermis and dermis and ultimately achieving systemic delivery of aptamers to osteosclerin.
  • topical administration is used as a means of selectively delivering an aptamer against osteosclerosis to the epidermis or dermis of an animal or a particular layer thereof.
  • the aptamer for osteosclerin may be formulated as a pharmaceutically acceptable ointment, cream, lotion, ophthalmic ointment, eye drop, ear drop, impregnated dressing, and aerosol,
  • a medicated powder, a medicated binder, a foam may contain suitable conventional additives or excipients, including, for example, preservatives or solvents and ointments that aid in the penetration of the drug, gels, and softeners in creams.
  • Such topical formulations may also contain compatible conventional carriers such as ethanol or oleyl alcohol for use in emulsions. Such carriers may constitute from about 1% to about 98% by weight of the formulation, and more typically such carriers will constitute up to about 80% by weight of the formulation. Specific formulations for topical delivery of aptamers have been described in the prior art.
  • an aptamer against osteosclerin is prepared with a carrier that prevents rapid removal from the body.
  • controlled release formulations can be used, including implanted and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods of preparing such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions (including liposomes with monoclonal antibodies directed against viral antigens that target infected cells) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • a suspension of the aptamer against osteosclerin can be prepared as a suitable oily injection suspension.
  • suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes.
  • Non-lipid polycationic amino acid polymers can also be used for delivery.
  • the suspension may also include suitable stabilizers or agents to increase the solubility of the compound and allow for the preparation of highly concentrated solutions.
  • Dosage unit form as used herein refers to physically discrete units suitable as a single dose for the subject to be treated; each unit contains a predetermined amount of aptamer against osteosclerin calculated to produce the desired therapeutic effect. And the desired pharmaceutical carrier.
  • the description of the dosage unit form of the aptamer described herein for osteosclerin is dependent on and directly dependent on the unique characteristics of the aptamer specific to the osteosclerin and the particular therapeutic effect to be achieved and the formulation of such treatment for treatment The inherent limitations of the art of individual active agents.
  • a pharmaceutical composition comprising at least one aptamer for osteosclerin may comprise one or more pharmaceutical excipients.
  • excipients include, but are not limited to, binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrating agents, effervescent agents, and Other excipients. Such excipients are known in the art.
  • Exemplary excipients include: (1) binding agents include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose (such as Avicel PH101 and Avicel PH102), silicified microcrystalline cellulose (ProSolv SMCC TM), yellow Silicone and gelatin; (2) fillers, such as various starches, lactose, lactose monohydrate, anhydrous lactose; (3) disintegrants such as alginic acid, Primogel, corn starch, lightly crosslinked polyethylene Pyrrolidine, potato starch, corn starch and modified starch, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof; (4) lubricants, including those acting on the powder to be compressed Flowing agents, including magnesium stearate, colloidal silica (such as Aerosil 200, talc), stearic acid, calcium stearate, and silica gel; (5) glidants, such as colloidal silica; 6) preservative
  • the sequence in the ssDNA library consists of a 18 nt conserved region at both ends and a central random region. Two ssDNA libraries of random sequences of different lengths were used.
  • the sequence in the long ssDNA library contains a 40 nt random region (5'-CGTACGGTCGACGCTAGC-(N) 40 -CACGTGGAGCTCGGATCC-3'), and the sequence in the short ssDNA library contains a 25 nt random region (5'-CGTACGGTCGACGCTAGC-(N) 25 -CACGTGGAGCTCGGATCC- 3').
  • a synthetic forward primer (FP: 5'-CGTACGGTCGACGCTAGC-3') and a biotinylated reverse primer (Bio-RP: 5'-biotin-GGATCCGAGCTCCACGTG-3') were used to amplify ssDNA during selection. All oligonucleotides were purified by HPLC after synthesis.
  • the protein SELEX method was performed to identify high affinity aptamers (Ellington and Szostak, 1990; Tuerk and Gold, 1990). 100-30 picomoles of His 6- labeled osteosclerosis protein was immobilized on NTA magnetic beads for 1 hour at 4 ° C (Murphy, Fuller et al., 2003). The 1 nanomolar ssDNA library was denatured at 95 ° C for 5 minutes and rapidly cooled to 4 ° C, followed by incubation with immobilized osteosclerin at room temperature for 0.5-1 hour. Unbound sequences were removed with wash buffer. After washing, the bound DNA-protein-NTA was collected, resuspended in H 2 O/Tween 20, and applied to PCR amplification.
  • PCR amplification with unmodified forward primer and biotinylated reverse primer (step 1: initial denaturation at 95 ° C for 1 minute; step 2: denaturation at 95 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds , repeat 12 cycles; and step 3: 72 ° C last extended 5 minutes).
  • the PCR product was applied to streptavidin magnetic beads by biotin-streptavidin binding.
  • the regenerated single stranded sequence was treated by treatment with 0.2 M NaOH. Negative selection was made for other His 6- tagged irrelevant proteins immobilized on NTA magnetic beads. A total of 20 rounds of SELEX were performed for each selection.
  • the DNA pool from the last round was subjected to a high-throughput second-generation sequence (NGS).
  • NGS high-throughput second-generation sequence
  • Figure 1 shows the increase in affinity of the DNA pool for osteosclerin after the 10th and 20th rounds of selection, indicating that high affinity osteosclerin aptamers are enriched by SELEX.
  • aptamer candidates for osteosclerosis representative aptamer candidates and random sequences (RS) (negative controls) were synthesized using N-terminal biotinylation modifications.
  • Each aptamer/RS was assayed for specificity to osteosclerosis by enzyme-linked oligonucleotide assay (ELONA) using 1 ⁇ M.
  • ELONA enzyme-linked oligonucleotide assay
  • 160 ng of purified recombinant human osteosclerosis protein was coated with 96-well microtiter plates by incubating overnight in 100 ⁇ l of PBS at 4 °C.
  • the plates were then blocked with blocking buffer (PBS, 0.1% Tween 20 and 1% BSA) for 1 hour at room temperature and washed with SELEX B&W buffer (PBS, 1 mM MgCl 2 , 0.1% Tween 20 and 0.1% BSA) 4 Times.
  • the aptamer candidates were denatured at 95 ° C for 10 minutes and rapidly cooled on ice for 10 minutes before use. 1 ⁇ M biotin-labeled aptamer was added to each well, and then SELEX B&W buffer was added to 100 ⁇ l, and incubated at room temperature for 45 min, and gently shaken continuously.
  • the absorbance at 450 nm was measured with a microplate reader (Stoltenburg, Krafcikova et al., 2016).
  • the characterization step was similar to ELONA. 300,000 cells were incubated with each aptamer candidate, washed and separated by centrifugation.
  • aptamer candidates identified from long and short ssDNA libraries show high selectivity for human osteosclerosis when compared to binding to hepatocytes and PBMC (Fig. 2).
  • Enzyme-linked oligonucleotide assays were performed to determine the binding affinity of aptamer candidates to osteosclerosis (Drolet, Moon-McDermott et al., 1996).
  • an enzyme-linked immunosorbent assay was performed to determine the binding affinity of anti-osteosperm antibody (Romosozumab, a humanized therapeutic antibody against osteosclerin, purchased from Creative Biolabs) to human osteosclerosis (Engvall and Perlmann) , 1971).
  • 160 ng of purified recombinant human sclerostin was coated with 96-well microtiter plates by incubating overnight in 100 ⁇ l of PBS at 4 °C.
  • the plates were then blocked with blocking buffer (PBS, 0.1% Tween 20 and 1% BSA) for 1 hour at room temperature and washed with SELEX B&W buffer (PBS, 1 mM MgCl 2 , 0.1% Tween 20 and 0.1% BSA) 4 Times.
  • the 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, then SELEX B&W buffer was added to 100 ⁇ l and incubated for 45 minutes at room temperature with gentle shaking.
  • the absorbance at 450 nm was measured with a microplate reader (Stoltenburg, Krafcikova et al., 2016). Data were analyzed using Origin software (OriginLab, Northampton, MA).
  • Origin software OlinLab, Northampton, MA.
  • aptscl 6, 9, 15, 46, 56, and 132 have high affinity for osteosclerin with dissociation constant (Kd) values at nanomolar levels (Kd)
  • Kd dissociation constant
  • the values are 4.2, 3.4, 45.6, 43.1, and 42.2 nM, respectively ( Figure 3).
  • aptscl 36, 140, 136 and random sequence (RS) failed to fit.
  • aptscl 32, 29, 22, 16, 3, 2, and 1 have higher binding affinities for osteosclerins with Kd values of 0.18, 0.28, and 0.76, respectively. 0.02, 0.04, 0.006 and 0.02 nM (Fig. 4). Random sequences show low binding ability to osteosclerosis and cannot be fitted. In contrast, the Kd value of the osteosclerosis antibody to osteosclerosis protein was 3.55 nM.
  • Example 4 in vitro evaluation of the ability of candidate osteosclerosis protein aptamers to inhibit osteosclerosis protein activity
  • MC3T3-E1 cells were seeded in 24-well plates, and on day 2, the corresponding reporter plasmid (100 ng), Wnt3a plasmid (800 ng) and osteosclerosis protein pellet (800 ng) were transfected using FuGENE HD Transfection Reagent (Promega). After 10 hours of transfection, the medium was changed to fresh medium and the cells were treated with aptamers/antibodies. After 24 hours of treatment, cells in each well were lysed with 100 ⁇ l of passive lysis buffer and 20 ⁇ l was taken for analysis.
  • Luciferase Assay Reagent II and Stop&Glo Reagent were automatically prepared by SpectraMax i3x Multi-Mode Detection Platform (Molecular Device) according to the manufacturer's protocol (Promega), and the data were analyzed accordingly (Grentzmann, Ingram et al., 1998; McNabb) , Reed et al., 2005).
  • aptscl 56, aptscl 6, aptscl 3 and anti-osteoclast protein antibodies potently inhibited the antagonism of osteosclerosis to Wnt signaling and released Wnt-induced luciferase activity.
  • the inhibition of osteosclerin was dose-dependent, and the response was stable when the concentrations of aptscl 56 and aptscl6 reached 25 ⁇ g/ml and 47.4 ⁇ g/ml, respectively. When treated with antibodies, the response was still unstable with increasing concentrations to 20 mg/ml.
  • the inhibition ability of aptscl56, aptscl 6 and aptscl 3 was analyzed by nonlinear curve fitting. The EC50 of Aptscl 56, aptscl 6 and aptscl 3 were 19.7 ⁇ g/ml, 36.8 ⁇ g/ml and 18.2 ⁇ g/ml, respectively.
  • the present inventors have selected DNA aptamers for osteosclerosis and eventually developed two truncated aptamers, called aptscl56 and aptscl3-5, which specifically bind tightly to osteosclerosis and have a low nanomolar range.
  • the dissociation constant inside. 2'-O-methyl (2'-OMe) modifications of aptamers have previously been used as post-selective modifications because of their enhanced nuclease resistance and elevated duplex melting temperatures, such as clinical trials. Shown in (Fine, Martin et al. 2005; Gupta, Hirota et al. 2014).
  • the 3' end capping of the reverse dT is also a common strategy for aptamers for disease treatment in ongoing or completed clinical trials (Padilla, Sousa et al. 1999; Ruckman, Green et al. 1998). Therefore, this example evaluates whether the serum stability of aptscl56 and aptscl3-5 can be improved by 2'-OMe and 3'-end inverted dT (3'-idT) modification.
  • Modified nucleotides are introduced during the synthesis.
  • the serum metabolic stability of the modified and unmodified aptamers was evaluated in freshly prepared mouse sera. All aptamer samples were incubated with 10% and 100% mouse serum for 0, 2, 4, 8, 12, 24, 36, 48 and 72 hours at 37 °C, respectively. At the indicated times, aptamer samples were snap frozen in a dry ice bath and then stored at -80 °C until all samples were harvested for evaluation. The stability of all aptamer samples is expressed as the band density of the intact aptamer remaining after incubation, which can be determined by agarose gel electrophoresis.
  • the modified aptscl56 sequence was CGGG G TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CT GCCC -idT, and the underlined nucleotide was modified with 2'-OMe.
  • the modified sequence of aptscl3-5 is GCTA G CTGTT GTACA TCGCC TTACG CA CGT G -idT, and the underlined nucleotide is 2'-OMe modified.
  • aptscl56 For aptscl56, the unmodified aptamer completely degraded after 48 hours in 10% serum and remained only 8 hours in 100% serum. 2'-OMe and 3'-idT modified aptscl56 were maintained in 10% mouse serum for 72 hours and degraded after 12 hours in 100% mouse serum. At 72 hours, a small amount of modified aptamer remained ( Figure 7).
  • aptscl3-5 unmodified aptamers degraded 24 hours after incubation in 10% mouse serum. 2'-OMe and 3'-idT modified aptscl3-5 were maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl3-5 degraded rapidly and completely after 8 hours, while modified aptscl3-5 maintained integrity after 72 hours ( Figure 8).
  • the chemically modified aptscl56 and aptscl3-5 showed high binding affinity to osteosclerosis with Kd values of 6.55 and 0.54 nM, respectively.
  • chemically modified aptscl56 and aptscl3-5 were effective in relieving the inhibitory effect of osteosclerosis on Wnt signaling in cells with potencies of 14 and 11 ⁇ g/ml, respectively (Figure 9).
  • Modification of aptscl56 and aptscl3-5 into therapeutic nuclease resistant aptamers can be further facilitated by modification with 2'-OMe and 3'-idT.
  • the present invention determines the plasma pharmacokinetics of PEG-modified and non-PEG-modified aptamers (PEG40K-aptscl56 and aptscl56) against osteosclerin after subcutaneous administration in rats.
  • the aptscl56 sequence is CGGGG TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CTGCCC-idT, and the nucleotides of the starting CGGG and the terminal GCCC are modified with 2'-OMe.
  • PEG40K-aptscl56 further linked PEG40K (PEG with a molecular weight of 40,000) at the 5'-end.
  • aptscl56 and PEG40K-aptscl56 were performed in 6-month-old female primary Sprague-Dawley rats fed ad libitum to a standard laboratory diet and maintained under controlled conditions (12-hour photoperiod) , 20 ° C). Rats were treated with 6.1 mg/kg aptscl56 and 25 mg/kg PEG40K-aptscl56, respectively, by a single subcutaneous injection. Aptscl56 and PEG40K-aptscl56 were dissolved in saline at concentrations of 1.6 mg/ml and 6.2 mg/ml, respectively (Judith M. Healy, Ryan M. Boomer et al., 2004).
  • aptscl56 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h; PEG40K-aptscl56: 30min, 1h, 2h, 4h, 8h, 12h, 24h, 30h, 36h, 48h, 54h
  • Plasma preparation Approximately 800 ⁇ l of blood was collected from each rat through the orbital vein and collected into a tube containing sodium-heparin as an anticoagulant (1.8 ml vacuum container, BD Biosciences) and immediately placed on wet ice (Healy) , Lewis et al. 2004, Perschbacher, Smestad et al. 2015). Plasma was separated by centrifugation at 6000 g for 10 minutes at 4 °C for 1 hour after collection and stored at -80 °C until analysis (Healy, Lewis et al. 2004, Siller-Matula, Merhi et al. 2012, Gao, Shen Et al.2016).
  • HPLC quantification The HPLC system was equipped with a C4 column to quantify PEG40K-aptscl56 in plasma samples collected at different time points, while the C18 column was used to quantify aptscl56.
  • This method uses a mobile phase elution gradient made of phase A (TEAA [pH 7.0]) and phase B (acetonitrile). When the oven temperature was set at 50 ° C, the flow rate was 1.0 mL / min. The injection volume was determined to be 20 uL.
  • Standards were prepared in blank rat plasma containing different concentrations of sodium-heparin of aptscl56 and PEG40K-aptscl56 (Gao, Shen et al, 2016). All reported concentrations of aptscl56 and PEG40K-aptscl56 were based on the mass of aptscl56. The aptamer concentration in the plasma samples was calculated from the standard curve.
  • the lower limit of HPLC quantification of aptscl56 is 10 ul/mL, and the linear concentration range is from 10 ⁇ g/mL to 360 ug/mL.
  • the mean elimination half-life (Elim. T1/2) of aptscl56 aptamers in Sprague-Dawley rats after subcutaneous administration was 1.8 hours.
  • the average value of C max was 265.5 ⁇ g/ml, and T max was 0.5 hours (Fig. 10, Table 3).
  • the lower limit of HPLC quantification of PEG40K-aptscl56 was 7.5 ⁇ g/mL, and the linear concentration ranged from 7.5 ⁇ g/mL to 240 ⁇ g/mL.
  • the mean elimination half-life (Elim. T1/2) of PEG40K-aptscl56 in Sprague-Dawley rats after subcutaneous administration was 66.9 hours.
  • the average value of C max was 152.8 ⁇ g/ml, and T max was 72 hours (Fig. 11, Table 4).
  • the dosing interval for multiple doses of aptamer can be defined based on its elimination half-life and the dose ratio of the loading dose to the maintenance dose (Birkett 1996, Jambhekar 2012).
  • the elimination half-life of PEG40K-aptscl56 was 66.9 hours. If the dose ratio is 2, the dosing interval is equal to the elimination half-life (T1/2). If the dose ratio is less than 2, the dosing interval should be longer than T1/2. In the pharmacodynamic study of PEG40K-aptscl56, the recommended dose ratio is 1 (loading dose equals maintenance dose). Therefore, the PEG40K-aptscl56 dosing interval should be longer than 66.9 h.
  • Example 8 Evaluation of bone anabolic efficacy of PEGylated aptamer candidates in osteoporotic rats induced by ovariectomy
  • Micro-CT analysis Analysis of the right distal femoral metaphysis, right tibia proximal metaphysis, fifth lumbar vertebrae, and right femur central axis was performed using micro-CT (version 6.5, vivaCT40, SCANCO Medical AG, Bassersdorf, Switzerland). Images of vertebrae and femur were reconstructed and calibrated with isotropic voxel sizes of 12.5 and 17.5 ⁇ m (70 kVp, 114 ⁇ A, 200 ms integration time, 260 threshold, 1200 mg HA/cm 3 ), respectively. The same filtering and segmentation values are used for each measurement.
  • a region of interest is defined for the cortical and trabecular parameters using the Scanco evaluation software.
  • the entire femur or tibia will be reoriented, with the medial backbone parallel to the z-axis, and the bone length measured as the femur between the proximal and distal transverse plates. distance. Starting from the most proximal end of the growth plate, the trabecular region on 100 consecutive sections at a distance of 1.4 mm from the growth plate was selected. The trabeculae were analyzed by manual contouring to exclude cortical bone.
  • a central region corresponding to 70% of the height of the vertebral body and extending from the proximal end of the distal growth plate to the end toward the vertebral body is selected.
  • the trabecular ROI was drawn freehand on 100 serial sections to ensure it was within the intraosseous capsule.
  • an automatic threshold algorithm was used to measure 100 slices at the exact center and distal 50% femur length. The trabecular contact with the cortical bone was manually removed from the ROI.
  • trabecular bone parameters including trabecular volume per volume (Tb.BV/TV), trabecular volume mineral density (Tb.vBMD), trabecular thickness (Tb.Th), trabecular number (Tb.N) ), trabecular spacing (Tb.Sp), trabecular structure model index (Tb.SMI), trabecular connection density (Tb.conn.D).
  • Tb.BV/TV trabecular volume per volume
  • Tb.vBMD trabecular volume mineral density
  • Tb.Th trabecular thickness
  • Tb.N trabecular number
  • Tb.Sp trabecular spacing
  • Tb.SMI trabecular structure model index
  • Tb.conn.D trabecular connection density
  • OVX rats were treated with PEG40K-aptscl56/aptscl3, a random sequence or six regular injections of the vehicle at weekly intervals (designated OVX+aptscl56, OVX+aptscl3, OVX+RS and OVX+Veh, respectively).
  • Micro-CT analysis of the metaphyseal region of the distal femur showed significantly higher BMD and BV/TV, indicating a significant increase in trabecular bone quality in OVX+aptscl56 rats compared to OVX-BS (P ⁇ 0.005).
  • the trabecular structure of OVX+aptscl56 was also significantly improved compared to OVX-BS (Fig. 13b).
  • the OVX+aptscl3 group did not show significantly increased bone mass and improved structure compared to OVX-BS.
  • micro-CT data indicate that PEG40K-aptscl56 promotes bone formation in OVX-induced osteoporosis rats, improves bone microstructure, and increases bone mass.
  • RNA-based aptamers to the 165-amino acid Form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.”J Biol Chem 273(32): 20556-20567.

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Abstract

针对骨硬化蛋白的适体及其用途，特别是其用于治疗骨硬化蛋白相关疾病例如骨质疏松症中的用途。

Description

针对骨硬化蛋白的适体及其用途 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及针对骨硬化蛋白(sclerostin)的适体及其用途，特别是其用于治疗骨硬化蛋白相关疾病例如骨质疏松症中的用途。
发明背景
骨质疏松症是一种骨量减少和骨强度降低的疾病，导致骨折风险增加(Hamersma,Gardner et al.,2003)。用于治疗骨质疏松症的各种药物主要是抗再吸收剂，其抑制骨再吸收以防止进一步的骨损失(Russell,Watts et al.,2008；Pennypacker,Duong et al.,2011)。甲状旁腺激素(PTH)是唯一可用于刺激骨形成以逆转已建立的骨质疏松症的合成代谢剂(Compston 2007；Greenspan,Bone et al.,2007)。然而，使用PTH的长期治疗有导致骨肉瘤的风险(Whitfield 2001；Orwoll,Scheele et al.,2003)。因此，迫切需要促进骨形成而没有副作用的替代性合成代谢药物。
骨硬化蛋白是开发骨质疏松症药物的有希望的靶标(Rey and Ellies,2010)。据报道抗人骨硬化蛋白的人源化单克隆抗体在临床试验中促进骨形成和增加骨量，具有良好的耐受性。然而，治疗性抗体有几个主要问题，包括高免疫原性(Padhi,Jang et al.,2011；Padhi,Allison et al.,2014)，生产成本高(Baker,2015；Bradbury and Pluckthun 2015；Groff,Brown et al.,2015)，不稳定，需要连续冷链运输和储存(Jayasena,1999)。因此，对于骨合成代谢疗法而言，期望无免疫原性、容易生产、低成本和高稳定性的替代性抗骨硬化蛋白剂。
适体是通过构象互补而与其靶标结合的短单链寡核苷酸(Ellington and Szostak,1990；Tuerk and Gold,1990)。与治疗性抗体相比，适体具有相似的亲和力和特异性，但是具有一些重要的优点。对于免疫原性，适体不会被免疫系统识别为外来的，并且因为低分子量而不刺激负面的免疫应答(Keefe,Pai et al.,2010)。就生产和成本而言，适体可以在各种选择条件下在体外鉴 定，并且可以通过化学方法容易地合成，因此生产成本更低，风险更低(Banerjee,2010)。就稳定性而言，适体具有无限的保存期限，因为它们温度不敏感，并且在运输过程中没有任何冷却的特殊要求，因此不需要连续的冷链(Jayasena,1999)。Pegaptanib，一种针对血管内皮生长因子(VEGF)的适体，已成功用于临床治疗年龄相关性黄斑变性(Jellinek,Green et al.,1994；Ruckman,Green et al.,1998；Ng and Adamis 2006；Que-Gewirth and Sullenger2007)。
因此，期望开发针对骨硬化蛋白的适体来代替单克隆抗体用于治疗骨质疏松症。
发明简述
在一方面，本发明提供一种针对骨硬化蛋白的适体，所述适体
i)包含与SEQ ID NO:1-17中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性或至少大约95％相同性的核苷酸序列，或者，
ii)包含SEQ ID NO:1-17中的任一中的至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸，
优选地，所述适体包含SEQ ID NO:1-17或19-24中任一的序列，其中所述适体特异性结合骨硬化蛋白。
在一些实施方案中，所述适体对骨硬化蛋白具有小于100nM，优选小于50nM，优选小于40nM，优选小于30nM，优选小于20nM，优选小于10nM或更小的K d。
在一些实施方案中，所述适体能够抑制骨硬化蛋白的生物学活性。在一些实施方案中，所述适体能在基于细胞的Wnt信号测试法中阻断骨硬化蛋白的拮抗作用。在一些实施方案中，所述适体以小于100μg/ml，优选小于50μg/ml，优选小于40μg/ml，优选小于30μg/ml，优选小于20μg/ml，优选小于10μg/ml或更小的EC50值抑制骨硬化蛋白的生物学活性，例如抑制骨硬化蛋白对Wnt信号通路的拮抗作用。
在一些实施方案中，所述适体还包含一或多种赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。在一些实施方案中，所述修饰包括3’反向脱氧胸苷(3’idT)修饰。在一些实施方案中，所述修饰包 括用经修饰的核苷酸取代一或多个天然存在的核苷酸，所述经修饰的核苷酸选自2’-氟、2’-甲氧乙基、2’-甲氧基或2’丙烯氧基修饰的核苷酸，优选2’-甲氧基修饰的核苷酸。在一些实施方案中，所述修饰包括核苷酸间的修饰，例如核苷酸间的硫代磷酸酯键修饰。在一些实施方案中，所述修饰包括PEG修饰。在一些实施方案中，所述适体包含2’-甲氧基(2’-OMe)修饰、3’反向脱氧胸苷(3’idT)修饰和/或PEG修饰。
在另一方面，本发明提供一种治疗骨硬化蛋白相关疾病的方法，该方法包括给有需要的对象施用治疗有效量的本发明的针对骨硬化蛋白的适体，所述对象例如是人。
在一些实施方案中，所述骨硬化蛋白相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
在另一方面，本发明提供一种药物组合物，其包含至少一种本发明的针对骨硬化蛋白的适体，和药学上可接受的载体或赋形剂。
在另一方面，本发明提供本发明所述的针对骨硬化蛋白的适体或本发明所述的药物组合物在制备药物中的用途，其中所述药物用于治疗骨硬化蛋白相关疾病。
在一些实施方案中，所述骨硬化蛋白相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
附图简述
图1示出通过SELEX富集骨硬化蛋白高亲和力适体。(A)富集的ssDNA和未选择的文库对硬骨素的结合亲和力。(B)富集的ssDNA文库和未选择的文库与对照蛋白质的结合亲和力。
图2示出适体候选物的特异性表征。相比于与肝细胞和PBMC的结合，适体候选物显示对人骨硬化蛋白的高选择性。
图3示出从具有40nt随机区域的ssDNA文库鉴定的适体候选物对重组人骨硬化蛋白的亲和力。通过非线性曲线拟合分析计算适体候选物和抗体对骨硬化蛋白的解离常数(Kd)。各适体候选物对骨硬化蛋白的Kd值分别是：aptscl 6为4.2nM，aptscl 9为3.4nM，aptscl 15为45.6nM，aptscl 46为43.1 nM，aptscl 56为43.1nM和aptscl 132为42.2nM。抗骨硬化蛋白抗体的Kd值为3.55nM。
图4示出从具有25nt随机区域的ssDNA文库鉴定的适配体候选物与重组人骨硬化蛋白的结合亲和力。通过非线性曲线拟合分析计算各候选物的解离常数(Kd)。各适体候选物和抗体的Kd值分别为：aptscl 32为0.18nM，aptscl 29为0.28nM，aptscl 22为0.76nM，aptscl 16为0.22，aptscl 3为0.04nM，aptscl 2为0.006nM，aptscl 1为0.02nM。抗骨硬化蛋白抗体的Kd值为3.55nM。
图5示出使用TOP-Wnt诱导的萤光素酶报道基因测定评估适体候选物和抗体的抑制能力。(A)与抗体比较，用适体候选物处理的MC3T3-E1细胞中Wnt信号传导介导的荧光素酶活性。Aptscl 56、aptscl 6、aptscl 3和抗骨硬化蛋白抗体能有效抑制骨硬化蛋白对Wnt信号传导的拮抗作用，并释放Wnt诱导的荧光素酶活性。当aptscl 56和aptscl 6的浓度分别达到25μg/ml和47.4μg/ml时，响应达到稳定。而当用抗体处理时，在该实验中浓度增加到20mg/ml时，响应仍未稳定。(B)aptscl 56的抑制能力分析，aptscl 56的EC50为19.7μg/ml。(C)aptscl 6的抑制能力分析，aptscl 6的EC50 36.8μg/ml。(D)aptscl 3的抑制能力分析，aptscl 3的EC50 18.2μg/ml。
图6示出截短的aptscl3与骨硬化蛋白结合的表征。(A)aptscl3-1、-2、-3、-4和-5对骨硬化蛋白保持高亲和力，而aptscl3-6显示对硬化蛋白的低结合能力并且不能拟合亲和力分析曲线；(B)截短的aptscl3-5仍保持高结合亲和力，保留了对骨硬化蛋白拮抗细胞中Wnt信号传导途径的高抑制效力(EC50＝28.4μg/ml)。
图7示出与未修饰的aptscl56相比，修饰的aptscl56的血清稳定性评估。所有适体均用10％和100％小鼠血清处理0至72小时。在10％小鼠血清中孵育48小时后，几乎所有未修饰的aptscl56降解。2’-OMe和3’-idT修饰的aptscl56可在10％小鼠血清中保持48小时。在100％血清中，未修饰的aptscl56在8小时后迅速完全降解；在72小时，仍然保留少量的修饰的适体。
图8示出与未修饰的aptscl3-5相比，修饰的aptscl3-5的血清稳定性评估。所有适体均用10％和100％小鼠血清处理0至72小时。在10％小鼠血清中孵育24小时后，Aptscl3-5降解。2’-OMe和3’idT修饰的aptscl3-5可 在10％小鼠血清中保持48小时。在100％血清中，未修饰的aptscl3-5在8小时后迅速完全降解，而修饰的aptscl3-5在72小时后仍保持完整性。
图9示出化学修饰的aptscl56和aptscl3-5的亲和力和抑制效力。在化学修饰后，aptscl56和aptscl3-5都保持对骨硬化蛋白的高亲和力和体外抑制效力。
图10示出在6只大鼠中单次皮下注射Aptscl56的药代动力学：通过软件DAS拟合的药代动力学曲线(左)、实际药代动力学曲线(右)。
图11示出在6只大鼠中单次皮下注射PEG40K-aptscl56的药代动力学：通过软件DAS拟合的药代动力学曲线(左)、实际药代动力学曲线(右)。
图12示出分别在大鼠中单次皮下注射Aptscl56(蓝色)和PEG40K-aptscl56(橙色)的药代动力学。
图13示出PEG40K-aptscl56/aptscl3在卵巢切除术诱导的骨质疏松大鼠骨代谢效力的评估。(a)显示该研究的实验设计的示意图。(b)代表性3D微架构图像和每组股骨远端骨小梁的micro-CT参数。(c)代表性3D微架构图像和每组胫骨近端骨小梁的micro-CT参数。(d)代表性3D微架构图像和每组中第五椎骨的微CT参数。注：BMD：骨密度；Tb.BV/TV：骨小梁相对骨量；Tb.vBMD：小梁体积矿物质密度；Tb.Th：小梁厚度；Tb.N：小梁数；Tb.Sp：小梁间距；Tb.conn.D：小梁连接密度；Tb.SMI：小梁结构模型指数；MAR：矿物沉积率；BFR/BS：骨形成率；Ob.S/BS：成骨细胞表面；和Ob.N/B.Pm：成骨细胞数；PINP：原胶原I完整的N末端；OPG：骨保护作用。SHAM-BS：SHAM基线；OVX-BS：治疗前的OVS基线；SHAM+Veh：用载体治疗的SHAM大鼠；OVX+Veh：用载体治疗的OVX大鼠；OVX+aptscl56：用PEG40K-aptscl56处理的OVX大鼠；OVX+RS：用PEG40K-随机序列处理的OVX大鼠。随机序列：5'-ATGCAAGCACATTGTGATCGCTTCAAATGTCTTCCGTCCG-3'。数据表示为平均值±标准差与OVX-BS。每组n＝10。*P<0.05；**P<0.01；***P<0.005；****P<0.0001。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al., “Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
定义
如本文所用，术语“核苷酸”是指核糖核苷酸或脱氧核糖核苷酸，或其修饰的形式及其类似物。核苷酸包括种类，其包括嘌呤(例如，腺嘌呤、次黄嘌呤、鸟嘌呤和它们的衍生物和类似物)以及嘧啶(例如，胞嘧啶、尿嘧啶、胸腺嘧啶和它们的衍生物和类似物)。
在本文中，互换地使用“核酸”、“寡核苷酸”和“多核苷酸”来指核苷酸的聚合物，并包括DNA、RNA、DNA/RNA杂交体和这些种类的核酸、寡核苷酸和多核苷酸的修饰，其中包括在所述核苷酸单位的任何位置上附加各种实体或部分。术语“多核苷酸”、“寡核苷酸”和“核酸”包括双或单链分子。核酸、寡核苷酸和多核苷酸是比术语适体更宽的术语，因此术语核酸、寡核苷酸和多核苷酸包括适体但不限于适体。
如本文所用，“适体”指具有期望的对靶分子的作用的非天然存在的核酸。期望的作用包括但不限于与所述靶结合、催化改变所述靶、以修饰或改变所述靶或所述靶的功能活性的方式与靶反应、共价地连接所述靶和促进所述靶与其他分子间的反应。在一些实施方案中，所述作用是针对靶分子(例如骨硬化蛋白)的特异性结合亲和力，这样的靶分子是三维化学结构而不是多核苷酸，其通过不依赖于Watson/Crick碱基配对或三重螺旋形成的机制来结合所述适体，其中所述适体不是具有被所述靶分子结合的已知生理功能的核酸。在本上下文中，适体针对其靶(例如骨硬化蛋白)的“特异性结合”是指所述适体通常以比其结合至混合物或样品中其他的非靶组分高得多的亲和力来结合至其靶。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：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)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。许多算法可用于确定序列相同性百分比。适合于确定序列相同性百分比的算法的一个实例是在基本局部比对搜索工具(以下为“BLAST”)中使用的算法，见例如Altschul等人，J.Mol.Biol.215:403-410,1990和Altschul等人，Nucleic Acids Res.,15:3389-3402,1997。进行BLAST分析的软件是通过国家生物技术信息中心(以下为“NCBI”)公开地可获得的。在使用从NCBI可获得的软件(如针对核酸序列的BLASTN)来确定序列相同性所使用的默认参数在McGinnis等人Nucleic Acids Res.,32:W20-W25,2004中有所描述。
针对骨硬化蛋白的适体
本发明人基于蛋白质-SELEX技术，采用骨硬化蛋白作为靶蛋白正向筛选，并采用不相关蛋白进行负筛选，最终选择出以高亲和力特异性地结合骨硬化蛋白的适体。本文所述骨硬化蛋白优选是人骨硬化蛋白，例如，氨基酸序列示于SEQ ID NO:18的骨硬化蛋白。
示例性人骨硬化蛋白氨基酸序列：
Figure PCTCN2019074764-appb-000001
因此，在一方面，本发明提供针对骨硬化蛋白的适体，所述适体包含与SEQ ID NO:1-17中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少 大约98％相同性或至少大约99％相同性的核苷酸序列，或者，所述适体包含SEQ ID NO:1-17中的任一中的至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸。在一些实施方案中，所述适体特异性结合骨硬化蛋白。在一些优选实施方案中，所述适体包含SEQ ID NO:1-17和19-24中的任一的核苷酸序列，更优选地，所述适体包含SEQ ID NO:1、3、10或19-23之一的核苷酸序列。
在一些实施方案中，本发明的所述适体对骨硬化蛋白具有小于100nM，优选小于50nM，优选小于40nM，优选小于30nM，优选小于20nM，优选小于10nM或更小的K d(解离常数)。所述K d例如通过酶联寡核苷酸测定法(ELONA)测定。
在一些实施方案中，本发明的所述适体抑制骨硬化蛋白的生物学活性。“抑制”是指与不存在所述适体相比，骨硬化蛋白的生物学活性在所述适体存在下被降低，例如降低至少约10％，至少约20％，至少约30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％，甚至至少约90％。
如本文所用，术语“生物学活性”表示对一或多种细胞或细胞外的过程的作用，其可以影响生理或病生理过程。骨硬化蛋白的生物学活性包括但不限于拮抗Wnt信号通路。
在一些实施方案中，本发明的所述适体能够抑制骨硬化蛋白对Wnt信号通路的拮抗作用。例如，本发明的所述适体能在基于细胞的Wnt信号测试法中阻断骨硬化蛋白的拮抗作用。
在一些实施方案中，本发明的所述适体以小于100μg/ml，优选小于50μg/ml，优选小于40μg/ml，优选小于30μg/ml，优选小于20μg/ml，优选小于10μg/ml或更小的EC50值抑制骨硬化蛋白的生物学活性，例如抑制骨硬化蛋白对Wnt信号通路的拮抗作用。在一些实施方案中，所述EC50值在体外通过成骨细胞中的TOP-Wnt诱导萤光素酶报告基因测定法测定。
在一些实施方案中，本发明的所述适体还可以包含一或多种修饰。例如，所述修饰是赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。
所述修饰包括例如3’和5’的修饰，如3’和5’加帽。在一些实施方案中，所述适体在3’末端用反向脱氧胸苷加帽，即3’反向脱氧胸苷(3’idT)修饰。
所述修饰还可以包括用经修饰的核苷酸取代一或多个天然存在的核苷酸。例如，所述经修饰的核苷酸包括但不限于2’-氟、2’-甲氧乙基、2’-甲氧基和/或2’丙烯氧基修饰的核苷酸(即核糖的2’位置羟基被氟、甲氧乙基、甲氧基或丙烯氧基等取代)。所述经修饰的核苷酸还可以包括C-5修饰的嘧啶。术语“C-5修饰的嘧啶”是指C-5位上有修饰的嘧啶。C-5修饰的嘧啶能够增强寡核苷酸的核酸酶抗性，且是本领域已知的，例如可以参见国际专利申请WO 2011/130195及其引用的文献。在一些优选实施方式中，所述修饰是2’-甲氧基(2’-OMe)修饰。在一些实施方式中，在适体的5’和/或3’端的一或多个，例如4个核苷酸进行所述修饰，例如2’-甲氧基(2’-OMe)修饰。
所述修饰还包括核苷酸间的修饰，例如具有不带电荷的键(例如甲基膦酸酯、磷酸三酯、磷酸胺酯、氨基甲酸酯等)的核苷酸间修饰和具有带电荷的键(例如硫代磷酸酯、二硫代磷酸酯等)的核苷酸间修饰，有嵌入剂(例如吖啶、补骨脂素等)的核苷酸间修饰，含有螯合剂(例如金属、放射性金属、硼、氧化性金属等)的核苷酸间修饰，含有烷化剂的核苷酸间修饰和有修饰的键(例如阿尔法异头核酸等)的核苷酸间修饰。
所述修饰还可以包括聚乙二醇化修饰(PEG修饰)。通过与PEG缀合可以延长适体的半衰期例如体内半衰期。在一些实施方式中，所述PEG的分子量为大约1kDa至大约100kDa，例如大约10kDa至大约80kDa，大约20kDa至大约60kDa，大约30kDa至大约50kDa，大约40kDa。在一些实施方案中，所述PEG可以缀合至适体的5’端。在一些实施方案中，所述PEG可以缀合至适体的3’端。
在一些实施方案中，所述适体可以包含上述多种修饰的组合。例如，所述适体可以包含2’-甲氧基(2’-OMe)修饰、3’反向脱氧胸苷(3’idT)修饰和/或PEG修饰。优选地，所述PEG分子量为大约40kDa。
疾病治疗
在另一方面，本发明提供通过本发明的针对骨硬化蛋白的适体治疗疾病的方法，该方法包括给有需要的对象施用治疗有效量的本发明的针对骨硬化蛋白的适体。
通过本发明的针对骨硬化蛋白的适体治疗的疾病例如是骨硬化蛋白相关疾病，例如骨硬化蛋白介导的疾病。
如本文所使用，“骨硬化蛋白相关疾病”包括其中骨矿质密度(BMD)相对于健康对象不正常和/或病理上低的病症。由低BMD和/或骨易碎性表征的疾病包括但不限于：原发性和继发性骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性坏死(骨坏死)、骨折和植入物愈合(牙种植体和髋植入物)、由于其它病症的骨丧失(例如，与HIV感染、癌症和关节炎相关)。其它“骨硬化蛋白相关疾病”包括但不限于：类风湿性关节炎、骨关节炎、关节炎，以及溶骨性病变。
如本文所使用，“骨硬化蛋白相关疾病”包括骨硬化蛋白相关癌症，例如骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
“骨硬化蛋白相关疾病”还可包括至少由骨硬化蛋白在肾脏和在心血管中表达而引起的肾病及心血管疾病。所述病症包括但不限于诸如以下的肾病：肾小球疾病(例如，急性和慢性肾小球性肾炎，急进性肾小球性肾炎，肾病综合征，局灶增生性肾小球肾炎，与诸如系统性红斑狼疮、古德帕斯彻氏综合征、多发性骨髓瘤、糖尿病、多囊肾病、瘤形成、镰刀形红细胞病、以及慢性炎症等全身疾病相关的肾小球损害)、肾小管疾病(例如，急性肾小管坏死和急性肾衰竭、多囊肾病、髓质海绵肾、髓质囊性病、肾原性糖尿病，以及肾小管性酸中毒)、小管间质性疾病(例如，肾盂肾炎、药和毒素诱导的小管间质性肾炎、高钙性肾病，以及低钾血症性肾病)、急性和急进性肾衰竭、慢性肾衰竭、肾结石、痛风、血管疾病(例如，高血压和肾硬化、微血管病性溶血性贫血、动脉粥样硬化栓塞肾病、扩散皮层的坏死以及肾梗塞)，或肿瘤(例如，肾细胞癌和肾胚细胞瘤)。
所述疾病还包括但不限于诸如以下的心血管疾病：缺血性心脏病(例如，心绞痛、心肌梗死，以及慢性缺血性心脏病)、高血压心脏病、肺心病、心脏瓣膜病(例如，风湿热和风湿性心脏病、心内膜炎、二尖瓣脱垂以及主动脉瓣狭窄)、先天性心脏病(例如，瓣和血管堵塞性损害、房或室中隔缺损、以及久存性动脉导管)，或心肌病(例如，心肌炎、充血型心肌病，以及肥厚型心肌病)。
所述对象可以是任何动物(驯养的、家畜或野生的)，其包括但不限于猫、狗、马、猪和牛，并优选人类对象。如本文中所用，术语患者、个体和对 象可以互换地使用。
对象可以是男性或女性。优选地，人类对象处于骨折的风险中，更优选地人类对象处于骨质疏松的风险中或患有骨质疏松症。人类对象优选为女性，并更优选为处于闭经后骨质疏松风险或患有闭经后骨质疏松的女性。预期本发明的方法可对骨质疏松症任何阶段的对象有益。
如本文所用，“治疗”患有疾病的对象表示所述对象的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。如本文所用，“疗效”表示由对象的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
按照多种因素选择利用所述针对骨硬化蛋白的适体的剂量方案，所述因素包括例如，所述患者的类型、种类、年龄、体重、性别和医疗病症，；所要治疗的病症的严重性；施用途径；所述患者的肾与肝的功能；和所使用的特定的针对骨硬化蛋白的适体或其盐。普通熟练的医生可以容易地确定并指定预防、对抗或抑制所述病症进展所需要的组合物的有效量。
通常，所述针对骨硬化蛋白的适体的剂量方案为每天大约1μg/kg体重至约100mg/kg体重。
示例性的治疗方案需要每天给药一次、每两天给药一次、每周给药一次、每周给药两次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。给药频率和间隔可以由本领域技术人员根据适体的药代动力学参数确定。
药物组合物
在另一方面，本发明还提供药物组合物，其包括至少一种本发明的针对骨硬化蛋白的适体，和药学上可接受的载体或赋形剂。所述药物组合物例如用于治疗骨硬化蛋白相关的疾病。
本文所描述的适体可以任何药学上可接受的剂量形式使用，包括但不 限于以可注射剂量形式、液体分散体、凝胶、喷雾、软膏、霜剂、冻干制剂、干粉、片剂、胶囊、控制释放制剂、速熔制剂、延迟释放制剂、延长释放制剂、脉动释放制剂、混合的立即释放和控制释放制剂等。具体地，本文所描述的适体可以配制为：(a)通过选自口腔、肺、静脉内、动脉内、鞘内、内关节、直肠、眼、结肠、胃肠外、脑池内、阴道内、腹腔内、局部、颊、鼻和局部施用中的任一项来施用；(b)选自液体分散体、凝胶、喷雾、软膏、霜剂、片剂、香囊和胶囊中任一项的剂量形式；(c)选自冻干制剂、干粉、速熔制剂、控制释放制剂、延迟释放制剂、延长释放制剂、脉动释放制剂和混合的立即释放和控制释放制剂中任一项的剂量形式；或(d)它们的任意组合。
用于肠胃外、皮内或皮下的施用的溶液或悬浮液可以包含一或多种的以下组分：(1)无菌稀释剂，例如用于注射的水、盐溶液、固定油、聚乙二醇、甘油、丙二醇或其他合成溶剂；(2)抗菌剂，例如苯甲醇或对羟苯甲酯；(3)抗氧化剂，例如抗坏血酸或亚硫酸钠；(4)螯合试剂，例如乙二胺四乙酸；(5)缓冲液，例如乙酸盐、柠檬酸盐或磷酸盐；和(5)用于调节张力的物质，例如氯化钠或葡萄糖。用酸或碱(如盐酸或氢氧化钠)可以调整pH。肠胃外的制剂可以封装在玻璃或塑料制成的安瓿瓶、一次性注射器或多剂量小瓶中。
适于注射使用的药物组合物可以包括无菌的水性溶液(其中是水溶的)或分散体和用于临时制备无菌注射溶液或分散体的无菌粉末。对于静脉内施用，适合的载体包括生理盐水、抑菌水、或磷酸盐缓冲盐水(PBS)。在所有情况下，所述组合物应该是无菌的并且其流动程度应该为易于注射。在制造和储存的条件下所述药物组合物应该是稳定的并应当被保护以防微生物如细菌和真菌的污染作用。如本文中所用的术语“稳定的”意为保持在适合于给患者施用的状态或条件。
所述载体可以是溶剂或分散介质，包括例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)及其适合的混合物。例如通过使用如卵磷脂的包衣，通过在分散体的情况下维持所需的颗粒大小和通过使用表面活性剂可以保持适当的流动性。通过各种抗菌和抗真菌的试剂，例如，对羟基苯甲酯、氯丁醇、苯酚、抗坏血酸，硫柳汞等可以实现防止微生物的作用。在许多情况下，优选在所述组合物中包括等渗试剂，例如糖、多元醇(如 甘露醇或山梨醇)和无机盐(如氯化钠)。通过在所述组合物中包括延迟吸收的物质如单硬脂酸铝和明胶可以带来可注射组合物的延长的吸收。
可以通过在适当的溶剂中用以上所列举的成分的一种或组合(如需要的)以需要的量并入活性剂(例如，针对骨硬化蛋白的适体)接着过滤灭菌来制备无菌的可注射溶液。通常，通过向含有基本分散介质和任何其他所需成分的无菌载体并入至少一种针对骨硬化蛋白的适体来制备分散体。在用于制备无菌可注射溶液的无菌粉末的情况下，制备的示例性方法包括真空干燥和冷冻干燥，这两者会获得所述针对骨硬化蛋白的适体的粉末以及来自之前其无菌过滤溶液的任何额外的所需的成分。
口服组合物通常包括惰性的稀释剂或可食用的载体。例如，可以将它们装入明胶胶囊或压成片剂。针对口服治疗施用的目的，所述针对骨硬化蛋白的适体可以并入赋形剂并以片剂、锭剂或胶囊的形式使用。可以包括药物相容的结合剂和/或佐剂材料作为所述组合物的一部分。
对于通过吸入的施用，以来自含有适合的推进物(例如气体(如二氧化碳)、雾化的液体或来自适合的装置的干粉末)的加压容器或分配器的气溶胶喷雾的形式来递送所述化合物。对于经粘膜或经皮的施用，在制剂中使用对要渗透的屏障是适当的渗透剂。这样的渗透剂通常是本领域已知的，并包括例如，用于经粘膜施用的去污剂、胆汁盐和夫西地酸衍生物。通过使用鼻腔喷雾剂或栓剂可以实现经粘膜的施用。对于经皮的施用，所述活性剂配制成如本领域所熟知的软膏、油膏、凝胶或霜剂。也可以栓剂(例如用常规的栓剂基质，如可可脂和其它甘油酯)或用于直肠递送的保留灌肠剂的形式来制备所述试剂。
在一个实施方案中，针对骨硬化蛋白的适体配制为用于局部施用。如本文所用，“局部施用”是指通过将(直接地或其他方式)包含所述针对骨硬化蛋白的适体的制剂与动物的全部或部分皮肤(表皮)接触来递送针对骨硬化蛋白的适体至所述动物。该术语包括了几种施用途径，包括但不限于局部施用和经皮施用。针对这些施用模式的普遍需求是有效递送至靶组织或层。一方面，使用局部施用作为渗透所述表皮和真皮的方式并最终实现针对骨硬化蛋白的适体的全身性递送。另一方面，使用局部施用作为选择性地递送针对骨硬化蛋白的适体至动物的表皮或真皮或其特定的层的方式。
对于局部施用，所述针对骨硬化蛋白的适体可以配制成药学上可接受 的软膏剂、霜剂、洗剂、眼用软膏剂、滴眼剂、滴耳剂、浸渍敷料、和气雾剂、含药粉末、含药粘合剂、泡沫，并且可以含有适当的常规添加剂或赋形剂，包括，例如防腐剂或帮助药物渗透的溶剂和软膏剂、凝胶和霜剂中的软化剂。这种局部制剂也可以含有兼容的常规载体，例如用于乳液的乙醇或油醇。这样的载体可能构成所述制剂的大约1％至大约98％的重量，更通常地，这样的载体会构成所述制剂的高达大约80％的重量。针对适体的局部递送的具体制剂在现有技术中已有描述。
在一个实施方案中，用会防止从身体迅速除去的载体制备针对骨硬化蛋白的适体。例如，可以使用控制释放制剂，包括植入和微囊化的递送系统。可以使用可生物降解的、生物兼容的聚合物，例如乙烯乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。这种制剂的制备方法对本领域技术人员会是显而易见的。
脂质体悬浮液(包括以被感染的细胞为靶的具有针对病毒抗原的单克隆抗体的脂质体)也可以用作药学上可接受的载体。这些可以根据本领域技术人员已知的方法制备。
此外，可以制备所述针对骨硬化蛋白的适体的悬浮液作为适当的油性注射悬浮液。适合的亲脂性溶剂或载体包括脂肪油(如芝麻油)或合成脂肪酸酯(如油酸乙酯、甘油三酯)或脂质体。非脂的聚阳离子氨基酸聚合物也可以用于递送。任选地，所述悬浮液还可以包括适合的稳定剂或试剂以增加所述化合物的溶解度并允许用于制备高浓缩的溶液。
在某些情况下，以剂量单位来配制口服或胃肠外的组合物对便于施用和剂量均一可能是特别有利的。如本文中所用的剂量单位形式是指物理上离散的单位，适合作为用于要治疗的对象的单一剂量；每单位含有经计算能产生期望的治疗效果的预定量的针对骨硬化蛋白的适体以及所需的药物载体。本文所描述的针对骨硬化蛋白的适体的剂量单位形式的说明决定于并直接依赖于特定针对骨硬化蛋白的适体的唯一的特性和所要实现的特定的治疗效果和配制此类用于治疗个体的活性剂的技术的固有限制。
包含至少一种针对骨硬化蛋白的适体的药物组合物可以包括一或多种药物赋形剂。这样的赋形剂的实施例包括但不限于，结合剂、填充剂、润滑剂、悬浮剂、甜味剂、调味剂、防腐剂、缓冲剂、润湿剂、崩解剂、泡腾剂和其它赋形剂。这样的赋形剂在本领域中是已知的。示例性赋形剂包 括：(1)结合剂，包括各种纤维素和交联聚乙烯吡咯烷酮、微晶纤维素(如Avicel PH101和Avicel PH102)、硅化微晶纤维素(ProSolv SMCC TM)、黄蓍胶和明胶；(2)填充剂，例如各种淀粉、乳糖，乳糖单水合物、无水乳糖；(3)崩解剂如藻酸、Primogel、玉米淀粉、轻度交联的聚乙烯基吡咯烷、马铃薯淀粉、玉米淀粉和修饰的淀粉、交联羧甲基纤维素钠、交聚维酮、乙醇酸淀粉钠及它们的混合物；(4)润滑剂，包括作用于所要压缩的粉末的流动性的试剂，包括硬脂酸镁、胶状二氧化硅(如Aerosil 200、滑石)、硬脂酸、硬脂酸钙和硅胶；(5)助流剂，如胶状二氧化硅；(6)防腐剂，如山梨酸钾、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸及其盐、其他对羟基苯甲酸的酯(如对羟基苯甲酸丁酯)、醇(如乙醇或苯甲醇)、酚类化合物(如苯酚)或季铵化合物(如氯化苯甲烃胺)；(7)稀释剂，如药学上可接受的惰性填料，如微晶纤维素、乳糖、磷酸氢钙、糖类和/或任何以上的混合物；稀释剂的实施例包括微晶纤维素，如Avicel PH101和Avicel PH102；乳糖如乳糖单水合物、无水乳糖和Pharmatose DCL21；磷酸氢钙如Emcompress甘露醇、淀粉、山梨醇、蔗糖和葡萄糖；(8)甜味剂，包括任何天然或人造的甜味剂，如蔗糖、糖精蔗糖、木糖醇、糖精钠、甜蜜素、阿斯巴甜和安赛蜜；(9)调味剂，如薄荷、水杨酸甲酯、橙调味剂、Magnasweet(商标MAFCO)、泡泡糖香料、水果香料等；和(10)泡腾剂，包括泡腾剂对，如有机酸和碳酸盐或重碳酸盐。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1、通过SELEX富集和筛选针对骨硬化蛋白的高亲和力适体
ssDNA文库中的序列由两端18nt保守区域和一个中央随机区域组成。使用了两个不同长度随机序列的ssDNA文库。长ssDNA文库中的序列包含40nt随机区(5’-CGTACGGTCGACGCTAGC-(N) 40-CACGTGGAGCTCGGATCC-3’)，短ssDNA文库中的序列含有25nt随机区(5’-CGTACGGTCGACGCTAGC-(N) 25-CACGTGGAGCTCGGATCC-3’)。合 成正向引物(FP：5’-CGTACGGTCGACGCTAGC-3’)和生物素化反向引物(Bio-RP：5’-生物素-GGATCCGAGCTCCACGTG-3’)用于在选择期间扩增ssDNA。所有寡核苷酸在合成后通过HPLC纯化。
进行蛋白SELEX方法以鉴定高亲和力适体(Ellington and Szostak,1990；Tuerk and Gold,1990)。100-30皮摩尔的His 6标记的骨硬化蛋白在4℃下1小时固定在NTA磁珠上(Murphy,Fuller et al.,2003)。1纳摩尔ssDNA文库在95℃变性5分钟并迅速冷却至4℃，随后与固定化的骨硬化蛋白在室温孵育0.5-1小时。用洗涤缓冲液除去未结合的序列。洗涤后，收集结合的DNA-蛋白质-NTA，用H 2O/吐温20重悬，并应用于PCR扩增。用未修饰的正向引物和生物素化的反向引物进行PCR扩增(步骤1：95℃初始变性1分钟；步骤2：95℃变性30秒，56℃退火30秒，72℃延伸30秒，重复12个循环；和步骤3：72℃最后延伸5分钟)。通过生物素-链霉亲和素结合将PCR产物应用于链霉亲和素磁珠。通过用0.2M NaOH处理再生单链序列。针对固定在NTA磁珠上的其他His 6标记的不相关蛋白进行负选择。每种选择共进行20轮SELEX。来自最后一轮的DNA池进行高通量二代序列(NGS)。
图1示出第10轮和第20轮选择之后DNA池对骨硬化蛋白的亲和力增加，表明通过SELEX富集了高亲和力骨硬化蛋白适体。
实施例2、候选骨硬化蛋白适体的特异性鉴定
根据NGS结果，合成具有高出现频率的代表性适体进行特异性测定。这些适体候选物的详细序列列于表1中。
为了确定适体候选物对骨硬化蛋白的特异性，使用N-末端生物素化修饰合成代表性适体候选物和随机序列(RS)(阴性对照)。每种适体/RS使用1μM通过酶联寡核苷酸测定法(ELONA)测定对骨硬化蛋白的特异性。将160ng纯化的重组人骨硬化蛋白通过在100μl PBS中4℃温育过夜而包被96孔微量滴定板。然后将板用封闭缓冲液(PBS，0.1％吐温20和1％BSA)在室温封闭1小时，并用SELEX B&W缓冲液(PBS，1mM MgCl 2，0.1％吐温20和0.1％BSA)洗涤4次。适体候选物在95℃变性10分钟，在使用前在冰上快速冷却10分钟。每孔加入1μM生物素标记的适体，然后加入SELEX B&W缓冲液至100μl，室温孵育45min，连续温和摇动。结合后，将板用 SELEX B&W缓冲液洗涤4次以除去非特异性和非常弱的结合，然后用PBST+0.1％BSA洗涤4次。向每个孔中加入100μl链霉亲和素-HRP/山羊抗人IgG Fc-HRP(1:10000稀释到PBST+0.1％BSA中)并孵育30/60分钟，并用PBST+0.1％BSA洗涤4次。向每个孔中加入50μl TMB并温育20分钟。加入50μl 2M H 2SO 4终止反应。用酶标仪测量450nm处的吸光度(Stoltenburg,Krafcikova et al.,2016)。为了确定适体候选物与肝细胞/PBMC的结合能力，表征步骤与ELONA类似。将300,000个细胞与每种适体候选物一起温育，通过离心进行洗涤和分离。
结果表明，从长和短ssDNA文库中鉴定的适体候选物在与肝细胞和PBMC的结合比较时显示对人骨硬化蛋白的高选择性(图2)。从长ssDNA文库鉴定的适体候选物aptscl 6、9、15、27、34、36、46、51、56、132和140，以及从短ssDNA文库鉴定的aptscl 1、2、3、5、8、12、16、22、29和32，显示出对骨硬化蛋白的高结合特异性，因此被选择用于以下亲和力表征。
实施例3、候选骨硬化蛋白适体的亲和力鉴定
进行酶联寡核苷酸测定(ELONA)以确定适体候选物与骨硬化蛋白的结合亲和力(Drolet,Moon-McDermott et al.,1996)。类似地，进行酶联免疫吸附测定(ELISA)以确定抗骨硬化蛋白抗体(Romosozumab，针对骨硬化蛋白的人源化治疗性抗体，购自Creative Biolabs)对人骨硬化蛋白的结合亲和力(Engvall and Perlmann,1971)。将160ng纯化的重组人硬化蛋白通过在100μl PBS中4℃温育过夜而包被96孔微量滴定板。然后将板用封闭缓冲液(PBS，0.1％吐温20和1％BSA)在室温封闭1小时，并用SELEX B&W缓冲液(PBS，1mM MgCl 2，0.1％吐温20和0.1％BSA)洗涤4次。适体候选物在95℃变性10分钟，在使用前在冰上快速冷却10分钟。将适当浓度的生物素化的适体/抗体加入到每个孔中，然后加入SELEX B&W缓冲液至100μl中，并在室温下温育45分钟，连续温和摇动。结合后，将板用SELEX B&W缓冲液洗涤4次以除去非特异性和非常弱的结合，然后用PBST+0.1％BSA洗涤4次。向每个孔中加入100μl链霉亲和素-HRP/山羊抗人IgG Fc-HRP(1:10000稀释到PBST+0.1％BSA中)并孵育30/60分钟，并用PBST+0.1％BSA洗涤4次。向每个孔中加入50μl TMB并温育20分钟。加 入50μl 2M H 2SO 4终止反应。用酶标仪测量450nm处的吸光度(Stoltenburg,Krafcikova et al.,2016)。用Origin软件(OriginLab，Northampton，MA)分析数据。使用非线性曲线拟合模型Hyperbl绘制结合曲线Hyperbl模型的方程是y＝P1*x/(P2+x)，P2是Kd值。
对于从含有40nt随机区的ssDNA文库鉴定的适体候选物，aptscl 6、9、15、46、56和132对骨硬化蛋白具有高亲和力，具有纳摩尔水平的解离常数(Kd)值(Kd值分别为4.2、3.4、45.6、43.1和42.2nM)(图3)。而aptscl 36、140、136和随机序列(RS)未能拟合。对于从含有25nt随机区的ssDNA文库中鉴定的适体候选物，aptscl 32、29、22、16、3、2和1对骨硬化蛋白的结合亲和力更高，Kd值分别为0.18、0.28、0.76、0.02、0.04、0.006和0.02nM(图4)。随机序列显示对骨硬化蛋白的低结合能力并无法拟合。相比之下，抗骨硬化蛋白抗体对骨硬化蛋白的Kd值是3.55nM。
实施例4、体外评估候选骨硬化蛋白适体对骨硬化蛋白活性的抑制能力
在成骨细胞MC3T3-E1细胞中使用TOP-Wnt诱导的萤光素酶报告基因测定法(van Bezooijen,Svensson et al.,2007；Shum,Chan et al.,2011)，研究适体候选物抑制骨硬化蛋白对Wnt信号传导的拮抗作用的能力。
将MC3T3-E1细胞接种于24孔板，在第2天使用FuGENE HD转染试剂(Promega)，转染相应的报告基因质粒(100ng)、Wnt3a质粒(800ng)和骨硬化蛋白质粒(800ng)。转染10小时后，将培养基更换为新鲜培养基，并用适体/抗体处理细胞。处理24小时后，每个孔的细胞用100μl被动裂解缓冲液裂解，并取20μl用于分析。根据制造商的方案(Promega)，通过SpectraMax i3x Multi-Mode Detection Platform(Molecular Device)自动制备萤光素酶测定试剂II和Stop&Glo试剂，并且相应地分析数据(Grentzmann,Ingram et al.,1998；McNabb,Reed et al.,2005)。
结果如图5所示，aptscl 56、aptscl 6、aptscl 3和抗骨硬化蛋白抗体可有效抑制骨硬化蛋白对Wnt信号传导的拮抗作用，并释放Wnt诱导的荧光素酶活性。对于骨硬化蛋白的抑制作用具有剂量依赖性，当aptscl 56、aptscl6的浓度分别达到25μg/ml和47.4μg/ml时，响应稳定。当用抗体处理时，浓度增加到20mg/ml响应仍不稳定。此外，用非线性曲线拟合分析了aptscl56、aptscl 6和aptscl 3的抑制能力。Aptscl 56、aptscl 6和aptscl 3的EC50 分别为19.7μg/ml、36.8μg/ml和18.2μg/ml。
表1、骨硬化蛋白适体候选物的序列
Figure PCTCN2019074764-appb-000002
实施例5、aptscl3的截短和表征
显示对硬化蛋白具有高亲和力和抑制效力的aptscl3被截短(表2)。使用与前述的研究中相同的方案进行结合亲和力和体外抑制效力检测。aptscl3-1、aptscl3-2，aptscl3-3、aptscl3-4和aptscl3-5保持对硬化蛋白的高结合亲和力，Kd值分别为0.86、0.52、0.2和0.22nM。aptscl3-6在该浓度范围内无法拟合结合曲线，表明对硬化蛋白的结合能力低(图6a)。此外，aptscl3-5保留了对硬化蛋白对Wnt信号传导的拮抗作用的高抑制效力(EC50＝28.4μg/ml(图6b))。
表2.适体候选物aptscl3的序列截短
Figure PCTCN2019074764-appb-000003
实施例6、化学修饰的适体候选物的血清稳定性评估
本发明人已经选择出针对骨硬化蛋白的DNA适体，并最终开发两种截短的适体，称为aptscl56和aptscl3-5，其特异性地与骨硬化蛋白紧密结合，具有低纳摩尔范围内的解离常数。核酸适体的2'-O-甲基(2'-OMe)修饰先前已被用作选择后修饰，因为它们具有增强的核酸酶抗性和升高的双链体解链温度，如临床实验中所示(Fine,Martin et al.2005；Gupta,Hirota et al.2014)。反向dT的3'末端封端也是正在进行或已完成的临床试验中用于疾病治疗的适体的常用策略(Padilla,Sousa et al.1999；Ruckman,Green et al.1998)。因此，本实施例评估aptscl56和aptscl3-5的血清稳定性是否可以通过2'-OMe和3'-末端反向dT(3'-idT)修饰来改善。
实验设计：
在合成过程中引入修饰的核苷酸。在新鲜制备的小鼠血清中评估修饰的和未修饰的适体的血清代谢稳定性。将所有适体样品与10％和100％小鼠血清在37℃下分别孵育0、2、4、8、12、24、36、48和72小时。在指定的时间，将适体样品在干冰浴中快速冷冻，然后储存在-80℃直至收获所有样品用于评估。所有适体样品的稳定性表示为孵育后剩余的完整适体的条带密度，其可通过琼脂糖凝胶电泳确定。
DNA合成方案：
使用市售的5'-O-DMT-2'-脱氧核苷(ABz、CAc、GiBu和T)亚磷酰胺单体、5'-O-DMT-2'-O-甲基核苷(ABz、CAc、GiBu和T)亚磷酰胺单体和/或5'-O-DMT-2'-F-核苷(ABz、CAc、GiBu和T)亚磷酰胺单体在K&A H8标准DNA/RNA合成仪上以1μmole规模合成修饰的和未修饰的DNA序列(Beaucage和Caruthers 2001)。修饰的aptscl56序列为 CGGGG TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CT GCCC-idT，下划线核苷酸用2'-OMe修饰。aptscl3-5的修饰序列是 GCTAG CTGTT GTACA TCGCC TTACG CA CGT G-idT，下划线核苷酸是2'-OMe修饰的。
评估方案：
通过分子成像仪(Bio-Rad)测定所有适体样品的条带密度(Klussmann,Nolte et al.1996,Siller-Matula,Merhi et al.2012)。使用与前述研究中相同的方案进行结合亲和力和体外抑制效力测定。
结果：
对于aptscl56，未修饰的适体在10％血清中48小时后完全降解，并且在100％血清中仅保持8小时。2'-OMe和3'-idT修饰的aptscl56在10％小鼠血清中保持72小时，并在100％小鼠血清中12小时后才降解。在72小时，仍然保留少量修饰的适体(图7)。
对于aptscl3-5，未修饰的适体在10％小鼠血清中孵育后24小时降解。2'-OMe和3'-idT修饰的aptscl3-5可在10％小鼠血清中保持48小时。在100％血清中，未修饰的aptscl3-5在8小时后迅速和完全降解，而修饰的 aptscl3-5在72小时后可保持完整性(图8)。
化学修饰的aptscl56和aptscl3-5显示出对骨硬化蛋白的高结合亲和力，Kd值分别为6.55和0.54nM。此外，化学修饰的aptscl56和aptscl3-5可有效解除骨硬化蛋白对细胞中Wnt信号的抑制作用，其效力分别为14和11μg/ml(图9)。
结论：
用2'-OMe和3'-idT修饰可以进一步促进将aptscl56和aptscl3-5开发成治疗性核酸酶抗性适体。
实施例7、PEG修饰的适体
本发明确定在大鼠皮下给药后，针对骨硬化蛋白的进行PEG修饰和不进行PEG修饰的适体(PEG40K-aptscl56和aptscl56)的血浆药代动力学。其中aptscl56序列为CGGGG TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CTGCCC-idT，起始CGGG和末端GCCC各核苷酸用2'-OMe修饰。PEG40K-aptscl56在此基础上进一步在5'-末端连接PEG40K(分子量40000的PEG)。
实验设计：
适体aptscl56和PEG40K-aptscl56的药代动力学研究在6个月大的雌性原始Sprague-Dawley大鼠中进行，该大鼠随意喂食标准实验室饮食并在受控条件下饲养(12小时光周期，20℃)。通过单次皮下注射分别用6.1mg/kg aptscl56和25mg/kg PEG40K-aptscl56处理大鼠。将aptscl56和PEG40K-aptscl56分别以1.6mg/ml和6.2mg/ml的浓度溶解在盐水中(Judith M.Healy，Ryan M.Boomer等人，2004)。在不同时间点(aptscl56：5min、15min、30min、1h、2h、4h、8h、12h、24h；PEG40K-aptscl56：30min、1h、2h、4h、8h、12h、24h、30h、36h、48h、54h、62h、70h、76h、84h、96h、107h)分别从各组的重复大鼠(n＝6)收集血样，分离血浆。在用蛋白酶K处理后，通过HPLC定量血浆中剩余的aptscl56和PEG40K-aptscl56。
评估方案：
样品制备：从每只大鼠通过眼眶静脉采集大约800μl血液，并收集到含有钠-肝素作为抗凝血剂的管中(1.8ml真空容器，BD Biosciences)，然后立即置于湿冰上(Healy,Lewis et al.2004,Perschbacher,Smestad et al.2015)。通过在收集后1小时内在4℃下以6000g离心10分钟来分离血浆，并在-80℃下储存直至分析(Healy,Lewis et al.2004,Siller-Matula,Merhi et al.2012,Gao,Shen et al.2016)。在分析之前，25μl消化缓冲液(60mM Tris-HCl，pH8.0,100mM EDTA和0.5％SDS)和75μl蛋白酶溶液(在10mM Tris HCl中1mg/mL蛋白酶K，pH 7.5，20mM CaCl 2，10％甘油v/v)加入50μl血浆样品中。然后将样品在55℃下振荡培养过夜。温育后，将样品离心(14000rpm；4℃；15分钟)并取出100μl上清液并转移至HPLC注射小瓶(Siller-Matula，Merhi等人，2012)。
HPLC定量：HPLC系统配备有C4柱以定量在不同时间点收集的血浆样品中的PEG40K-aptscl56，而C18柱用于定量aptscl56。该方法均使用由A相(TEAA[pH 7.0])和B相(乙腈)制成的流动相洗脱梯度。当柱温箱温度设定在50℃时，流速均为1.0mL/min。测定注射体积为20uL。在含有不同浓度的aptscl56和PEG40K-aptscl56的钠-肝素的空白大鼠血浆中制备标准品(Gao，Shen等人，2016)。所有报告的aptscl56和PEG40K-aptscl56的浓度均基于aptscl56的质量。根据标准曲线计算血浆样品中的适体浓度。
药代动力学分析：绘制Aptscl56和PEG40K-aptscl56浓度对时间曲线，并通过软件DAS 3.0(BioGuider Co.，Shanghai，China)分析每只大鼠。将得到的药代动力学参数取平均值。根据适体消除一半最大血浆浓度所需的时间计算适体的半衰期(t1/2)(Grieken和Bruin 1994)。根据药代动力学曲线获得最大血浆浓度(C max)和最大血浆浓度时间(T max)。从药物施用开始计算曲线下面积(AUC)，并且当血浆中的浓度可忽略不计时结束(Rowland,Benet et al.1973,Toutain and Bousquet-Melou 2004,Siller-Matula,Merhi et al.2012)。随后，根据下式计算多剂量PEG40K-aptscl56的给药间隔：D＝1/1-e -Ke·t(Ke：消除常数，Ke＝ln2/T1/2；t：给药间隔；D：给药比＝负荷剂量/维持剂量)(Birkett 1996,Jambhekar 2012)。
结果：
aptscl56的HPLC定量下限为10ul/mL，线性浓度范围为10μg/mL至 360ug/mL。皮下给药后Sprague-Dawley大鼠中aptscl56适体的消除半衰期(Elim.T1/2)的平均值为1.8小时。C max的平均值为265.5μg/ml，T max为0.5小时(图10，表3)。
PEG40K-aptscl56的HPLC定量的下限为7.5μg/mL，线性浓度范围为7.5μg/mL至240μg/mL。皮下给药后Sprague-Dawley大鼠中PEG40K-aptscl56的消除半衰期(Elim.T1/2)的平均值为66.9小时。C max的平均值为152.8μg/ml，T max为72小时(图11，表4)。
表3.皮下施用的aptscl56的药代动力学参数
Figure PCTCN2019074764-appb-000004
表4.皮下施用的PEG40K-aptscl56的药代动力学参数
Figure PCTCN2019074764-appb-000005
讨论：
本实施例研究了在S.D.大鼠皮下施用的PEG40K-aptscl56和aptscl56的药代动力学特征。与aptscl56的1.8小时消除半衰期相比，PEG40K-aptscl56的半衰期显著延长了65.1小时。这表明PEG对延长aptscl56的体内停留时间具有显著作用。然而，血浆中PEG40K-aptscl56的浓度整体低于aptscl56的浓度(图12)。许多研究表明PEG化的大小增加导致血液吸收的系统性降低(Caliceti 2003,Kaminskas,Kota et al.2009)。聚乙二醇化可能阻碍aptscl56对血液的吸收。
在PEG40K-aptscl56的药代动力学研究中，吸收需要60小时，但消除需要35小时。然而，消除大多数适体-PEG所需的时间总是比吸收长得多 (Christopher E.Tucker 1999,Siller-Matula,Merhi et al.2012)。一些研究表明PEG化的大小增强通过被动增强的渗透和保留机制促进了渗透组织的积累(Caliceti 2003)。将PEG40K-aptscl56累积到可渗透组织中可能是药代动力学曲线消除阶段快速下降的原因之一。进一步研究PEG40K-aptscl56的这种药代动力学现象将作出清楚的解释。
多剂适体的给药间隔可以基于其消除半衰期和负荷剂量与维持剂量的剂量比来定义(Birkett 1996,Jambhekar 2012)。PEG40K-aptscl56的消除半衰期为66.9小时。如果剂量比为2，则给药间隔等于消除半衰期(T1/2)。如果剂量比小于2，则给药间隔应长于T1/2。在PEG40K-aptscl56的药效学研究中，建议的剂量比为1(加载剂量等于维持剂量)。因此，PEG40K-aptscl56给药间隔应长于66.9h。
实施例8、评估聚乙二醇化适体候选物在通过卵巢切除术诱导的骨质疏松大鼠中的骨合成代谢功效
PEG40K缀合的适体在骨质疏松大鼠中的治疗方案。为了评估化学修饰的aptscl56/aptscl3与PEG40K组合在卵巢切除诱导的骨质疏松大鼠中的骨合成代谢功效，将70只3月龄雌性Sprague Dawley大鼠进行切除卵巢(OVX，n＝50)或假手术(SHAM，n＝20)，并且不处理2个月。在治疗前将10只OVX大鼠和10只SHAM操作的大鼠安乐死作为基线(OVX-BS和SHAM-BS)。其余的SHAM或OVX大鼠每周一次皮下注射载体(Veh)、PEG40K-aptscl56/aptscl3(25mg/kg)或PEG40K-随机序列(RS，25mg/kg)，进行6周(n＝每组10个)。在第一次注射后6周对所有动物实施安乐死。在安乐死之前，所有动物分别在第13天和第3天腹膜内注射钙黄绿素(20mg/kg)。安乐死后，收集右股骨，通过显微CT检查骨微结构。收集左股骨用于未脱钙切片和进一步的组织形态学分析。所有实验均按照相关指南和法规进行，所有实验程序均经香港浸会大学动物伦理委员会和实验安全委员会批准。
Micro-CT分析。使用micro-CT(版本6.5，vivaCT40，SCANCO Medical AG，Bassersdorf，Switzerland)进行右股骨远端干骺端、右胫骨近端干骺端、第五腰椎和右股骨中轴的分析。椎骨和股骨的图像分别以12.5和17.5μm的各向同性体素尺寸(70kVp，114μA，200ms积分时间，260阈值，1200mg HA/cm 3)重建和校准。每次测量都使用相同的过滤和分段值。使用Scanco 评估软件，为皮质和小梁参数定义感兴趣区域(ROI)。对于右侧远端股骨干骺端和右侧胫骨近端干骺端，整个股骨或胫骨将重新定向，中间骨干平行于z轴，骨长度测量为最近端和远端横向板之间含有股骨的距离。从生长板的最近端开始，选择在离生长板1.4mm的距离处的100个连续切片上的小梁区域。通过手动轮廓排除皮质骨来分析小梁。对于第五腰椎，选择相当于椎体高度的70％并且从远端生长板的近端到末端朝向椎体延伸的中心区域。在100个连续切片上徒手绘制了小梁ROI，以确保它在骨内包膜内。对于右股骨中轴，使用自动阈值算法在准确的中心和远端50％的股骨长度处测量100个切片。从ROI手动移除与皮质骨接触的小梁。计算骨小梁参数，包括每总体积的小梁体积(Tb.BV/TV)，小梁体积矿物质密度(Tb.vBMD)，小梁厚度(Tb.Th)，小梁数(Tb.N)，小梁间距(Tb.Sp)，小梁结构模型指数(Tb.SMI)，小梁连接密度(Tb.conn.D)。
统计分析。使用GraphPad Prism(版本8；GraphPad Software，Inc)进行统计分析。所有数值数据均表示为平均值±标准偏差。单因素ANOVA和Tukey的事后检验用于所有参数。P<0.05被认为具有统计学意义。
结果：
对于骨小梁，微型计算机断层扫描(micro-CT)分析显示，相比于卵巢切除(OVX)基线(OVX-BS)大鼠，SHAM基线(SHAM-BS)大鼠的股骨远端、胫骨近端和第五腰椎的小梁骨质量明显较高，骨小梁结构性能较好(图13)。将PEG40K-aptscl56/aptscl3，随机序列或载体的六次定期注射分别以每周一次的间隔对OVX大鼠进行处理(分别命名为OVX+aptscl56，OVX+aptscl3，OVX+RS和OVX+Veh)。SHAM大鼠每周一次用载体治疗6周(SHAM+Veh)(图13a)。股骨远端，胫骨近端和第五腰椎的小梁区域的数量分析显示，与SHAM+Veh对照相比，OVX+Veh的骨量显著减少(图13b、c和d)。这些数据表明OVX大鼠成功诱导了骨质疏松症。
股骨远端干骺端区域的显微CT分析显示BMD和BV/TV显著较高，表明与OVX-BS相比，OVX+aptscl56大鼠的骨小梁骨质量明显增加(P<0.005)。与OVX-BS相比，OVX+aptscl56的小梁结构也得到了显著改善(图13b)。然而，与OVX+Veh和OVX+RS组相同，与OVX-BS相比，OVX+aptscl3组未显示显著增加的骨量和改善的结构。
对于第五腰椎，与OVX-BS相比，OVX大鼠中PEG40K-aptscl56治疗6周完全恢复了骨量和骨结构(图13d)。而与OVX-BS相比，像OVX+RS和OVX+Veh那样，OVX+aptscl3没有改变骨量和骨骼结构。
综上，micro-CT数据表明，PEG40K-aptscl56可促进OVX诱导的骨质疏松大鼠骨形成，改善骨微结构，增加骨量。
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Claims (16)

  1. 一种针对骨硬化蛋白的适体，所述适体
    i)包含与SEQ ID NO:1-17中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性或至少大约95％相同性的核苷酸序列，或者，
    ii)包含SEQ ID NO:1-17中的任一中的至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸，
    优选地，所述适体包含SEQ ID NO:1-17或19-24中的任一的序列，
    其中所述适体特异性结合骨硬化蛋白。
  2. 权利要求1的适体，其中所述适体对骨硬化蛋白具有小于100nM，优选小于50nM，优选小于40nM，优选小于30nM，优选小于20nM，优选小于10nM或更小的K d。
  3. 权利要求1的适体，其中所述适体能够抑制骨硬化蛋白的生物学活性。
  4. 权利要求1的适体，其中所述适体能在基于细胞的Wnt信号测试法中阻断骨硬化蛋白的拮抗作用。
  5. 权利要求1的适体，其中所述适体以小于100μg/ml，优选小于50μg/ml，优选小于40μg/ml，优选小于30μg/ml，优选小于20μg/ml，优选小于10μg/ml或更小的EC50值抑制骨硬化蛋白的生物学活性，例如抑制骨硬化蛋白对Wnt信号通路的拮抗作用。
  6. 权利要求1的适体，其中所述适体还包含一或多种赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。
  7. 权利要求6的适体，其中所述修饰包括3’反向脱氧胸苷(3’idT)修饰。
  8. 权利要求6的适体，其中所述修饰包括用经修饰的核苷酸取代一或多个天然存在的核苷酸，所述经修饰的核苷酸选自2’-氟、2’-甲氧乙基、2’-甲氧基和/或2’丙烯氧基修饰的核苷酸，优选2’-甲氧基修饰的核苷酸。
  9. 权利要求6的适体，其中所述修饰包括核苷酸间的修饰，例如核苷酸间硫代磷酸酯键修饰。
  10. 权利要求6的适体，其中所述修饰包括PEG修饰，优选地，所述PEG的分子量为大约1kDa至大约100kDa，例如大约40kDa。
  11. 权利要求6的适体，其中所述适体包含2’-甲氧基(2’-OMe)修饰、3’反向脱氧胸苷(3’idT)修饰和/或PEG修饰。
  12. 一种治疗骨硬化蛋白相关疾病的方法，该方法包括给有需要的对象施用治疗有效量的权利要求1-11中任一项的针对骨硬化蛋白的适体，所述对象例如是人。
  13. 权利要求12的方法，其中所述骨硬化蛋白相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
  14. 一种药物组合物，其包含至少一种权利要求1-11中任一项的针对骨硬化蛋白的适体，和药学上可接受的载体或赋形剂。
  15. 权利要求1-11中任一项的针对骨硬化蛋白的适体或权利要求14的药物组合物在制备药物中的用途，其中所述药物用于治疗骨硬化蛋白相关疾病。
  16. 权利要求15的用途，其中所述骨硬化蛋白相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
PCT/CN2019/074764 2018-02-12 2019-02-11 针对骨硬化蛋白的适体及其用途 Ceased WO2019154410A1 (zh)

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