WO2020241493A1 - Médicament pour le traitement de troubles liés à des anomalies dans la formation osseuse et le métabolisme osseux - Google Patents

Médicament pour le traitement de troubles liés à des anomalies dans la formation osseuse et le métabolisme osseux Download PDF

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WO2020241493A1
WO2020241493A1 PCT/JP2020/020294 JP2020020294W WO2020241493A1 WO 2020241493 A1 WO2020241493 A1 WO 2020241493A1 JP 2020020294 W JP2020020294 W JP 2020020294W WO 2020241493 A1 WO2020241493 A1 WO 2020241493A1
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nucleotide
bone
cell death
nucleotides
agent according
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PCT/JP2020/020294
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Japanese (ja)
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義一 中村
将寿 藤原
石川　大
智志 松本
舞 中村
透 上杉
典央 児玉
健二 前田
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株式会社リボミック
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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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • the present invention relates to a therapeutic agent for a disease related to abnormalities in bone formation and bone metabolism, which contains nucleotides as an active ingredient.
  • Bone tissue is reconstructed (remodeled) by constantly repeating bone resorption and bone formation. By maintaining the balance of bone metabolism (bone resorption and bone formation), bone mass and structure are maintained and homeostasis is maintained. This imbalance causes various diseases.
  • microRNAs are involved in the regulation of osteoblast differentiation, which plays a major role in bone formation, and some miRNAs can induce selective apoptosis of osteoblasts. It has been reported (Non-Patent Documents 1 and 2).
  • Midkine (hereinafter abbreviated as "MK” if necessary) was discovered as a product of a gene transiently expressed in the process of inducing differentiation of embryonic tumor (EC: embryonal carcinoma) cells by retinoic acid. It is a growth / differentiation factor that is rich in basic amino acids and cysteine and has a molecular weight of 13 kDa, and is known to have various biological activities. For example, increased expression of midkine in human cancer cells is thought to promote cancer cell survival and migration, promote angiogenesis, and aid in cancer progression. On the other hand, in relation to bone metabolism, it has been reported that bone density increases in midkine-deficient mice (Non-Patent Document 3).
  • RNA aptamers for midkine are also known.
  • Patent Documents 1 and 2 disclose an aptamer showing an inhibitory activity against midkine, and this aptamer is a drug or diagnosis for various diseases such as autoimmune diseases, cancer, postoperative adhesions, and endometriosis. It is said to be useful as a reagent for medicines.
  • Patent Document 3 also discloses another cholesterol-modified aptamer that exhibits an inhibitory activity against midkine and exhibits a similar effect.
  • Non-Patent Document 4 shows that the midkine aptamer shown in Patent Document 1 or 2 reduces the symptoms of experimental autoimmune encephalomyelitis (EAE), the midkine aptamer is also used. There is no description suggesting that it induces bone metabolism or cell death such as osteoblasts.
  • An object of the present invention is to provide nucleotides that affect the survival of cells involved in bone formation, thereby providing a novel therapeutic means for diseases associated with abnormalities in bone formation and bone metabolism.
  • the present inventors have diligently studied to solve the above problems. As a result, it was discovered that among the nucleotides known as aptamers that bind to midkine and inhibit its activity, there are specific nucleotides that induce cell death. This cell death was induced specifically for cells producing type I collagen and was not induced for other cells. As shown in Non-Patent Document 3 above, it is known that bone density increases in mice lacking midkine, suggesting that inhibition of midkine rather favors bone formation balance in bone metabolism. Therefore, it was considered that these nucleotides induce cell-specific cell death that produces type I collagen by a route different from the action by inhibition of midkine.
  • the present invention provides the following inventions and the like.
  • nucleotide comprises the nucleotide sequence represented by any of SEQ ID NOs: 1 to 11.
  • nucleoside is substituted with either a hydrogen atom, a fluorine atom and an -O-alkyl group at the 2'position of ribose.
  • Agent. [4] The agent according to any one of [1] to [3], wherein the nucleotide is terminally modified with cholesterol and / or inverted dT.
  • [6] The agent according to any one of [1] to [5], wherein the type I collagen-producing cell is a cell involved in bone formation.
  • the agent according to [9] wherein the disease associated with bone dysplasia / bone metabolism disorder is osteosclerosis or posterior longitudinal ligament ossification.
  • nucleic acid containing the nucleotide sequence represented by any of SEQ ID NOs: 1 to 11, wherein the terminal thereof is modified with cholesterol and / or inverted dT.
  • the nucleic acid according to [12] wherein cholesterol is linked with a C12 alkyl linker.
  • at least one nucleoside is substituted with either a hydrogen atom, a fluorine atom and an -O-alkyl group at the 2'position of ribose. Nucleic acid.
  • the nucleotide of the present invention induces cell death of cells producing type I collagen, and may be useful as a cell death inducer. Since the nucleotide of the present invention induces cell death of type I collagen-producing cells in particular, it can be an active ingredient of a therapeutic agent for diseases related to abnormal bone dysplasia and abnormal bone metabolism.
  • A Shows cell death of type I collagen-producing cells. Brown indicates caspase 3-positive cells, and blue indicates type I collagen staining. Yellow arrows indicate both stains. This indicates that many dead cells are type I collagen-producing cells.
  • B Shows cell death of osteocalcin-positive cells (osteocytes, osteoblasts). Brown indicates caspase 3-positive cells, and blue indicates osteocalcin staining. Yellow arrows indicate both stains. This indicates that some dead cells are osteocalcin-producing cells.
  • A indicates a dose-dependent decrease in total bone mineral density
  • B indicates a dose-dependent decrease in cancellous bone mineral density. It is a figure which shows the effect which the osteosclerotic state of NOG-D1-Tg mouse was improved by the nucleotide of this invention by CT analysis.
  • A has decreased total bone mineral density
  • B has decreased cancellous bone mineral density
  • C has decreased cortical bone mineral density. It is a figure which shows the effect which the osteosclerotic state of NOG-D1-Tg mouse improved by the nucleotide of this invention by pathological analysis.
  • A shows the result of using a model in which zoledronic acid was administered at 0.05 mg / kg and osteosclerosis was used
  • B shows the result of using a model in which zoledronic acid was administered at 0.5 mg / kg and was osteosclerotic.
  • the bone density was reduced by the nucleotide of this invention in an oc / oc mouse.
  • Top panel Hematoxylin and eosin (HE) stained image.
  • Middle panel Immunostaining image of osteocalcin-positive cells.
  • Bottom panel Immunostaining image of type I collagen positive cells.
  • nucleotides of the present invention provides nucleotides that induce cell death of cells that produce type I collagen (sometimes abbreviated as "nucleotides of the present invention” in the present specification). .. It also provides a cell death inducer having the nucleotide.
  • the nucleotide of the present invention is not particularly limited as long as it is a nucleotide that induces cell death of cells that produce type I collagen.
  • the nucleotides of the present invention can be nucleic acids such as RNA, DNA, modified nucleic acids or mixtures thereof.
  • the nucleotide of the present invention is preferably a nucleotide containing, for example, the nucleotide sequence represented by any of SEQ ID NOs: 1 to 11 shown in Table 1. By including these nucleotide sequences, the nucleotides of the present invention exert a function of inducing cell death of cells producing type I collagen.
  • sequence specified by “SEQ ID NO:” means the sequence of each nucleotide.
  • “nucleotide of SEQ ID NO: 1” is a natural nucleotide sequence represented by SEQ ID NO: 1. It means a nucleic acid composed of a nucleic acid, a modified nucleic acid, or both
  • the "nucleotide containing the nucleotide of SEQ ID NO: 1" is a natural nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 1, a modified nucleic acid, or both. It means a nucleic acid to be composed.
  • nucleotide sequence refers to the 3'end of each constituent nucleoside residue and the 5'end of the next constituent nucleotide residue via a linking group (eg, phosphate diester bond, phosphorothioate bond, etc.). It means a concatenated sequence, and does not include, for example, a sequence in which 3'ends are bound to each other, such as an inverted dT added to the 3'end.
  • a linking group eg, phosphate diester bond, phosphorothioate bond, etc.
  • nucleotides of the present invention are the following (a) to (c).
  • A Nucleotide sequence represented by any of SEQ ID NOs: 1 to 11;
  • B A sequence in which one or several nucleotides are substituted, deleted, inserted or added in the above (a);
  • C In (a) or (b) above, a sequence in which the 2'-position group of ribose of one or several nucleotides is replaced with another group; Any of the sequences of is included in part or in whole.
  • the number of nucleotides to be substituted, deleted, inserted or added is not particularly limited as long as it retains the function of inducing cell death of cells producing type I collagen, but is, for example, 1 to 5, and more. It can be preferably 1 to 3, most preferably 1 or 2.
  • Table 2 is an alignment diagram of the nucleotide sequences shown in Table 1.
  • the position of the nucleotide to be substituted, deleted, inserted or added is not particularly limited, but is preferably deleted or substituted with reference to the nucleotide sequence of SEQ ID NO: 1 in the alignment diagram of Table 2. However, it is a position where the activity is maintained.
  • Examples of such a position include the 1st to 19th nucleosides, the 22nd to 28th nucleosides, the 31st to 32nd nucleosides, and the 37th to 38th nucleosides (note that the "Xth nucleoside” in this paragraph is used. Means the Xth nucleoside in the nucleotide sequence of SEQ ID NO: 1). At these positions, nucleotide substitutions, deletions, insertions or additions are possible as long as they retain the function of inducing cell death of type I collagen-producing cells.
  • the other group substituted at the 2'position of ribose of one or several nucleotides is not particularly limited. However, it is desirable that such substitutions be carried out in order to maintain or improve the activity of the nucleotides of the present invention, and the substitutions made for such purposes are preferably hydroxyl groups, hydrogen atoms, fluorine atoms and. It can be a group selected from the group consisting of —O—alkyl groups (eg, —O—Me groups).
  • the number of nucleotides in which the 2'-position group of ribose is replaced with another group is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5. obtain.
  • the nucleotides of the present invention also include a plurality of linkages of the nucleotide sequence of (a) above, a plurality of linkages of the nucleotide sequence of (b) above, a plurality of linkages of the nucleotide sequence of (c) above, and the above (a).
  • conjugates can also induce cell death in cells that produce type I collagen.
  • the connection can be made by tandem bonding.
  • a linker may be used for the connection.
  • the linkers include nucleotide chains (eg, 1 to about 20 nucleotides), non-nucleotide chains (eg,-(CH 2 ) n -linker,-(CH 2 CH 2 O) n -linker, hexaethylene glycol linker, TEG linker. , A linker containing a peptide, a linker containing an —S—S-link, a linker containing a -CONH-bond, a linker containing a -OPO 3 -bond).
  • the plurality in the plurality of conjugates is not particularly limited as long as it is two or more, but may be, for example, two, three, or four.
  • nucleotides of the present invention one or several nucleotides, for example, 1 to 2, 1 to 3, 1 to 4, and 1 to 5 nucleotides are cross-linked nucleic acids for the purpose of stabilizing or improving activity. It may be replaced with a Bridged Nucleic Acid (BNA). Examples of such a bridged nucleic acid include 2', 4'-BNA (Locked Nucleic Acid (LNA)), 2'-O, 4'-C-ethylene-bridged Nucleic Acid (ENA), and the like. Not limited to.
  • a part or all of the chemical structure of the phosphate diester bond of the nucleotide may be modified or substituted with an arbitrary substituent.
  • the phosphate diester bond can be replaced with a phosphodiester bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or the like.
  • Each nucleoside contained in the nucleotides of the present invention is the same or different, and is a nucleoside containing a hydroxyl group at the 2'position of ribose (eg, ribose of pyrimidine nucleotide, ribose of purimidine nucleotide) (that is, an unsubstituted nucleoside). Or it can be a nucleoside in which the hydroxyl group is replaced by any atom or group at the 2'position of ribose.
  • ribose eg, ribose of pyrimidine nucleotide, ribose of purimidine nucleotide
  • Such arbitrary atoms or groups include, for example, hydrogen atom, fluorine atom or -O-alkyl group (eg, -O-Me group), -O-acyl group (eg, -O-CHO group), amino.
  • Groups eg, -NH 2 groups
  • hydrogen atoms, fluorine atoms, and -O-alkyl groups are preferred.
  • the nucleotides of the present invention preferably contain at least one (eg, 1, 2, 3 or 4) nucleotides at the 2'position of ribose, a hydroxyl group, or any atom or group described above, eg, a hydrogen atom.
  • the nucleotide of the present invention is composed of RNA (that is, the sugar group is ribose), and the mode of modification to the sugar group in the nucleotide will be described.
  • RNA that is, the sugar group is ribose
  • the nucleotide of the present invention is composed of DNA
  • the replacement of the hydroxyl group at the 2'position of ribose with X is read as the replacement of one hydrogen atom at the 2'position of deoxyribose with X.
  • the nucleotide of the present invention may be modified with at least one sugar residue, as shown in Examples described later (note that the "X-th nucleoside” in this paragraph is the nucleotide of SEQ ID NO: 1. It means the Xth nucleoside in the sequence. In the nucleotide sequences of other SEQ ID NOs, it means the Xth nucleoside of SEQ ID NO: 1 when aligned as shown in Table 2). For example, in each nucleoside constituting the nucleotide of the present invention, it may be desirable from the viewpoint of improving activity that one of the purine nucleosides is methoxylated at the 2'position of ribose.
  • At least one of the 3, 4, 7, 10, 11, 16, 18 and 37th A is methoxylated, or 12, 14, 20-22, 25, 26, 28, 33. And it may be desirable in terms of improving activity that at least one of the 35th G is methoxylated at the 2'position of ribose.
  • At least one is fluorophorized at the 2'position of ribose in terms of improving activity may be desirable. If this is the case, if the substituted nucleoside is a purine nucleoside, it may be methoxylated at the 2'position of ribose, and if the substituted nucleoside is a pyrimidine nucleoside, it may be fluoromorphized at the 2'position of ribose. May be desirable). Alternatively, both the above-mentioned methoxylation and fluorolation may be performed.
  • Table 3 shows a desirable modification mode for the nucleotides of the present invention in the nucleotides of each SEQ ID NO: in the alignment diagram of Table 2.
  • Preferred nucleotides of the present invention are nucleotides having the sequences and modifications shown in Table 3.
  • nucleosides that should be methoxylated (3, 4, 7, 10, 11, 16, 18 and 37 A, or 12, 14, 20-22, 25, 26, 28, 33 and Underline the 35th G) and bold the nucleosides (15, 17, 30, 36 and 38th C, or 13, 23, 24, 27, 29 and 34th U) that should be fluorosylated. Indicated. That is, “nucleotide X” in Table 3 indicates a desirable modification mode of the sugar residue in the nucleotide of SEQ ID NO: X.
  • the nucleotides of the present invention are also nucleic acid bases (eg, for the purpose of improving the activity of inducing cell death of cells producing type I collagen, the drug delivery property of the nucleotide itself, the stability of the nucleotide itself in blood, and the like.
  • Purine, pyrimidine may be modified (eg, chemically substituted). Such modifications include, for example, 5-position pyrimidine modification, 6- and / or 8-position purine modification (such as O-methyl modification), modification with an extracyclic amine, substitution with 4-thiouridine, 5-bromo or 5-. Substitution with iodo-uracil, 5-amino acid type modification, 5-tryptophan side chain modification.
  • the phosphate group contained in the nucleotide of the present invention may be modified so as to be resistant to nucleases and hydrolysis.
  • the phosphate moiety of the nucleotide of the present invention is P (O) S (thioate), P (S) S (dithioate), P (O) NR 2 (amidate), P (O) R, P (O) OR.
  • Modifications are further made of polyethylene glycol (hereinafter sometimes referred to as "PEG"), amino acids, peptides, inverted dT, Myristoyl, Lithocolic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleoyl, other lipids, steroids, etc.
  • PEG polyethylene glycol
  • This can be done by adding cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin, etc. to the ends.
  • cholesterol and inverted dT are desirable as the nucleotide modification of the present invention.
  • the linker used when adding cholesterol to the nucleotide of the present invention is not particularly limited, and the number of carbon chains, functional groups and the like can be appropriately selected according to the binding site and the like.
  • Examples of such a linker include an alkyl linker having 12 carbon atoms (C12 alkyl linker) when cholesterol is added to the 5'terminal side.
  • the nucleotide of the present invention can be produced by any nucleic acid synthesis method known in the art. Examples of such a method include the methods described in WO2008 / 059877 or WO2009 / 063998.
  • the base length of the nucleotide of the present invention is not particularly limited as long as it maintains the activity of inducing cell death of cells producing type I collagen, but is usually 10 to 100 base length, preferably 15 to 70 base length. Yes, more preferably 20 to 50 bases in length, and most preferably 20 to 40 bases in length. In particular, it is desirable that the nucleotide of the present invention has a length of 40 bases or less. When the length exceeds 40 bases, unpredictable side effects may occur in addition to the cell death-inducing activity of cells producing type I collagen. If it is shorter than 10 bases in length, it is possible that the desired activity cannot be obtained.
  • nucleotide of the present invention Includes any of the sequences listed in Table 1: Nucleotides of 40 or less: and the following characteristics (i) At least one nucleoside constituting a nucleotide has a hydroxyl group at the 2'position of ribose. Substituted with either a hydrogen atom, a fluorine atom and an -O-alkyl group; (Ii) Inverted dT or cholesterol is added to the end; or the requirements of (iii) (i) and (ii) are met; A nucleotide having the above can be mentioned as a preferable specific example.
  • nucleotide of the present invention includes any of the sequences listed in Table 3; It has the modification mode at the ribose 2'position shown in Table 3; Nucleotides having a nucleotide length of 40 or less; and having inverted dT or cholesterol added to the end can be mentioned as preferred specific examples.
  • nucleotide of the present invention includes any of the sequences listed in Table 3; It has the modifications at the ribose 2'position shown in Table 3; Nucleotides having a nucleotide length of 40 or less; with inverted dT added to the 3'end and cholesterol added to the 5'end can be mentioned as more preferred specific examples.
  • nucleotide of the present invention Consists of any of the sequences listed in Table 3;
  • the most preferable specific example is a nucleotide having the modification mode at the ribose 2'position shown in Table 3; and having inverted dT added to the 3'end and cholesterol added to the 5'end.
  • the nucleotides of the present invention may have an activity of inducing cell death of cells producing type I collagen. Therefore, the nucleotide of the present invention is useful as a cell death inducer for cells producing type I collagen.
  • the cells that produce type I collagen are not particularly limited, but in the present specification, such cells include, for example, cells involved in bone formation.
  • the cells involved in bone formation refer to cells derived from mesenchymal stem cells that are produced in bone marrow and are involved in bone formation, specifically, osteoblasts, osteoblasts, anterior osteoblasts, and the like. Examples include osteoblast precursor cells.
  • the cell that produces type I collagen may be a cell that further produces osteocalcin. Specific examples of such cells include bone cells.
  • the cells that produce type I collagen of the present invention do not include chondrocytes, adipocytes, etc. derived from the same mesenchymal stem cells as the cells involved in bone formation described above.
  • osteoclasts produced in bone marrow and cells derived from hematopoietic stem cells such as erythrocytes, lymphocytes, macrophages, and platelets are not included.
  • the nucleotides of the present invention induce cell death of cells involved in such bone formation.
  • the induced cell death includes various cell deaths, and is not particularly limited, but the form of cell death can be preferably caspase-positive cell death, and more preferably apoptosis.
  • cell death after a single intravenous administration of the nucleotide of the present invention using a normal mouse (ICR) was observed with a microscope after HE (hematoxylin / eosin) staining and immunostaining. Among them, those with 10 or less immunostaining positive cells are caspase-negative (-), 11 to 19 are slightly caspase-positive ( ⁇ ), and those with 20 to 49 are weakly caspase-positive (+).
  • the nucleotide-induced cell death of the present invention in which the osteoblast viability in the same field is evaluated to be less than 10% by observation with a microscope by HE staining is the above-mentioned (+) or more cell death.
  • the induction of cell death of the nucleotides of the present invention is cell-selective, such as cells that produce type I collagen or cells that further produce osteocalcin, which are directly involved in bone formation. Therefore, even if these cells are derived from the same mesenchymal stem cells, they do not induce cell death in, for example, chondrocytes and adipocytes. Similarly, it does not induce cell death in osteoclasts or cells derived from hematopoietic stem cells such as erythrocytes, lymphocytes, macrophages, and platelets, which are produced in bone marrow.
  • the induction of cell death by the nucleotides of the present invention is carried out within 24 hours, preferably within 12 hours, more preferably within 8 hours, for example, when intravenously administered to a mammal at an amount of 100 mg / kg.
  • the mammal is not particularly limited, and preferred examples thereof include humans, mice, and rabbits, and more preferably humans.
  • the induction of cell death depends on the subject to be administered, but in the case of mammals, for example, even if the administration is interrupted, it is maintained for a certain period of time. The period is, for example, 7 to 50 days, preferably 25 to 40 days, and more preferably 35 to 40 days. Bone remodeling in mice is completed in about 2 weeks, whereas in humans it is completed in 6-9 months (J.
  • the nucleotide of the present invention induces cell death of the target bone formation-related cells.
  • the cell death of this bone formation-related cell does not depend on the presence or absence of osteoclasts. That is, even in a place where osteoclasts do not exist, the function of the nucleotide of the present invention results in suppression of an increase in bone density or a decrease in bone density.
  • the subject to which it is administered is not particularly limited, but for example, primates (eg, humans, monkeys), guinea pigs, etc. Examples include teeth (eg, mice, rats, guinea pigs), as well as pets, livestock and working animals (eg, dogs, cats, horses, cows, goats, sheep, pigs).
  • the nucleotide of the present invention has an activity of inducing cell death of cells producing type I collagen, and examples of such cells include cells involved in bone formation. Therefore, the drug containing the nucleotide of the present invention as an active ingredient can be used as a therapeutic agent for diseases related to bone dysplasia / abnormal bone metabolism (hereinafter, also referred to as “therapeutic agent of the present invention”).
  • diseases related to abnormal bone dysplasia and abnormal bone metabolism in the present invention include diseases related to an increase in abnormal bone formation and / or a predominant bone formation balance due to insufficient bone resorption.
  • diseases related to an increase in abnormal bone formation and / or a predominant bone formation balance due to insufficient bone resorption include osteoblastoma, thoracic yellow ligament ossification, thoracic posterior longitudinal ligament ossification, cervical posterior longitudinal ligament ossification, cervical yellow ligament ossification, yellow ligament ossification, lumbar posterior longitudinal Ligament ossification, anterior longitudinal ligament ossification, posterior longitudinal ligament ossification, thoracic anterior longitudinal ligament ossification, cervical anterior longitudinal ligament ossification, lumbar yellow ligament ossification, femoral traumatic ossification Myitis, progressive ossifying myitis, progressive ossifying fibrous dysplasia, ossifying myitis associated with paralysis, oss
  • the therapeutic agents of the present invention are also used in combination with bone marrow transplantation.
  • it can be used as a therapeutic agent for diseases in which osteoclasts are dysfunctional or decreased. That is, by administering the nucleotide of the present invention to a subject, the increase in the bone density of the subject is suppressed or the bone density decreases.
  • osteoclasts are dysfunctional or decreased.
  • the therapeutic effect of the nucleotide of the present invention can be enhanced even for diseases associated with dysfunction or decrease of osteoclasts.
  • the therapeutic agent of the present invention may be a formulation of the nucleotide of the present invention as it is, together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone.
  • Excipients such as gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, disintegrant such as calcium citrate, magnesium stearate , Lubricants such as aerodyl, talc, sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid , Stabilizers such as sodium citrate, acetic acid, suspending agents such as methyl cellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, orange juice, cacao butter, polyethylene.
  • the therapeutic agent of the present invention can be coated by a method known per se for the purpose of sustainability, if necessary.
  • the coating agent used for coating include hydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, and hydroxymethyl cellulose acetate succinate.
  • Eudragit manufactured by Roam, Germany, methacrylic acid / acrylate copolymer
  • dyes eg, red iron oxide, titanium dioxide, etc.
  • the drug may be either a rapid-release preparation or a sustained-release preparation.
  • the sustained-release substrate include liposomes, atelocollagen, gelatin, hydroxyapatite, PLGA and the like.
  • the administration form of the therapeutic agent of the present invention is not particularly limited, but it is preferably administered parenterally.
  • parenteral administration include intravenous administration, subcutaneous administration, intramuscular administration, local administration, intraperitoneal administration, nasal administration, and pulmonary administration, and intravenous administration or topical administration is preferable.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injectables, even if they contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Good.
  • aqueous and non-aqueous sterile suspensions may be mentioned, which may include suspending agents, solubilizing agents, thickeners, stabilizers, preservatives and the like.
  • the preparation can be encapsulated in a container in units of doses or multiple doses, such as ampoules and vials.
  • the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state where it can be dissolved or suspended in a suitable sterile solvent immediately before use.
  • sustained-release preparations can also be mentioned as suitable preparations.
  • Sustained-release preparations include artificial bone, biodegradable or non-degradable sponges, bags, drug pumps, osmotic pumps, and other sustained-release forms from carriers or containers embedded in the body, or continuous or intermittent from outside the body. Examples include devices that are delivered internally or locally.
  • biodegradable substrate examples include liposomes, cationic liposomes, Poly (lactic-co-glycolic) acid (PLGA), atelocollagen, gelatin, hydroxyapatite, and polysaccharide schizophyllan.
  • liposomes examples include liposomes, cationic liposomes, Poly (lactic-co-glycolic) acid (PLGA), atelocollagen, gelatin, hydroxyapatite, and polysaccharide schizophyllan.
  • examples of the surfactant include oleic acid, lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl trioleate, glyceryl monolaurate, glyceryl monooleate, and glyceryl monosteer.
  • the dose of the therapeutic agent of the present invention varies depending on the type / activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc., but is usually 1 day for adults.
  • the amount of active ingredient per unit can be 0.0001 to 100 mg / kg.
  • the preferred dose can be 0.01 to 1000 mg / kg, more preferably 0.05 to 500 mg / kg, even more preferably 1 to 500 mg / kg, and most preferably 30. It can be up to 300 mg / kg.
  • nucleotide of the present invention (sometimes referred to as “nucleotide of the present invention” or “nucleic acid that specifically induces cell death in osteoblasts", “nucleic acid of the present invention", etc.)
  • Nucleotide No. 1 (nucleotide 1) was made by the method described in WO2008 / 059877 or WO2009 / 063998.
  • nucleotides of SEQ ID NOs: 2 to 11 (nucleotides 2 to 11 respectively) and the nucleotides of SEQ ID NO: 1 (nucleotides 12) were prepared by purifying with a hydrophobic column using a nucleic acid synthesizer based on SEQ ID NO: 1.
  • the nucleotide sequences of the nucleotides obtained by this method are as shown below.
  • the nucleic acid shown in the nucleotide sequence uses the nucleic acid code used in the international nucleotide sequence database (Cornish-Bowden, A. Nucl Acid Res 13, 3021-3030 (1985) and Feature Table Definition: 7.4.1 Nucleotide base. codes (IUPAC)).
  • the parentheses in the nucleotide indicate the modification of the ribose at the 2'position, M indicates that it is modified with an O-methyl group, and F indicates a fluorine atom (the same applies hereinafter).
  • Nucleotide 1 ch Reserved-GGA (M) A (M) GGA (M) GGA (M) A (M) G (M) U (F) G (M) C (F) A (M) C (F) A (M) GG (M) G (M) G (M) U (F) U (F) G (M) G (M) U (F) C (F) GGG (M) U ( F) G (M) C (F) A (M) C (F) -idT (SEQ ID NO: 1)
  • This nucleotide is a 38-nucleotide (not containing inverted dT; the same applies hereinafter) nucleic acid consisting of repeating sequences of A, G, C and U, with cholesterol added to the 5'end and inverted dT added to the 3'end. ing. Cholesterol and nucleic acid are linked by a C12 alkyl linker.
  • nucleotides 2 to 12 The modified portion (nucleotides 2 to 12) of the modified product will be described below.
  • cholesterol is added to the 5'end (excluding nucleotide 12), and the 3'end is inverted dT. Cholesterol and nucleic acid are linked by a C12 alkyl linker.
  • Nucleic acid 2 A 27-nucleotide nucleic acid sequence obtained by removing 11 nucleotides on the 5'terminal side based on RNA represented as nucleotide 1.
  • Nucleotide 3 A 31-nucleotide nucleic acid sequence obtained by removing the internal 7 nucleotides from the RNA represented as nucleotide 1.
  • Nucleotide 4 A 34-nucleotide nucleic acid sequence obtained by removing 2 nucleotides from each of the two internal locations based on the RNA represented as nucleotide 1.
  • Nucleotide 5 A 35-nucleotide nucleic acid sequence obtained by removing 3 nucleotides on the 5'terminal side based on the RNA represented as nucleotide 1.
  • Nucleic acid 6 A 29-nucleotide nucleic acid sequence obtained by removing 9 nucleotides on the 5'-terminal side based on RNA represented as nucleotide 1.
  • Nucleic acid 7 A 34-nucleotide nucleic acid sequence based on RNA represented as nucleotide 1, excluding idT, with 2 nucleotides on the 3'-terminal side removed, and 2 nucleotides inside removed.
  • Nucleotide 8 A 32-nucleotide nucleic acid sequence obtained by removing the internal 6 nucleotides based on the RNA represented as nucleotide 1.
  • Nucleotide 9 A 28-nucleotide nucleic acid sequence obtained by removing the internal 10 nucleotides based on the RNA represented as nucleotide 1.
  • Nucleotide 10 A 28-nucleotide nucleic acid sequence obtained by trimming the internal 10 nucleotides based on the RNA represented as nucleotide 1.
  • Nucleotide 11 A 38-nucleotide nucleic acid sequence in which the 10th to 15th AAGUGC sequences are replaced with GGACAU based on the RNA represented as nucleotide 1.
  • Nucleotide 12 A 38-nucleotide nucleic acid sequence in which the 5'end adduct of nucleotide 1 is replaced with inverted dT.
  • Example 2 Cell death of osteoblasts after administration of the nucleotide of the present invention
  • the nucleotide of the present invention was administered once from the tail vein of a mouse at 100 mg / kg and 300 mg / kg. Forty-eight hours later, after exsanguination under isoflurane anesthesia, the femur and tibia were excised and fixed in 10% neutral buffered formalin. The next day, after decalcification of formic acid, pathological analysis was performed. Osteoblasts, Osteoblast progenitor cells, and preosteoblasts are stained with anti-type I collagen antibody, chondrocytes are stained with Arcian blue, and osteoclasts (Osteoclasts). Osteoclast) was stained with anti-osteoclast antibody (Fig. 1). The test was conducted using nucleotide 1 as the nucleotide of the present invention.
  • cell death of anti-type I collagen-expressing cells occurred in a nucleotide dose-dependent manner by administering the nucleotide of the present invention.
  • the number of osteoclasts did not change with or without administration.
  • Cell death by the nucleotide of the present invention further induced cell death in osteocalcin-positive cells, and a part of the cell death by the nucleotide of the present invention was caspase 3 positive (Fig. 2).
  • Example 3 Time-dependent changes in osteoblast death after administration of the nucleotide of the present invention
  • Nucleotide 1 was administered at 100 mg / kg from the tail vein of a mouse. After 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration, the femur and tibia were exsanguinated and fixed in 10% neutral buffered formalin after exsanguination under isoflurane anesthesia. .. The next day, after decalcification of formic acid, HE staining and immunostaining were performed, and pathological analysis was performed (Fig. 3).
  • the pathologically stained images of the femur and tibia were observed under a microscope for each individual, and the number of caspase 3 positive cells was counted.
  • the average number was qualitatively determined according to the following. (-): 10 or less, ( ⁇ ): 10 to 19, (+): 20 to 49, (++): 50 to 99, (++++): 100 or more.
  • cell death was induced at least 8 hours after the administration of the nucleotide of the present invention.
  • Example 4 Examination of recovery period of osteoblasts after administration of nucleotides of the present invention
  • Nucleotides 1 were administered single intravenously (iv) at 100 mg / kg and 300 mg / kg from the tail vein of mice. After lethal death under isoflurane anesthesia on Day2, Day5, Day7, and Day14, the femur and tibia were excised and stained with anti-type I collagen antibody to examine the recovery of osteoblasts (Fig. 4). ..
  • Example 5 Decrease in bone mineral density of normal mice after administration of nucleotides of the present invention
  • ICR Claire Japan
  • Normal mice male, 12-18 g
  • the body weight of each individual was measured on each administration day.
  • Vehicle and the nucleotides of the invention were administered as a single intravenous (iv) dose on Day0, Day3, Day7, Day10, Day14, Day17, Day21, Day24, Day28, and Day31. Vehicle used physiological saline.
  • the nucleotides of the present invention were adjusted before use and administered at a rate of 5 mL / kg (100 ⁇ L / 20.0 g).
  • the femur and tibia were excised and fixed in 10% neutral buffered formalin after exsanguination under isoflurane anesthesia.
  • the next day bone density was analyzed using an X-ray CT device (LaTheata LCT-200, Hitachi Aloka). (Fig. 5). As a result, the bone mineral density decreased in a dose-dependent manner by administering the nucleotide of the present invention.
  • Example 6 Test using osteosclerosis model NOG-D1-Tg mouse Test 1 NOG-D1-Tg mouse (Central Institute for Experimental Animals), which is an osteosclerosis model, was obtained as a pregnant animal and delivered by cesarean section. After that, the genotype was determined by the PCR method. Based on genotype, sex and body weight, they were divided into Vehicle group, 30 mg / kg nucleotide administration group, 100 mg / kg nucleotide administration group, and 300 mg / kg nucleotide administration group. Saline (Vehicle) or nucleotide 1 (30, 100 or 300 mg / kg) set forth in SEQ ID NO: 1 was administered twice weekly into the tail vein for 4 weeks. The femur and tibial tissues were excised, CT analysis and pathological analysis were performed, and the osteosclerosis improving effect of nucleotide 1 shown in SEQ ID NO: 1 was evaluated (FIG. 6).
  • NOG-D1-Tg mouse Central Institute for Experimental Animals
  • mice NOG-D1-Tg mice were divided into a Vehicle group, a 100 mg / kg nucleotide administration group, and a 300 mg / kg nucleotide administration group based on genotype, sex, and body weight.
  • Saline (Vehicle) or nucleotide 1 (100 or 300 mg / kg) set forth in SEQ ID NO: 1 was administered once weekly into the tail vein for 4 weeks.
  • the femur and tibial tissues were removed, and CT analysis and pathological analysis were performed to evaluate the osteosclerosis-improving effect of nucleotide 1 (Fig. 7).
  • nucleotide 1 suppressed the increase in bone mineral density of whole bone, cancellous bone, and cortical bone in a dose-dependent manner, and decreased from the normal level at 300 mg / kg (Fig. 6). ).
  • Pathological analysis also confirmed that nucleotide 1 improved bone mineral density (Fig. 7). That is, it was shown that the nucleotide of the present invention can improve the osteosclerotic state.
  • Example 7 Test using a bisphosphonate-induced osteosclerosis model
  • Bisphosphonate (zoledronic acid: ZA) has an action of specifically adsorbing on the bone surface and suppressing the action of osteoclasts by various mechanisms of action.
  • the first day of zoledronic acid administration was set to Day 0.
  • Index of bone mineral density in the metaphyseal region of the tibia the day after the final administration of zoledronic acid (Day 7) for mice in which osteosclerosis was induced by subcutaneous administration of zoledronic acid at 0.05 or 0.5 mg / kg three times a week for one week.
  • Zoledronic acid-treated animals were divided into 3 groups (Vehicle group, 100 mg / kg nucleotide administration group, and 300 mg / kg nucleotide administration group). From the day after grouping, nucleotide 1 was intravenously administered 3 times a week for 4 weeks. Bone mineral density in the metaphyseal region of the tibia was monitored once a week by CT analysis, mice were dissected after the final administration of nucleotide 1, and femur and tibial tissue were removed. CT analysis and pathological analysis were performed on the excised tissue to evaluate the osteosclerosis improving effect of nucleotide 1.
  • nucleotide 1 suppresses the increase in bone mineral density in a dose-dependent manner, and especially in the 300 mg / kg dose group, the bone mineral density is improved to be lower than the bone mineral density at the time of grouping.
  • nucleotide 1 improved the bone mineral density, but it was weaker than the effect of the 0.05 mg / kg zoledronic acid model.
  • Fig. 8B That is, it was suggested that the nucleotide of the present invention can suppress an abnormally high bone density state in cooperation with osteoclasts.
  • OC / OC mouse is one of human osteopetrosis.
  • Osteoclast recessive osteoptrosis 1 is a mouse having the same gene mutation (Tcirg1 gene mutation) and lacking osteoclast function, and is a model mouse for osteopetrosis that spontaneously develops a high bone mass pathology.
  • Pregnant mice obtained by embryo transfer of fertilized eggs obtained by mating OC / + heterozygous mice and wild-type mice (B6C3F1) were obtained.
  • Genotyping of newborns (F1) born by spontaneous delivery was determined to obtain male and female OC / + heteropairs.
  • F2 generation oc / oc homomouses obtained by mating this OC / + hetero and spontaneously delivering were used in the test.
  • mice were intermittently administered saline solution (5 mL / kg) or nucleotide 1 (300 mg / kg) intravenously 7 times every other day.
  • saline solution 5 mL / kg
  • nucleotide 1 300 mg / kg
  • mice were laparotomized under isoflurane anesthesia, the abdominal large arteries and veins were incised, and phlebotomy was euthanized.
  • the femurs and tibias of both hind limbs were removed, fixed with neutral formalin, and bone density was measured using an X-ray CT device (LaTheata LCT-200, Hitachi Aroca) or pathologically observed.
  • the cured state was analyzed.
  • the administration of a single nucleotide in this model did not show any effect of improving the pathological condition such as improving the survival rate. That is, according to the nucleotide of the present invention, it was shown that osteoblast death is induced without affecting the function of osteoclasts. That is, according to the nucleotide of the present invention, when combined with the results of Example 7, the progression of high bone mass pathology such as osteopetrosis such as osteoclast dysfunction may be suppressed by the combined use with bone marrow transplantation. It has been shown.
  • Example 9 Nucleotides that induce osteoblast death
  • Each of the nucleotides of the present invention produced in this example was administered once from the tail vein of a mouse at 300 mg / kg. Twenty-four hours later, after death by bleeding under isoflurane anesthesia, the femur and tibia were excised, immersed in 10% neutral buffered formalin, fixed, immunostained with anti-type I collagen antibody and anti-caspase 3 antibody, and osteoblasted. Cell death was analyzed in comparison with the Vehicle-administered group.
  • the nucleotide of the present invention can selectively induce cell death in cells producing type I collagen, it is possible to suppress an increase in the number of cells in a cell group involved in bone formation and suppress the activity of the cell group. It is extremely useful for improving various pathological conditions in which the balance of bone metabolism is predominantly bone formation, that is, for treating diseases involving bone dysplasia / bone metabolism or higher.

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Abstract

L'objectif de cette invention est de fournir un nucléotide qui a un impact sur la survie de cellules mises en jeu dans la formation osseuse, et fournit ainsi une nouvelle méthode pour le traitement de troubles associés à des anomalies dans la formation osseuse et le métabolisme osseux.　Un inducteur de mort cellulaire pour des cellules productrices de collagène de type I, ayant, en tant que principe actif, un nucléotide contenant l'une des séquences (a) à (c) : (a) une séquence nucléotidique représentée par l'une des SEQ ID NO : 1-11 ; (b) une séquence obtenue par substitution, délétion, insertion ou ajout d'un ou plusieurs nucléotides à (a) ; (c) une séquence obtenue par substitution du groupe en position 2' du ribose d'un ou plusieurs nucléotides dans (a) ou (b) avec un autre groupe.
PCT/JP2020/020294 2019-05-24 2020-05-22 Médicament pour le traitement de troubles liés à des anomalies dans la formation osseuse et le métabolisme osseux WO2020241493A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008059877A1 (fr) * 2006-11-14 2008-05-22 Ribomic Inc. Aptamère contre la midkine et son utilisation
WO2009063998A1 (fr) * 2007-11-14 2009-05-22 Ribomic Inc. Acide nucléique sur lequel est ajoutée une substance hydrophobe, et son utilisation
JP2019510080A (ja) * 2016-03-01 2019-04-11 セルミド リミティッド 骨疾患、障害及び／または傷害の治療方法及びそのための試薬

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008059877A1 (fr) * 2006-11-14 2008-05-22 Ribomic Inc. Aptamère contre la midkine et son utilisation
WO2009063998A1 (fr) * 2007-11-14 2009-05-22 Ribomic Inc. Acide nucléique sur lequel est ajoutée une substance hydrophobe, et son utilisation
JP2019510080A (ja) * 2016-03-01 2019-04-11 セルミド リミティッド 骨疾患、障害及び／または傷害の治療方法及びそのための試薬

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