WO2024043523A1 - Synovium-targeting compound and uses thereof - Google Patents

Synovium-targeting compound and uses thereof Download PDF

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WO2024043523A1
WO2024043523A1 PCT/KR2023/009907 KR2023009907W WO2024043523A1 WO 2024043523 A1 WO2024043523 A1 WO 2024043523A1 KR 2023009907 W KR2023009907 W KR 2023009907W WO 2024043523 A1 WO2024043523 A1 WO 2024043523A1
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compound
pharmaceutically acceptable
solvate
hydrate
present
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PCT/KR2023/009907
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French (fr)
Korean (ko)
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김도경
김재훈
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경희대학교 산학협력단
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic

Definitions

  • the present invention relates to compounds that specifically target the synovial membrane and their uses.
  • Arthritis is a general term for a condition that affects joints and surrounding tissues. Joints are located in the body where bones come together, such as the knees, wrists, fingers, toes, and hips. The two most common types of arthritis are osteoarthritis and rheumatoid arthritis. Osteoarthritis (OA) is the most common type of joint disease, affecting more than 20 million individuals in the United States alone. It is the leading cause of chronic disability in people over 70 years of age and costs more than $185 billion annually in the United States. This is a painful, degenerative joint disease that often involves the small joints of the hips, knees, neck, lower back, or hands.
  • Osteoarthritis usually develops in joints that are damaged by repeated overuse, such as performing special tasks, playing a favorite sport, or carrying excessive weight. Osteoarthritis can be considered a degenerative disorder that causes biochemical destruction of synovial intra-articular (hyaline) cartilage.
  • hyaline synovial intra-articular
  • current views maintain that arthritis involves not only the articular cartilage but also the entire joint organ, including the subchondral bone and synovium.
  • Rheumatoid arthritis is an autoinflammatory disease that commonly involves various joints in the fingers, thumbs, wrists, elbows, shoulders, knees, feet, and ankles, and is caused by inflammation of the tissue called the synovium that surrounds the joints.
  • rheumatoid arthritis is a systemic disease that can cause inflammation in extra-articular tissues throughout the body, including the skin, blood vessels, heart, lungs, and muscles. .
  • Gout is a disease that results from the deposition of monosodium urate crystals in synovial fluid and other tissues and the formation of uric acid stones in the kidneys. Gout typically occurs during middle age and is not common before the age of 30. Women rarely suffer attacks of gouty arthritis before menopause.
  • Synovium (synovium) is the tissue closest to articular cartilage and is the only tissue capable of producing hyaline cartilage in malignant conditions such as synovial chondromatosis and osteochondral spurs. Cells from the synovium are extracted to repair cartilage damage. There was a report that he was being recruited.
  • the object of the present invention is to provide a substance targeting the synovial membrane.
  • an object of the present invention is to provide a material that specifically visualizes the synovial membrane.
  • an object of the present invention is to provide a method for producing the above materials.
  • an object of the present invention is to provide a synovium targeting drug delivery system.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis diseases.
  • an object of the present invention is to provide a synovial contrast agent composition.
  • the present invention provides a compound targeting the synovial membrane, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a compound that specifically visualizes the synovial membrane, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method for producing the above compound.
  • the present invention provides a synovium-targeting drug delivery system in which a pharmaceutically active ingredient is conjugated to the material.
  • the present invention provides a pharmaceutical composition for preventing or treating arthritis disease, comprising the drug carrier.
  • the present invention provides a synovial contrast agent composition.
  • the compound prepared according to the present invention does not exhibit toxicity in vitro and in vivo , is stable in the biological environment, and can specifically target synovial cells and synovial areas in vivo, so it can be used in drug development and medical fields. It can be useful in the diagnosis and treatment of synovial-related diseases.
  • Figure 1 is a diagram showing the results of confirming the absorption and fluorescence emission characteristics of the DRL-TAM of the present invention under various solvent conditions (distilled water, saline solution, and cell culture medium).
  • Figure 2 is a diagram showing the results of confirming the absorption and fluorescence emission characteristics of the DRL-TAM of the present invention under various pH conditions (pH 3, 5, 7, and 9).
  • Figure 3 is a diagram showing the results of confirming the photostability of DRL-TAM of the present invention.
  • Figure 4 shows the results of confirming the cytotoxicity of DRL-TAM at a concentration of 1 to 100 ⁇ M in three types of cells:
  • End3 endothelial cells derived from mouse brain tissue
  • HEK293 human embryonic kidney-derived epithelial cells
  • Figure 5 is a diagram showing the results of analyzing the hemolysis reaction of DRL-TAM (1 to 100 ⁇ M) of the present invention (**** p ⁇ 0.0001 compared to Triton X-100 treatment group).
  • Figure 6 is a diagram showing the results of confirming the in vitro synovium targeting effect and visual effects of the DRL-TAM of the present invention:
  • End3 endothelial cells derived from mouse brain tissue
  • HEK293 human embryonic kidney-derived epithelial cells
  • RSPC mouse synovium-derived primary cells
  • Figure 7 is a diagram showing in vitro fluorescence images confirming the distribution process in vivo over time after administration of PBS (pH 7.4), Rho, and DRL-TAM (i.v., 5 mg/kg) (here: 530-570) nm, emission channel: 575-640 nm).
  • Figure 8 is an in vitro fluorescence image confirming the distribution process in body organs and parts after administration of PBS (pH 7.4), Rho and DRL-TAM (i.v., 5 mg/kg) and blood circulation for 10 minutes, and a graph quantifying the radiation efficiency. (compared to the PBS treatment group, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001; compared to the Rho treated group, ##p ⁇ 0.01, ###p ⁇ 0.001, # ###p ⁇ 0.0001).
  • Figure 9 is a diagram showing the chemical formula of DRL-TAM of the present invention, which can specifically visualize the synovial membrane (D: aspartic acid, R: arginine, L: leucine).
  • amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
  • the present invention relates to a compound represented by the following formula (1), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:
  • the compound of the present invention may be used for targeting the synovial membrane.
  • the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt is highly stable under various solvent conditions, pH conditions, and light irradiation conditions, and is proportional to concentration even under various conditions such as above. As a result, the efficacy can be shown stably.
  • the compound of the present invention is non-cytotoxic and does not exhibit a hemolytic reaction in vivo, so it may be safe.
  • the compound represented by Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine.
  • the acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone or acetonitrile. It can be manufactured by ordering. Additionally, this mixture can be prepared by evaporating the solvent or excess acid and drying it, or by suction-filtering the precipitated salt. Additionally, a pharmaceutically acceptable metal salt can be prepared using a base.
  • a water-miscible organic solvent for example, methanol, ethanol, acetone or acetonitrile. It can be manufactured by ordering. Additionally, this mixture can be prepared by evaporating the solvent or excess acid and drying it, or by suction-filtering the precipitated salt. Additionally, a pharmaceutically acceptable metal salt can be prepared using a base.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering off the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, calun, or calcium salts as metal salts. Additionally, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).
  • a suitable silver salt e.g., silver nitrate
  • the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt may further include a labeling substance, and the labeling substance may be a chromogenic enzyme, a radioisotope, or a chromophore. ), coloring materials, light-emitting materials, fluorescers, superparamagnetic particles, and ultraparamagnetic particles.
  • the chromogenic enzyme is streptavidin-conjugated horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, and luciferase.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase ⁇ -galactosidase
  • luciferase acetylcholinesterase
  • urease catalase
  • asparginase ribonuclease
  • malate dehydrogenase staphylococcal
  • It may be a nuclease (staphylococcal nuclease), triose phosphate isomerase, glucose oxidase, cytochrome P450 and peroxidase compounds.
  • the fluorescent material may be a fluorescent protein, photoprotein, luciferase, fluorescent dye, or time-resolved fluorescence (TRF),
  • the fluorescent substances are Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rubpy (tris(2,2-bipyridyl)ruthenium(II) ), FITC (fluoresein Isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (5(6)-Carboxytetramethylrhodamine), Texas Red, DAPI (4,6-diamidino-2) -phenylindole) and coumarin. It is more preferable that it is at least one selected from the group consisting of, and TAMRA is most preferable.
  • the compound represented by Formula 1, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a synovium targeting composition, and the targeting composition is selected from solvents, acids, bases, and buffer solutions. One or more may be included.
  • the targeting composition can be prepared by adding the above-described compound to a solvent, buffer solution, or a mixture thereof, and adding an acid or base thereto. Additionally, the targeting composition may additionally include other additives that can be used in the art. The contents of the solvent, acid, base, and buffer solution contained in the composition can be appropriately adjusted depending on the required performance.
  • the solvent may include water, tetrahydrofuran (THF), methanol, ethanol, HI aqueous solution, N,N-dimethylformamide (DMF), or a combination thereof.
  • the composition may include combinations with other drugs and chemical molecules.
  • the drug may include, but is not limited to, a drug that is effective in a specific area, and any drug that can be used as a chemical sample such as a drug, contrast medium, nutrient, or fluorescent substance in the art is possible.
  • the compound represented by Formula 1, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a composition for diagnosing arthritis disease.
  • the arthritic disease may be an arthritic disease associated with the synovium and may include degenerative arthritis, rheumatoid arthritis, etc., but is not limited thereto.
  • a new peptide capable of specifically targeting a specific part of the body was developed, and this new peptide was conjugated with the TAMRA fluorophore using a lysine and 6-aminohexanoic acid linker, resulting in DRL-TAM. was developed (see Example 1 and FIG. 9; D: aspartic acid, R: arginine, L: leucine).
  • the new peptides DRL and DRL-TAM do not show toxicity in vitro and in vivo , can be stably used in biological aqueous solutions (pH 7.4) and cell cultures, and have excellent pharmacokinetic properties.
  • mice It is distributed within the mouse body in a short period of time (10 to 20 minutes), and can specifically target and visualize synovial cells or synovial areas in vivo (sternum, spinal cord, leg bones, feet, etc., where synovium is abundant). It was confirmed that it can act on both the cellular and biological levels.
  • targeting refers to the ability to target a specific cell or body part, and refers to the property of a substance being specifically delivered to the corresponding part when distributed in the body.
  • Targeting characteristics are characteristics that appear only in a specific biological part and may mean that a significantly larger amount is delivered compared to the remaining parts excluding that part.
  • the present invention relates to a nucleic acid encoding the compound of Formula 1 (DRL peptide) of the present invention, a vector containing the nucleic acid, or a host cell containing the vector.
  • vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc.
  • Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is a bacterium of the Escherichia sp., and the ⁇ -amylase signal sequence and subtilis when the host is a bacterium of the genus Bacillus sp.
  • the host is yeast, the MF ⁇ signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. can be used.
  • the vector may also contain a selection marker for selecting host cells containing the vector and, if the vector is replicable, an origin of replication.
  • the vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • the vectors can be delivered in vivo or into cells through electroporation, lipofection, viral vectors, nanoparticles, as well as PTD (Protein translocation domain) fusion protein methods, respectively.
  • PTD Protein translocation domain
  • vector refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence.
  • Nucleic acid sequences may be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). Those skilled in the art can construct vectors by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).
  • expression control sequences such as a promoter, terminator, enhancer, etc. are added depending on the type of host cell to produce the compound of Formula 1 (DRL peptide).
  • sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways depending on the purpose.
  • the host cell is selected from the genus Escehreichia sp., Salmonellae sp., Yersinia sp., Shigella sp., and Enterobacter genus. sp.), Pseudomonas sp., Proteus sp., or Klebsiella sp., and Escherichia coli of the Escherichia genus is used for mass production of fusion proteins. It is more desirable.
  • the present invention relates to a fusion protein comprising the compound of Formula 1 (DRL peptide), an analog thereof, or a variant thereof, or a nucleic acid encoding the same.
  • fusion protein used in the present invention refers to a recombinant protein in which two or more proteins or domains responsible for a specific function within a protein are linked so that each protein or domain performs its original function.
  • a linker peptide which typically has a flexible structure, may be inserted between the two or more proteins or domains.
  • peptide used in the present invention refers to an amino acid polymer and may include not only natural amino acids but also non-proteinaceous amino acids as components.
  • variant refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) when compared to a reference material.
  • a “variant” has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions when compared to a reference sequence.
  • analogs may include analogs substituted with one or more other functional groups for the side chain of an amino acid or the alpha-amino acid backbone.
  • side chain or backbone modified peptide analogs include, but are not limited to, hydroxyproline or N-methyl glycine “peptoids” in which the pyrrolidine ring is replaced with a hydroxy group.
  • Types of protein analogs are known in the art.
  • Peptide variants according to the present invention are interpreted to also include variants in which amino acid residues are conservatively substituted at specific amino acid residue positions.
  • “conservative substitution” refers to a modification of a variant that involves substituting one or more amino acids with an amino acid having similar biochemical properties that does not cause loss of biological or biochemical function of the peptide variant.
  • a “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain.
  • Classes of amino acid residues with similar side chains are defined and well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), and amino acids with uncharged polar side chains (e.g., glycine).
  • amino acids with non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains amino acids with aromatic side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • the peptide or fusion protein containing the same according to the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method in the art.
  • the DRL peptide of Formula 1 of the present invention can be prepared by sequentially reacting aspartic acid, arginine, and leucine.
  • the peptide or fusion protein containing the same according to the present invention can be synthesized by a number of methods, including, for example, the following methods:
  • the present invention relates to a nucleic acid encoding the peptide of the present invention or a fusion protein containing the same, or a plasmid vector containing the nucleic acid.
  • the present invention relates to a compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, for specifically visualizing the synovial membrane, represented by the following formula (2):
  • the compound of Formula 2 (DRL-TAM) according to the present invention is a TAMRA fluorophore conjugated to a peptide library containing the compound of Formula 1 (DRL peptide) of the present invention using lysine and 6-aminohexanoic acid linkers. It may be possible. At this time, the carboxylic acid group at the para position of TAMRA may be bonded to the 6-amino group of lysine. The carboxylic acid group in lysine was linked to the DRL peptide using 6-aminohexanoic acid, which allows the final structure to retain hydrophobic features with structural flexibility that fully liberates the activity of the peptide.
  • the compound represented by Formula 2, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a composition for diagnosing arthritis disease.
  • the present invention relates to a pharmaceutically active ingredient conjugated to a compound of Formula 1 (DRL peptide) or a compound of Formula 2 (DRL-TAM), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof. It relates to a conjugated, synovial targeting drug delivery system.
  • the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM) of the present invention targets the synovium by binding to the cell membrane through competition with the ligand of the synovial microvascular endothelium. may be possible.
  • the pharmaceutically active ingredient may be RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule.
  • the drug is a peptide drug, protein drug, desensitizing agent, antigen, non-steroidal anti-inflammatory agent, anti-inflammatory drug, anesthetic agent, antioxidant, anti-infective agent, chemotherapeutic agent, anti-nociceptive agent, DMOAD, anabolic agent, It may be an anti-catabolic agent, an autophagy regulator, an anti-osteoclast-mediated bone loss agent, a nutraceutical agent, analgesic agent, biologics, or a mixture thereof.
  • the non-steroidal anti-inflammatory agent is etofenamate, celecoxib, apricoxib, rofecoxib, nabumetone, benorylate, etoricoxib, ampyroxicam, aminophemazone, val.
  • Decoxib Decoxib, acetominophen, bupexamak, nimesulide, parecoxib, mefenamic acid, dexibuprofen, ibuprofen, flurbiprofen, aspirin, dexdetoprofen, diclofenac, diflunisal, etodolac, Fenoprofen, firocoxib, flurbiprofen, indomethacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, loxomak, lumiracoxib, meclofenamic acid, mel It may be Roxicam, naproxen, naprosyn, nimalox, oxaporozin, piroxicam, salsalate, sulindac, tenoxicam, tolfenamic acid, ropivicaine, or mixtures thereof.
  • the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM) of the present invention and the pharmaceutically active ingredient may be linked/combined via a linker, and the linker may be a peptide, ligand, or antibody. , can bind through the amine group, carboxyl group, or sulfhydryl group of proteins such as antibody fragments, or the phosphate group or hydroxyl group of nucleic acids such as aptamers. Any linker having a functional group may be used.
  • the linker may be selected and used appropriately depending on the drug.
  • a linker having an aldehyde reactive group may be connected to the drug and bound to the N-terminal amino group of the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM).
  • linkers include isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, and glyoxal ( glyoxal, epoxide, oxirane, carbonate, arylhalide, imidoester, carbodiimide, anhydride, fluorophenyl ester It may be (fluorophenyl ester), hydroxymethyl phosphine, maleimide, haloacetyl, pyridyldisulfide, thiosulfonate, or vinylsulfone. .
  • the linker may be cleavable by protease, cleavable under acid or base conditions, cleavable by high temperature or light irradiation, cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions. It may be.
  • cleavable linkers include hydrazone linkers that are cleaved under acidic conditions, peptide linkers that are cleaved by proteases, and linkers with a disulfide functional group that are cleaved under reducing conditions.
  • linkers examples include: Maleimidomethyl cyclohexane-1-carboxylate (MCC) linker, maleimidocaproyl (MC) linker, or a derivative thereof such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linker or sulfosuccine. and imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).
  • MMCC Maleimidomethyl cyclohexane-1-carboxylate
  • sMCC maleimidocaproyl
  • sMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • sulfo-sMCC imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • the linker may be a self-immol
  • Self-immolative linkers include, for example, the linker disclosed in U.S. Pat. No. 9,089,614, entitled “Hydrophilic self-immolative linkers and conjugates thereof,” and the linker, entitled “SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES. THEREOF", the linker disclosed in International Publication No.
  • linkers that leave no trace after cutting include phenylhydrazide linker, aryl-triazene linker, and the literature [Blaney, et al., "Traceless solid-phase organic synthesis,” Chem Rev. 102: 2607-2024 (2002)], etc.
  • Biocompatible polymers refer to polymers that have tissue compatibility and anticoagulant properties that do not cause tissue necrosis or blood coagulation in contact with biological tissue or blood.
  • Synthetic polymers as biocompatible polymers include polyester, polyhydroxyalkanoate (PHAs), poly( ⁇ -hydroxyacid), poly( ⁇ -hydroxyacid), and poly(3-hydrosybutyrate-co).
  • PHBV poly(3-hydroxypropionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxy acid), poly(4-hydroxy butyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycoside) ride; PLGA), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), polycyano Acrylate, poly(trimethylene carbonate), poly(iminocarbonate), poly(tyrosine carbonate), polycarbonate, poly(tyrosine arylate), polyalkylene oxalate, polyphosphazene, PHA-PEG, ethylene vinyl Alcohol copolymers (EVOH), polyurethane
  • Vinyl ketone polyvinyl aromatics, polystyrene, polyvinyl ester, polyvinyl acetate, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer, polyamide, alkyd resin.
  • polyoxymethylene, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-co-maleic acid or polyaminoamine, and natural polymers include chitosan, dextran, cellulose, heparin, hyaluronic acid, and alginate. , inulin, starch or glycogen.
  • the synovial targeting drug carrier of the present invention can be prepared as a pharmaceutical composition in oral formulation or parenteral formulation depending on the route of administration by a conventional method known in the art including a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
  • the present invention relates to a pharmaceutical composition for preventing or treating arthritis disease containing the synovium targeting drug delivery system of the present invention as an active ingredient.
  • the arthritic condition is synovitis, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, It could be arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.
  • the composition may further include a marker, and in this case, arthritis disease can be diagnosed and treated simultaneously.
  • the pharmaceutical composition of the present invention may further include known therapeutic agents for arthritis diseases in addition to torsemide and/or cromolyn, or salts thereof, as active ingredients, and may be used in combination with other known treatments for the treatment of these diseases. there is.
  • prevention refers to any action that inhibits or delays the occurrence, spread, and recurrence of arthritis disease by administering the pharmaceutical composition according to the present invention
  • treatment refers to all actions that inhibit or delay the occurrence, spread, and recurrence of arthritis disease by administering the pharmaceutical composition according to the present invention. It refers to all actions that improve or beneficially change the symptoms of arthritis disease.
  • Korean Medical Association etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.
  • the term "therapeutically effective amount” used in combination with an active ingredient in the present invention refers to an amount effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention is determined by several factors, such as the method of administration. , may vary depending on the target area, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type of arthritic disease, cause of arthritic disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, combination or drugs used simultaneously, and It may be determined based on other factors well known in the medical field.
  • composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products.
  • a carrier diluent, excipient, or a combination of two or more commonly used in biological products.
  • pharmaceutically acceptable means that the composition exhibits non-toxic properties to cells or humans exposed to the composition.
  • the carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc.
  • saline solution sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added.
  • diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets.
  • it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays, with oral or injectable formulations being more preferable.
  • the term "administration” means providing a predetermined substance to an individual or patient by any appropriate method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally) according to the desired method. Alternatively, it can be applied topically as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease.
  • Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
  • the pharmaceutical composition of the present invention may be administered by any device capable of transporting the active agent to target cells.
  • Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection.
  • Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.).
  • stabilizers to prevent deterioration
  • emulsifiers e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.
  • buffers for pH adjustment e.g., buffers for pH adjustment
  • agents to prevent microbial growth e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.
  • emulsifiers e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.
  • emulsifiers e.g., buffers for pH adjustment
  • agents to prevent microbial growth e.g., buffers for pH adjustment, and
  • the term "individual” refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, including humans who have or may develop the arthritic disease. Or, it refers to all animals including guinea pigs, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
  • the pharmaceutical composition of the present invention can be administered in combination with existing therapeutic agents.
  • the pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc may be used.
  • the pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
  • the present invention relates to a method for preventing or treating arthritic diseases, comprising administering to a subject the pharmaceutical composition for preventing or treating arthritic diseases of the present invention in a therapeutically effective amount.
  • the method for preventing or treating the arthritic disease may be administering a composition for preventing or treating the arthritic disease to an individual in need of treatment.
  • the present invention provides a compound of Formula 1 (DRL peptide) or a compound of Formula 2 (DRL-TAM) of the present invention, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, or a synovial membrane targeting agent of the present invention. It relates to a synovial contrast agent composition containing a drug carrier as an active ingredient.
  • the composition may further include a labeling substance.
  • the contrast medium composition can be used for diagnosing arthritis disease.
  • the present invention includes the steps of treating a human or animal with the synovial contrast agent composition of the present invention and irradiating an excitation light source; and measuring the change in emitted fluorescence. It relates to a method for diagnosing arthritis disease, including.
  • the step of measuring the fluorescence change may include a method of confirming that the fluorescence emission intensity of the joint to be measured is increased compared to the fluorescence intensity before the synovial contrast agent composition is treated.
  • the present invention relates to a method of imaging the synovial membrane using the synovial contrast agent composition of the present invention.
  • the method for imaging the synovial membrane of the present invention includes treating a human or animal with the synovial contrast agent composition of the present invention and irradiating an excitation light source; And measuring the change in fluorescence emitted; may include targeting and imaging the synovial membrane.
  • the present invention relates to a method for producing a compound represented by the following formula (1), comprising the step of sequentially reacting aspartic acid, arginine, and leucine:
  • the method for preparing the compound represented by Formula 1 includes adding aspartic acid and arginine to N,N-dimethylformamide and performing a primary reaction; And it may include adding N,N-dimethylformamide to perform a secondary reaction with leucine to form a peptide.
  • the step of forming the peptide may further include recovering the reacted material, drying and concentrating it.
  • a purification process using high-performance liquid chromatography may be further included after the drying and concentrating steps.
  • the present invention is represented by the following formula 2, comprising the step of reacting a compound represented by the following formula 1, lysine-conjugated TAMRA (5(6)-Carboxytetramethylrhodamine) and diisopropylethylamine. It relates to a method of preparing a compound that is:
  • reaction of the method for producing the compound represented by Formula 2 may be carried out using N,N-dimethylformamide as a solvent.
  • the method for producing the compound represented by Formula 2 may further include the step of recovering, drying and concentrating the reacted material after the reacting step, and after the drying and concentrating step, high performance A purification process using liquid chromatography may be additionally included.
  • the compound (DRL peptide) of the present invention In order to confirm the efficacy of the compound (DRL peptide) of the present invention, the compound (DRL peptide), lysine-conjugated 5-Carboxytetramethylrhodamine (TAMRA) and diisopropylethylamine were mixed with N,N-dimethylform. It was added to amide and reacted overnight to prepare DRL-TAM (Formula 2), in which Tamra was conjugated to DRL peptide as a labeling material. The formed material was separated and purified using high-performance liquid chromatography.
  • TAMRA 5-Carboxytetramethylrhodamine
  • DRL-TAM Form 2
  • DRL-TAM absorbance and fluorescence emission graphs were measured to determine whether the DRL-TAM prepared in Example 1 was stably maintained under various solvent conditions and various pH conditions. Fluorescence stability was confirmed. Specifically, to ensure stability when using cells and living organisms, distilled water (deionized water; DI.H 2 O), physiological saline (phosphate-buffered saline; PBS, pH 7.4), and cell culture medium (Dulbecco's modified Eagle's media; DMEM media) ) The absorbance and fluorescence emission of DRL-TAM (10 ⁇ M) were confirmed under the conditions.
  • DRL-TAM (10 ⁇ M) were confirmed under pH 3, 5, 7, and 9 conditions.
  • light with a wavelength of 530 nm (50 mW/cm 2 ) was irradiated for 60 minutes, and the fluorescence intensity was checked at 10-minute intervals.
  • a UV/Vis spectrophotometer (Agilent Technologies Cary 8454, USA) was used to analyze the absorption spectrum (UV/Vis absorption spectra), and a fluorescence photometer (SHIMADZU CORP. RF-6000) was used to analyze the fluorescence spectrum.
  • the absorption spectrum increases with the maximum absorption wavelength at 554 nm in all solutions, and the fluorescence emission spectrum increases between 580 nm and 600 nm (Figure 1), which is suitable for use in cells and living organisms. It was confirmed that the fluorescence intensity was strong enough to be applied to bio-imaging within cells and organs/tissues. Additionally, under various pH conditions, the absorption spectrum increases between 550 nm and 560 nm for all solutions, and the fluorescence emission spectrum appears to show a maximum fluorescence intensity at 583 nm ( Figure 2), which supports fluorescence-based screening under multiple in vivo conditions. It was confirmed that it exhibits a strong fluorescence intensity that can be used for bio-imaging. In addition, the fluorescence intensity showed almost no change for 60 minutes upon light irradiation ( Figure 3), confirming that it exhibits high photostability.
  • Rat synovium-derived primary cells were isolated from the hind limb synovium of the 10-week-old male SD rat (DBL Co., Ltd., Incheon, Korea).
  • Mouse brain tissue-derived endothelial cells mouse cerebrovascular endothelial carcinoma cell line, B.End3
  • human embryonic kidney-derived epithelial cells HEK293
  • DMEM Dulbecco's modified Eagle's media
  • fetal bovine serum Hyclone, US
  • penicillinstreptomycin Gabco, US
  • cytotoxic response was confirmed through Cell Counting Kit (CCK)-8 (Dojindo, Japan) assay using DRL-TAM.
  • RSPC rat synovial membrane-derived primary cells
  • B.End3 mouse brain tissue-derived endothelial cells
  • HEK293 human embryonic kidney-derived epithelial cells
  • DPBS Dulbecco's phosphate buffered saline
  • each cell was treated with DRL-TAM at a concentration of 1 to 100 ⁇ M and cultured in a 5% CO 2 incubator at 37°C for 24 hours, and then the cells were washed twice with phosphate-buffered saline (PBS, pH 7.4). After that, 10 ⁇ L of CCK-8 solution was added and cultured at 37°C for 1 hour. Next, the optical density was measured at a wavelength of 450 nm using a microplate reader (Multiskan FC, Thermo Fisher, MA, US), and the cell viability was measured using the absorbance value. Cell survival rate (%) was calculated as (test group/control group) ⁇ 100 and expressed as mean ⁇ standard error of the mean (SEM).
  • DRL-TAM (1-100 ⁇ M) did not exhibit significant toxicity in both primary cells derived from mouse synovium, endothelial cells derived from mouse brain tissue, and epithelial cells derived from human embryonic kidney (FIG. 4).
  • DRL-TAM In order to confirm whether the compound of the present invention (DRL peptide) exhibits a toxic reaction when used in vivo, the hemolytic reaction in mouse blood was confirmed using DRL-TAM. Specifically, blood collected directly from the heart of an anesthetized mouse was anticoagulated, and the plasma was washed twice with physiological saline (cold 1 ⁇ PBS) using a centrifuge (4°C, 1300rpm, 3 minutes) to produce red blood cells (RBCs). ) were isolated purely. Purified red blood cells (8%, v/v cold 1 ⁇ PBS) were treated with DRL-TAM at a concentration of 1-100 ⁇ M and incubated for 1 hour in a shaking incubator (300 rpm, 37°C).
  • rat synovium-derived primary cells (RSPC) of Preparation Example 1 and mouse brain tissue-derived endothelial cells (B.End3) as a control were used.
  • mouse brain tissue-derived endothelial cells B.End3
  • human embryonic kidney-derived epithelial cells (HEK293) were seeded on a 35 mm glass bottom confocal dish (SPL Life Science, Korea Rep.) and cultured at 37°C for 24 hours.
  • Each cell was treated with DRL-TAM (10 ⁇ M) for 24 hours in a 5% CO 2 incubator at 37°C, washed with PBS (pH 7.4), removed, and DAPI (4' ,6-diamidino-2-phenylindole) and CellMask Green plasma membrane stain were treated with 2 ⁇ L each (1000 ⁇ working concentration) for 20 minutes in a 5% CO 2 incubator and serum-free medium.
  • the cells were washed three times with PBS (pH 7.4), treated with 4% formaldehyde for 6 minutes, washed again with PBS (pH 7.4) to remove the solution, and then analyzed using a confocal laser scanning microscope ( The images of the cells were confirmed using CLSM, LSM-800, Carl Zeiss, Germany), and the fluorescence intensity of DRL-TAM was compared and analyzed in each cell.
  • the excitation and emission channels were red (560nm, 565-700nm), green (488nm, 520-550nm), and blue (405nm, 450-520nm).
  • DRL-TAM showed significantly higher fluorescence intensity specifically in synovium-derived primary cells than in the two cells used as controls, and DRL-TAM showed fluorescence intensity at the same location as CellMask Green plasma membrane stain, which stains the cell membrane. As it was clearly shown to be present in the cell membrane of mouse synovial membrane-derived cells ( Figure 6), it was confirmed that DRL-TAM has a synovial targeting effect and can be visualized.
  • DRL peptide DRM-TAM and rhodamine B (Rho), a structural analogue of Tamra, were administered to 6-week-old mice as a control. It was administered into the vagus vein at a concentration of 5 mg/kg, and the biodistribution process of the substance over time was confirmed through fluorescence tracking. The biodistribution of DRL-TAM and Rho was examined through whole-body fluorescence imaging using a fluorescence tissue imaging system (FTIS, VISQUE® InVivo Elite, Vieworks Co. Ltd, Korea) (530-570 nm excitation, 575-640 nm detection channel). Monitored.
  • FTIS fluorescence tissue imaging system
  • the compound of the present invention shows a specific targeting effect on a specific area in the body, that is, the synovial membrane, and that it can be used to visualize specific areas rich in the body's synovial membrane.

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Abstract

The present invention relates to a compound specifically targeting synovium, and uses thereof. The compound prepared according to the present invention exhibits no toxicity in vitro and in vivo, is stable in biological environments, can specifically target and visualize synovial cells and synovial areas in the body, and thus can be used in drug development and medical fields and be effectively used in the diagnosis and treatment of synovium-associated diseases.

Description

활막 표적화 화합물 및 이의 용도Synovial membrane targeting compounds and uses thereof
본 발명은 활막을 특이적으로 표적화하는 화합물 및 이의 용도에 관한 것이다.The present invention relates to compounds that specifically target the synovial membrane and their uses.
관절염은 관절 및 주변 조직에 영향을 미치는 상태에 대한 일반적인 용어이다. 관절은 무릎, 손목, 손가락, 발가락, 및 엉덩이와 같이, 뼈들이 함께 있는 신체 내에 위치한다. 관절염의 2가지 가장 일반적인 유형은 골관절염 및 류마티스 관절염이다. 골관절염(Osteoarthritis, OA)은 미국에서만 2천만명 이상의 개인에게 영향을 미치는 가장 일반적인 유형의 관절 질환이다. 이는 70세 이상의 노인에게서 만성 장애의 주된 원인이며, 미국에서 매년 1,850억 달러 이상을 소비한다. 이는 엉덩이, 무릎, 목, 등 아래, 또는 손의 작은 관절을 흔히 포함하는 고통스러운, 퇴행성 관절 질환이다. 골관절염은 특수한 업무를 수행하거나 좋아하는 운동을 하거나 과도한 체중를 지니고 다님으로써 반복된 과다사용으로 인해 손상되는 관절에서 일반적으로 진행된다. 골관절염은 활막 관절내 관절(유리질) 연골의 생화학적 파괴를 유발하는 퇴행성 장애로 고려될 수 있다. 그러나, 현재의 관점은 관절염이 관절 연골 뿐만 아니라 연하골 및 활막을 포함하는 전체 관절 기관을 포함하는 것도 유지하고 있다. 류마티스 관절염(rheumatoid arthritis, RA)은 손가락, 엄지손가락, 손목, 팔꿈치, 어깨, 무릎, 발, 및 발목에서 다양한 관절을 일반적으로 포함하는 자가염증성 질환으로, 관절 주위를 둘러싸고 있는 활막이라는 조직의 염증 때문에 일어나며, 전체 인구의 약 1~2% 가량에서 발병하는 것으로 알려진 질환이다(Alamanosa and Drosos, Autoimmun. Rev., 4:130-136(2005)). 류마티스 관절염의 발병은 유전적-환경적 인자가 작용하며, 유전적인 요인이 대략 60% 정도 기여하는 것으로 알려져 있다. 또한, 관절 내 부위 또는 관절 주변 부위에만 영향을 미치는 다른 관절염 상태들과는 달리, 류마티스 관절염은 피부, 혈관, 심장, 폐 및 근육을 포함하는 신체에 걸쳐 관절 외 조직에서 염증을 야기할 수 있는 전신성 질환이다. 통풍은 활액 및 다른 조직 속에서 요산일나트륨 결정의 침착 및 신장에서 요산 결석의 형성으로부터 생성되는 질환이다. 통풍은 전형적으로 중년기 동안에 일어나며 30세 이전에는 일반적이지 않다. 여성은 폐경기 전에는 통풍 관절염 공격을 거의 받지 않는다.Arthritis is a general term for a condition that affects joints and surrounding tissues. Joints are located in the body where bones come together, such as the knees, wrists, fingers, toes, and hips. The two most common types of arthritis are osteoarthritis and rheumatoid arthritis. Osteoarthritis (OA) is the most common type of joint disease, affecting more than 20 million individuals in the United States alone. It is the leading cause of chronic disability in people over 70 years of age and costs more than $185 billion annually in the United States. This is a painful, degenerative joint disease that often involves the small joints of the hips, knees, neck, lower back, or hands. Osteoarthritis usually develops in joints that are damaged by repeated overuse, such as performing special tasks, playing a favorite sport, or carrying excessive weight. Osteoarthritis can be considered a degenerative disorder that causes biochemical destruction of synovial intra-articular (hyaline) cartilage. However, current views maintain that arthritis involves not only the articular cartilage but also the entire joint organ, including the subchondral bone and synovium. Rheumatoid arthritis (RA) is an autoinflammatory disease that commonly involves various joints in the fingers, thumbs, wrists, elbows, shoulders, knees, feet, and ankles, and is caused by inflammation of the tissue called the synovium that surrounds the joints. It is a disease known to occur in approximately 1-2% of the total population (Alamanosa and Drosos, Autoimmun. Rev., 4:130-136 (2005)). The onset of rheumatoid arthritis is influenced by genetic and environmental factors, and genetic factors are known to contribute approximately 60%. Additionally, unlike other arthritic conditions that only affect intra-articular or peri-articular areas, rheumatoid arthritis is a systemic disease that can cause inflammation in extra-articular tissues throughout the body, including the skin, blood vessels, heart, lungs, and muscles. . Gout is a disease that results from the deposition of monosodium urate crystals in synovial fluid and other tissues and the formation of uric acid stones in the kidneys. Gout typically occurs during middle age and is not common before the age of 30. Women rarely suffer attacks of gouty arthritis before menopause.
활막(활액 또는 활액막)(synovium)은 관절연골에 가장 근접해 있는 조직으로서 활막 연골종증 및 골연골 스퍼스와 같은 악성 상태에서 유리연골을 생성할 수 있는 유일한 조직이며, 연골손상 복구를 위하여 활막으로부터 세포들이 리크루팅된다는 보고가 있었다.Synovium (synovium) is the tissue closest to articular cartilage and is the only tissue capable of producing hyaline cartilage in malignant conditions such as synovial chondromatosis and osteochondral spurs. Cells from the synovium are extracted to repair cartilage damage. There was a report that he was being recruited.
한편, 특정 부위를 표적화하는 방법에 대한 연구는 여러 질병의 정밀 치료 및 정밀 진단과 같은 의료 기술 개발과 함께 지속적으로 관심 받아온 분야이다. 특정 부위의 표적화하는 방법으로는 유전자 및 폴리에틸렌글리콜, 펩타이드 등과 같은 특정 물질을 이용하여 연구 개발되어지고 있다. 그 중에서 펩타이드를 이용한 표적화 방법은 낮은 면역원성, 저비용, 장기 보관 및 쉬운 취급과 같은 장점을 가지고 있어서 많은 각광을 받아온 분야이다(Accardo, A., Tesauro, D., & Morelli, G. Polymer journal, 2013, 45(5), 481-493.).Meanwhile, research on methods to target specific areas is an area that has continued to receive attention along with the development of medical technologies such as precise treatment and precise diagnosis of various diseases. Methods for targeting specific areas are being researched and developed using specific substances such as genes, polyethylene glycol, and peptides. Among them, targeting methods using peptides have received a lot of attention because they have advantages such as low immunogenicity, low cost, long-term storage, and easy handling (Accardo, A., Tesauro, D., & Morelli, G. Polymer journal, 2013, 45(5), 481-493.).
활막 부위내에 약제학적으로 활성인 유효성분을 보유함으로써 약물이 장기간에 걸쳐 전달되도록 하는, 포유동물내 관절에 약제학적으로 활성인 유효성분을 전달하는 것이 당해 기술에서 여전히 요구되고 있다.There is still a need in the art to deliver pharmaceutically active ingredients to joints in mammals, allowing the drug to be delivered over a long period of time by retaining the pharmaceutically active ingredients within the synovial region.
본 발명의 목적은 활막을 표적화하는 물질을 제공하는 것이다.The object of the present invention is to provide a substance targeting the synovial membrane.
또한, 본 발명의 목적은 활막을 특이적으로 시각화하는 물질을 제공하는 것이다.Additionally, an object of the present invention is to provide a material that specifically visualizes the synovial membrane.
또한, 본 발명의 목적은 상기 물질들의 제조방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for producing the above materials.
또한, 본 발명의 목적은 활막 표적화 약물 전달체를 제공하는 것이다.Additionally, an object of the present invention is to provide a synovium targeting drug delivery system.
또한, 본 발명의 목적은 관절염 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis diseases.
아울러, 본 발명의 목적은 활막 조영제 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a synovial contrast agent composition.
상기 목적의 달성을 위해, 본 발명은 활막을 표적화하는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염을 제공한다.To achieve the above object, the present invention provides a compound targeting the synovial membrane, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
또한, 본 발명은 활막을 특이적으로 시각화하는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염을 제공한다.Additionally, the present invention provides a compound that specifically visualizes the synovial membrane, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
또한, 본 발명은 상기 화합물의 제조방법을 제공한다.Additionally, the present invention provides a method for producing the above compound.
또한, 본 발명은 상기 물질에 약제학적 활성 성분이 접합된, 활막 표적화 약물 전달체를 제공한다.In addition, the present invention provides a synovium-targeting drug delivery system in which a pharmaceutically active ingredient is conjugated to the material.
또한, 본 발명은 상기 약물 전달체를 포함하는 관절염 질환의 예방 또는 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing or treating arthritis disease, comprising the drug carrier.
아울러, 본 발명은 활막 조영제 조성물을 제공한다.In addition, the present invention provides a synovial contrast agent composition.
본 발명에 따라 제조된 화합물은 in vitro 및 in vivo에서 독성을 나타내지 않고, 생물학적 환경에서 안정적이었으며, 활막 세포 및 생체 내 활막 부위를 특이적으로 표적화할 수 있으므로, 이를 약물 개발 및 의료 분야에 이용할 수 있으며, 활막 관련 질환의 진단 및 치료에 유용하게 활용될 수 있다.The compound prepared according to the present invention does not exhibit toxicity in vitro and in vivo , is stable in the biological environment, and can specifically target synovial cells and synovial areas in vivo, so it can be used in drug development and medical fields. It can be useful in the diagnosis and treatment of synovial-related diseases.
도 1은 다양한 용매 조건(증류수, 생리 식염수 및 세포 배양액)에서 본 발명의 DRL-TAM의 흡광 및 형광 방출 특성을 확인한 결과를 나타낸 도이다. Figure 1 is a diagram showing the results of confirming the absorption and fluorescence emission characteristics of the DRL-TAM of the present invention under various solvent conditions (distilled water, saline solution, and cell culture medium).
도 2는 다양한 pH 조건(pH 3, 5, 7 및 9)에서 본 발명의 DRL-TAM의 흡광 및 형광 방출 특성을 확인한 결과를 나타낸 도이다.Figure 2 is a diagram showing the results of confirming the absorption and fluorescence emission characteristics of the DRL-TAM of the present invention under various pH conditions ( pH 3, 5, 7, and 9).
도 3은 본 발명의 DRL-TAM의 광안정성을 확인한 결과를 나타낸 도이다.Figure 3 is a diagram showing the results of confirming the photostability of DRL-TAM of the present invention.
도 4는 3 종류의 세포에서 1 내지 100 μM 농도의 DRL-TAM의 세포 독성을 확인한 결과를 나타낸 도이다:Figure 4 shows the results of confirming the cytotoxicity of DRL-TAM at a concentration of 1 to 100 μM in three types of cells:
B.End3: 마우스 뇌조직 유래 내피 세포;B.End3: endothelial cells derived from mouse brain tissue;
HEK293: 인간 배아 신장 유래 상피 세포; 및 HEK293: human embryonic kidney-derived epithelial cells; and
RSPC; 마우스 활막 유래 일차 세포.RSPC; Mouse synovium-derived primary cells.
도 5는 본 발명의 DRL-TAM(1 내지 100 μM)의 용혈 반응을 분석한 결과를 나타낸 도이다(**** Triton X-100 처리군 대비 p < 0.0001).Figure 5 is a diagram showing the results of analyzing the hemolysis reaction of DRL-TAM (1 to 100 μM) of the present invention (**** p < 0.0001 compared to Triton X-100 treatment group).
도 6은 본 발명의 DRL-TAM의 in vitro 활막 표적화 효과 및 시각과 효과를 확인한 결과를 나타낸 도이다:Figure 6 is a diagram showing the results of confirming the in vitro synovium targeting effect and visual effects of the DRL-TAM of the present invention:
B.End3: 마우스 뇌조직 유래 내피 세포;B.End3: endothelial cells derived from mouse brain tissue;
HEK293: 인간 배아 신장 유래 상피 세포;HEK293: human embryonic kidney-derived epithelial cells;
RSPC; 마우스 활막 유래 일차 세포;RSPC; mouse synovium-derived primary cells;
a: 공초점 현미경 이미지(축척 막대: 50μm);a: Confocal microscopy image (scale bar: 50 μm);
b: DRL-TAM의 형광 세기; 및b: Fluorescence intensity of DRL-TAM; and
c: CellMask Green 세포막 염색(CellMask Green plasma membrane stain)과 동일한 위치에서의 DRL-TAM의 형광 세기(** RSPC 그룹 대비 p < 0.01).c: CellMask Green plasma membrane stain and fluorescence intensity of DRL-TAM at the same location (** p < 0.01 compared to RSPC group).
도 7은 PBS(pH 7.4), Rho 및 DRL-TAM(i.v., 5 mg/kg) 투여후 시간 경과에 따른 생체 내에서의 분포 과정을 확인한 생체외 형광 이미지를 나타낸 도이다(여기: 530-570 nm, 방출 채널: 575-640 nm).Figure 7 is a diagram showing in vitro fluorescence images confirming the distribution process in vivo over time after administration of PBS (pH 7.4), Rho, and DRL-TAM (i.v., 5 mg/kg) (here: 530-570) nm, emission channel: 575-640 nm).
도 8은 PBS(pH 7.4), Rho 및 DRL-TAM(i.v., 5 mg/kg) 투여 및 10분 혈액 순환 후 신체 장기 및 부위에서의 분포 과정을 확인한 생체외 형광 이미지 및 복사 효율을 정량화한 그래프를 나타낸 도이다(PBS 처리군 대비, **p<0.01, ***p<0.001, ****p<0.0001; Rho 처리군 대비, ##p < 0.01, ###p < 0.001, ####p < 0.0001).Figure 8 is an in vitro fluorescence image confirming the distribution process in body organs and parts after administration of PBS (pH 7.4), Rho and DRL-TAM (i.v., 5 mg/kg) and blood circulation for 10 minutes, and a graph quantifying the radiation efficiency. (compared to the PBS treatment group, **p<0.01, ***p<0.001, ****p<0.0001; compared to the Rho treated group, ##p < 0.01, ###p < 0.001, # ###p < 0.0001).
도 9는 활막을 특이적으로 시각화시킬 수 있는 본 발명의 DRL-TAM의 화학식을 나타낸 도이다(D: 아스파르트산, R: 아르기닌, L: 류신).Figure 9 is a diagram showing the chemical formula of DRL-TAM of the present invention, which can specifically visualize the synovial membrane (D: aspartic acid, R: arginine, L: leucine).
이하, 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 이에 의해 본 발명이 제한되지는 않으며 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail through embodiments of the present invention. However, the following embodiments are provided as examples of the present invention, and the present invention is not limited thereto, and the present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom. .
달리 정의되지 않는 한, 본원에서 사용된 모든 기술적 및 과학적 용어는 본 발명이 속하는 분야의 당업자가 통상적으로 이해하는 것과 동일한 의미를 갖는다. 본원에 기술된 것들과 유사하거나 등가인 임의의 방법 및 재료가 본 발명을 테스트하기 위한 실행에서 사용될 수 있지만, 바람직한 재료 및 방법이 본원에서 기술된다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the invention, the preferred materials and methods are described herein.
본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대한 통상의 1문자 및 3문자 코드가 사용될 뿐만 아니라 Aib(
Figure PCTKR2023009907-appb-img-000001
-아미노이소부티르산), Sar(N-methylglycine) 등과 같은 다른 아미노산에 대해 일반적으로 허용되는 3문자 코드가 사용된다. 또한 본 발명에서 약어로 언급된 아미노산은 하기와 같이 IUPAC-IUB 명명법에 따라 기재되었다:Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as Aib (
Figure PCTKR2023009907-appb-img-000001
For other amino acids, such as -aminoisobutyric acid), Sar (N-methylglycine), etc., generally accepted three-letter codes are used. Additionally, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
알라닌: A, 아르기닌: R, 아스파라긴: N, 아스파르트산: D, 시스테인: C, 글루탐산: E, 글루타민: Q, 글리신: G, 히스티딘: H, 이소류신: I, 류신: L, 리신: K, 메티오닌: M, 페닐알라닌: F, 프롤린: P, 세린: S, 트레오닌: T, 트립토판: W, 티로신: Y 및 발린: V. Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.
일 측면에서, 본 발명은 하기 화학식 1로 표시되는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염에 관한 것이다:In one aspect, the present invention relates to a compound represented by the following formula (1), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:
[화학식 1][Formula 1]
Figure PCTKR2023009907-appb-img-000002
Figure PCTKR2023009907-appb-img-000002
일 구현예에서, 본 발명의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 활막 표적화용일 수 있다.In one embodiment, the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt may be used for targeting the synovial membrane.
일 구현예에서, 본 발명의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 다양한 용매 조건 및 pH 조건, 및 빛 조사 조건에서 안정성이 높으며, 상기와 같은 다양한 조건에서도 농도에 비례하여 효능을 안정적으로 나타낼 수 있다.In one embodiment, the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt is highly stable under various solvent conditions, pH conditions, and light irradiation conditions, and is proportional to concentration even under various conditions such as above. As a result, the efficacy can be shown stably.
일 구현예에서, 본 발명의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 세포 독성이 없고 생체 내에서 용혈 반응을 나타내지 않아 안전할 수 있다.In one embodiment, the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt is non-cytotoxic and does not exhibit a hemolytic reaction in vivo, so it may be safe.
일 구현예에서, 본 발명의 화학식 1로 표시되는 화합물은 약학적으로 허용 가능한 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디아이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, 하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.In one embodiment, the compound represented by Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Ide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Includes malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면, 상기 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한, 이 혼합물에서 용매나 과량의 산을 증발시켜서 건조하거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다. 또한, 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이 때, 금속염으로는 나트륨, 칼륜 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone or acetonitrile. It can be manufactured by ordering. Additionally, this mixture can be prepared by evaporating the solvent or excess acid and drying it, or by suction-filtering the precipitated salt. Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering off the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, calun, or calcium salts as metal salts. Additionally, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).
일 구현예에서, 본 발명의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 표지 물질을 추가로 포함할 수 있으며, 표지 물질은 발색효소, 방사성동위원소, 크로모포어(chromophore), 발색 물질, 발광 물질, 형광 물질(fluorescer), 상자성 입자(superparamagnetic particles) 및 초상자성입자(ultrasuper paramagnetic particles)로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment, the compound of the present invention, its hydrate, its solvate, or its pharmaceutically acceptable salt may further include a labeling substance, and the labeling substance may be a chromogenic enzyme, a radioisotope, or a chromophore. ), coloring materials, light-emitting materials, fluorescers, superparamagnetic particles, and ultraparamagnetic particles.
일 구현예에서, 발색효소는 스트렙트아비딘(streptavidin)이 결합된 홀스래디시 퍼옥시다아제(horseradish peroxidase; HRP), 알칼라인 포스파타아제(Alkaline phosphatase; AP), β-갈락토시다아제(galactosidase), 루시페라아제(luciferase), 아세틸콜린스테라제, 유레아제(urease), 카탈라아제(catalase), 아스파르기나아제(asparginase), 리보뉴클레아제(ribonuclease), 말레이트 데하이드로지나아제(malate dehydrogenase), 스타필로코칼 뉴클레아제(staphylococcal nuclease), 트리오스 포스페이트 이소머라아제(triose phosphate isomerase), 글루코오스산화효소, 사이토크롬 P450 및 퍼옥시다아제 화합물일 수 있다.In one embodiment, the chromogenic enzyme is streptavidin-conjugated horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and luciferase. (luciferase), acetylcholinesterase, urease, catalase, asparginase, ribonuclease, malate dehydrogenase, staphylococcal It may be a nuclease (staphylococcal nuclease), triose phosphate isomerase, glucose oxidase, cytochrome P450 and peroxidase compounds.
일 구현예에서, 형광 물질은 형광 단백질(fluorescent protein), 발광 단백질(photoprotein), 루시퍼라제(luciferase), 형광염료(fluorescent dye) 또는 시분해 형광입자(Time-resolved Fluorescence, TRF)일 수 있으며, 형광 물질은 알렉사 플루오르 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, 루피(Rubpy)(tris(2,2-bipyridyl)ruthenium(Ⅱ)), FITC(fluoresein Isothiocyanate), 로다민 6G(rhodamine 6G), 로다민 B(rhodamine B), TAMRA(5(6)-Carboxytetramethylrhodamine), 텍사스 레드(Texas Red), DAPI(4,6-diamidino-2-phenylindole) 및 쿠마린으로 이루어진 군으로부터 선택된 어느 하나 이상인 것이 더욱 바람직하고, TAMRA인 것이 가장 바람직하다.In one embodiment, the fluorescent material may be a fluorescent protein, photoprotein, luciferase, fluorescent dye, or time-resolved fluorescence (TRF), The fluorescent substances are Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rubpy (tris(2,2-bipyridyl)ruthenium(Ⅱ) ), FITC (fluoresein Isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (5(6)-Carboxytetramethylrhodamine), Texas Red, DAPI (4,6-diamidino-2) -phenylindole) and coumarin. It is more preferable that it is at least one selected from the group consisting of, and TAMRA is most preferable.
일 구현예에서, 상기 화학식 1로 표시되는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 활막 표적화 조성물로 사용될 수 있으며, 표적화 조성물은 용매, 산, 염기, 및 버퍼 용액 중에서 선택된 하나 이상을 더 포함할 수 있다. 상기 표적화 조성물은 용매, 버퍼 용액 또는 이들의 혼합물에 전술한 화합물을 첨가하고, 여기에 산 또는 염기를 첨가하여 준비될 수 있다. 또한 상기 표적화 조성물은 당해 기술분야에서 사용할 수 있는 다른 첨가제를 추가적으로 포함할 수 있다. 상기 조성물이 포함하는 용매, 산, 염기, 및 버퍼 용액의 함량은 요구되는 성능에 따라 적절히 조절될 수 있다. 상기 용매는 물, 테트라하이드로퓨란(tetrahydrofuran, THF), 메탄올, 에탄올, HI 수용액, N,N-디메틸포름아미드(N, N-dimethylformamide, DMF), 또는 이들의 조합을 포함할 수 있다. 또는 상기 조성물은 다른 약물 및 화학 분자와의 조합을 포함할 수 있다. 해당 약물은 특정 부위에 효능을 가지는 약물을 포함할 수 있으나, 이에 제한되지 않으며, 당해 기술 분야에서 약물, 조영제 영양제, 형광체 등 화학 시료로 사용할 수 있는 것이라면 모두 가능하다.In one embodiment, the compound represented by Formula 1, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a synovium targeting composition, and the targeting composition is selected from solvents, acids, bases, and buffer solutions. One or more may be included. The targeting composition can be prepared by adding the above-described compound to a solvent, buffer solution, or a mixture thereof, and adding an acid or base thereto. Additionally, the targeting composition may additionally include other additives that can be used in the art. The contents of the solvent, acid, base, and buffer solution contained in the composition can be appropriately adjusted depending on the required performance. The solvent may include water, tetrahydrofuran (THF), methanol, ethanol, HI aqueous solution, N,N-dimethylformamide (DMF), or a combination thereof. Alternatively, the composition may include combinations with other drugs and chemical molecules. The drug may include, but is not limited to, a drug that is effective in a specific area, and any drug that can be used as a chemical sample such as a drug, contrast medium, nutrient, or fluorescent substance in the art is possible.
일 구현예에서, 상기 화학식 1로 표시되는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 관절염 질환의 진단용 조성물로 사용될 수 있다.In one embodiment, the compound represented by Formula 1, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a composition for diagnosing arthritis disease.
일 구현예에서, 상기 관절염 질환은 활막과 연관된 관절염 질환일 수 있고, 퇴행성 관절염, 류마티스 관절염 등을 포함하는 것일 수 있으나, 이에 제한되지는 않는다.In one embodiment, the arthritic disease may be an arthritic disease associated with the synovium and may include degenerative arthritis, rheumatoid arthritis, etc., but is not limited thereto.
본 발명의 일 실시예에서, 신체 내 특정 부분을 특이적으로 표적화할 수 있는 새로운 펩타이드를 개발하였으며, 이 신규 펩타이드를 라이신 및 6-아미노헥산산 링커를 사용하여 TAMRA 형광단과 접합시킴으로써, DRL-TAM을 개발하였다(실시예 1 및 도 9 참조; D: 아스파르트산, R: 아르기닌, L: 류신). 본 발명의 일 실시예에 따르면, 신규 펩타이드 DRL 및 DRL-TAM은 in vitro 및 in vivo에서 독성을 나타내지 않았으며, 생물학적 조성인 수용액(pH 7.4)과 세포 배양액에서 안정적으로 사용 가능하고, 약물 동태학적으로 마우스의 체내에서 짧은 시간 안에 분포되었으며(10분 내지 20분), 특이적으로 활막 세포 또는 생체 내 활막 부위(활막이 많이 존재하는 흉골, 척수, 다리뼈, 발 등)를 표적화 및 시각화할 수 있어, 세포 단위와 생체 단위 모두에서 작용 가능할 수 있음을 확인하였다. In one embodiment of the present invention, a new peptide capable of specifically targeting a specific part of the body was developed, and this new peptide was conjugated with the TAMRA fluorophore using a lysine and 6-aminohexanoic acid linker, resulting in DRL-TAM. was developed (see Example 1 and FIG. 9; D: aspartic acid, R: arginine, L: leucine). According to one embodiment of the present invention, the new peptides DRL and DRL-TAM do not show toxicity in vitro and in vivo , can be stably used in biological aqueous solutions (pH 7.4) and cell cultures, and have excellent pharmacokinetic properties. It is distributed within the mouse body in a short period of time (10 to 20 minutes), and can specifically target and visualize synovial cells or synovial areas in vivo (sternum, spinal cord, leg bones, feet, etc., where synovium is abundant). It was confirmed that it can act on both the cellular and biological levels.
본 발명에서 용어, "표적화"는 특정 세포 또는 생체 내 부위를 표적화하는 능력을 말하며, 물질의 체내 분포 시 특이적으로 해당 부분으로 전달되는 특성을 의미한다. 표적화 특성은 특정 생체 부분에 한하여 나타나는 특성으로 해당 부분을 제외한 나머지 부분과 비교하여 유의적으로 많은 양이 전달되는 특성을 의미할 수 있다.In the present invention, the term “targeting” refers to the ability to target a specific cell or body part, and refers to the property of a substance being specifically delivered to the corresponding part when distributed in the body. Targeting characteristics are characteristics that appear only in a specific biological part and may mean that a significantly larger amount is delivered compared to the remaining parts excluding that part.
일 측면에서, 본 발명은 본 발명의 화학식 1의 화합물(DRL 펩타이드)을 암호화하는 핵산, 상기 핵산을 포함하는 벡터 또는 상기 벡터를 포함하는 숙주세포에 관한 것이다.In one aspect, the present invention relates to a nucleic acid encoding the compound of Formula 1 (DRL peptide) of the present invention, a vector containing the nucleic acid, or a host cell containing the vector.
일 구현예에서, 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 상기 시그널 서열에는 숙주가 에스케리치아 속(Escherichia sp.) 박테리아인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속(Bacillus sp.) 박테리아인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모(yeast)인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널 서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다. 또한 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고, 복제 가능한 벡터인 경우 복제 기원을 포함한다. 예를 들어, 상기 벡터는 "플라스미드"이며, 환형의 이중 가닥 DNA 루프를 지칭하는 것으로서, 여기에 예컨대, 표준 분자 클로닝 기법에 의해 추가적인 DNA 세그먼트가 삽입될 수 있다.In one embodiment, vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is a bacterium of the Escherichia sp., and the α-amylase signal sequence and subtilis when the host is a bacterium of the genus Bacillus sp. For new signal sequences, etc., if the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. However, it is not limited to this. The vector may also contain a selection marker for selecting host cells containing the vector and, if the vector is replicable, an origin of replication. For example, the vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
상기 벡터는 각각 전기천공법(electroporation), 리포펙션, 바이러스 벡터, 나노파티클(nanoparticles) 뿐만 아니라, PTD(Protein translocation domain) 융합 단백질 방법 등을 통해 생체 내 또는 세포 내로 전달될 수 있다.The vectors can be delivered in vivo or into cells through electroporation, lipofection, viral vectors, nanoparticles, as well as PTD (Protein translocation domain) fusion protein methods, respectively.
본 발명에서 용어, "벡터"는 핵산서열을 복제할 수 있는 세포로의 도입을 위해서 핵산서열을 삽입할 수 있는 전달체를 의미한다. 핵산서열은 외생(exogenous) 또는 이종(heterologous)일 수 있다. 벡터로서는 플라스미드, 코스미드 및 바이러스(예를 들면 박테리오파지)를 들 수 있으나, 이에 제한되지 않는다. 당업자는 표준적인 재조합 기술에 의해 벡터를 구축할 수 있다(Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; 및 Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994 등).In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). Those skilled in the art can construct vectors by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).
일 구현예에서, 상기 벡터의 제작 시, 상기 화학식 1의 화합물(DRL 펩타이드)을 생산하고자 하는 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In one embodiment, when constructing the vector, expression control sequences such as a promoter, terminator, enhancer, etc. are added depending on the type of host cell to produce the compound of Formula 1 (DRL peptide). , sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways depending on the purpose.
일 구현예에서, 숙주세포는 에스케리치아 속(Escehreichia sp.), 살모넬라 속(Salmonellae sp.), 예르시니아 속(Yersinia sp.), 쉬겔라 속(Shigella sp.), 엔테로박터 속(Enterobacter sp.), 슈도모나스 속(Pseudomonas sp.), 프로테우스 속(Proteus sp.) 또는 클레브시엘라 속(Klebsiella sp.)의 박테리아일 수 있으며, 에스케리치아 속의 Escherichia coli인 것이 융합 단백질의 대량 생산에 더욱 바람직하다. In one embodiment, the host cell is selected from the genus Escehreichia sp., Salmonellae sp., Yersinia sp., Shigella sp., and Enterobacter genus. sp.), Pseudomonas sp., Proteus sp., or Klebsiella sp., and Escherichia coli of the Escherichia genus is used for mass production of fusion proteins. It is more desirable.
일 측면에서, 본 발명은 본 발명의 화학식 1의 화합물(DRL 펩타이드), 이의 유사체 또는 이의 변이체를 포함하는 융합단백질 또는 이를 암호화하는 핵산에 관한 것이다.In one aspect, the present invention relates to a fusion protein comprising the compound of Formula 1 (DRL peptide), an analog thereof, or a variant thereof, or a nucleic acid encoding the same.
본 발명에서 사용되는 용어 "융합단백질"은 둘 이상의 단백질 또는 단백질 내 특정 기능을 담당하는 도메인이 각각의 단백질 또는 도메인이 본연의 기능을 담당하도록 연결된 재조합 단백질(recombinant protein)을 의미한다. 상기 둘 이상의 단백질 또는 도메인 사이에는 통상적으로 유연한 구조를 갖는 링커 펩타이드(linker peptide)가 삽입될 수 있다.The term "fusion protein" used in the present invention refers to a recombinant protein in which two or more proteins or domains responsible for a specific function within a protein are linked so that each protein or domain performs its original function. A linker peptide, which typically has a flexible structure, may be inserted between the two or more proteins or domains.
본 발명에서 사용된 용어 "펩타이드"는 아미노산 중합체로서, 천연 아미노산 뿐 아니라, 비단백질성 아미노산도 구성요소로 포함할 수 있다.The term “peptide” used in the present invention refers to an amino acid polymer and may include not only natural amino acids but also non-proteinaceous amino acids as components.
본 발명에서 사용된, 용어 "변이체"는 기준 물질과 비교하였을 때 최소한 한개의 아미노산 차이(치환, 삽입 또는 결손)를 포함하는 대응하는 아미노산 서열을 말한다. 특정 구체예들에 있어서 "변이체"는 기준 서열과 비교하였을 때 높은 아미노산 서열 상동성(homology) 및/또는 보존적 아미노산 치환, 결손 및/또는 삽입을 가진다. As used herein, the term “variant” refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) when compared to a reference material. In certain embodiments, a “variant” has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions when compared to a reference sequence.
본 발명에서 사용된, 용어 "유사체"는 아미노산의 측쇄 또는 알파-아미노산 백본에 대하여 하나 이상의 다른 기능기로 치환된 유사체를 포함할 수 있다. 측쇄 또는 백본 개질화 펩타이드 유사체의 예로는 피롤리딘 고리가 하이드록시기로 치환된 하이드록시프롤린이나, N-메틸 글리신 "펩토이드"를 들 수 있으나, 이로 제한되지 않는다. 단백질 유사체의 종류에 대해서는 당업계에 공지되어 있다.As used herein, the term “analog” may include analogs substituted with one or more other functional groups for the side chain of an amino acid or the alpha-amino acid backbone. Examples of side chain or backbone modified peptide analogs include, but are not limited to, hydroxyproline or N-methyl glycine “peptoids” in which the pyrrolidine ring is replaced with a hydroxy group. Types of protein analogs are known in the art.
본 발명에 따른 펩타이드 변이체는 특정 아미노산 잔기 위치에서 아미노산 잔기가 보존적으로 치환된 변이체도 포함하는 의미로 해석된다.Peptide variants according to the present invention are interpreted to also include variants in which amino acid residues are conservatively substituted at specific amino acid residue positions.
본 발명에서 "보존적 치환"이란 1개 이상의 아미노산을 해당 펩타이드 변이체의 생물학적 또는 생화학적 기능의 손실을 야기하지 않는 유사한 생화학적 특성을 갖는 아미노산으로 치환하는 것을 포함하는 변이체의 변형을 의미한다. "보존적 아미노산 치환"은 아미노산 잔기를 유사한 측쇄를 갖는 아미노산 잔기로 대체시키는 치환이다. 유사한 측쇄를 갖는 아미노산 잔기 부류는 해당 기술분야에 규정되어 있으며, 잘 알려져 있다. 이들 부류는 염기성 측쇄를 갖는 아미노산(예를 들어, 라이신, 아르기닌, 히스티딘), 산성 측쇄를 갖는 아미노산(예를 들어, 아스파르트산, 글루탐산), 대전되지 않은 극성 측쇄를 갖는 아미노산(예를 들어, 글리신, 아스파라진, 글루타민, 세린, 트레오닌, 티로신, 시스테인), 비-극성 측쇄를 갖는 아미노산(예를 들어, 알라닌, 발린, 류신, 이소류신, 프롤린, 페닐알라닌, 메티오닌, 트립토판), 베타-분지된 측쇄를 갖는 아미노산(예를 들어, 트레오닌, 발린, 이소류신) 및 방향족 측쇄를 갖는 아미노산(예를 들어, 티로신, 페닐알라닌, 트립토판, 히스티딘)을 포함한다.In the present invention, “conservative substitution” refers to a modification of a variant that involves substituting one or more amino acids with an amino acid having similar biochemical properties that does not cause loss of biological or biochemical function of the peptide variant. A “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are defined and well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), and amino acids with uncharged polar side chains (e.g., glycine). , asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains amino acids with aromatic side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
본 발명에 따른 펩타이드 또는 이를 포함하는 융합단백질은 표준 합성 방법, 재조합 발현 시스템, 또는 임의의 다른 당해 분야의 방법에 의해 제조될 수 있다. 예컨대, 본 발명의 화학식 1의 DRL 펩타이드는 아스파틱산 및 아르기닌, 류신을 순차적으로 반응시켜 제조할 수 있다. 본 발명에 따른 펩타이드 또는 이를 포함하는 융합단백질은, 예를 들어 하기를 포함하는 방법을 포함하는 다수의 방법으로 합성될 수 있다:The peptide or fusion protein containing the same according to the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method in the art. For example, the DRL peptide of Formula 1 of the present invention can be prepared by sequentially reacting aspartic acid, arginine, and leucine. The peptide or fusion protein containing the same according to the present invention can be synthesized by a number of methods, including, for example, the following methods:
(a) 단백질을 고체상 또는 액체상 방법의 수단으로 단계적으로 또는 단편 조립에 의해 합성하고, 최종 단백질 생성물을 분리 및 정제하는 방법; 또는(a) a method of synthesizing a protein stepwise or by fragment assembly by means of a solid phase or liquid phase method, and isolating and purifying the final protein product; or
(b) 단백질을 인코딩하는 핵산 작제물을 숙주세포 내에서 발현시키고, 발현 생성물을 숙주 세포 배양물로부터 회수하는 방법; 또는(b) a method of expressing a nucleic acid construct encoding a protein in a host cell and recovering the expression product from the host cell culture; or
(c) 단백질을 인코딩하는 핵산 작제물의 무세포 시험관 내 발현을 수행하고, 발현 생성물을 회수하는 방법; 또는(c) a method of performing cell-free in vitro expression of a nucleic acid construct encoding a protein and recovering the expression product; or
(a),(b) 및(c)의 임의의 조합으로 단백질 단편을 수득하고, 이어서 단편을 연결시켜 단백질을 수득하고, 당해 단백질을 회수하는 방법.A method of obtaining a protein fragment by any combination of (a), (b), and (c), and then linking the fragments to obtain a protein, and recovering the protein.
일 측면에서, 본 발명은 본 발명의 펩타이드 또는 이를 포함하는 융합단백질을 암호화하는 핵산 또는 상기 핵산을 포함하는 플라스미드 벡터에 관한 것이다.In one aspect, the present invention relates to a nucleic acid encoding the peptide of the present invention or a fusion protein containing the same, or a plasmid vector containing the nucleic acid.
일 측면에서, 본 발명은 하기 화학식 2로 표시되는, 활막을 특이적으로 시각화하기 위한, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염에 관한 것이다:In one aspect, the present invention relates to a compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, for specifically visualizing the synovial membrane, represented by the following formula (2):
[화학식 2][Formula 2]
Figure PCTKR2023009907-appb-img-000003
Figure PCTKR2023009907-appb-img-000003
본 발명에 따른 화학식 2의 화합물(DRL-TAM)은 TAMRA 형광단이 라이신 및 6-아미노헥산산 링커를 사용하여 본 발명의 화학식 1의 화합물(DRL 펩타이드)을 포함하는 펩타이드 라이브러리에 접합(conjugate)되는 것일 수 있다. 이때, TAMRA의 파라 위치에 있는 카르복실산 그룹은 라이신의 6-아미노 그룹과 결합되는 것일 수 있다. 라이신 내의 카르복실산 그룹은 6-아미노헥산산을 사용하여 DRL 펩타이드에 연결되었으며, 이를 통해 최종 구조에서 펩타이드의 활성이 온전하게 자유로워지는 구조적 유연성을 가진 소수성 특징을 보유할 수 있다.The compound of Formula 2 (DRL-TAM) according to the present invention is a TAMRA fluorophore conjugated to a peptide library containing the compound of Formula 1 (DRL peptide) of the present invention using lysine and 6-aminohexanoic acid linkers. It may be possible. At this time, the carboxylic acid group at the para position of TAMRA may be bonded to the 6-amino group of lysine. The carboxylic acid group in lysine was linked to the DRL peptide using 6-aminohexanoic acid, which allows the final structure to retain hydrophobic features with structural flexibility that fully liberates the activity of the peptide.
일 구현예에서, 상기 화학식 2로 표시되는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염은 관절염 질환의 진단용 조성물로 사용될 수 있다.In one embodiment, the compound represented by Formula 2, its hydrate, its solvate, or its pharmaceutically acceptable salt can be used as a composition for diagnosing arthritis disease.
일 측면에서, 본 발명은 본 발명의 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM), 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염에 약제학적 활성 성분이 접합(conjugate)된, 활막 표적화 약물 전달체에 관한 것이다.In one aspect, the present invention relates to a pharmaceutically active ingredient conjugated to a compound of Formula 1 (DRL peptide) or a compound of Formula 2 (DRL-TAM), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof. It relates to a conjugated, synovial targeting drug delivery system.
일 구현예에서, 본 발명의 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM)은 활막 혈관 내피(synovial microvascular endothelium)의 리간드와 경쟁작용을 통해 세포막에 결합함을 통하여 활막 표적화가 가능한 것일 수 있다.In one embodiment, the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM) of the present invention targets the synovium by binding to the cell membrane through competition with the ligand of the synovial microvascular endothelium. may be possible.
일 구현예에서, 약제학적 활성 성분은 RNA, DNA, 항체, 이펙터, 약물, 전구약물, 독소, 펩타이드 또는 전달 분자일 수 있다.In one embodiment, the pharmaceutically active ingredient may be RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule.
일 구현예에서, 약물은 펩타이드 약물, 단백질 약물, 탈감작화제, 항원, 비-스테로이드성 소염제, 소염 약물, 마취제, 항산화제, 항감염제, 화학치료제, 항-통각반응제, DMOAD, 동화작용제, 항-이화작용제, 자가소화작용 조절제, 항-파골세포-매개된 골 손실제, 기능식품제(nutraceutical agent), 진통제, 생물제제(biologics) 또는 이들의 혼합물일 수 있다.In one embodiment, the drug is a peptide drug, protein drug, desensitizing agent, antigen, non-steroidal anti-inflammatory agent, anti-inflammatory drug, anesthetic agent, antioxidant, anti-infective agent, chemotherapeutic agent, anti-nociceptive agent, DMOAD, anabolic agent, It may be an anti-catabolic agent, an autophagy regulator, an anti-osteoclast-mediated bone loss agent, a nutraceutical agent, analgesic agent, biologics, or a mixture thereof.
일 구현예에서, 상기 비-스테로이드성 소염제는 에토페나메이트, 셀레콕시브, 아프리콕시브, 로페콕시브, 나부메톤, 베노릴레이트, 에토리콕시브, 암피록시캄, 아미노페마존, 발데콕시브, 아세토미노펜, 부펙사막, 니메술리드, 파레콕시브, 메페남산, 덱시부프로펜, 이부프로펜, 플루르비프로펜, 아스피린, 덱스데토프로펜, 디클로페낙, 디플루니살, 에토돌락, 페노프로펜, 피로콕시브, 플루르비프로펜, 인도메타신, 케토프로펜, 케토록락, 로르녹시캄, 록소프로펜, 록소막, 루미라콕시브, 메클로페남산, 멜록시캄, 나프록센, 나프로신, 니말록스, 옥사포로진, 피록시캄, 살살레이트, 술린닥, 테녹시캄, 톨페남산, 로피비카인 또는 이의 혼합물일 수 있다.In one embodiment, the non-steroidal anti-inflammatory agent is etofenamate, celecoxib, apricoxib, rofecoxib, nabumetone, benorylate, etoricoxib, ampyroxicam, aminophemazone, val. Decoxib, acetominophen, bupexamak, nimesulide, parecoxib, mefenamic acid, dexibuprofen, ibuprofen, flurbiprofen, aspirin, dexdetoprofen, diclofenac, diflunisal, etodolac, Fenoprofen, firocoxib, flurbiprofen, indomethacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, loxomak, lumiracoxib, meclofenamic acid, mel It may be Roxicam, naproxen, naprosyn, nimalox, oxaporozin, piroxicam, salsalate, sulindac, tenoxicam, tolfenamic acid, ropivicaine, or mixtures thereof.
일 구현예에서, 본 발명의 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM)과 약제학적 활성 성분은 링커를 매개로 연결/결합될 수 있으며, 링커는 펩타이드나 리간드, 항체, 항체 단편 등의 단백질의 아민기(amine group), 카르복시기(carboxyl group) 또는 설프히드릴기(sulfhydryl group)나 압타머 등의 핵산의 인산기(phosphate group, 히드록시기(hydroxyl group)를 통해 결합할 수 있는 작용기를 가진 임의의 링커를 사용할 수 있다. In one embodiment, the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM) of the present invention and the pharmaceutically active ingredient may be linked/combined via a linker, and the linker may be a peptide, ligand, or antibody. , can bind through the amine group, carboxyl group, or sulfhydryl group of proteins such as antibody fragments, or the phosphate group or hydroxyl group of nucleic acids such as aptamers. Any linker having a functional group may be used.
일 구현예에서, 상기 링커는 약물에 따라 적절한 것을 선택하여 사용할 수 있다. 예컨대 약물에, 알데하이드 반응기를 가지는 링커를 연결하고 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM)의 N 말단의 아미노기에 결합시킬 수 있다.In one embodiment, the linker may be selected and used appropriately depending on the drug. For example, a linker having an aldehyde reactive group may be connected to the drug and bound to the N-terminal amino group of the compound of Formula 1 (DRL peptide) or the compound of Formula 2 (DRL-TAM).
이러한 링커의 작용기는 아이소티오시아네이트(isothiocyanate), 아이소시아네이트(isocyanates), 아실 아자이드(acyl azide), NHS 에스터(NHS ester), 설포닐 클로라이드(sulfonyl chloride), 알데하이드(aldehyde), 글리옥살(glyoxal), 에폭사이드(epoxide), 옥시레인(oxirane), 칼보네이트(carbonate), 아릴 할라이드(arylhalide), 이미도에스터(imidoester), 카보이미드(carbodiimide), 안하이드라이드(anhydride), 플루오로페닐 에스터(fluorophenyl ester), 히드록시메틸포스핀(hydroxymethyl phosphine), 말레이미드(maleimide), 할로아세틸(haloacetyl), 피리딜디설파이드(pyridyldisulfide), 티오술포네이트(thiosulfonate), 또는 비닐술폰(vinylsulfone) 등일 수 있다. 링커는 프로테아제에 의해서 절단 가능하거나, 산이나 염기 조건에서 절단 가능하거나, 고온이나 광조사에 의해서 절단 가능하거나 또는 환원 또는 산화 조건에서 절단 가능한 링커일 수 있고, 또는 이러한 조건들에서 절단 가능하지 않은 링커일 수도 있다. 절단 가능한 링커로서는 예컨대 산성 조건에서 절단되는 히드라존(hydrazone) 링커, 프로테아제에 의해 절단되는 펩타이드 링커, 환원 조건에서 절단되는 디설파이드(disulfide) 작용기를 갖는 링커 등을 들 수 있고, 절단 가능하지 않은 링커로서는 MCC(Maleimidomethyl cyclohexane-1-carboxylate) 링커, MC(maleimidocaproyl) 링커, 또는 그 유도체로서 석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sMCC) 링커나 설포석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sulfo-sMCC)를 들 수 있다. 또한 링커는 자가 희생 링커(self-immolative linker) 또는 절단 후 흔적을 남기지 않는 링커(traceless linker)일 수 있다. 자가 희생 링커는 예컨대 발명의 명칭이 "Hydrophilic self-immolative linkers and conjugates thereof "인 미국 특허 제9,089,614호에 개시된 링커, 명칭이 "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF"인 국제공개 제WO2015038426호에 개시된 링커를 들 수 있으며, 절단 후 흔적을 남기지 않는 링커로서는 페닐하이드라지드 링커, 아릴-트리아젠 링커, 문헌[Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024(2002)]에 개시된 링커 등일 수 있다.The functional groups of these linkers include isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, and glyoxal ( glyoxal, epoxide, oxirane, carbonate, arylhalide, imidoester, carbodiimide, anhydride, fluorophenyl ester It may be (fluorophenyl ester), hydroxymethyl phosphine, maleimide, haloacetyl, pyridyldisulfide, thiosulfonate, or vinylsulfone. . The linker may be cleavable by protease, cleavable under acid or base conditions, cleavable by high temperature or light irradiation, cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions. It may be. Examples of cleavable linkers include hydrazone linkers that are cleaved under acidic conditions, peptide linkers that are cleaved by proteases, and linkers with a disulfide functional group that are cleaved under reducing conditions. Examples of linkers that are not cleaved include: Maleimidomethyl cyclohexane-1-carboxylate (MCC) linker, maleimidocaproyl (MC) linker, or a derivative thereof such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linker or sulfosuccine. and imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). Additionally, the linker may be a self-immolative linker or a traceless linker that leaves no trace after cleavage. Self-immolative linkers include, for example, the linker disclosed in U.S. Pat. No. 9,089,614, entitled “Hydrophilic self-immolative linkers and conjugates thereof,” and the linker, entitled “SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES. THEREOF", the linker disclosed in International Publication No. WO2015038426, and linkers that leave no trace after cutting include phenylhydrazide linker, aryl-triazene linker, and the literature [Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)], etc.
통상의 기술분야에서는 상기 예시한 바의 링커 이외에도 본 발명에 적용 가능한 수많은 링커가 상당한 수의 문헌을 통해 공지되어 있다. 그러한 문헌으로서 구체적으로 문헌[Castaneda, et al, "Acid-cleavable thiomaleamic acid linker for homogeneous antibodydrug conjugation," Chem Commun. 49: 8187-8189(2013)], 문헌[Lyon, et al, "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat Biotechnol. 32(10):1059-1062(2014)], 문헌[Dawson, et al "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779], 문헌[Dawson, et al "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am Chem Soc. 1997, 119, 4325-4329] 문헌[Hackeng, et al "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc Natl Acad Sci USA 1999, 96, 10068-10073], 문헌[Wu, et al "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew Chem Int Ed 2006, 45, 4116-4125], 문헌[Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R Liss, Inc, 1988, pp 199-218], 문헌[Fields, G and Noble, R(1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int J Peptide Protein Res 35:161-214] 등이나, 미국 특허 제6,884,869호, 미국 특허 제7,498,298호, 미국 특허 제8,288,352호, 미국 특허 제8,609,105호, 미국 특허 제8,697,688호, 미국 특허공개 제2014/0127239호, 미국 특허공개 제2013/028919호, 미국 특허공개 제2014/286970호, 미국 특허공개 제2013/0309256호, 미국 특허공개 제2015/037360호, 미국 특허공개 제2014/0294851호, 국제 특허공개 WO2015057699, 국제 특허공개 WO2014080251, 국제 특허공개 제WO2014197854, 국제 특허공개 WO2014145090, 국제 특허공개 WO2014177042 등을 참조할 수 있다.In the ordinary technical field, in addition to the linkers exemplified above, numerous linkers applicable to the present invention are known through a considerable number of literature. Such documents specifically include Castaneda, et al, “Acid-cleavable thiomaleamic acid linker for homogeneous antibodydrug conjugation,” Chem Commun. 49: 8187-8189 (2013), Lyon, et al, “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat Biotechnol. 32(10):1059-1062 (2014), Dawson, et al "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779, Dawson, et al "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am Chem Soc. 1997, 119, 4325-4329] Hackeng, et al "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc Natl Acad Sci USA 1999, 96, 10068-10073], Wu, et al “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew Chem Int Ed 2006, 45, 4116-4125, Geiser et al “Automation of solid-phase peptide synthesis” in Macromolecular Sequencing and Synthesis, Alan R Liss, Inc, 1988, pp 199-218], Fields, G and Noble, R (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int J Peptide Protein Res 35:161-214. etc., US Patent No. 6,884,869, US Patent No. 7,498,298, US Patent No. 8,288,352, US Patent No. 8,609,105, US Patent No. 8,697,688, US Patent Publication No. 2014/0127239, US Patent Publication No. 2013/028919. , US Patent Publication No. 2014/286970, US Patent Publication No. 2013/0309256, US Patent Publication No. 2015/037360, US Patent Publication No. 2014/0294851, International Patent Publication WO2015057699, International Patent Publication WO2014080251, International Patent Publication No. Reference may be made to WO2014197854, International Patent Publication WO2014145090, International Patent Publication WO2014177042, etc.
본 발명의 약물 전달체의 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM)과 약제학적 활성 성분은 생체 적합성 고분자를 매개로 또는 캐리어로 하여 결합될 수도 있다. 생체적합성 고분자는 생체조직 또는 혈액과 접촉하여 조직을 괴사시키거나 혈액을 응고시키지 않는 조직적합성(tissue compatibility) 및 항응혈성(blood compatibility)을 가지고 있는 고분자를 의미한다. 상기 생체적합성 고분자로서의 합성 중합체는 폴리에스테르, 폴리하이드 록시알카노에이트(PHAs), 폴리(α-하이드록시액시드), 폴리(β-하이드록시액시드), 폴리(3-하이드로식부티레이트-co-발러레이트; PHBV), 폴리(3-하이드록시프로프리오네이트; PHP), 폴리(3-하이드록시헥사노에이트; PHH), 폴리(4-하이드록시액시드), 폴리(4-하이드록시부티레이트), 폴리(4-하이드록시발러레이트), 폴리(4-하이드록시헥사노에이트), 폴리(에스테르아마이드), 폴리카프로락톤, 폴리락타이드, 폴리글리코라이드, 폴리(락타이드-co-글리코라이드; PLGA), 폴리디옥사논, 폴리오르토에스테르, 폴리언하이드라이드, 폴리(글리콜산-co-트리메틸렌카보네이트), 폴리포스포에스테르, 폴리포스포에스테르 우레탄, 폴리(아미노산), 폴리사이아노아크릴레이트, 폴리(트리메틸렌 카보네이트), 폴리(이미노카보네이트), 폴리(타이로신 카보네이트), 폴리카보네이트, 폴리(타이로신 아릴레이트), 폴리알킬렌 옥살레이트, 폴리포스파젠스, PHA-PEG, 에틸렌 비닐 알코올 코폴리머(EVOH), 폴리우레탄, 실리콘, 폴리에스테르, 폴리올레핀, 폴리이소부틸렌과 에틸렌-알파올레핀 공중합체, 스틸렌-이소브틸렌-스틸렌 트리블록 공중합체, 아크릴 중합체 및 공중합체, 비닐 할라이드 중합체 및 공중합체, 폴리비닐 클로라이드, 폴리비닐 에테르, 폴리비닐 메틸에테르, 폴리비닐리덴 할라이드, 폴리비닐리덴 플루오라이드, 폴리비닐리덴 클로라이드, 폴리플루오로알켄, 폴리퍼플루오로알켄, 폴리아크릴로니트릴, 폴리비닐 케톤, 폴리비닐 아로마틱스, 폴리스틸렌, 폴리비닐 에스테르, 폴리비닐 아세테이트, 에틸렌-메틸 메타크릴레이트 공중합체, 아크릴로니트릴-스틸렌 공중합체, ABS 수지와 에틸렌-비닐 아세테이트 공중합체, 폴리아마이드, 알키드 수지, 폴리옥시메틸렌, 폴리이미드, 폴리에테르, 폴리아크릴레이트, 폴리메타크릴레이트, 폴리아크릴산-co-말레산 또는 폴리아미노아민이며, 천연 중합체는 키토산, 덱스트란, 셀룰로오스, 헤파린, 히알루론산, 알기네이트, 이눌린, 녹말 또는 글리코겐이다.The compound of formula 1 (DRL peptide) or the compound of formula 2 (DRL-TAM) of the drug carrier of the present invention and the pharmaceutically active ingredient may be combined through a biocompatible polymer or carrier. Biocompatible polymers refer to polymers that have tissue compatibility and anticoagulant properties that do not cause tissue necrosis or blood coagulation in contact with biological tissue or blood. Synthetic polymers as biocompatible polymers include polyester, polyhydroxyalkanoate (PHAs), poly(α-hydroxyacid), poly(β-hydroxyacid), and poly(3-hydrosybutyrate-co). -valerate; PHBV), poly(3-hydroxypropionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxy acid), poly(4-hydroxy butyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycoside) ride; PLGA), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), polycyano Acrylate, poly(trimethylene carbonate), poly(iminocarbonate), poly(tyrosine carbonate), polycarbonate, poly(tyrosine arylate), polyalkylene oxalate, polyphosphazene, PHA-PEG, ethylene vinyl Alcohol copolymers (EVOH), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alphaolefin copolymers, styrene-isobutylene-styrene triblock copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, polyvinyl chloride, polyvinyl ether, polyvinyl methyl ether, polyvinylidene halide, polyvinylidene fluoride, polyvinylidene chloride, polyfluoroalkene, polyperfluoroalkene, polyacrylonitrile, poly. Vinyl ketone, polyvinyl aromatics, polystyrene, polyvinyl ester, polyvinyl acetate, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer, polyamide, alkyd resin. , polyoxymethylene, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-co-maleic acid or polyaminoamine, and natural polymers include chitosan, dextran, cellulose, heparin, hyaluronic acid, and alginate. , inulin, starch or glycogen.
본 발명의 활막 표적화 약물 전달체는 약제학적으로 허용되는 담체를 포함하여 당업계에 공지된 통상의 방법으로 투여 경로에 따라 경구용 제형 또는 비경구용 제형의 약제학적 조성물로 제조될 수 있다. 여기서 "약학적으로 허용가능한 담체"는 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The synovial targeting drug carrier of the present invention can be prepared as a pharmaceutical composition in oral formulation or parenteral formulation depending on the route of administration by a conventional method known in the art including a pharmaceutically acceptable carrier. Here, “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
일 측면에서, 본 발명은 본 발명의 활막 표적화 약물 전달체를 유효성분으로 함유하는 관절염 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating arthritis disease containing the synovium targeting drug delivery system of the present invention as an active ingredient.
일 구현예에서, 관절염 질환은 활막염, 류마티스 관절염(RA), 청소년 류마티스 관절염, 골관절염(OA), 통풍, 가통풍(pseudogout), 척추관절염(SpA), 건선성 관절염, 강직성 척추염, 폐혈성 관절염, 관절염, 청소년 특발성 관절염, 둔상(blunt trauma), 관절 교체 또는 스틸(Still) 질환일 수 있다.In one embodiment, the arthritic condition is synovitis, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, It could be arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.
일 구현예에서, 상기 조성물은 표지 물질을 추가로 포함할 수 있으며, 이 경우 관절염 질환을 진단과 동시에 치료할 수 있다.In one embodiment, the composition may further include a marker, and in this case, arthritis disease can be diagnosed and treated simultaneously.
본 발명의 약학적 조성물은 유효성분으로서 토르세미드 및/또는 크로몰린, 또는 이의 염 외에 공지된 관절염 질환 치료제를 추가로 포함할 수 있고, 이들 질환의 치료를 위해 공지된 다른 치료와 병용될 수 있다. The pharmaceutical composition of the present invention may further include known therapeutic agents for arthritis diseases in addition to torsemide and/or cromolyn, or salts thereof, as active ingredients, and may be used in combination with other known treatments for the treatment of these diseases. there is.
본 발명에서, 용어 "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 관절염 질환의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미하고, "치료"란 본 발명의 조성물의 투여로 관절염 질환의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of arthritis disease by administering the pharmaceutical composition according to the present invention, and "treatment" refers to all actions that inhibit or delay the occurrence, spread, and recurrence of arthritis disease by administering the pharmaceutical composition according to the present invention. It refers to all actions that improve or beneficially change the symptoms of arthritis disease. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.
본 발명에서 유효성분과 결합하여 사용된 "치료학적으로 유효한 양"이란 용어는 대상 질환을 예방 또는 치료하는데 유효한 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어있다.The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to an amount effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention is determined by several factors, such as the method of administration. , may vary depending on the target area, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 관절염 질환의 종류, 관절염 질환의 발병 원인, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type of arthritic disease, cause of arthritic disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, combination or drugs used simultaneously, and It may be determined based on other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 약학적 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 본 발명에서 사용되는 용어, "약학적으로 허용가능한"이란 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다. 상기 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
일 구현예에서, 상기 약학적 조성물은 경구형 제형, 외용제, 좌제, 멸균 주사용액 및 분무제를 포함하는 군으로부터 선택되는 하나 이상의 제형일 수 있으며, 경구형 또는 주사 제형이 더욱 바람직하다. In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays, with oral or injectable formulations being more preferable.
본 발명에서 사용되는 용어, "투여"란, 임의의 적절한 방법으로 개체 또는 환자에게 소정의 물질을 제공하는 것을 의미하며, 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 주사 제형으로 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다. 본 발명의 약학적 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수도 있다. 바람직한 투여방식 및 제제는 정맥 주사제, 피하 주사제, 피내주사제, 근육 주사제, 점적 주사제 등이다. 주사제는 생리식염액, 링겔액 등의 수성 용제, 식물유, 고급 지방산 에스테르(예, 올레인산에칠 등), 알코올 류(예, 에탄올, 벤질알코올, 프로필렌글리콜, 글리세린 등) 등의 비수성 용제 등을 이용하여 제조할 수 있고, 변질 방지를 위한 안정화제(예, 아스코르빈산, 아황산수소나트륨, 피로아황산나트륨, BHA, 토코페롤, EDTA 등), 유화제, pH 조절을 위한 완충제, 미생물 발육을 저지하기 위한 보존제(예, 질산페닐수은, 치메로살, 염화벤잘코늄, 페놀, 크레솔, 벤질알코올 등) 등의 약학적 담체를 포함할 수 있다.As used in the present invention, the term "administration" means providing a predetermined substance to an individual or patient by any appropriate method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally) according to the desired method. Alternatively, it can be applied topically as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device capable of transporting the active agent to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be manufactured using stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and agents to prevent microbial growth. It may contain pharmaceutical carriers such as preservatives (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).
본 발명에서 사용되는 용어, "개체"란, 상기 관절염 질환이 발병하였거나 발병할 수 있는 인간을 포함한 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 동물을 의미하고, 본 발명의 약학적 조성물을 개체에게 투여함으로써 상기 질환들을 효과적으로 예방 또는 치료할 수 있다. 본 발명의 약학적 조성물은 기존의 치료제와 병행하여 투여될 수 있다.As used in the present invention, the term "individual" refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, including humans who have or may develop the arthritic disease. Or, it refers to all animals including guinea pigs, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention can be administered in combination with existing therapeutic agents.
본 발명의 약학적 조성물은 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약제학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
일 측면에서, 본 발명은 본 발명의 관절염 질환의 예방 또는 치료용 약학적 조성물을 치료학적 유효량으로 개체에 투여하는 것을 포함하는 관절염 질환의 예방 또는 치료 방법에 관한 것이다.In one aspect, the present invention relates to a method for preventing or treating arthritic diseases, comprising administering to a subject the pharmaceutical composition for preventing or treating arthritic diseases of the present invention in a therapeutically effective amount.
상기 관절염 질환의 예방 또는 치료 방법은 상기 관절염 질환의 예방 또는 치료용 조성물을 치료가 필요한 개체에게 투여하는 것일 수 있다.The method for preventing or treating the arthritic disease may be administering a composition for preventing or treating the arthritic disease to an individual in need of treatment.
일 측면에서, 본 발명은 본 발명의 화학식 1의 화합물(DRL 펩타이드) 또는 화학식 2의 화합물(DRL-TAM), 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염, 또는 본 발명의 활막 표적화 약물 전달체를 유효성분으로 함유하는 활막 조영제 조성물에 관한 것이다.In one aspect, the present invention provides a compound of Formula 1 (DRL peptide) or a compound of Formula 2 (DRL-TAM) of the present invention, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, or a synovial membrane targeting agent of the present invention. It relates to a synovial contrast agent composition containing a drug carrier as an active ingredient.
일 구현예에서, 상기 조성물은 표지 물질을 추가로 포함할 수 있다.In one embodiment, the composition may further include a labeling substance.
일 구현예에서, 상기 조영제 조성물은 관절염 질환의 진단용으로 사용될 수 있다.In one embodiment, the contrast medium composition can be used for diagnosing arthritis disease.
일 측면에서, 본 발명은 본 발명의 활막 조영제 조성물을 인간 또는 동물에 처리하고, 여기광원을 조사하는 단계; 및 방출되는 형광 변화를 측정하는 단계;를 포함하는, 관절염 질환의 진단방법에 관한 것이다.In one aspect, the present invention includes the steps of treating a human or animal with the synovial contrast agent composition of the present invention and irradiating an excitation light source; and measuring the change in emitted fluorescence. It relates to a method for diagnosing arthritis disease, including.
일 구현예에서, 상기 형광 변화를 측정하는 단계는 측정 대상의 관절의 형광 발광 세기가 활막 조영제 조성물이 처리되기 전의 형광 발광 세기에 비해 증가된 것을 확인할 수 있는 방법을 포함할 수 있다.In one embodiment, the step of measuring the fluorescence change may include a method of confirming that the fluorescence emission intensity of the joint to be measured is increased compared to the fluorescence intensity before the synovial contrast agent composition is treated.
일 측면에서, 본 발명은 본 발명의 활막 조영제 조성물을 이용한 활막을 영상화하는 방법에 관한 것이다.In one aspect, the present invention relates to a method of imaging the synovial membrane using the synovial contrast agent composition of the present invention.
일 구현예에서, 본 발명의 활막을 영상화 하는 방법은 본 발명의 활막 조영제 조성물을 인간 또는 동물에 처리하고, 여기광원을 조사하는 단계; 및 방출되는 형광 변화를 측정하는 단계;를 포함하는 것일 수 있고, 활막을 표적화하여 영상화 하는 것일 수 있다.In one embodiment, the method for imaging the synovial membrane of the present invention includes treating a human or animal with the synovial contrast agent composition of the present invention and irradiating an excitation light source; And measuring the change in fluorescence emitted; may include targeting and imaging the synovial membrane.
일 측면에서, 본 발명은 아스파틱산 및 아르기닌, 류신을 순차적으로 반응시키는 단계를 포함하는, 하기 화학식 1로 표시되는 화합물의 제조방법에 관한 것이다:In one aspect, the present invention relates to a method for producing a compound represented by the following formula (1), comprising the step of sequentially reacting aspartic acid, arginine, and leucine:
[화학식 1][Formula 1]
Figure PCTKR2023009907-appb-img-000004
Figure PCTKR2023009907-appb-img-000004
일 구현예에서, 상기 화학식 1로 표시되는 화합물의 제조방법은 N,N-디메틸포름아미드에 아스파틱산과 아르기닌을 첨가하여 1차 반응시키는 단계; 및 N,N-디메틸포름아미드를 추가하여 류신을 2차 반응시켜 펩타이드를 형성시키는 단계를 포함할 수 있다. In one embodiment, the method for preparing the compound represented by Formula 1 includes adding aspartic acid and arginine to N,N-dimethylformamide and performing a primary reaction; And it may include adding N,N-dimethylformamide to perform a secondary reaction with leucine to form a peptide.
일 구현예에서, 상기 펩타이드를 형성시키는 단계 이후에 반응시킨 물질을 회수하여 건조 및 농축하는 단계를 추가로 포함할 수 있다.In one embodiment, the step of forming the peptide may further include recovering the reacted material, drying and concentrating it.
일 구현예에서, 상기 건조 및 농축하는 단계 이후에 고성능 액체 크로마토그래피를 이용하여 정제하는 과정을 추가로 포함할 수 있다.In one embodiment, a purification process using high-performance liquid chromatography may be further included after the drying and concentrating steps.
일 측면에서, 본 발명은 하기 화학식 1로 표시되는 화합물, 라이신이 접합된 TAMRA(5(6)-Carboxytetramethylrhodamine) 및 디이소프로필에틸아민(diisopropylethylamine)을 반응시키는 단계를 포함하는, 하기 화학식 2로 표시되는 화합물의 제조방법에 관한 것이다:In one aspect, the present invention is represented by the following formula 2, comprising the step of reacting a compound represented by the following formula 1, lysine-conjugated TAMRA (5(6)-Carboxytetramethylrhodamine) and diisopropylethylamine. It relates to a method of preparing a compound that is:
[화학식 1][Formula 1]
Figure PCTKR2023009907-appb-img-000005
Figure PCTKR2023009907-appb-img-000005
[화학식 2][Formula 2]
Figure PCTKR2023009907-appb-img-000006
Figure PCTKR2023009907-appb-img-000006
일 구현예에서, 상기 화학식 2로 표시되는 화합물의 제조방법의 반응은 N,N-디메틸포름아미드를 용매로 이용하여 수행되는 것일 수 있다.In one embodiment, the reaction of the method for producing the compound represented by Formula 2 may be carried out using N,N-dimethylformamide as a solvent.
일 구현예에서, 상기 화학식 2로 표시되는 화합물의 제조방법은 상기 반응시키는 단계 이후에 반응시킨 물질을 회수하여 건조 및 농축하는 단계를 추가로 포함할 수 있고, 상기 건조 및 농축하는 단계 이후에 고성능 액체 크로마토그래피를 이용하여 정제하는 과정을 추가로 포함할 수 있다.In one embodiment, the method for producing the compound represented by Formula 2 may further include the step of recovering, drying and concentrating the reacted material after the reacting step, and after the drying and concentrating step, high performance A purification process using liquid chromatography may be additionally included.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.
실시예 1. 표적화 기능을 가진 화합물의 합성 및 정제Example 1. Synthesis and purification of compounds with targeting function
활막(synovium) 표적능을 가지는 화합물을 개발하기 위해, N,N-디메틸포름아미드에 Fmoc로 아민기를 보호한 아스파틱산, 아르기닌 및 염(HOBT, HBTU 및 DIPEA)을 2시간 동안 반응시켜 1차 반응물을 형성하고, 1차 반응으로 합성된 물질에 동일한 방법으로 류신을 첨가하여 2차 반응으로 화합물을 형성하였다. 형성된 화합물에서 Fmoc를 제거하기 위해 20% 피페리딘(piperidine)을 N,N-디메틸포름아미드에서 10분간 반응시켜 활막을 표적화하는 화합물로서 DRL(D; aspartic acid, R; arginine, L; leucine)의 아미노산 서열(서열번호 1)로 이루어진 화학식 1의 디알엘 트리펩타이드(DRL tripeptide)(표 1)를 형성하였다. In order to develop a compound with synovium targeting ability, aspartic acid, arginine and salts (HOBT, HBTU and DIPEA) with an amine group protected with Fmoc were reacted with N,N-dimethylformamide for 2 hours to form the first reactants. was formed, and leucine was added to the material synthesized in the first reaction in the same manner to form a compound in the second reaction. To remove Fmoc from the formed compound, 20% piperidine is reacted in N,N-dimethylformamide for 10 minutes to form DRL (D; aspartic acid, R; arginine, L; leucine), a compound targeting the synovial membrane. A DRL tripeptide (Table 1) of Formula 1 consisting of the amino acid sequence (SEQ ID NO: 1) was formed.
본 발명의 화합물(DRL 펩타이드)의 효능을 확인하기 위해, 상기 화합물(DRL 펩타이드), 라이신이 접합된 탐라(5-Carboxytetramethylrhodamine, TAMRA) 및 디이소프로필에틸아민(diisopropylethylamine)을 N,N-디메틸포름아미드에 넣고 하룻밤 동안 반응시켜 DRL 펩타이드에 표지 물질로서 탐라를 접합한 DRL-TAM(화학식 2)을 제조하였다. 형성된 물질을 고성능 액체 크로마토그래피를 이용하여 분리 및 정제하였다.In order to confirm the efficacy of the compound (DRL peptide) of the present invention, the compound (DRL peptide), lysine-conjugated 5-Carboxytetramethylrhodamine (TAMRA) and diisopropylethylamine were mixed with N,N-dimethylform. It was added to amide and reacted overnight to prepare DRL-TAM (Formula 2), in which Tamra was conjugated to DRL peptide as a labeling material. The formed material was separated and purified using high-performance liquid chromatography.
[화학식 1][Formula 1]
Figure PCTKR2023009907-appb-img-000007
Figure PCTKR2023009907-appb-img-000007
[화학식 2][Formula 2]
Figure PCTKR2023009907-appb-img-000008
Figure PCTKR2023009907-appb-img-000008
펩타이드 이름peptide name 서열(아미노에서 카르복시 방향)Sequence (amino to carboxy direction) 서열번호sequence number
DRLDRL DRLDRL 1One
실시예 2. DRL 펩타이드의 안정성 확인Example 2. Confirmation of stability of DRL peptide
본 발명의 화합물(DRL 펩타이드)의 안정성을 확인하기 위해, 상기 실시예 1에서 제조한 DRL-TAM이 다양한 용매 조건, 다양한 pH 조건에서 안정적으로 여러 조건에서 유지되는지를 흡광 및 형광 방출 그래프를 측정함으로써 형광 안정성을 확인하였다. 구체적으로, 세포 및 생체의 사용시 안정적인지 확인하기 위해, 증류수(deionized water; DI.H2O), 생리 식염수(phosphate-buffered saline; PBS, pH 7.4) 및 세포 배양액(Dulbecco's modified Eagle's media; DMEM media)의 조건하에서 DRL-TAM(10μM)의 흡광 및 형광 방출을 확인하였다. 또한, 다양한 pH에서의 안정성을 확인하기 위해, pH 3, 5, 7, 9 조건하에서 DRL-TAM(10μM)의 흡광 및 형광 방출을 확인하였다. 아울러, 빛 조사에 따른 DRL-TAM(10 μM)의 광안정성을 확인하기 위해, 530 nm 파장의 빛(50 mW/cm2)을 60분 동안 조사하였고, 10분 간격으로 형광 세기를 확인하였다. 이 때, 흡광 스펙트럼(UV/Vis absorption spectra) 분석을 위해서는 UV/Vis 분광광도계(Agilent Technologies Cary 8454, USA)을 사용하였으며, 형광 스펙트럼(fluorescence spectra) 분석을 위해서는 형광 광도계(SHIMADZU CORP. RF-6000, Japan)을 사용하였고, 각 기기에 본 화합물을 넣어주는 셀(cell)은 1 cm 두께의 표준 석영 셀(standard quartz cell, internal volume=0.1 cm)을 이용하였다.In order to confirm the stability of the compound (DRL peptide) of the present invention, absorbance and fluorescence emission graphs were measured to determine whether the DRL-TAM prepared in Example 1 was stably maintained under various solvent conditions and various pH conditions. Fluorescence stability was confirmed. Specifically, to ensure stability when using cells and living organisms, distilled water (deionized water; DI.H 2 O), physiological saline (phosphate-buffered saline; PBS, pH 7.4), and cell culture medium (Dulbecco's modified Eagle's media; DMEM media) ) The absorbance and fluorescence emission of DRL-TAM (10 μM) were confirmed under the conditions. Additionally, to confirm stability at various pH, the absorption and fluorescence emission of DRL-TAM (10 μM) were confirmed under pH 3, 5, 7, and 9 conditions. In addition, to confirm the photostability of DRL-TAM (10 μM) according to light irradiation, light with a wavelength of 530 nm (50 mW/cm 2 ) was irradiated for 60 minutes, and the fluorescence intensity was checked at 10-minute intervals. At this time, a UV/Vis spectrophotometer (Agilent Technologies Cary 8454, USA) was used to analyze the absorption spectrum (UV/Vis absorption spectra), and a fluorescence photometer (SHIMADZU CORP. RF-6000) was used to analyze the fluorescence spectrum. , Japan) was used, and a 1 cm thick standard quartz cell (internal volume=0.1 cm) was used as the cell into which this compound was placed in each device.
그 결과, 세포 및 생체 조건에서, 흡광 스펙트럼은 모든 용액에서 554 nm에서 최대 흡수 파장으로 증가하고, 형광 방출 스펙트럼은 580 nm에서 600 nm 사이에서 증가하는 것으로 나타나(도 1), 세포 및 생체의 사용에 있어 세포 및 장기/조직 내 바이오 이미징에 적용하기에 충분히 강한 형광 세기를 보이는 것이 확인되었다. 또한, 다양한 pH 조건에서, 흡광 스펙트럼은 모든 용액에서 550 nm에서 560 nm 사이에서 증가하고, 형광 방출 스펙트럼은 583 nm에서 최대 형광 강도를 보이는 것으로 나타나(도 2), 여러 생체 내의 조건에서 형광 기반 스크리닝 및 바이오 이미징에 활용 가능한 강한 형광 세기를 보이는 것이 확인되었다. 아울러, 빛 조사시 60분 동안 거의 형광 세기가 거의 변화하지 않는 것으로 나타나(도 3), 높은 광안정성을 보이는 것이 확인되었다. As a result, in cellular and living conditions, the absorption spectrum increases with the maximum absorption wavelength at 554 nm in all solutions, and the fluorescence emission spectrum increases between 580 nm and 600 nm (Figure 1), which is suitable for use in cells and living organisms. It was confirmed that the fluorescence intensity was strong enough to be applied to bio-imaging within cells and organs/tissues. Additionally, under various pH conditions, the absorption spectrum increases between 550 nm and 560 nm for all solutions, and the fluorescence emission spectrum appears to show a maximum fluorescence intensity at 583 nm (Figure 2), which supports fluorescence-based screening under multiple in vivo conditions. It was confirmed that it exhibits a strong fluorescence intensity that can be used for bio-imaging. In addition, the fluorescence intensity showed almost no change for 60 minutes upon light irradiation (Figure 3), confirming that it exhibits high photostability.
준비예 1. 동물 모델 및 세포 준비Preparation Example 1. Animal model and cell preparation
9주령 수컷 SD 랫트와 6주령 수컷 balb/c nu/nu 마우스는(주)디비엘(인천, 한국)에서 구입하였다. 랫트는 케이지당 2마리(42Х26Х18 cm), 마우스는 케이지당 5마리(27Х22Х14 cm)씩 음식과 물에 자유롭게 접근할 수 있도록하면서 일정한 온도(23±1℃) 및 상대 습도(60±10%)에서 12시간 명/암 주기(07:30-19:30에 점등)로 보관하였다. 랫트와 마우스는 모든 실험 전에 일주일 동안 동물실에 보관되었다. 경희대학교에서 발행한 동물 관리 및 사용 지침에 따라 동물 치료 및 유지 관리를 수행하였으며, 모든 실험 프로토콜은 경희대학교 동물실험실 관리위원회의 승인을 받아 진행하였다(승인번호, KHSASP-22-024).9-week-old male SD rats and 6-week-old male balb/c nu/nu mice were purchased from DBL Co., Ltd. (Incheon, Korea). Two rats (42Х26Х18 cm) per cage and five mice (27Х22Х14 cm) per cage were kept at a constant temperature (23 ± 1°C) and relative humidity (60 ± 10%) with free access to food and water. It was stored under a 12-hour light/dark cycle (lights on from 07:30 to 19:30). Rats and mice were kept in the animal room for one week before all experiments. Animal treatment and maintenance were performed in accordance with the animal care and use guidelines issued by Kyung Hee University, and all experimental protocols were approved by the Kyung Hee University Animal Laboratory Management Committee (approval number, KHSASP-22-024).
상기 생후 10주령의 수컷 SD 랫트(DBL Co., Ltd., 인천, 한국)의 뒷다리 활액막으로부터 랫트 윤활막 유래 1차 세포(Rat synovium-derived primary cells; RSPC)를 분리하였다. 마우스 뇌조직 유래 내피 세포(마우스 뇌혈관 내피암 세포주, B.End3) 및 인간 배아 신장 유래 상피 세포(HEK293)는 대한세포주은행(서울, 한국)에서 입수하였다. 각 세포는 10% fetal bovine serum(Hyclone, US)과 1% penicillinstreptomycin(Gibco, US)이 첨가된 Dulbecco's modified Eagle's media(DMEM, Hyclone, US)에서 배양하였고, 37℃및 5% CO2가 포함된 가습 분위기에서 보관하였다.Rat synovium-derived primary cells (RSPC) were isolated from the hind limb synovium of the 10-week-old male SD rat (DBL Co., Ltd., Incheon, Korea). Mouse brain tissue-derived endothelial cells (mouse cerebrovascular endothelial carcinoma cell line, B.End3) and human embryonic kidney-derived epithelial cells (HEK293) were obtained from the Korean Cell Line Bank (Seoul, Korea). Each cell was cultured in Dulbecco's modified Eagle's media (DMEM, Hyclone, US) supplemented with 10% fetal bovine serum (Hyclone, US) and 1% penicillinstreptomycin (Gibco, US), at 37°C and containing 5% CO 2 . It was stored in a humidified atmosphere.
실시예 3. DRL 펩타이드의 독성 분석Example 3. Toxicity analysis of DRL peptides
3-1. 세포 독성 확인3-1. Confirmation of cytotoxicity
본 발명의 화합물(DRL 펩타이드)이 세포에 적용하기에 안전한지 확인하기 위해 DRL-TAM을 이용하여 Cell Counting Kit(CCK)-8(Dojindo, Japan) 분석법을 통해 세포 독성 반응을 확인하였다. 구체적으로, 상기 준비예 1의 랫트 활막 유래 일차 세포(RSPC)와 대조군으로써 마우스의 뇌조직 유래 내피 세포(B.End3) 및 인간 배아 신장 유래 상피 세포(HEK293)를 96-웰 플레이트(SPL, Korea Rep.)에 24시간 동안 시딩(웰당 1Х104)한 후, 각 세포를 Dulbecco의 인산염 완충 식염수(DPBS, 1x)로 세척하였다. 그 다음, 상기 각 세포에 DRL-TAM을 1 내지 100 μM 농도로 처리하여 37℃의 5% CO2 인큐베이터에서 24시간 동안 배양한 다음, 세포를 인산완충식염수(PBS, pH 7.4)로 2회 세척한 후 CCK-8 용액 10μL를 첨가하고 37℃에서 1시간 배양하였다. 다음으로, 마이크로플레이트 판독기(Multiskan FC, Thermo Fisher, MA, US)를 사용하여 450 nm의 파장에서 광학 밀도를 측정하여, 흡광값으로 세포 생존률을 측정하였다. 세포 생존률(%)은 (시험군/대조군)Х100 으로 계산하여, 평균±평균의 표준오차(SEM)로 나타내었다.To confirm whether the compound of the present invention (DRL peptide) is safe to apply to cells, the cytotoxic response was confirmed through Cell Counting Kit (CCK)-8 (Dojindo, Japan) assay using DRL-TAM. Specifically, the rat synovial membrane-derived primary cells (RSPC) of Preparation Example 1, mouse brain tissue-derived endothelial cells (B.End3), and human embryonic kidney-derived epithelial cells (HEK293) as a control were plated in a 96-well plate (SPL, Korea). Rep.) for 24 hours (1Х10 4 per well), each cell was washed with Dulbecco's phosphate buffered saline (DPBS, 1x). Next, each cell was treated with DRL-TAM at a concentration of 1 to 100 μM and cultured in a 5% CO 2 incubator at 37°C for 24 hours, and then the cells were washed twice with phosphate-buffered saline (PBS, pH 7.4). After that, 10 μL of CCK-8 solution was added and cultured at 37°C for 1 hour. Next, the optical density was measured at a wavelength of 450 nm using a microplate reader (Multiskan FC, Thermo Fisher, MA, US), and the cell viability was measured using the absorbance value. Cell survival rate (%) was calculated as (test group/control group) Х100 and expressed as mean ± standard error of the mean (SEM).
그 결과, 마우스 활막 유래 일차 세포 및 마우스의 뇌조직 유래 내피 세포, 인간 배아 신장 유래 상피 세포 모두에서 DRL-TAM(1-100 μM)가 유의미한 독성을 나타내지 않는 것으로 나타났다(도 4).As a result, it was shown that DRL-TAM (1-100 μM) did not exhibit significant toxicity in both primary cells derived from mouse synovium, endothelial cells derived from mouse brain tissue, and epithelial cells derived from human embryonic kidney (FIG. 4).
3-2. 생체 내 독성 확인3-2. Confirmation of in vivo toxicity
본 발명의 화합물(DRL 펩타이드)이 생체 내 사용에 있어 독성 반응을 나타내는지 확인하기 위해, DRL-TAM을 이용하여 마우스의 혈액에서의 용혈 반응을 확인하였다. 구체적으로, 마취된 마우스의 심장으로부터 바로 채혈한 혈액을 항응고 처리를 한 후 원심분리기(4℃, 1300rpm, 3분)를 이용하여 생리 식염수(차가운 1ХPBS)로 2번 혈장을 세척하여 적혈구(RBCs)만을 순수하게 분리하였다. 정제된 적혈구(8%, v/v 차가운 1ХPBS)에 DRL-TAM을 1-100 μM 농도로 처리하여 진탕 배양기(300 rpm, 37℃)에서 1시간 동안 배양하였으며, 용혈 반응이 외부 요소에 의한 것이 아님을 입증하기 위하여, 음성 대조군으로써 생리 식염수(PBS, pH 7.4) 및 양성 대조군으로써 계면 활성제인 Triton X-100(0.1%)을 사용하였다. 이후 원심분리기(4℃, 3000 rpm, 3분)를 이용하여 상층액을 분리하였고, 각 군에서 DRL-TAM과 대조군 처리 후 원심분리 전 및 후의 450 nm 여기파장에서 흡광도를 측정하여 용혈 반응을 그래프화하였다. 측정 결과는 평균±SEM으로 나타내었다.In order to confirm whether the compound of the present invention (DRL peptide) exhibits a toxic reaction when used in vivo, the hemolytic reaction in mouse blood was confirmed using DRL-TAM. Specifically, blood collected directly from the heart of an anesthetized mouse was anticoagulated, and the plasma was washed twice with physiological saline (cold 1ХPBS) using a centrifuge (4°C, 1300rpm, 3 minutes) to produce red blood cells (RBCs). ) were isolated purely. Purified red blood cells (8%, v/v cold 1ХPBS) were treated with DRL-TAM at a concentration of 1-100 μM and incubated for 1 hour in a shaking incubator (300 rpm, 37°C). It was confirmed that the hemolysis reaction was not caused by external factors. To prove this, physiological saline (PBS, pH 7.4) was used as a negative control and Triton X-100 (0.1%), a surfactant, was used as a positive control. Afterwards, the supernatant was separated using a centrifuge (4°C, 3000 rpm, 3 minutes), and the hemolysis reaction was graphed by measuring the absorbance at an excitation wavelength of 450 nm before and after centrifugation after treatment with DRL-TAM and the control group in each group. I got angry. Measurement results were expressed as mean ± SEM.
그 결과, 생리 식염수를 처리한 군과 DRL-TAM을 처리한 군 사이에서는 유의미한 차이가 관찰되지 않았으며, 계면 활성제를 처리한 군과 대비하여 나머지 모든 군에서 유의미한 차이가 관찰되었다(도 5). 이를 통해, DRL 펩타이드가 생체 내에서 용혈 반응을 일으키지 않으며, 생체 내 적용에 있어서 충분히 안전성을 갖는 것을 확인하였다.As a result, no significant difference was observed between the group treated with physiological saline and the group treated with DRL-TAM, and significant differences were observed in all remaining groups compared to the group treated with surfactant (Figure 5). Through this, it was confirmed that the DRL peptide does not cause a hemolytic reaction in vivo and is sufficiently safe for in vivo application.
실시예 4. DRL 펩타이드의 활막 표적화 효능 및 시각화 특성 확인Example 4. Confirmation of synovium targeting efficacy and visualization characteristics of DRL peptide
4-1. 세포 수준에서의 활막 표적화 효능 및 시각화 특성4-1. Synovial membrane targeting efficacy and visualization properties at the cellular level
본 발명의 화합물(DRL 펩타이드)의 세포 내에서의 표적화 효능 및 시각화 특성을 확인하기 위해, 상기 준비예 1의 랫트 활막 유래 일차 세포(RSPC)와 대조군으로써 마우스의 뇌조직 유래 내피 세포(B.End3) 및 인간 배아 신장 유래 상피 세포(HEK293)를 35 mm 유리 바닥 공초점 접시(SPL Life Science, Korea Rep.)에 시딩하고 37℃에서 24시간 동안 배양하였다. 37℃의 5% CO2 인큐베이터에서 각 세포에 DRL-TAM(10 μM)을 24시간 동안 처리한 뒤 PBS(pH 7.4)로 세척하여 제거하고, 세포 소기관인 핵과 세포막을 구별하기 위하여 DAPI(4',6-diamidino-2-phenylindole) 및 CellMask Green plasma membrane stain를 각각 2 μL(1000Х작업 농도)씩 5% CO2 인큐베이터 및 무혈청 배지에서 20분 동안 세포에 처리하였다. 이 후, 세포를 PBS(pH 7.4)로 3회 세척한 다음, 4% 포름알데하이드(formaldehyde)를 6 분 동안 처리하고 다시 PBS(pH 7.4)로 세척하여 용액을 제거한 뒤, 공초점 레이저 스캐닝 현미경(CLSM, LSM-800, Carl Zeiss, Germany)으로 세포의 이미지를 확인하고, DRL-TAM의 형광 세기를 각 세포에서 비교 분석하였다. 여기 및 방출 채널은 빨간색(560nm, 565-700nm), 녹색(488nm, 520-550nm) 및 파란색(405nm, 450-520nm)으로 하였다. 또한, 각 세포에서 DRL-TAM 채널의 형광 세기(강도) 및 RSPC의 CellMask와 DRL-TAM에 표시된 패널(도 6a)의 흰색 점선 부분에서의 형광 세기(강도)를 정량화하여 플롯으로 나타내었으며, 데이터는 평균±SEM으로 나타내었다.In order to confirm the intracellular targeting efficacy and visualization characteristics of the compound of the present invention (DRL peptide), rat synovium-derived primary cells (RSPC) of Preparation Example 1 and mouse brain tissue-derived endothelial cells (B.End3) as a control were used. ) and human embryonic kidney-derived epithelial cells (HEK293) were seeded on a 35 mm glass bottom confocal dish (SPL Life Science, Korea Rep.) and cultured at 37°C for 24 hours. Each cell was treated with DRL-TAM (10 μM) for 24 hours in a 5% CO 2 incubator at 37°C, washed with PBS (pH 7.4), removed, and DAPI (4' ,6-diamidino-2-phenylindole) and CellMask Green plasma membrane stain were treated with 2 μL each (1000Х working concentration) for 20 minutes in a 5% CO 2 incubator and serum-free medium. Afterwards, the cells were washed three times with PBS (pH 7.4), treated with 4% formaldehyde for 6 minutes, washed again with PBS (pH 7.4) to remove the solution, and then analyzed using a confocal laser scanning microscope ( The images of the cells were confirmed using CLSM, LSM-800, Carl Zeiss, Germany), and the fluorescence intensity of DRL-TAM was compared and analyzed in each cell. The excitation and emission channels were red (560nm, 565-700nm), green (488nm, 520-550nm), and blue (405nm, 450-520nm). In addition, the fluorescence intensity (intensity) of the DRL-TAM channel in each cell and the fluorescence intensity (intensity) in the white dotted area of the panel (Figure 6a) shown in CellMask and DRL-TAM of RSPC were quantified and plotted, data is expressed as mean ± SEM.
그 결과, 대조군으로 사용한 두 세포에서보다 활막 유래 일차 세포에서 특이적으로 DRL-TAM이 현저히 높은 형광 세기를 나타냈으며, DRL-TAM이 세포막을 염색하는 CellMask Green plasma membrane stain과 동일한 위치에서 형광 세기를 나타내, 마우스의 활막 유래 세포의 세포막에 존재하는 것을 명확히 나타냈으므로(도 6), DRL-TAM이 활막 표적화 효과를 가지고 이를 시각화할 수 있음을 확인하였다.As a result, DRL-TAM showed significantly higher fluorescence intensity specifically in synovium-derived primary cells than in the two cells used as controls, and DRL-TAM showed fluorescence intensity at the same location as CellMask Green plasma membrane stain, which stains the cell membrane. As it was clearly shown to be present in the cell membrane of mouse synovial membrane-derived cells (Figure 6), it was confirmed that DRL-TAM has a synovial targeting effect and can be visualized.
4-2. 생체 내 활막 표적화 효능 및 시각화 특성4-2. In vivo synovium targeting efficacy and visualization properties
본 발명의 화합물(DRL 펩타이드)의 생체 내에서의 표적화 효능 및 시각화 특성을 확인하기 위해, DRM-TAM과 대조군으로써 탐라와 구조적 유사체인 로다민 B(rhodamine B; Rho)를 6주령의 마우스에 각각 5 mg/kg 농도로 미주 정맥 투여하고, 시간에 따른 물질의 생체 분포 과정을 형광 추적을 통하여 확인하였다. DRL-TAM과 Rho의 체내분포는 형광 조직 이미징 시스템(FTIS, VISQUE® InVivo Elite, Vieworks Co. Ltd, Korea)(530-570 nm 여기, 575-640 nm 검출 채널)을 사용하여 전신형광영상을 통해 모니터링하였다.In order to confirm the in vivo targeting efficacy and visualization characteristics of the compound of the present invention (DRL peptide), DRM-TAM and rhodamine B (Rho), a structural analogue of Tamra, were administered to 6-week-old mice as a control. It was administered into the vagus vein at a concentration of 5 mg/kg, and the biodistribution process of the substance over time was confirmed through fluorescence tracking. The biodistribution of DRL-TAM and Rho was examined through whole-body fluorescence imaging using a fluorescence tissue imaging system (FTIS, VISQUE® InVivo Elite, Vieworks Co. Ltd, Korea) (530-570 nm excitation, 575-640 nm detection channel). Monitored.
FTIS를 사용한 전신형광영상을 통해 시간에 따른 물질의 생체 분포 과정을 추적한 결과, 미주 정맥 투여 10분 후, DRL-TAM을 투여한 군에서 가장 높은 형광 세기를 보이는 것으로 나타났으며(도 7), 이는 생체 내에서 DRL-TAM이 10분 동안 가장 많이 분포하는 것을 의미한다.As a result of tracking the biodistribution process of the substance over time through whole-body fluorescence imaging using FTIS, it was found that the group administered DRL-TAM showed the highest fluorescence intensity 10 minutes after vagus intravenous administration (Figure 7). , This means that DRL-TAM is most widely distributed in vivo for 10 minutes.
또한, DRL-TAM의 생체 내 형광 세기가 가장 높은 시간인 투여 10분 후에 마우스를 마취시키고 PBS(pH 7.4)로 경심 관류시키고 4% 파라포름알데히드로 고정시켰다. 마우스를 희생시켜 신체 장기 및 부위(흉골, 척추, 다리뼈, 발, 뇌, 심장, 폐, 간, 신장 및 비장)를 적출하여 추가 생체외 연구를 수행하였다. 암실에서 VISQUE® InVivo Elite(Vieworks Co., Ltd., Korea)를 이용하여 생체외 조직 형광 이미징을 통해 각각의 형광 세기를 대조군인 로다민 B와 비교 분석하였다. 데이터는 평균±SEM으로 나타내었다.Additionally, 10 minutes after administration, which is the time when the in vivo fluorescence intensity of DRL-TAM is highest, the mouse was anesthetized, perfused transcardially with PBS (pH 7.4), and fixed with 4% paraformaldehyde. Mice were sacrificed and body organs and parts (sternum, spine, leg bones, feet, brain, heart, lungs, liver, kidneys, and spleen) were removed for further in vitro studies. Each fluorescence intensity was compared and analyzed with the control rhodamine B through ex vivo tissue fluorescence imaging using VISQUE® InVivo Elite (Vieworks Co., Ltd., Korea) in a dark room. Data are expressed as mean ± SEM.
적출한 마우스의 신체 장기 및 부위에서 활막이 존재한다고 알려져 있는 부위와 활막이 존재하지 않는 부위를 나누어 형광 이미지를 확인한 결과, 활막이 존재하는 부위인 흉골, 척추, 다리뼈, 발에서는 대조군인 로다민 B에 대비하여 DRL-TAM이 유의적으로 높은 복사 효율을 가지는 것으로 나타났다. 반면에 뇌, 심장, 간, 신장 등 활막이 존재하지 않는 부위의 복사 효율은 대조군인 로다민 B가 DRL-TAM보다 높게 나타났다(도 8). As a result of confirming the fluorescence images by dividing the organs and parts of the extracted mouse body into areas known to have synovium and areas where synovium is not present, the control group, rhodamine, was used in the areas where synovium was present, such as the sternum, spine, leg bones, and feet. Compared to B, DRL-TAM was found to have significantly higher radiation efficiency. On the other hand, the radiation efficiency of the control group Rhodamine B in areas where synovium is not present, such as the brain, heart, liver, and kidneys, was higher than that of DRL-TAM (Figure 8).
이를 통해, 본 발명의 화합물(DRL 펩타이드)이 생체 내 특정 부위, 즉, 활막에 대해 특이적인 표적화 효능을 보이며, 이를 이용하여 신체 활액막이 풍부한 특정 부위들을 시각화할 수 있음을 확인하였다. 또한, 류마티스 관절염을 포함한 다양한 활막 질환을 치료하기 위한 진단 및 치료 용도의 여러 제형으로의 개발에 유용할 수 있음을 확인하였다.Through this, it was confirmed that the compound of the present invention (DRL peptide) shows a specific targeting effect on a specific area in the body, that is, the synovial membrane, and that it can be used to visualize specific areas rich in the body's synovial membrane. In addition, it was confirmed that it could be useful in the development of various formulations for diagnostic and therapeutic purposes to treat various synovial diseases, including rheumatoid arthritis.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention stated above is for illustrative purposes, and a person skilled in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (19)

  1. 하기 화학식 1로 표시되는 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염:A compound represented by the following formula (1), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:
    [화학식 1][Formula 1]
    Figure PCTKR2023009907-appb-img-000009
    Figure PCTKR2023009907-appb-img-000009
  2. 제 1항에 있어서, 활막을 특이적으로 표적화하기 위한, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염.The compound, its hydrate, its solvate or its pharmaceutically acceptable salt according to claim 1, for specifically targeting the synovial membrane.
  3. 제 1항에 있어서, 표지 물질을 추가로 포함하는, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염.The compound, its hydrate, its solvate, or its pharmaceutically acceptable salt according to claim 1, further comprising a labeling substance.
  4. 제 3항에 있어서, 표지 물질은 발색효소, 방사성동위원소, 크로모포어(chromophore), 발색 물질, 발광 물질, 형광 물질(fluorescer), 상자성 입자(superparamagnetic particles) 및 초상자성입자(ultrasuper paramagnetic particles)로 이루어진 군에서 선택되는 하나 이상인, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염.The method of claim 3, wherein the labeling substances include chromogenic enzymes, radioisotopes, chromophores, coloring substances, luminescent substances, fluorescers, superparamagnetic particles and ultrasuper paramagnetic particles. One or more compounds selected from the group consisting of, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
  5. 제 4항에 있어서, 형광 물질은 형광 단백질(fluorescent protein), 발광 단백질(photoprotein), 루시퍼라제(luciferase), 형광염료(fluorescent dye) 또는 시분해 형광입자(Time-resolved Fluorescence, TRF)인, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염.The compound according to claim 4, wherein the fluorescent substance is a fluorescent protein, photoprotein, luciferase, fluorescent dye, or time-resolved fluorescence (TRF). , its hydrate, its solvate or its pharmaceutically acceptable salt.
  6. 제 4항에 있어서, 형광 물질은 알렉사 플루오르 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, 루피(Rubpy)(tris(2,2-bipyridyl)ruthenium(Ⅱ)), FITC(fluoresein Isothiocyanate), 로다민 6G(rhodamine 6G), 로다민 B(rhodamine B), TAMRA(5(6)-Carboxytetramethylrhodamine), 텍사스 레드(Texas Red), DAPI(4,6-diamidino-2-phenylindole) 및 쿠마린으로 이루어진 군으로부터 선택된 어느 하나 이상인, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염.The method of claim 4, wherein the fluorescent material is Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rubpy (tris(2,2- bipyridyl)ruthenium(Ⅱ)), FITC(fluoresein Isothiocyanate), rhodamine 6G, rhodamine B, TAMRA(5(6)-Carboxytetramethylrhodamine), Texas Red, DAPI(4) ,6-diamidino-2-phenylindole) and coumarin, a compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.
  7. 제 1항의 화합물을 암호화하는 핵산.A nucleic acid encoding the compound of claim 1.
  8. 제 8항의 핵산을 포함하는 벡터.A vector containing the nucleic acid of claim 8.
  9. 하기 화학식 2로 표시되는, 활막을 특이적으로 시각화하기 위한, 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염:A compound, its hydrate, its solvate, or its pharmaceutically acceptable salt for specifically visualizing the synovial membrane, represented by the following formula (2):
    [화학식 2][Formula 2]
    Figure PCTKR2023009907-appb-img-000010
    Figure PCTKR2023009907-appb-img-000010
  10. 제 1항의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염 또는 제 9항의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염에 약제학적 활성 성분이 접합(conjugate)된, 활막 표적화 약물 전달체.The pharmaceutically active ingredient is conjugated to the compound of claim 1, its hydrate, solvate or pharmaceutically acceptable salt or the compound of claim 9, its hydrate, solvate or pharmaceutically acceptable salt. , synovium targeting drug carrier.
  11. 제 10항에 있어서, 약제학적 활성 성분은 RNA, DNA, 항체, 이펙터, 약물, 전구약물, 독소, 펩타이드 또는 전달 분자인, 활막 표적화 약물 전달체.11. The synovium targeting drug delivery system of claim 10, wherein the pharmaceutically active ingredient is RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule.
  12. 제 10항에 있어서, 약물은 펩타이드 약물, 단백질 약물, 탈감작화제, 항원, 비-스테로이드성 소염제, 소염 약물, 마취제, 항산화제, 항감염제, 화학치료제, 항-통각반응제, DMOAD, 동화작용제, 항-이화작용제, 자가소화작용 조절제, 항-파골세포-매개된 골 손실제, 기능식품제(nutraceutical agent), 진통제, 생물제제(biologics) 또는 이들의 혼합물인, 활막 표적화 약물 전달체.The method of claim 10, wherein the drug is a peptide drug, a protein drug, a desensitizing agent, an antigen, a non-steroidal anti-inflammatory drug, an anti-inflammatory drug, an anesthetic, an antioxidant, an anti-infective agent, a chemotherapeutic agent, an anti-nociceptive agent, DMOAD, an anabolic agent. , anti-catabolic agents, autophagy regulators, anti-osteoclast-mediated bone loss agents, nutraceutical agents, analgesics, biologics, or mixtures thereof.
  13. 제 10항의 활막 표적화 약물 전달체를 유효성분으로 함유하는 관절염 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of arthritis disease containing the synovial membrane targeting drug carrier of claim 10 as an active ingredient.
  14. 제 13항에 있어서, 관절염 질환은 활막염, 류마티스 관절염(RA), 청소년 류마티스 관절염, 골관절염(OA), 통풍, 가통풍(pseudogout), 척추관절염(SpA), 건선성 관절염, 강직성 척추염, 폐혈성 관절염, 관절염, 청소년 특발성 관절염, 둔상(blunt trauma), 관절 교체 또는 스틸(Still) 질환인, 관절염 질환의 예방 또는 치료용 약학적 조성물.The method of claim 13, wherein the arthritis disease is synovitis, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, and septic arthritis. A pharmaceutical composition for preventing or treating arthritic diseases, such as arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.
  15. 제 1항의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염, 제 9항의 화합물, 이의 수화물, 이의 용매화물 또는 이의 약학적으로 허용가능한 염, 또는 제 10항의 활막 표적화 약물 전달체를 유효성분으로 함유하는 활막 조영제 조성물.The compound of claim 1, its hydrate, its solvate or a pharmaceutically acceptable salt thereof, the compound of claim 9, its hydrate, its solvate or a pharmaceutically acceptable salt thereof, or the synovial membrane targeting drug delivery system of claim 10 is effective. A synovial contrast agent composition containing as an ingredient.
  16. 제 15항의 활막 조영제 조성물을 이용한 활막을 영상화하는 방법.A method of imaging the synovial membrane using the synovial contrast agent composition of claim 15.
  17. 아스파틱산 및 아르기닌, 류신을 순차적으로 반응시키는 단계를 포함하는, 하기 화학식 1로 표시되는 화합물의 제조방법:A method for producing a compound represented by Formula 1 below, comprising the step of sequentially reacting aspartic acid, arginine, and leucine:
    [화학식 1][Formula 1]
    Figure PCTKR2023009907-appb-img-000011
    Figure PCTKR2023009907-appb-img-000011
  18. 제 16항에 있어서, 상기 제조방법은 N,N-디메틸포름아미드에 아스파틱산과 아르기닌을 첨가하여 1차 반응시키는 단계; 및 N,N-디메틸포름아미드를 추가하여 류신을 2차 반응시켜 펩타이드를 형성시키는 단계;를 포함하는, 제조방법.The method of claim 16, wherein the preparation method includes adding aspartic acid and arginine to N,N-dimethylformamide and performing a primary reaction; And adding N,N-dimethylformamide to cause a secondary reaction with leucine to form a peptide.
  19. 하기 화학식 1로 표시되는 화합물, 라이신이 접합된 TAMRA(5(6)-Carboxytetramethylrhodamine) 및 디이소프로필에틸아민(diisopropylethylamine)을 반응시키는 단계를 포함하는, 하기 화학식 2로 표시되는 화합물의 제조방법:A method for producing a compound represented by the following Formula 2, comprising the step of reacting a compound represented by the following Formula 1, lysine-conjugated TAMRA (5(6)-Carboxytetramethylrhodamine) and diisopropylethylamine:
    [화학식 1][Formula 1]
    Figure PCTKR2023009907-appb-img-000012
    Figure PCTKR2023009907-appb-img-000012
    [화학식 2][Formula 2]
    Figure PCTKR2023009907-appb-img-000013
    Figure PCTKR2023009907-appb-img-000013
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