WO2020045570A1 - Auto-assemblage pour activer une immunité - Google Patents

Auto-assemblage pour activer une immunité Download PDF

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WO2020045570A1
WO2020045570A1 PCT/JP2019/033920 JP2019033920W WO2020045570A1 WO 2020045570 A1 WO2020045570 A1 WO 2020045570A1 JP 2019033920 W JP2019033920 W JP 2019033920W WO 2020045570 A1 WO2020045570 A1 WO 2020045570A1
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self
assembly
compound
formula
shows
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PCT/JP2019/033920
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Japanese (ja)
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志成 上杉
ブー・フエ・ティ
大樹 吉田
尚孝 野田
ジン・シュウユウ
晶 山▲崎▼
徹 島根
石井　健
仰 日置
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国立大学法人京都大学
国立大学法人大阪大学
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Publication of WO2020045570A1 publication Critical patent/WO2020045570A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link

Definitions

  • the present invention relates to a novel compound forming a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
  • Self-assembly which is the spontaneous self-assembly of molecules, plays an important role in the formation of biological hierarchical structures in living organisms.
  • DNA, lipid membranes, cytoskeletal fibers, and the like are examples of natural self-assemblies.
  • a single compound molecule has a single function
  • a self-assembly has complex physical or chemical properties and functions depending on the surrounding environment (Non-Patent Document 1). Therefore, the synthesis of a new self-assembly has attracted attention as one of new methods for drug development that regulates the function and biological activity of biological systems.
  • Non-Patent Document 2 synthetic oligonucleotides containing unmethylated CG sequences (CpG- ODN) is known (Non-Patent Document 2). As described above, several self-assemblies that mimic the natural self-assembly structure have been reported, but most of them use peptides as constituent units of the self-assembly.
  • Non-Patent Documents 3 to 10 Although some examples of self-assembly of non-peptide synthetic chemical molecules are also known, their biological activities are mainly related to cytotoxicity and cell death (Non-Patent Documents 3 to 10). Therefore, new and useful self-assembly of synthetic chemical molecules that exhibit biological activity is desired.
  • One of the problems to be solved by the present invention is to provide a novel compound which forms a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, a small molecule compound having a partial structure in which a specific chemical modification has been made to bile acid forms a new self-assembly, and the self-assembly concerned Showed that they exhibited an immunostimulatory effect, and thus completed the present invention.
  • the self-assembled substance formed by linking the compounds of the formula (I) can exhibit a biological activity, for example, an immunostimulatory action, and can be useful as an adjuvant.
  • FIG. 1 shows the hydrodynamic diameter (nm) (vertical axis) of a compound (Hit @ compounds) that formed a self-assembly in the compound library.
  • FIG. 2 shows the hydrodynamic radii (average R h (nm)) (vertical axis) of self-assemblies of the compounds of Example 1, Reference Example 2 and Reference Example 3 in DMEM solutions at various concentrations ( ⁇ M).
  • FIG. 3 shows the IL-6 production amount (ng / mL) (vertical axis) of the self-assembly of the compound of Example 1 in DMEM solutions at various concentrations ( ⁇ M).
  • FIG. 1 shows the hydrodynamic diameter (nm) (vertical axis) of a compound (Hit @ compounds) that formed a self-assembly in the compound library.
  • FIG. 2 shows the hydrodynamic radii (average R h (nm)) (vertical axis) of self-assemblies of the compounds of Example 1, Reference
  • FIG. 4 shows the IL-6 production amount (ng / mL) (vertical axis) of the compounds of Example 1 and Reference Examples 1 to 3 in a 30 ⁇ M @DMEM solution.
  • DCA shows the result for deoxycholic acid.
  • NC is a negative control of 1% (v / v) DMSO, and LPS is a positive control.
  • FIG. 5 shows the relative value (vertical axis) of the amount of IL-6 produced by the self-assembly of each example compound in a 30 ⁇ M @DMEM solution when the negative control (DMSO) is set to 1.
  • FIG. 6 shows the particle size distribution (%) of the self-assembly of the compound of Example 1 in a 30 ⁇ M @DMEM solution (right side).
  • FIG. 9 shows the results of an IL-6 ⁇ ELISA assay (ng / mL) using FcR ⁇ / MyD88 double knockout BMDC (right) and wild-type BMDC (WT) (left).
  • FIG. 12 shows IL-6 production (pg / mL) of wild-type (WT) and TLR7 knockout (Tlr7 ⁇ / ⁇ ) mice.
  • the bar on the left side of each test substance shows the result of the wild type, and the bar on the right side shows the result of the TLR7 knockout mouse.
  • FIG. 18 shows the production amount (ng / mL) of the anti-OVA antibody (IgE). Each bar in each test substance shows the results at 1, 10 and 100 ⁇ g from the left, respectively.
  • FIG. 20A shows the relationship between the number of days after influenza infection (horizontal axis) and the weight loss rate (%) (vertical axis).
  • ⁇ indicates a self-assembly of the compound of Example 1
  • ⁇ indicates a self-assembly of the compound of Example 7
  • ⁇ indicates a self-assembly of the compound of Example 10
  • ⁇ indicates DCA
  • ⁇ indicates 1% DMSO
  • ⁇ indicates an aluminum salt.
  • FIG. 20B shows the relationship between the number of days after influenza infection (horizontal axis) and the survival rate (%) (vertical axis).
  • ⁇ indicates a self-assembly of the compound of Example 1
  • ⁇ indicates a self-assembly of the compound of Example 7
  • ⁇ indicates data when DCA
  • ⁇ indicates 1% DMSO
  • FIG. 21A shows the number of days after influenza SV injection (horizontal axis) and weight loss rate (%) (vertical axis) for each dose ( ⁇ : 1 ⁇ g, ⁇ : 10 ⁇ g, ⁇ : 100 ⁇ g) of the self-assembly of the compound of Example 10. Shows the relationship.
  • ⁇ indicates data when 1% DMSO was used, and ⁇ indicates data when aluminum salt was used.
  • FIG. 23 shows the results of a SEAP assay for null and no TLR9 using HEK293 cells.
  • FIG. 24 shows the results of a SEAP assay for TLR2 and TLR4 using HEK293 cells without an agonist (null).
  • FIG. 25 shows the results of the IL-6 quantitative PCR assay (qPCR).
  • Item 2 The self-assembly according to Item 1, wherein R x is the same as R y .
  • R x is of the formula (II ′): Is a group represented by R y has the formula (III ′): Item 3.
  • Item 4 The self-assembly according to any one of Items 1 to 3, wherein R 3 and R 4 are each independently C 1-3 alkyl.
  • Item 3 The self-assembly according to Item 1 or 2, wherein R x and R y are both H.
  • Item 6 The self-assembly according to any one of Items 1 to 5, wherein n is an integer of 5 to 10.
  • Item 7 The self-assembly according to any one of Items 1 to 6, which forms an aggregate having a hydrodynamic size of 40 nm to 12,000 nm.
  • Item 8 An immunostimulator comprising the self-assembly according to any one of Items 1 to 7.
  • Item 9 The immunostimulant according to Item 8, which is an adjuvant.
  • Item 10 A vaccine composition comprising the immunostimulating agent according to Item 9 and an antigen.
  • Item 11 The vaccine composition according to Item 10, for treating and / or preventing a disease selected from the group consisting of cancer, infectious disease, allergic disease, autoimmune disease, hypertension and Alzheimer's disease.
  • R x is H or formula (II): Is a group represented by R y is H or formula (III): Is a group represented by R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl; Wavy lines indicate binding sites, n is an integer of 1 to 12]
  • C 1-12 alkyl refers to a linear or branched saturated hydrocarbon group having 1 to 12 carbon atoms.
  • “C 1-12 alkyl” includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 2,2-dimethylpentyl , 2,2,3-trimethylbutyl, n-octyl, 3,4-dimethylhexyl, 2,2,4-trimethylpentyl, n
  • C 1-6 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl and the like.
  • C 1-12 haloalkyl refers to a C 1-12 alkyl in which one or more optional hydrogen atoms have been replaced with the same or different halogen.
  • C 1-12 haloalkyl includes difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2-chloroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 1,1 -Difluoropropyl, 3,3,3-trifluoropropyl, 2,2,2-trifluoro-1-methyl-ethyl, 1,1-difluoro-2-methylpropyl, 4-chlorobutyl, 5-fluoropentyl and the like No.
  • halo or “halogen” includes fluoro, chloro, bromo, iodo and the like.
  • self-assembly refers to an assembly formed by self-assembling, defined as the spontaneous self-assembly of a molecule, and comprising the molecule as a constituent unit, and two or more molecules. Is a micellar, fibrous, crystalline, colloidal, or particulate aggregate formed by non-covalent bonding.
  • non-covalent bond include a hydrophobic bond, ⁇ - ⁇ stacking, a hydrogen bond, a van der Waals bond, and an arbitrary combination thereof.
  • the molecules constituting the self-assembly include small molecule compounds having a partial structure in which bile acid is chemically modified, and preferably a compound of the formula (I).
  • the self-assembly may have a plurality of hydrophobic pockets, and through this hydrophobic pocket, various substances such as other molecules and antigens can be incorporated into the assembly structure.
  • a self-assembly may also be an aggregate of compounds of formula (I) having different structures based on the differences in R x , R y and R 1 -R 4 , and compounds other than compounds of formula (I) Mixed together to form an aggregate.
  • the structure and properties of the self-assembly can be appropriately controlled by chemical modification of a molecule serving as a structural unit.
  • Self-assemblies may exhibit immunostimulatory effects through interaction with membrane proteins.
  • the hydrodynamic size of the self-assembly is not particularly limited and may depend on the surrounding environment such as the concentration in the medium, the concentration in the blood and the concentration in the tissue, but is, for example, 40 to 12,000 nm, and is preferably It is 45 to 5,000 nm, more preferably 100 to 1,000 nm, further preferably 140 to 800 nm, and particularly preferably 150 to 500 nm.
  • the term "hydrodynamic size" refers to the hydrodynamic diameter, which may be measured, for example, using dynamic light scattering (DLS).
  • the self-assembly may also have an increased immunostimulatory effect as its concentration in the medium, blood or tissue increases.
  • immunostimulatory effect refers to endogenous or exogenous agonist activity for a pattern recognition receptor on the cell surface.
  • agonist activity antigen presenting cells such as macrophages and dendritic cells can be activated to produce cytokines such as interleukins.
  • the immunostimulatory activity is preferably an agonist activity for a Toll-like receptor (TLR), more preferably a TLR7 agonist activity or a TLR9 agonist activity, and particularly preferably a TLR7 agonist activity.
  • TLR Toll-like receptor
  • Immunostimulatory effects can also be increased as the hydrodynamic size of the self-assembly increases.
  • TLR7 stimulates the production of interferon and the like by sensing single-stranded RNA or synthetic small molecule ligand derived from virus. Therefore, TLR7 agonist has an immunostimulatory effect, for example, various diseases such as infectious diseases and autoimmune diseases. May be useful as therapeutics for Since TLR9 is commonly found in bacteria and viruses and plays a role in stimulating innate and adaptive immune responses to unmethylated DNA, TLR9 agonists can improve the immunogenicity of antigens used in vaccine compositions.
  • immunostimulant refers to a substance or composition that exhibits an immunostimulatory effect, and can be used for immunotherapy of various diseases.
  • the immunostimulant may contain a self-assembly.
  • an immunostimulant is used in combination with an antigen or a vaccine composition containing the antigen, immunogenicity to the antigen can be obtained or enhanced, and the duration of immunity can be prolonged.
  • the immunostimulant may be administered alone, may be used to be administered separately, sequentially or simultaneously at an interval from the antigen or vaccine composition comprising the antigen, or May be used as a component of the product. Examples of the immunostimulant include an adjuvant.
  • the amount of the self-assembly contained in the immunostimulant is not particularly limited as long as it is an immunologically acceptable amount, but is, for example, 0.1 to 20 w / w%, preferably 0.2 to 20 w / w%. It is 15 w / w%, more preferably 0.3 to 10 w / w%, particularly preferably 0.5 to 5 w / w%.
  • antigen refers to a substance that provokes an immune response, such as a peptide or protein.
  • Antigens include cancer antigens, viral antigens, bacterial antigens, fungal antigens, protozoal or parasite antigens, allergy-related antigens, and disease-related antigens.
  • the term “vaccine composition” refers to a composition containing an antigen, and can be used for immunotherapy of various diseases.
  • the vaccine composition may be administered alone, may be used so that it is administered separately, sequentially or simultaneously at an interval from the immunostimulant, or a vaccine containing the immunostimulant. It may be used as a composition.
  • the vaccine composition may also include a pharmaceutically acceptable carrier.
  • Such pharmaceutically acceptable carriers include additives commonly used in the art, such as excipients, isotonic agents, buffers, pH adjusters, suspending agents, preservatives, and the like.
  • the vaccine composition may be administered by administration routes commonly used in the art.
  • administration route examples include oral administration; and parenteral administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intrathecal administration, and transdermal administration.
  • parenteral administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intrathecal administration, and transdermal administration.
  • the amount of each component contained in the vaccine composition is not particularly limited as long as it is an immunologically acceptable amount usually used in the art.
  • disease includes cancer (eg, leukemia, lymphoma, neurological tumor, melanoma, breast cancer, lung cancer, head and neck cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, vaginal cancer , Testicular cancer, prostate cancer, penile cancer, bone tumor, vascular tumor, lip cancer, nasopharyngeal cancer, pharyngeal cancer, esophageal cancer, rectal cancer, gallbladder cancer, bile duct cancer, laryngeal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor Thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma, etc., infectious diseases (eg, influenza, human immunodeficiency syndrome (HIV), hepatitis such as viral hepatitis, viral infections such as Ebola hemorrhagic fever; pertussis, diphtheria, tetanus, tubercul
  • infectious diseases
  • -Bacterial infections caused by H. pylori, pneumococci, etc . fungal infections such as Candida; protozoal or parasite infections such as malaria; allergic diseases (eg, atopic dermatitis, allergic nose) , Asthma, etc.), autoimmune diseases (including, for example, diabetes mellitus (type 1 diabetes and type 2 diabetes), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.), hypertension, Alzheimer's disease.
  • allergic diseases eg, atopic dermatitis, allergic nose
  • Asthma Asthma
  • autoimmune diseases including, for example, diabetes mellitus (type 1 diabetes and type 2 diabetes), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.
  • hypertension Alzheimer's disease.
  • R x and R y are both H.
  • R 1 and R 2 are preferably the same.
  • R 1 and R 2 are preferably H, C 1-3 alkyl or C 1-3 haloalkyl, more preferably H or isopropyl. Particularly preferably, R 1 and R 2 are both H.
  • R 3 and R 4 are preferably the same.
  • R 3 and R 4 are preferably C 1-3 alkyl or C 1-3 haloalkyl, more preferably C 1-3 alkyl, and particularly preferably methyl or ethyl.
  • ⁇ n is preferably from 2 to 12, more preferably from 5 to 10, and particularly preferably from 5 to 8.
  • the compound of formula (I) preferably has the formula (I-1): [Wherein, R x , R y and n have the same meanings as in formula (I)] It is a compound shown by these.
  • R a -COOH is a bile acid, such as a primary bile acid or a secondary bile acid, preferably deoxycholic acid
  • R b is C 1-12 alkyl, preferably C 2-10 alkyl, more preferably C 5-10 alkyl, particularly preferably C 5-8 alkyl
  • R c is H, C 1-6 alkyl or C 1-6 haloalkyl
  • the compounds of formula (I) can be obtained by subjecting compounds (a), (b), (c) and (d) to a condensation reaction in a solvent.
  • the condensation reaction may be performed by mixing the compounds (b) and (c) to obtain an imine intermediate, and then adding the compounds (a) and (d) to the mixture.
  • the solvent include alcohols such as methanol and ethanol, and aprotic polar solvents such as DMF, and ethanol is preferable.
  • the reaction temperature is from room temperature to 60 ° C.
  • the compound of formula (I) can also be prepared when R x and R y are both H, for example according to the following method.
  • the compound of formula (I) can be obtained by subjecting compound (a), compound (b) and the dehydrating condensing agent to a condensation reaction in a solvent in the presence of a base as appropriate.
  • the dehydrating condensing agent include carbodiimides such as WSCI ⁇ HCl and N, N′-dicyclohexylcarbodiimide.
  • the base include DMAP.
  • the solvent include a non-polar solvent such as dichloromethane.
  • the reaction temperature is from room temperature to 40 ° C.
  • the self-assembly of the compound of the formula (I) can be produced from the compound of the formula (I) produced as described above, for example, according to the following method. After dissolving the compound of formula (I) in an aprotic polar solvent (eg, DMSO), the solution is added to a tissue culture medium (eg, DMEM) containing an additive (eg, serum) as appropriate. Mix. The mixed solution is allowed to stand to obtain a self-assembly of the compound of the formula (I).
  • an aprotic polar solvent eg, DMSO
  • DMEM tissue culture medium
  • an additive eg, serum
  • the formation of the self-assembly can be confirmed by mixing the self-assembly with the environment-sensitive fluorescent probe in an aqueous solvent (for example, water) and measuring the fluorescence intensity of the mixture.
  • aqueous solvent for example, water
  • Environmentally sensitive fluorescent probes are essentially non-fluorescent in aqueous solvents, but show fluorescence in less polar solvents or in hydrophobic environments.
  • Hydrophobic environments include, for example, self-assembled hydrophobic pockets.
  • Examples of the environmentally sensitive fluorescent probe include 8-anilino-1-naphthalenesulfonic acid (ANS) and Nile Red.
  • Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 ⁇ l, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours.
  • the EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (10/1).
  • the silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (1.3 mg, 0.0011 mmol).
  • WSCI ⁇ HCl 230 mg, 1.2 mmol
  • DMAP 122.17 mg, 1.0 mmol
  • extraction was performed three times with chloroform.
  • the organic layer was washed with a 2M aqueous NaOH solution, 1M hydrochloric acid and saturated saline, and the solvent was distilled off under reduced pressure.
  • the crude product was purified by column chromatography, the solution was concentrated under reduced pressure, and dried under vacuum to obtain the title compound (160 mg, 0.19 mmol).
  • Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 ⁇ l, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours.
  • the EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (20/1).
  • the silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (25 mg, 0.046 mmol).
  • Each self-assembly was produced from the above example compound and reference example compound according to the following method.
  • Each compound was dissolved in DMSO to prepare a 10 mM stock solution. After preparing a 3 mM DMSO solution (20 ⁇ l) of each compound from the stock solution, each solution was added to DMEM (2 ml) containing 10% FBS and 1% penicillin-streptomycin solution at room temperature, and mixed by pipetting. The mixed solution was allowed to stand for 1 hour to obtain a self-assembly of each compound.
  • mice MyD88-deficient mice (Oriental Yeast Co., Ltd.) and C57BL / 6 (CLEA Japan) background FcR ⁇ -deficient mice (RIKEN) were used.
  • cell RAW-Blue TM cells (InvivoGen) expressed the secreted fetal alkaline phosphatase (SEAP) reporter gene by NF- ⁇ B and AP-1 activation.
  • SEAP secreted fetal alkaline phosphatase
  • Bone marrow-derived dendritic cells were prepared according to the method described in Ishikawa et al., Cell Host & Microbe, April 17, 2013, 13, 477-488.
  • bone marrow cells were cultured in RPMI1640 medium supplemented with 10% (v / v) FBS and ⁇ -mercaptoethanol together with GM-CSF released from MGM-5 at 37 ° C. for 10 days, Bone marrow-derived dendritic cells were prepared.
  • RAW 264.7 cells American Type Culture Collection
  • DMEM DMEM containing 10% FBS and 1% penicillin-streptomycin solution.
  • Trehalose-6,6′-dimicolate (reagent) Trehalose-6,6′-dimicolate (TDM; Sigma) was dissolved at 1 mg / mL in a chloroform: methanol solution (2: 1 (v / v)) and diluted with 2-propanol to a final concentration of 50 ⁇ g / mL. After coating on a 96-well plate at 20 ⁇ L / well, the solvent was distilled off. The amount of TDM coated was 1 ⁇ g / well.
  • Lipopolysaccharide (LPS; L4516; Sigma) and curdlan (InvivoGen) were diluted with RPMI or DMEM containing 5% or 10% FBS and used for immunostimulation property measurement.
  • FIG. 1 shows the hydrodynamic diameter of a compound that formed a self-assembly in the compound library.
  • Test Example 1-2 Measurement of hydrodynamic size of self-assembly (2) For each example compound, a self-assembly was formed according to the method of Test Example 1-1. Samples were prepared at various concentrations in DMEM medium containing 10% FBS (Biowest) and 1% penicillin-streptomycin (Nacalai Tesque Inc.) at 25 ° C., and 100 ⁇ L in a 96-well UV plate (Corning Inc.). / Well.
  • the hydrodynamic radius of the self-assembly was measured using a DynaPro Plate reader II (Wyatt Technology Corp .; 165 °; automatic decay mode at 25 ° C .; laser wavelength 830 nm; scattering angle 58 °; data acquisition time 10 seconds / well; 10 acquisitions / measurements).
  • Hydrodynamic diameter converted from the self-assembly of 30 [mu] M DMEM solution measured hydrodynamic radius in (R h) and the R h values of the Example compounds are as follows.
  • FIG. 2 shows the results of the Rh values of the self-assembly of the compound of Example 1 and the compound of Reference Example in a DMEM solution.
  • the compound of Reference Example 1 did not form a self-assembly.
  • Immunostimulation properties (1) The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. 20,000 RAW 264.7 cells / well were seeded in 96-well tissue culture plates in DMEM containing 10% FBS at 37 ° C. for 24 hours. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% (v / v) DMSO and 100 pg / mL LPS were used as negative and positive controls, respectively.
  • FIG. 4 shows the result of comparison between the self-assembly of the compound of Example 1 and the compound of Reference Example.
  • FIG. 5 shows the results of the self-assembly of each example compound.
  • the particle size distribution of the self-assembly of the compound of Example 1 in a 30 ⁇ M DMEM solution is shown in FIG.
  • Immunostimulation properties (3) The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. After seeding BMDCs at 1 ⁇ 10 6 cells / well in RPMI 1640 containing 5% FBS in 96-well tissue culture plates, compounds of Examples 1, 7, 7 and 10 at 5, 10, 30, 50 and 100 ⁇ M respectively Of a self-assembled RPMI solution (containing 5% FBS) was added.
  • the mouse IL-6 concentration in the supernatant was measured using a Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex (Bio-Rad Laboratories, Inc.).
  • Deoxycholic acid (DCA) and DMSO (1%) were used as negative controls, and LPS (20 ng) and R848 (2 ⁇ g) were used as positive controls.
  • DCA Deoxycholic acid
  • DMSO DMSO
  • LPS (20 ng) and R848 (2 ⁇ g) were used as positive controls.
  • the result is shown in FIG.
  • the results show that the immunostimulatory effect induced by the self-assembly is TLR7-dependent, as is the positive control R848 (TLR7 agonist).
  • the test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 ⁇ M, respectively, were added to a 96-well plate, and 1 ⁇ 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA.
  • FIG. 13 shows the results.
  • Test examples to evaluate the agonistic activity on self-assembling of IL-6 production using T57-9 of T57-9 using C57BL / 6J (WT) or Tlr 9 ⁇ / ⁇ bone marrow-derived dendritic cells The test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 ⁇ M, respectively, were added to a 96-well plate, and 1 ⁇ 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA.
  • FIG. 14 shows the results.
  • Example 7 compound 1, 10 and 100 ⁇ g
  • Example 10 compound 1, 10 and 100 ⁇ g
  • deoxycholic acid 1, 10 and 100 ⁇ g
  • Deoxycholic acid and PBS were used as a negative control
  • aluminum salt Al salt
  • a 96-well plate was coated with 10 ⁇ g / mL OVA at 4 ° C. overnight. After washing, the plates were incubated with blocking buffer for 1 hour at room temperature. Plates were washed and incubated with diluted serum for 2 hours. The plates were then washed and horseradish peroxidase-conjugated anti-mouse total IgG, IgG1 or IgG2c antibodies (Southern Biotech) were each added to the plates. One hour later, the plate was washed and TMB Microwell Peroxidase Substrate System (KPL) was added to the wells. After 20 min incubation, the reaction was stopped by adding 2N H 2 SO 4.
  • KPL TMB Microwell Peroxidase Substrate System
  • the production amounts of anti-OVA total IgG, IgG1 and IgG2c are shown in FIGS. 15 to 17, respectively.
  • Anti-OVA total IgE was measured using a DS mouse IgE ELISA (OVA) kit (DS Pharma Biomedical).
  • FIG. 18 shows the amount of anti-OVA IgE produced.
  • mice were treated on day 0 and day 14 with influenza SV (1 ⁇ g), DMSO (final concentration: 1%), Example 1 compound (1, 10 and 100 ⁇ g), Example 7 compound (1, 10 And 100 ⁇ g) or a self-assembly of the compound of Example 10 (1, 10 and 100 ⁇ g), 100 ⁇ l / shot of a PBS solution containing either deoxycholic acid (1, 10 and 100 ⁇ g) or an aluminum salt (500 ⁇ g) subcutaneously.
  • mice were infected intranasally with 10 LD50 of clinically isolated A / Puerto Rico / 8/1934 influenza (H1N1) virus.
  • Anti-HA IgG1 and IgG2c in sera collected on day 21 were measured by ELISA.
  • the production amounts of anti-HA IgG1 and IgG2c are shown in FIGS. 19A and 19B. Mice were followed for body weight and survival 21 days after influenza infection on day 34.
  • Influenza SV (1 ⁇ g), self-assembly of DMSO (final concentration: 1%), Example 1 compound (100 ⁇ g), Example 7 compound (100 ⁇ g) or Example 10 compound (100 ⁇ g), deoxycholic acid (100 ⁇ g) Changes in body weight and viability for each mouse using either subcutaneous immunization, or with aluminum salt (500 ⁇ g) are shown in FIGS. 20A and 20B.
  • FIGS. 21A and 21B changes in body weight and survival rate are shown.
  • TLR293 cells In order to elucidate the SEAP assay signal pathway using HEK293 cells , several major TLRs were used using human embryonic kidney cells (HEK293) stably transfected with an NF- ⁇ B-responsive SEAP reporter gene.
  • HEK cells expressing TLR2, TLR4 or TLR9 are seeded at 3 ⁇ 10 4 cells / well in 96-well tissue culture plates in DMEM (100 ⁇ l) containing 10% FBS and self-assembled with the compound of Example 1 at 37 ° C. Stimulation was for 24 or 48 hours.
  • SEAP in the culture supernatant was stained with an alkaline phosphatase detection reagent QUANTI-Blue (InvivoGen) for 3 hours or 6 hours, and optical density (630 nm) was measured with a Multiskan microplate reader (Thermo Fisher Scientific Inc.). OD value) was measured.
  • DMSO was used as a negative control (NC)
  • oligodeoxynucleotide ODN1826; tlrl-1826; InvivoGen
  • TLR2 agonist Pam3 Pam3CSK4; InvivoGen
  • LPS Self-assembled SEAP responses were elevated in TLR9.
  • IL-6 Quantitative PCR assay RAW264.7 cells were seeded 24 hours 6 ⁇ 10 5 cells / well in DMEM containing 10% FBS at six-well tissue culture plates at 37 ° C.. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% DMSO and 100 ng / mL LPS were used as negative and positive controls, respectively. After 4 hours, the cells were washed twice with PBS. Cells were lysed with ISOGENE (1 mL / well; Nippon Gene Co., Ltd.) for 5 minutes. Then, chloroform (200 ⁇ L) was added.
  • the mixture was mixed for 15 minutes by inverting the test tube and then centrifuged at 14,000 rpm for 15 minutes at 4 ° C.
  • the upper 400 ⁇ L of the aqueous layer was collected in another 1.5 mL test tube and mixed with 500 ⁇ L of isopropanol.
  • the test tube was centrifuged again at 14,000 rpm, and the supernatant was removed.
  • the residue was washed twice with ethanol (700 ⁇ L), air-dried and redissolved in TE buffer (pH 5.2).
  • PrimeScript (TM) II 1 st strand cDNA Synthesis Kit Takara Bio Inc.
  • reverse transcribed total mRNA samples (2 [mu] g)
  • Quantitative real-time PCR was performed using the Fast CYBR Green Master Mix kit (Thermo Fisher Scientific Inc.). All assays were performed in triplicate and TNF- ⁇ and ⁇ -actin were used as internal standards. All primers for specific target genes are shown below: The results are shown in FIG.
  • a self-assembly obtained by linking the compounds of the formula (I) can be useful as an adjuvant, and is expected to be used as a vaccine composition for treating and / or preventing various diseases.

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Abstract

L'invention concerne : un nouveau composé qui forme un auto-assemblage ; l'auto-assemblage ; des procédés de production du composé et de l'auto-assemblage ; et l'utilisation pharmaceutique du composé et de l'auto-assemblage. Le composé est représenté par la formule (I) [dans la formule, Rx représente H ou un groupe représenté par la formule (II), Ry représente H ou un groupe représenté par la formule (III), R1, R2, R3, et R4 représentent chacun indépendamment H, un groupe alkyle en C1-6, ou halogénoalkyle en C1-6, une ligne ondulée représente un site de liaison, et n représente un nombre entier de 1 à 12]. L'auto-assemblage est formé par la composition. Les procédés de production concernent le composé et l'auto-assemblage, et l'utilisation pharmaceutique concerne le composé et l'auto-assemblage.
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Citations (3)

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US20050222115A1 (en) * 2004-03-30 2005-10-06 Salunke Deepak B Bile acid derived steroidal dimers with novel amphiphilic topology having antifungal activity
US20060003974A1 (en) * 2004-06-30 2006-01-05 Council Of Scientific And Industrial Research Bile acid derived steroidal dimers with amphiphilic topology having antiproliferative activity
WO2015079952A1 (fr) * 2013-11-29 2015-06-04 テルモ株式会社 Composition adjuvante, composition vaccinale la contenant et procédé de production associé

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US20050222115A1 (en) * 2004-03-30 2005-10-06 Salunke Deepak B Bile acid derived steroidal dimers with novel amphiphilic topology having antifungal activity
US20060003974A1 (en) * 2004-06-30 2006-01-05 Council Of Scientific And Industrial Research Bile acid derived steroidal dimers with amphiphilic topology having antiproliferative activity
WO2015079952A1 (fr) * 2013-11-29 2015-06-04 テルモ株式会社 Composition adjuvante, composition vaccinale la contenant et procédé de production associé

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