[go: up one dir, main page]

WO2023027006A1 - Activateur de perméation membranaire, procédé d'amélioration de perméation membranaire et procédé d'administration intracellulaire - Google Patents

Activateur de perméation membranaire, procédé d'amélioration de perméation membranaire et procédé d'administration intracellulaire Download PDF

Info

Publication number
WO2023027006A1
WO2023027006A1 PCT/JP2022/031507 JP2022031507W WO2023027006A1 WO 2023027006 A1 WO2023027006 A1 WO 2023027006A1 JP 2022031507 W JP2022031507 W JP 2022031507W WO 2023027006 A1 WO2023027006 A1 WO 2023027006A1
Authority
WO
WIPO (PCT)
Prior art keywords
membrane
substance
permeable
intracellular delivery
complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/031507
Other languages
English (en)
Japanese (ja)
Inventor
秀樹 酒井
一民 坂本
貴一 横山
優作 五十嵐
允顕 赤松
健一 酒井
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tokyo University of Science
Original Assignee
Tokyo University of Science
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tokyo University of Science filed Critical Tokyo University of Science
Publication of WO2023027006A1 publication Critical patent/WO2023027006A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention provides a membrane permeation enhancer that promotes the membrane permeability of a complex of a target substance and a membrane permeable substance, a membrane permeation enhancement method and an intracellular delivery method using the membrane permeation enhancer, and the membrane permeation enhancement. It relates to an intracellular delivery kit comprising an agent.
  • Biological membranes such as cell membranes have the role of separating the inside of the membrane from the outside world so that the inside surrounded by the membrane can exhibit normal biological functions.
  • This membrane does not simply separate the inside from the outside.
  • a cell membrane it is equipped with devices for exchanging substances, information, and energy with the outside of the cell and performing various metabolic reactions. Specifically, there are transporters and receptors in the cell membrane, and these functions selectively take in necessary substances and excrete various metabolites.
  • a membrane permeation enhancer containing an amino acid-based surfactant such as lauroyl-L-arginine ethyl hydrochloride (LAEHCl) enhances the membrane permeability of a membrane permeable substance complex.
  • LAEHCl lauroyl-L-arginine ethyl hydrochloride
  • a membrane permeable substance has the property of locally changing the curvature of the membrane in a convex direction with respect to the membrane permeable substance donor side.
  • a membrane permeation enhancer containing an amino acid-based surfactant can enhance the membrane permeability of the membrane permeable substance complex by reinforcing this property.
  • An object of the present invention is to provide a membrane permeation promoting method and an intracellular delivery method using an agent, and an intracellular delivery kit comprising the membrane permeation enhancing agent.
  • a membrane permeation enhancer that promotes permeation of a complex of a target substance and a membrane permeable substance through a lipid bilayer membrane, the membrane permeation enhancer containing a surfactant having a steroid skeleton.
  • the surfactant having a steroid skeleton is cholate, deoxycholate, taurocholate, taurodeoxycholate, glycodeoxycholate, 3-[(3-cholamidopropyl)dimethylammonium At least one selected from the group consisting of e]-1-propanesulfonate (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)
  • CHAPS -1-propanesulfonate
  • CHPA 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
  • ⁇ 3> The membrane permeation enhancer according to ⁇ 1> or ⁇ 2>, wherein the membrane permeable substance is a membrane permeable peptide.
  • ⁇ 4> The membrane permeation enhancer according to any one of ⁇ 1>
  • ⁇ 5> A method for promoting membrane permeation to promote permeation of a complex of a target substance and a membrane permeable substance through a lipid bilayer membrane, wherein the complex and any one of ⁇ 1> to ⁇ 4>
  • ⁇ 6> An intracellular delivery method for delivering a target substance into cells, wherein a complex of the target substance and a membrane permeable substance is formed in the presence of the membrane permeation enhancer according to ⁇ 1> or ⁇ 2>.
  • a method of intracellular delivery comprising contacting a cell with ⁇ 7> The intracellular delivery method according to ⁇ 6>, wherein the membrane-permeable substance is a membrane-permeable peptide.
  • the target substance is a low-molecular-weight compound having a molecular weight of 2000 or less, a protein, a nucleic acid, an antibody or a functional fragment thereof, an organelle, or a substance obtained by binding a labeled substance thereto, ⁇ 6> or ⁇ 7 > the intracellular delivery method described in .
  • An intracellular delivery kit for delivering a target substance into cells comprising a complex of the target substance and a membrane permeable substance and the membrane permeation enhancer according to ⁇ 1> or ⁇ 2>
  • a kit for intracellular delivery comprising:
  • a novel membrane permeation enhancer that is excellent in biocompatibility and capable of further promoting the membrane permeability of a membrane permeable substance complex, a membrane permeation enhancing method using the membrane permeation enhancer, and An intracellular delivery method and an intracellular delivery kit comprising the membrane permeation enhancer can be provided.
  • FIG. 4 is a diagram showing the degree of hemolysis when an erythrocyte solution and sodium cholate (NaC) solutions of various concentrations are mixed.
  • FIG. 4 is a diagram showing the degree of hemolysis when a red blood cell solution and lauroyl-L-arginine ethyl hydrochloride (LAEHCl) solutions of various concentrations are mixed.
  • FIG. 10 shows the results of flow cytometry when adding sodium cholate (NaC) together with FITC-labeled arginine tetramer (FITC-R4) to an erythrocyte solution.
  • FIG. 1 sodium cholate
  • FITC-R4 FITC-labeled arginine tetramer
  • FIG. 10 shows the results of flow cytometry when adding sodium cholate (NaC) together with FITC-labeled arginine octamer (FITC-R8) to an erythrocyte solution.
  • FIG. 3 shows the results of flow cytometry when adding sodium cholate (NaC) or lauroyl-L-arginine ethyl hydrochloride (LAEHCl) together with FITC-labeled arginine tetramer (FITC-R4) to a red blood cell solution.
  • FIG. 4 shows the results of flow cytometry when sodium cholate (NaC) or lauroyl-L-arginine ethyl hydrochloride (LAEHCl) was added to erythrocyte solution together with FITC-labeled arginine octamer (FITC-R8). .
  • the present invention is not limited to the following embodiments.
  • the numerical range indicated using “to” indicates the range including the numerical values before and after “to” as the minimum and maximum values, respectively.
  • the left side is the N-terminal side, and amino acid residues are represented by one-letter abbreviations (e.g., "R” for arginine residues) or three-letter abbreviations (for example, arginine residues) well known in the art. (for example, “Arg”).
  • the membrane permeation enhancer enhances the permeation of a complex of a target substance and a membrane-permeable substance (membrane-permeable substance complex) through a lipid bilayer membrane (hereinafter also simply referred to as “membrane”).
  • a facilitating membrane permeation enhancer comprising a surfactant having a steroid skeleton.
  • surfactants having a steroid skeleton are less likely to damage lipid bilayer membranes, so the added concentration can be increased, and as a result, the membrane permeability of the membrane-permeable substance complex can be further promoted. .
  • the lipid bilayer membrane may be a naturally occurring membrane or an artificial membrane.
  • naturally occurring membranes include biological membranes such as cell membranes (eg, membranes constituting blood cells such as erythrocytes), nuclear membranes, and membranes surrounding cell organelles.
  • cell membranes eg, membranes constituting blood cells such as erythrocytes
  • nuclear membranes e.g., nuclear membranes
  • membranes surrounding cell organelles e.g, a liposome etc. are mentioned as an artificial film
  • the lipid bilayer membrane may contain, in addition to lipids, other membrane constituents such as proteins and sugar chains.
  • a membrane-permeable substance complex is a complex of a target substance and a membrane-permeable substance.
  • a membrane-permeable substance is a substance that can permeate a lipid bilayer membrane without the mediation of transporters or receptors. It has the property of changing locally. It should be noted that changing the curvature of the membrane in a convex direction toward the membrane-permeable substance-donating side means that the membrane increases its curvature and protrudes toward the membrane-permeable substance-donating side. It includes both the meaning of reducing the curvature of the membrane which is concave to the side.
  • membrane-permeable substances examples include peptides or peptoids having 2 to 30 side chains and/or groups selected from the group consisting of guanidino side chains, amidino side chains, and amino groups, Oligomers having at least one bond selected from the group consisting of a bond, a urethane bond, a polyester bond, and a polyether bond are included.
  • membrane-permeable peptides CPP: Cell Penetrating Peptides
  • membrane-permeable peptides include polyarginines in which 3 to 13 consecutive arginines are linked (see, for example, Japanese Patent No. 6320469).
  • polyarginine examples include (Arg) 8 (SEQ ID NO: 1: RRRRRRRR) and (Arg) 4 (SEQ ID NO: 2: RRRR).
  • membrane-permeable peptides include Tat Peptide (48-60) (SEQ ID NO: 3: GRKKRRQRRRPPQ); Penetratin (SEQ ID NO: 4: RQIKIWFQNRRMKWKK); (Lys) 8 (SEQ ID NO: 5: KKKKKKKK ); HIV-1 Rev (34-50) (SEQ ID NO: 6: TRQARRNRRRRRWRERQR); FHV Coat (35-49) (SEQ ID NO: 7: RRRRNRTRRNRRRVR); BMV Gag (7-25) (SEQ ID NO: 8: KMTRAQRRAAARRNRWTAR); -II Rex (4-16) (SEQ ID NO: 9: TRRQRTRRARRNR); CCMV Gag (7-25) (SEQ ID NO: 10: KLTRAQRRAAAARKNKRNTR); P22 N (14-30) (SEQ ID NO: 11: NAKTRRHERRRKLAIER); 22) (SEQ ID NO: 12:
  • target substance examples of target substances that permeate lipid bilayer membranes include low-molecular-weight compounds with a molecular weight of 2000 g/mol or less (preferably molecular weight of 1500 g/mol or less), proteins, nucleic acids, antibodies or functional fragments thereof, cell organelles, and the like. and a labeling substance (fluorophore, affinity labeling agent, etc.) bound thereto. Proteins include enzymes, receptors, fluorescent proteins and the like. Nucleic acids include DNA, RNA, hybrid nucleic acids, aptamers, and the like. The isotype of the antibody is not particularly limited, and may be any of IgG, IgM, IgA, IgD, IgE, and the like.
  • the antibody may be either a polyclonal antibody or a monoclonal antibody.
  • Functional fragments of antibodies include Fab, Fab', F(ab') 2 , Fv, scFv and the like.
  • Examples of cell organelles include mitochondria.
  • the target substance may be directly covalently bonded to the membrane-permeable substance, or may be covalently bonded via a linker.
  • the membrane-permeable substance is a membrane-permeable peptide
  • the binding position of the target substance may be on the C-terminal side or the N-terminal side of the membrane-permeable peptide.
  • the complex of the target substance and the membrane-permeable substance can be a fusion protein.
  • a surfactant having a steroid skeleton is an amphipathic substance having a hydrophobic portion containing a steroid skeleton and a hydrophilic portion.
  • hydrophilic moieties include carboxylate groups, sulfonate groups, sulfate ester groups, phosphate ester groups, phosphonate groups, amine groups, quaternary ammonium groups, pyridinium groups, sulfonium groups, phosphonium groups, and polyethylenepolyamine groups.
  • surfactants having a steroid skeleton can be anionic surfactants, cationic surfactants, amphoteric surfactants, or nonionic surfactants, depending on the type of hydrophilic moiety.
  • Salts in the case where the surfactant having a steroid skeleton has an acidic functional group include alkali metal salts such as sodium salts and potassium salts. Examples of salts in the case where the surfactant having a steroid skeleton has a basic functional group include inorganic acid salts such as hydrochloride and bromate; organic acid salts such as acetate and lactate; and the like.
  • the surfactant having a steroid skeleton may be a naturally occurring surfactant or an artificial surfactant.
  • Specific examples of surfactants having a steroid skeleton include cholic acid or salts thereof, glycocholic acid or salts thereof, taurocholic acid or salts thereof, deoxycholic acid or salts thereof, glycodeoxycholic acid or salts thereof, tauro Deoxycholic acid or its salts, lithocholic acid or its salts, glycolitocholic acid or its salts, chenodeoxycholic acid or its salts, ursodeoxycholic acid or its salts, tauroursodeoxycholic acid or its salts, hyocholic acid or its salts, hyocholic acid Deoxycholic acid or its salts, polyoxyethylene cholesteryl ether, N,N-bis(3-D-gluconamidopropyl) cholamide (BIGCHAP), 3-[(3-cholamidopropyl)dimethylammoni
  • surfactants having a steroid skeleton may be used singly or in combination of two or more.
  • the membrane permeation enhancer according to this embodiment may contain various additives in addition to the surfactant having a steroid skeleton.
  • the application target of the membrane permeation enhancer according to the present embodiment is not particularly limited, and examples thereof include mammals (mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human, etc.) and animal cells. be done.
  • the method for promoting membrane permeation according to the present embodiment is a method for promoting permeation of a membrane permeable substance complex through a lipid bilayer membrane, and comprises the membrane permeable substance complex and the membrane permeation according to the present embodiment. Contacting the facilitating agent with the lipid bilayer.
  • the membrane permeable substance complex, the membrane permeation enhancer according to the present embodiment, and, if necessary, additives are mixed and administered in vivo as a preparation.
  • a method of promoting penetration of the membrane-permeable substance complex into the target cells by contacting with the target cells in vivo can be mentioned.
  • collected target cells for example, blood cells such as erythrocytes
  • a membrane permeable substance complex for example, a membrane permeable substance complex
  • the membrane permeation enhancer according to the present embodiment are combined into a solution.
  • the concentration of the surfactant having a steroid skeleton in the solution is, for example, preferably 5 ⁇ M to 50 mM, more preferably 10 ⁇ M to 30 mM, even more preferably 20 ⁇ M to 20 mM.
  • the intracellular delivery method according to the present embodiment is an intracellular delivery method for delivering a target substance into cells, wherein a complex of a target substance and a membrane permeable substance (membrane permeable substance complex) is Contacting the cell in the presence of a morphological membrane permeation enhancer.
  • target substances to be delivered into cells include low-molecular-weight compounds with a molecular weight of 2000 g/mol or less (preferably molecular weight of 1500 g/mol or less), proteins, nucleic acids, antibodies or functional fragments thereof, cell organelles, and labels thereof. Examples include substances bound with substances (fluorophores, affinity labeling agents, etc.). Proteins include enzymes, receptors, fluorescent proteins and the like. Nucleic acids include DNA, RNA, hybrid nucleic acids, aptamers, and the like. The isotype of the antibody is not particularly limited, and may be any of IgG, IgM, IgA, IgD, IgE, and the like.
  • the antibody may be either a polyclonal antibody or a monoclonal antibody.
  • Functional fragments of antibodies include Fab, Fab', F(ab') 2 , Fv, scFv and the like.
  • Examples of cell organelles include mitochondria.
  • the target substance may be directly covalently bonded to the membrane-permeable substance, or may be covalently bonded via a linker.
  • the membrane-permeable substance is a membrane-permeable peptide
  • the binding position of the target substance may be on the C-terminal side or the N-terminal side of the membrane-permeable peptide.
  • the complex of the target substance and the membrane-permeable substance can be a fusion protein.
  • the target substance may be for therapeutic purposes or for examination purposes.
  • the target substance may be a therapeutic substance or a test substance.
  • therapeutic substances include drugs such as low-molecular-weight drugs, nucleic acid drugs, and high-molecular-weight drugs.
  • drugs such as low-molecular-weight drugs, nucleic acid drugs, and high-molecular-weight drugs.
  • therapeutic substances include substances to be introduced into transplanted cells for cell medicine. For example, by complexing mitochondria with a membrane-permeable substance and bringing it into contact with transplanted cells together with the membrane permeation enhancer according to the present embodiment to permeate into the cells, the therapeutic effect of cell medicine can be further improved.
  • test substances include antibodies or functional fragments thereof, lectins, nucleic acids, and substances bound to labeled substances.
  • Specific examples of test substances include antibodies or functional fragments thereof against malaria-derived proteins (malaria aggravation markers) present in red blood cells of malaria-infected patients, cancer markers, and the like.
  • Such an antibody or a functional fragment thereof is conjugated with a membrane-permeable substance, and is brought into contact with the cell together with the membrane permeation enhancer according to the present embodiment to permeate into the cell, thereby improving the accuracy of the test for intracellular molecules. It can be improved further.
  • the delivery of the target substance into cells may be performed in vivo or in vitro.
  • the intracellular delivery kit according to the present embodiment is a kit for intracellular delivery that delivers a target substance into cells, comprising a complex of a target substance and a membrane permeable substance (membrane permeable substance complex), and a membrane permeation enhancer according to the present embodiment.
  • the intracellular delivery kit according to this embodiment can be suitably used for the intracellular delivery method described above.
  • the concentrations of sodium cholate in each solution after mixing with the red blood cell solution were 13.5 mM, 16.5 mM, 19 mM, 21.5 mM, 27 mM, 36 mM, 45 mM, 54 mM, 72 mM, 81 mM, 108 mM, and 162 mM. be. Thereafter, centrifugation (25° C., 3000 rpm, 5 min) was performed to recover the supernatant, and the degree of hemolysis was evaluated by measuring absorbance at a wavelength of 540 nm using a UV-visible spectrophotometer. Hemolysis% was calculated based on the absorbance of hemolysis with water (complete hemolysis state) as 100%.
  • lauroyl-L-arginine ethyl hydrochloride (54 ⁇ M, 75 ⁇ M, 110 ⁇ M, 160 ⁇ M, 210 ⁇ M, 540 ⁇ M) dissolved in 0.4% red blood cell solution (3 mL) and isotonic (140 mM) PBS solution ( LAEHCl) solution (3 mL) and incubated (25° C., 30 min).
  • the concentrations of LAEHCl in each solution after mixing with the erythrocyte solution are 27 ⁇ M, 37.5 ⁇ M, 55 ⁇ M, 80 ⁇ M, 105 ⁇ M and 270 ⁇ M.
  • FIG. 1A The results of adding the sodium cholate solution are shown in FIG. 1A, and the results of adding the LAEHCl solution are shown in FIG. 1B.
  • the horizontal axis of FIG. 1A indicates the concentration of the sodium cholate solution before mixing with the red blood cell solution
  • the horizontal axis of FIG. 1B indicates the concentration of the LAEHCl solution before mixing with the red blood cell solution.
  • FIGS. 1A and 1B there was approximately a 500-fold difference in the concentration that caused hemolysis between the addition of the sodium cholate solution and the addition of the LAEHCl solution. This result shows that sodium cholate has significantly better biocompatibility than LAEHCl.
  • FITC-labeled arginine tetramer (FITC-R4) and FITC-labeled arginine octamer (FITC-R8) were used as model molecules of the membrane permeable substance complex.
  • FITC-labeled arginine tetramer (FITC-R4) is a compound in which x is 4 in the following formula
  • FITC-labeled arginine octamer (FITC-R8) is a compound in which x is 8 in the following formula.
  • red blood cell solution 100 ⁇ L
  • 80 mM PBS solution 60 ⁇ L
  • 27 mM or 43 mM sodium cholate (NaC) solution 100 ⁇ M membrane permeable substance complex
  • FITC-labeled arginine tetramer FITC-R4
  • FITC-labeled arginine octamer FITC-R8
  • centrifuge After completion of incubation, centrifuge (4° C., 10500 rpm, 5 min) to remove the supernatant, add 80 mM PBS solution (500 ⁇ L) and 0.05% trypsin solution (20 ⁇ L), incubate (37° C., 10 min )bottom. Thereafter, centrifugation (4° C., 10500 rpm, 5 min) was performed twice to remove the supernatant, 80 mM PBS solution (500 ⁇ L) was added, and the sample was analyzed by flow cytometry.
  • the sodium cholate (NaC) solution was not added and the PBS solution was added in an amount of 360 ⁇ L, and the sodium cholate (NaC) solution and the membrane permeable substance complex were not added but the PBS solution was added.
  • An addition amount of 400 ⁇ L was prepared and analyzed by flow cytometry in the same manner.
  • FIG. 2B The results of flow cytometry when FITC-labeled arginine tetramer (FITC-R4) was added as the membrane-permeable substance complex are shown in FIG. 2B. As shown in FIGS. 2A and 2B, when the sodium cholate solution was added together with the membrane permeable substance complex, the peak of the histogram shifted to the right. These results show that sodium cholate promotes the membrane permeability of the membrane permeable substance complex.
  • LAEHCl lauroyl-L-arginine ethyl hydrochloride
  • FIG. 3B The results of flow cytometry when FITC-labeled arginine tetramer (FITC-R4) was added as the membrane-permeable substance complex are shown in FIG. 3B.
  • FIGS. 3A and 3B when 43 mM sodium cholate solution was added together with the membrane permeable substance complex, the histograms were significantly higher than when 75 ⁇ M LAEHCl solution was added together with the membrane permeable substance complex. The peak was shifted to the right.
  • sodium cholate has remarkably superior biocompatibility compared to LAEHCl, so the addition concentration can be increased, and as a result, the membrane permeability of the membrane-permeable substance complex is further promoted. be able to.
  • FITC-R4 membrane permeable substance complex
  • FITC-R8 FITC-labeled arginine octamer
  • Membrane permeation enhancers include sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC), sodium taurodeoxycholate (NaTDC), sodium glycodeoxycholate (NaGDC), 3-[ (3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) used.
  • NaC sodium cholate
  • NaDC sodium deoxycholate
  • NaTC sodium taurocholate
  • NaTDC sodium taurodeoxycholate
  • NaGDC sodium glycodeoxycholate
  • CHAPS 3-[ (3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
  • CHPA 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
  • centrifuge After completion of incubation, centrifuge (4° C., 10500 rpm, 5 min) to remove the supernatant, add 80 mM PBS solution (500 ⁇ L) and 0.05% trypsin solution (160 ⁇ L), incubate (37° C., 10 min )bottom. Thereafter, centrifugation (4° C., 10500 rpm, 5 min) was performed twice to remove the supernatant, 80 mM PBS solution (500 ⁇ L) was added, and the sample was analyzed by flow cytometry.
  • the membrane permeation amount of the membrane permeable substance complex was calculated from the analysis results by flow cytometry.
  • Table 1 below shows the amount of FITC-labeled arginine tetramer (FITC-R4) permeated by membrane permeation enhancers at various concentrations. are shown in Table 2 below. Tables 1 and 2 below show relative values based on the assumption that the membrane permeation amount of the membrane permeable substance complex when no membrane permeation enhancer is added is 1.00.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention fournit : un activateur de perméation membranaire qui contient un tensioactif ayant un squelette stéroïde, et améliore la perméation d'un complexe d'une substance voulue et d'une substance perméable à la membrane par l'intermédiaire d'une membrane bimoléculaire lipidique ; et un procédé d'amélioration de perméation membranaire utilisant l'activateur de perméation membranaire. La présente invention fournit également : un procédé d'administration intracellulaire pour administrer une substance voulue dans une cellule par mise en contact d'un complexe de la substance voulue et d'une substance perméable à la membrane avec la cellule en présence d'un activateur de perméation membranaire ; et un kit qui est destiné à une administration intracellulaire et qui est utilisé de manière appropriée dans le procédé d'administration intracellulaire.
PCT/JP2022/031507 2021-08-27 2022-08-22 Activateur de perméation membranaire, procédé d'amélioration de perméation membranaire et procédé d'administration intracellulaire Ceased WO2023027006A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021-138716 2021-08-27
JP2021138716 2021-08-27

Publications (1)

Publication Number Publication Date
WO2023027006A1 true WO2023027006A1 (fr) 2023-03-02

Family

ID=85322295

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/031507 Ceased WO2023027006A1 (fr) 2021-08-27 2022-08-22 Activateur de perméation membranaire, procédé d'amélioration de perméation membranaire et procédé d'administration intracellulaire

Country Status (1)

Country Link
WO (1) WO2023027006A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08507759A (ja) * 1993-02-22 1996-08-20 セラテック・インコーポレイテッド 高分子薬物の粘膜透過による投与
JP2005154413A (ja) * 2003-09-25 2005-06-16 Ajinomoto Co Inc 膜透過性物質の膜透過の制御方法及び膜透過性物質のスクリーニング方法
WO2021054351A1 (fr) * 2019-09-17 2021-03-25 学校法人東京理科大学 Amplificateur de perméation membranaire et procédé d'amélioration de la perméation membranaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08507759A (ja) * 1993-02-22 1996-08-20 セラテック・インコーポレイテッド 高分子薬物の粘膜透過による投与
JP2005154413A (ja) * 2003-09-25 2005-06-16 Ajinomoto Co Inc 膜透過性物質の膜透過の制御方法及び膜透過性物質のスクリーニング方法
WO2021054351A1 (fr) * 2019-09-17 2021-03-25 学校法人東京理科大学 Amplificateur de perméation membranaire et procédé d'amélioration de la perméation membranaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAZARI MOZHGAN, KURDI MUSTAFA, HEERKLOTZ HEIKO: "Classifying Surfactants with Respect to Their Effect on Lipid Membrane Order", BIOPHYSICAL JOURNAL, ELSEVIER, AMSTERDAM, NL, vol. 102, no. 3, 1 February 2012 (2012-02-01), AMSTERDAM, NL, pages 498 - 506, XP093039040, ISSN: 0006-3495, DOI: 10.1016/j.bpj.2011.12.029 *
YAMAMOTO AKIRA: "Improvement of transmucosal absorption of peptide and protein drugs", DRUG DELIVERY SYSTEM, TOKYO, vol. 15, no. 6, 1 November 2000 (2000-11-01), Tokyo, pages 507 - 519, XP093039036, DOI: 10.2745/dds.15.507 *

Similar Documents

Publication Publication Date Title
Futaki et al. Cell-surface interactions on arginine-rich cell-penetrating peptides allow for multiplex modes of internalization
Nakase et al. Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery
Kobayashi et al. Cytosolic targeting of macromolecules using a pH-dependent fusogenic peptide in combination with cationic liposomes
Nakase et al. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis
JP5269597B2 (ja) 血液―脳隔壁エピトープとその利用
Foerg et al. Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape
Lebreton et al. Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides
Boeneman et al. Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials
JP2022097596A (ja) 自己免疫疾患の診断、予防、および／または治療
Dhanvantari et al. Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion
Nitin et al. Tat peptide is capable of importing large nanoparticles across nuclear membrane in digitonin permeabilized cells
Rehmani et al. Orally-delivered insulin-peptide nanocomplexes enhance transcytosis from cellular depots and improve diabetic blood glucose control
US20240400628A1 (en) Polypeptides, nucleic acid molecules, compositions, and related methods
JP4838957B2 (ja) 無細胞タンパク質合成系を用いたタンパク質の製造方法
Tan et al. Rapid endolysosomal escape and controlled intracellular trafficking of cell surface mimetic quantum-dots-anchored peptides and glycopeptides
US20230025108A1 (en) RAPID ASSAY FOR DETECTION OF SARS-CoV-2 ANTIBODIES
Keum et al. Examination of effective buccal absorption of salmon calcitonin using cell-penetrating peptide-conjugated liposomal drug delivery system
Hong et al. Enhanced cellular uptake and transport of polyclonal immunoglobulin G and fab after their cationization
Greschner et al. PEGylation of a peptide-based amphiphilic delivery agent and influence on protein delivery to cells
Akita et al. Particle tracking analysis for the intracellular trafficking of nanoparticles modified with African swine fever virus protein p54-derived peptide
WO2023027006A1 (fr) Activateur de perméation membranaire, procédé d'amélioration de perméation membranaire et procédé d'administration intracellulaire
WO2018000324A1 (fr) Procédé d'introduction d'un anticorps dans une cellule
Cruz-Bustos et al. Functionalization of immunostimulating complexes (ISCOMs) with lipid vinyl sulfones and their application in immunological techniques and therapy
US20040063905A1 (en) Armed peptides
Deo et al. A model for targeting colon carcinoma cells using single-chain variable fragments anchored on virus-like particles via glycosyl phosphatidylinositol anchor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22861294

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22861294

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP