WO2016068228A1 - Véhicule pour libération prolongée de médicament, et procédé pour libération prolongée de médicament - Google Patents

Véhicule pour libération prolongée de médicament, et procédé pour libération prolongée de médicament Download PDF

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WO2016068228A1
WO2016068228A1 PCT/JP2015/080530 JP2015080530W WO2016068228A1 WO 2016068228 A1 WO2016068228 A1 WO 2016068228A1 JP 2015080530 W JP2015080530 W JP 2015080530W WO 2016068228 A1 WO2016068228 A1 WO 2016068228A1
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solution
drug
sustained
ringer
release
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PCT/JP2015/080530
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Japanese (ja)
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一朗 藤本
薫子 池田
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株式会社高研
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Priority to JP2016512715A priority Critical patent/JP6186572B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a drug sustained-release carrier or a drug sustained-release method for introducing a desired nucleic acid, protein, peptide, low molecular compound or the like into a target cell.
  • Drug sustained release carrier A plurality of drug sustained-release carriers have been reported as described in the following literature.
  • Patent Document 1 describes the use of RNA and an aqueous injection buffer in preparing an RNA injection solution to increase RNA transport and / or RNA translation in / in a host organism, Discloses the use comprising sodium, calcium and, optionally, potassium salts.
  • the injection buffer contains collagen or a collagen derivative” and “sustained release effect” are not specified.
  • Patent Document 2 discloses "a nucleic acid introduction promoter containing collagen or a collagen derivative”. However, it does not disclose or suggest that the nucleic acid introduction promoter contains a “Ringer solution”.
  • Patent Document 3 discloses a “sustained release pharmaceutical composition comprising a drug, collagen, and one or more sugars selected from monosaccharides, disaccharides, trisaccharides and tetrasaccharides”. However, there is no disclosure or suggestion that the sustained release pharmaceutical composition contains a “Ringer solution”.
  • the present invention provides a sustained-release carrier or drug sustained-release method that has higher gelation rate, sustained-release action and cell introduction efficiency than conventional drug-release carriers or sustained-release methods containing collagen. is there.
  • Drug sustained-release carrier comprising: (1) Collagen or collagen derivative (2) Solution containing potassium salt, calcium salt and sodium salt
  • the sustained-release drug carrier according to any one of 1 to 10 above, wherein the drug is a nucleic acid, protein, peptide, and / or low molecular weight compound. 12 12. The drug sustained-release carrier according to any one of items 4 to 11, wherein the Ringer's solution is a bicarbonated Ringer's solution and the drug is a nucleic acid. 13 12. The drug sustained-release carrier according to any one of items 4 to 11, wherein the Ringer's solution is a lactated Ringer's solution and the drug is a nucleic acid. 14 14. A medical device, wherein the drug sustained-release carrier according to any one of items 1 to 13 is applied to a surface. 15. 14.
  • a cell culture instrument wherein the drug sustained-release carrier according to any one of items 1 to 13 is applied to a cell culture surface. 16. 14. A drug comprising the drug sustained release carrier according to any one of 1 to 13 above. 17. Use of a drug sustained-release carrier according to any one of items 1 to 13 or a drug sustained-release carrier to administer the drug according to item 13 to mammals other than humans. 18. A method for sustained drug release comprising the following steps: (1) a step of mixing a drug and a solution containing collagen or collagen derivative and potassium salt, calcium salt and sodium salt to prepare a mixed solution; and (2) a patient who needs the drug for the mixed solution. The step of administering to. 19. 19. 19.
  • Potassium chloride 0.001mM to 5.00M Calcium chloride: 0.001mM to 10.00M Sodium chloride: 0.001mM to 10.00M Sodium bicarbonate: 0.001mM to 2.00M Magnesium chloride: 0.001mM to 5.00M Trisodium citrate: 0.001 mM to 2.00 M Sodium lactate: 0.001mM to 10.00M Sodium acetate: 0.001mM to 10.00M 26. 26.
  • the drug sustained-release carrier has gel-forming ability.
  • the drug sustained-release carrier or drug sustained-release method of the present invention has the following effects compared to conventional drug carriers or drug sustained-release methods. (1) Since the gel forming ability is high, it is possible to hold a high concentration transport object (drug) for a long time. (2) Since the sustained release action ability is high, the drug can be supplied to the target cells for a long time. (3) Since the introduction efficiency of the drug into the target cell is high, the drug effect is high. (4) The amount of drug outflow when administered locally can be suppressed.
  • Measurement result of gel forming ability External view of filter tube. Measurement results of sustained release action ability. Measurement results of introduction efficiency into target cells to be transported.
  • the “drug sustained release carrier” of the present invention is a carrier (carrier) containing a solution containing collagen or a collagen derivative and Ringer's solution.
  • the “drug sustained release carrier” of the present invention has an effect of transporting a transport target to a cell ⁇ target cell (a cell into which the transport target is introduced) ⁇ . Furthermore, the drug sustained-release carrier of the present invention not only simply transports the object to be transported to the cells, but also has a high gel-forming ability, so it can hold a high-concentration transport object (drug) for a long time, and has a sustained-release functioning ability. Therefore, the drug can be supplied to the target cells for a long time, and the drug has a high medicinal effect because the efficiency of introducing the drug into the target cells is high.
  • the “drug sustained release method” of the present invention is a method of using a collagen or collagen derivative and Ringer's solution to transport a transport target to a cell ⁇ target cell (cell into which the transport target is introduced) ⁇ . Furthermore, the sustained drug release method of the present invention not only simply transports the transport target to the cells, but also has a high gel-forming ability, so it can hold a high concentration transport target (medicine) for a long time, and has a sustained release function. Therefore, the drug can be supplied to the target cells for a long time, and the drug has a high medicinal effect because the efficiency of introducing the drug into the target cells is high.
  • the “solution containing potassium salt, calcium salt and sodium salt” of the present invention indicates a physiological electrolyte solution having an ionic composition, an osmotic pressure and a hydrogen ion concentration for use in extracellular fluid replacement, and has a specific composition.
  • Ringer's solution is preferable.
  • the potassium salt include potassium chloride, potassium iodide, potassium bromide, potassium carbonate, potassium hydrogen carbonate, potassium sulfate, and hydrates thereof, and potassium chloride is preferable.
  • Examples of calcium salts include calcium chloride, calcium iodide, calcium bromide, calcium carbonate, calcium sulfate, calcium hydroxide, and their hydrates, and calcium chloride is preferred.
  • Examples of the sodium salt include sodium chloride, sodium iodide, sodium bromide, sodium carbonate, sodium hydrogen carbonate, sodium sulfate, and hydrates thereof, and sodium chloride is preferable.
  • the pH value of this solution is 4.0 to 10.0, more preferably 6.0 to 8.5, and still more preferably 6.7 to 7.8.
  • this solution may contain a phosphate buffer, HEPES, Na 2 HPO 4 / NaH 2 PO 4, etc., as necessary, in order to adjust the pH value.
  • the Ringer's solution of the present invention can preferably be exemplified by Ringer's basic solution, bicarbonated Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, or those obtained by adding carbohydrates to the Ringer's solution, but is not particularly limited.
  • sugars include xylose, arabinose, ribose, glucose, mannose, galactose, fructose, fucose, inositol, glucosamine, and erythritol.
  • disaccharides include sucrose, maltose, lactose, trehalose, and isomaltose.
  • raffinose and tetrasaccharide include stachyose.
  • the Ringer base solution used in the present invention can be exemplified by the following concentration ranges including at least potassium chloride, calcium chloride and sodium chloride.
  • the concentration of potassium chloride is 0.001 mM to 5.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of calcium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of sodium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 5.00 M, more preferably 0.1 mM to 1.00 M, even more preferably 1.0 mM to 500 mM, and most preferably 50 mM to 200 mM.
  • the “lactic acid Ringer's solution” used in the present invention essentially includes sodium lactate, and may contain two or more or three or more from any of sodium chloride, potassium chloride, and calcium chloride. Although the density
  • the concentration of sodium lactate is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 500 mM, even more preferably 1.0 mM to 100 mM, and most preferably 10 mM to 50 mM.
  • the concentration of potassium chloride is 0.001 mM to 5.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of calcium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of sodium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 5.00 M, more preferably 0.1 mM to 1.00 M, even more preferably 1.0 mM to 500 mM, and most preferably 50 mM to 200 mM.
  • the “bicarbonate Ringer's solution” used in the present invention essentially comprises sodium bicarbonate, and may contain 3 or more or 4 or more from any of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and trisodium citrate.
  • concentration of each component is not specifically limited, The following can be illustrated.
  • the concentration of sodium bicarbonate is 0.001 mM to 2.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 500 mM, even more preferably 1.0 mM to 100 mM, and most preferably 10 mM to 50 mM.
  • the concentration of potassium chloride is 0.001 mM to 5.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of calcium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of sodium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 5.00 M, more preferably 0.1 mM to 1.00 M, even more preferably 1.0 mM to 500 mM, and most preferably 50 mM to 200 mM.
  • the concentration of magnesium chloride is 0.001 mM to 5.00 M, preferably 0.005 mM to 1.00 M, more preferably 0.01 mM to 500 mM, even more preferably 0.1 mM to 50 mM, and most preferably 0.5 mM to 5 mM.
  • the concentration of trisodium citrate is 0.001 mM to 2.00 M, preferably 0.005 mM to 1.00 M, more preferably 0.01 mM to 500 mM, even more preferably 0.1 mM to 50 mM, and most preferably 0.5 mM to 5 mM.
  • the “acetated Ringer's solution” used in the present invention essentially includes sodium acetate and may contain 2 or more or 3 or more from any of sodium chloride, potassium chloride, and calcium chloride. Although the density
  • the concentration of sodium acetate is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 500 mM, even more preferably 1.0 mM to 100 mM, and most preferably 10 mM to 50 mM.
  • the concentration of potassium chloride is 0.001 mM to 5.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of calcium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 1.00 M, more preferably 0.1 mM to 100 mM, even more preferably 0.5 mM to 50 mM, and most preferably 1.0 mM to 10 mM.
  • the concentration of sodium chloride is 0.001 mM to 10.00 M, preferably 0.01 mM to 5.00 M, more preferably 0.1 mM to 1.00 M, even more preferably 1.0 mM to 500 mM, and most preferably 50 mM to 200 mM.
  • the “collagen or collagen derivative” of the present invention means any “collagen or collagen derivative” usually used in the medical field, cosmetic field, industrial field and food field.
  • soluble or solubilized collagen is preferably used.
  • Soluble collagen is soluble in acidic or neutral water or salt solution, and solubilized collagen includes enzyme-solubilized collagen that is solubilized by enzymes, alkali-solubilized collagen that is solubilized by alkalis, In any case, it is preferable that the pore size can pass through a membrane filter of 1 micrometer.
  • Collagen can be used from any animal species, but is preferably extracted from vertebrates, more preferably extracted from mammals, birds and fish, more preferably a denaturation temperature.
  • Collagen extracted from high mammals and birds is desirable.
  • the collagen type may be any type of collagen, but types I to V are preferred in view of the abundance in the animal body. Specific examples include type I collagen extracted from the dermis of mammals, and more preferable examples include type I collagen extracted from calf dermis, type I collagen produced by genetic engineering, and the like. It is done. Further, from the viewpoint of safety, atelocollagen obtained by enzymatically removing a highly antigenic telopeptide or atelocollagen produced by genetic engineering is desirable, and atelocollagen having 3 or less tyrosine residues per 1000 residues is more preferable.
  • the preferred collagen or collagen derivative of the present invention is atelocollagen.
  • the following cell membrane-permeable peptide may be added to the collagen or collagen derivative.
  • the introduction rate of target cells can be improved.
  • TAT GRKKRRQRRRPQ: SEQ ID NO: 1
  • r8 ⁇ rrrrrrrr D form-R: SEQ ID NO: 2 ⁇
  • MPG-8 ⁇ AFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 3
  • the drug sustained-release carrier of the present invention can be prepared by mixing collagen or a collagen derivative, a solution containing potassium salt, calcium salt and sodium salt, and a drug by a method known per se.
  • the drug sustained-release carrier of the present invention can also contain biocompatible materials, additives and the like in addition to collagen or a collagen derivative and a solution containing potassium salt, calcium salt and sodium salt.
  • biocompatible materials include gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose, hydroxyapatite, polypropylene, polyethylene, polydimethylsiloxane, or glycolic acid, lactic acid or Examples thereof include polymers of amino acids or copolymers thereof, and mixtures of two or more kinds of these biocompatible materials.
  • the additive examples include an isotonic agent, a pH adjuster, a soothing agent when used as an injection, an excipient, a disintegrant, and a coating agent when used as a solid agent.
  • Specific examples include salts and saccharides used to maintain the pH at 6 to 8 or to keep isotonicity with cells.
  • the drug sustained-release carrier of the present invention may be solid or solution. When the drug sustained-release carrier of the present invention is in a solid state, it is used as it is or in the form of a solution using purified water, physiological saline, a buffer solution isotonic with a living body, and the like, and is introduced into desired cells.
  • the drug sustained-release carrier of the present invention may further contain one or more sugars selected from monosaccharides, disaccharides, trisaccharides, and tetrasaccharides in order to improve the sustained release action ability (see: Patents). Reference 3).
  • the use of the drug sustained-release carrier of the present invention is not particularly limited, it can be used for drugs, medical instruments, cell culture instruments, labeling agents and the like.
  • Methods for administering the drug sustained-release carrier of the present invention are oral, injection, eye drops , Nasal, pulmonary, and absorption through the skin, and preferably injection.
  • the administration site can be selected depending on the disease, but it can also be placed directly at the site required during surgery.
  • systemic administration of the drug sustained-release carrier of the present invention by intravenous injection (infusion, etc.), local administration by injecting into the affected area (cancer cells, etc.) and the like can be mentioned.
  • the “drug sustained release method” of the present invention has at least the following steps. (1) A step of preparing a mixed solution by mixing a drug and a solution containing collagen or a collagen derivative and potassium salt, calcium salt and sodium salt. (2) A step of administering the mixed solution to a patient in need of the drug.
  • a step of preparing a mixed solution by mixing a drug and a solution containing collagen or a collagen derivative and potassium salt, calcium salt and sodium salt (2) A step of administering the mixed solution to a patient in need of the drug.
  • the drug sustained-release carrier or drug sustained-release method of the present invention has higher gel forming ability than the drug carrier or drug sustained-release method containing known collagen (atelocollagen). Due to the high gel-forming ability of the drug sustained-release carrier or the drug sustained-release method of the present invention, it is possible to hold a high concentration transport target for a long time.
  • the drug sustained-release carrier or drug sustained-release method of the present invention has a higher sustained release action ability than a drug carrier or drug sustained-release method containing known collagen (atelocollagen).
  • the drug can be supplied for a long time due to the high sustained release action ability of the drug sustained-release carrier or drug sustained-release method of the present invention.
  • the drug sustained-release carrier or drug sustained-release method of the present invention has higher target cell introduction efficiency than the drug carrier or drug sustained-release method containing known collagen (atelocollagen). Due to the high cell introduction efficiency of the drug sustained-release carrier or drug sustained-release method of the present invention, the drug effect is high.
  • the “drug” in the present invention is not particularly limited, but includes nucleic acids, proteins, peptides, low molecular compounds and the like.
  • (protein) Examples include, but are not particularly limited to, agonists / antagonists for the enzyme and the target receptor, the receptor itself, and antibodies.
  • (peptide) Examples of the low molecular weight protein include those having enzyme activity, those obtained by synthesizing a part of a functional protein, and agonists / antagonists for the target receptor, but are not particularly limited.
  • (Low molecular compound) Examples of the anti-cancer agent that is a low-molecular-weight drug include those that are specifically effective for killing tumor cells, and those that enhance or suppress the physiological activity of cells, but are not particularly limited.
  • the “nucleic acid to be transported” in the present invention may be a polynucleotide or an oligonucleotide, and may be a DNA or RNA molecule.
  • a DNA molecule it may be plasmid DNA, cDNA, genomic DNA or synthetic DNA. Both DNA and RNA can be double-stranded or single-stranded. In the case of a single strand, it can be a coding strand or a non-coding strand.
  • Nucleic acid includes DNA derivatives or RNA derivatives, and these derivatives have phosphorothioate-bonded nucleic acids, or chemically modified at the phosphate moiety, sugar moiety, or base moiety of the internucleotide to avoid enzymatic degradation. Means nucleic acid.
  • the “nucleic acid” also includes viruses such as adenovirus and retrovirus. When the nucleic acid is a vector used for gene therapy such as plasmid DNA or virus, a form configured to express the encoded genetic information in the cell when introduced into the cell is preferable. A vector containing an element necessary for the expression of or containing an element enabling integration into a chromosome.
  • the present invention is also directed to a medical device or a cell culture device on which the drug sustained-release carrier of the present invention is applied.
  • a medical device or a cell culture device on which the drug sustained-release carrier of the present invention is applied.
  • the drug sustained-release carrier of the present invention is applied to the solid phase surface and the target cells are brought into contact therewith, the introduction efficiency of the transport target is improved compared to the case where the drug sustained-release carrier of the present invention is dropped from above the target cells.
  • the medical device of the present invention includes an artificial organ, more specifically, an artificial blood vessel, a medical device stent that reinforces a blood vessel, an adhesive sheet, or an artificial heart.
  • the cell culture instrument of the present invention include a petri dish, a flask, a 96-well microplate, a three-dimensional culture carrier and the like that are usually used for cell culture experiments.
  • the function of the gene or protein in the target cell can be easily examined.
  • a method for examining the function of a gene by introducing and expressing a plasmid DNA in which the gene whose function is to be examined is introduced into the cell, or an siRNA nucleic acid that suppresses the expression of the gene whose function is to be examined is introduced into the cell.
  • a method for examining the function of a gene by suppressing the expression of the gene is useful.
  • plasmid DNA that expresses a gene whose function is to be elucidated, adenovirus vector, or siRNA nucleic acid that suppresses expression of a gene whose function is to be elucidated and the drug sustained-release carrier of the present invention are mixed, and the culture plate is fixed. Apply and align on phase. After the applied drug sustained-release carrier is dried and fixed on the solid phase, the cells are seeded and cultured on a plate for several days. The applied drug sustained-release carrier is efficiently introduced into cells adhered to the applied part, and expresses a gene whose function is to be examined or suppresses its expression for a long period of time. After a few days, the function of the targeted gene can be determined by examining the cell growth rate, morphology (phenotype), state of gene expression in the cell (gene expression level), or the type and amount of protein produced from the cell. Can be revealed.
  • candidate substances capable of treating various diseases such as genetic diseases, cancer, AIDS, rheumatoid arthritis, lifestyle-related diseases and the like can be screened.
  • a candidate substance eg, nucleic acid
  • a drug sustained-release carrier of the present invention are mixed, and applied and aligned on a culture plate solid phase. After the applied drug sustained-release carrier is dried and fixed on the solid phase, the cells are seeded and cultured on a plate for several days.
  • the effect of a candidate substance eg, nucleic acid
  • the properties of the target cells can be changed.
  • an antagonist that suppresses the activity of a specific intracellular receptor is administered to a target cell as a delivery target
  • the target cell has a lower activity of the receptor compared to a target cell to which the antagonist is not administered.
  • the nature of the target cell can be altered.
  • siRNA capable of degrading a specific mRNA is administered to a target cell as a delivery target, a cell in which the mRNA is degraded and the expression level of the functional protein encoded by the mRNA is reduced can be obtained.
  • the present invention is also directed to a labeling agent comprising the drug sustained-release carrier of the present invention.
  • a target cell can be labeled by administering to a living body a transport target in which a labeling substance is bound to a substance that specifically recognizes the target cell or the like (a peptide or protein that specifically binds to the target cell). .
  • a preferred combination of the drug sustained-release carrier or the drug sustained-release method of the present invention is as follows, but is not particularly limited, and may further contain a biocompatible material and / or an additive as necessary.
  • the reagents used are as follows. ⁇ SiRNA: GL3 siRNA (siRNA for firefly luciferase) Sequence (sense side): 5'-CUUACGCUGAGUACUUCGA-3 '(SEQ ID NO: 4) ⁇ Atelocollagen (product of Koken, hereinafter referred to as AC) ⁇ Sodium chloride (Wako) ⁇ Potassium chloride (Wako) ⁇ Calcium chloride (Wako) ⁇ Magnesium chloride hexahydrate (Wako) ⁇ Sodium bicarbonate (Wako) ⁇ Trisodium citrate dihydrate (Wako) ⁇ Sodium lactate solution (50%) (Wako) ⁇ PBS (phosphate buffered saline, DS Pharma) ⁇ PH 7.2 phosphate buffer (hereinafter referred to as PB)
  • PB phosphate buffered saline
  • composition of the bicarbonate Ringer's solution is as follows.
  • composition of the lactated Ringer's solution is as follows. Sodium chloride: 103 mM Potassium chloride: 4 mM Calcium chloride: 3 mM Sodium lactate: 28 mM
  • Bicarbonate Ringer solution / siRNA fraction (0.5% AC / 5 ⁇ M GL3 siRNA / bicarbonate Ringer solution), lactated Ringer solution / siRNA fraction (0.5% AC / 5 ⁇ M GL3 siRNA / lactate Ringer solution) prepared in Example 1 and conventional buffer / siRNA
  • the fraction (0.5% AC / 5 ⁇ M GL3 siRNA / 50 mM PB / 0.5 ⁇ PBS) was dispensed into a 96-well plate at a total volume of 100 ⁇ L / well while stirring.
  • the measurement results of the gel-forming ability of the drug sustained-release carrier are shown in FIG.
  • the gel formation time of the bicarbonate Ringer solution / siRNA fraction and the lactate Ringer solution / siRNA fraction was about 10 minutes.
  • the gel formation time of the conventional buffer / siRNA fraction as a control was 60 minutes or more. That is, it was confirmed that the gel forming ability of the bicarbonate Ringer solution / siRNA fraction and the lactated Ringer solution / siRNA fraction was about 6 times that of the conventional buffer / siRNA fraction as a control.
  • High gel-forming ability means that “the gel is rapidly formed, so that the ratio of the object to be transported enclosed in the gel is high, and the object to be transported can be held for a long period of time”.
  • a low gel-forming ability means that “the gelation is slow, so that the object to be transported partially leaves the carrier within the gel-forming time, and the object to be transported cannot be held at a high concentration”.
  • the sustained-release drug carrier of the present invention can hold a high concentration transport target for a long period of time as compared with a conventional drug carrier.
  • a filter tube (Millipore, ultra-free-MC, 0.22 ⁇ m, PVDF, see FIG. 2) was used to measure the sustained release action ability.
  • the filter tube of the filter tube is placed in the bicarbonate Ringer solution / siRNA fraction (0.3% AC / 50 ⁇ M GL3 siRNA / bicarbonate Ringer solution) or the conventional buffer / siRNA fraction (0.3% AC / 50 ⁇ M GL3 siRNA / 50
  • the plate was allowed to stand at 37 ° C.
  • the concentration of siRNA in PBS is measured over time using a spectrophotometer (Thermo scientific, NanoDrop 2000). It was measured.
  • the measurement results of the sustained release action ability of the drug sustained release carrier are shown in FIG.
  • the bicarbonate Ringer's solution / siRNA fraction released less nucleic acid in the initial stage than the conventional buffer / siRNA fraction as a control, and gradually released the nucleic acid.
  • the time until the amount of nucleic acid released from the gel of the buffer Ringer's solution / siRNA fraction is halved the time until the amount of nucleic acid released from the gel of the buffer / siRNA fraction is reduced to half, It was about twice. That is, it was confirmed that the sustained release action ability of the drug sustained-release carrier of the present invention was about twice that of the conventional drug carrier.
  • mice and reagents used are as follows. ⁇ Mouse: C57BL / 6NCrSlc, 7-week-old, Sakai, Japan SLC ⁇ Cell: B16F10-C2 SV40 Dual Luc # 8-4 (a cell line that constantly expresses luciferase) -Medium: D-MEM + 10% FBS + 1 mg / ml G418 (Promega) D-MEM (Invitrogen) G418 Sulfate Solution (Invitrogen) ⁇ Dual-Luciferase Reporter Assay System (Promega) ⁇ 1 ml syringe (TERUMO) ⁇ 26 G injection needle (TERUMO) The drug carriers used are as follows.
  • Luciferase assay The inhibition rate of luciferase expression was measured using a Dual-Luciferase Reporter Assay System kit. Specifically, the tumor was homogenized in a cell lysis solution (attached to the kit), centrifuged, and the supernatant was collected to obtain a sample solution. 100 ⁇ L of the reaction substrate reagent (attached to the kit) was added to 20 ⁇ L of the sample solution, and immediately after mixing, the luminescence intensity was measured with a microplate reader. This luminous intensity was taken as the luminous intensity of the firefly.
  • FIG. 4 shows the result of confirming the efficiency of introduction into the target cell of the delivery target of the drug sustained release carrier of the present invention.
  • the inhibition rates of the bicarbonate Ringer solution fraction and the lactate Ringer solution fraction were 73.2% and 61.2%, respectively.
  • the inhibition rate of the PBS fraction was 49.7%.
  • the sustained-release drug carrier of the present invention showed a higher growth inhibitory effect (higher target cell introduction efficiency of the transport target) than the conventional drug carrier.
  • the target cell introduction efficiency of the bicarbonate Ringer solution fraction and the lactated Ringer solution fraction is 1.47 times and 1.23 times, respectively, compared to the target cell introduction efficiency of the conventional drug carrier delivery target. Met.
  • the outflow amount (fraction that did not enter the tumor) when the bicarbonate Ringer's solution fraction and lactate Ringer's solution fraction were administered to the tumor (local)
  • the PBS fraction was administered to the tumor (local) Compared to the amount of spillage, we were able to suppress it.
  • the drug sustained-release carrier or drug sustained-release method of the present invention had the following effects compared to the conventional drug carrier or drug sustained-release method. (1) Since the gel forming ability is high, it is possible to hold a high concentration transport object (drug) for a long time. (2) Since the sustained release action ability is high, the drug can be supplied to the target cells for a long time. (3) Since the introduction efficiency of the drug into the target cell is high, the drug effect is high. (4) The amount of drug outflow when administered locally can be suppressed.
  • a novel and useful drug sustained release carrier and drug sustained release method can be provided.

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Abstract

La présente invention vise à fournir un véhicule pour une libération prolongée de médicament, qui a une plus grande vitesse de gélification, une meilleure action de libération prolongée et une plus grande efficacité d'introduction dans des cellules par comparaison avec des véhicules classiques de libération prolongée qui comprennent du collagène. À cet effet, les inventeurs ont fait des recherches pour résoudre le problème ci-dessus et, en conséquence, ont découvert que le problème peut être résolu par un véhicule pour une libération prolongée de médicament qui comprend du collagène ou un dérivé de collagène conjointement avec un liquide de Ringer, ce qui permet la présente invention.
PCT/JP2015/080530 2014-10-29 2015-10-29 Véhicule pour libération prolongée de médicament, et procédé pour libération prolongée de médicament WO2016068228A1 (fr)

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ITUB20160857A1 (it) * 2016-02-18 2017-08-18 Bruno Silvestrini Una forma di dosaggio comprendente solfato di calcio, collagene e un farmaco e relativo uso in un metodo di rilascio modificato avente un meccanismo di regolazione insito nella sua composizione.
IT201700093635A1 (it) * 2017-08-14 2019-02-14 Bruno Silvestrini Una forma di dosaggio topica comprendente un sale di calcio, collagene e un farmaco e relativo uso in un metodo di rilascio modificato avente un meccanismo di regolazione insito nella sua composizione.
WO2019064807A1 (fr) * 2017-09-29 2019-04-04 祐徳薬品工業株式会社 Procédé pour la production de vitrigel de collagène, procédé pour la production de vitrigel de collagène purifié et vitrigel de collagène et vitrigel de collagène purifié produits par lesdits procédés
WO2023059921A3 (fr) * 2021-10-07 2023-05-19 Intelligent Optical Systems, Inc. Dosages à écoulement latéral améliorés et dispositifs de détection d'analytes dans des échantillons de sang

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITUB20160857A1 (it) * 2016-02-18 2017-08-18 Bruno Silvestrini Una forma di dosaggio comprendente solfato di calcio, collagene e un farmaco e relativo uso in un metodo di rilascio modificato avente un meccanismo di regolazione insito nella sua composizione.
WO2017141282A1 (fr) * 2016-02-18 2017-08-24 Bruno Silvestrini Forme pharmaceutique comprenant un sel de calcium, du collagène et un médicament et utilisation associée dans un procédé de libération contrôlée pourvue d'un mécanisme de régulation de libération de médicament à l'intérieur de la composition
IT201700093635A1 (it) * 2017-08-14 2019-02-14 Bruno Silvestrini Una forma di dosaggio topica comprendente un sale di calcio, collagene e un farmaco e relativo uso in un metodo di rilascio modificato avente un meccanismo di regolazione insito nella sua composizione.
WO2019064807A1 (fr) * 2017-09-29 2019-04-04 祐徳薬品工業株式会社 Procédé pour la production de vitrigel de collagène, procédé pour la production de vitrigel de collagène purifié et vitrigel de collagène et vitrigel de collagène purifié produits par lesdits procédés
WO2023059921A3 (fr) * 2021-10-07 2023-05-19 Intelligent Optical Systems, Inc. Dosages à écoulement latéral améliorés et dispositifs de détection d'analytes dans des échantillons de sang

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