WO2016068228A1 - Carrier for sustained drug release and method for sustained drug release - Google Patents
Carrier for sustained drug release and method for sustained drug release Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- solution
- drug
- sustained
- ringer
- release
- Prior art date
Links
Images
Classifications
-
- 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.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dispersion Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Transplantation (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
[Problem] To provide a carrier for sustained drug release that has a higher gelation speed, improved sustained release action and higher introduction efficiency into cells compared with conventional carriers for sustained release that comprise collagen.
[Solution] The present inventors studied to solve the above problem and, as a result, found that the problem can be solved by a carrier for sustained drug release that comprises collagen or a collagen derivative together with a Ringer's solution, thereby completing the present invention.
Description
本発明は、所望の核酸、タンパク質、ペプチド、低分子化合物等を標的細胞へ導入するための薬剤徐放担体又は薬剤徐放方法に関する。
本出願は、参照によりここに援用されるところの日本出願、特願2014-220101号優先権を請求する。 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.
This application claims the priority of Japanese Patent Application No. 2014-220101, which is incorporated herein by reference.
本出願は、参照によりここに援用されるところの日本出願、特願2014-220101号優先権を請求する。 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.
This application claims the priority of Japanese Patent Application No. 2014-220101, which is incorporated herein by reference.
（薬剤徐放担体）
薬剤徐放担体は、下記の文献に記載のように複数報告されている。 (Drug sustained release carrier)
A plurality of drug sustained-release carriers have been reported as described in the following literature.
薬剤徐放担体は、下記の文献に記載のように複数報告されている。 (Drug sustained release carrier)
A plurality of drug sustained-release carriers have been reported as described in the following literature.
（特許文献１）
特許文献１は、「宿主生物内／内部のＲＮＡ移送及び／またはＲＮＡ翻訳を増加させるためにＲＮＡ注入溶液を調製する際の、ＲＮＡ、及び水性の注入緩衝液の利用であって、注入緩衝液は、ナトリウム塩、カルシウム塩、及び必要に応じてカリウム塩を含有する、利用」を開示している。
しかし、該利用では、「該注入緩衝液は、コラーゲン又はコラーゲン誘導体を含むこと」及び「徐放効果」を明示していない。 (Patent Document 1)
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.
However, in this application, “the injection buffer contains collagen or a collagen derivative” and “sustained release effect” are not specified.
特許文献１は、「宿主生物内／内部のＲＮＡ移送及び／またはＲＮＡ翻訳を増加させるためにＲＮＡ注入溶液を調製する際の、ＲＮＡ、及び水性の注入緩衝液の利用であって、注入緩衝液は、ナトリウム塩、カルシウム塩、及び必要に応じてカリウム塩を含有する、利用」を開示している。
しかし、該利用では、「該注入緩衝液は、コラーゲン又はコラーゲン誘導体を含むこと」及び「徐放効果」を明示していない。 (Patent Document 1)
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.
However, in this application, “the injection buffer contains collagen or a collagen derivative” and “sustained release effect” are not specified.
（特許文献２）
特許文献２は、「コラーゲンまたはコラーゲン誘導体を含有する、標的細胞への核酸導入促進剤」を開示している。
しかし、該核酸導入促進剤は、「リンゲル液」を含むことを開示又は示唆していない。 (Patent Document 2)
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”.
特許文献２は、「コラーゲンまたはコラーゲン誘導体を含有する、標的細胞への核酸導入促進剤」を開示している。
しかし、該核酸導入促進剤は、「リンゲル液」を含むことを開示又は示唆していない。 (Patent Document 2)
However, it does not disclose or suggest that the nucleic acid introduction promoter contains a “Ringer solution”.
（特許文献３）
特許文献３は、「薬物、コラーゲン、ならびに、単糖類、二糖類、三糖類および四糖類から選択される１種以上の糖を含む、徐放性医薬組成物」を開示している。
しかし、該徐放性医薬組成物は、「リンゲル液」を含むことを開示又は示唆していない。 (Patent Document 3)
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”.
特許文献３は、「薬物、コラーゲン、ならびに、単糖類、二糖類、三糖類および四糖類から選択される１種以上の糖を含む、徐放性医薬組成物」を開示している。
しかし、該徐放性医薬組成物は、「リンゲル液」を含むことを開示又は示唆していない。 (Patent Document 3)
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”.
本発明では、従来のコラーゲンを含む薬剤徐放担体又は薬剤徐放方法と比較して、ゲル化速度、徐放作用及び細胞の導入効率が高い徐放担体又は薬剤徐放方法を提供することである。
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.
本発明者らは、上記課題を解決するために研究した結果、コラーゲン又はコラーゲン誘導体並びにリンゲル液を含む薬剤徐放担体は、上記課題を解決できることを見出して、本発明を完成した。
As a result of researches to solve the above problems, the present inventors have found that a drug sustained-release carrier containing collagen or a collagen derivative and Ringer's solution can solve the above problems, thereby completing the present invention.
本発明は以下の通りである。
「１．以下を含む薬剤徐放担体。
（１）コラーゲン又はコラーゲン誘導体
（２）カリウム塩、カルシウム塩及びナトリウム塩を含む溶液
２．前記カリウム塩は塩化カリウム、前記カルシウム塩は塩化カルシウム及び前記ナトリウム塩は塩化ナトリウムである前項１に記載の薬剤徐放担体。
３．前記溶液は、リンゲル液である前項１又は２に記載の薬剤徐放担体。
４．前記リンゲル液は、重炭酸リンゲル液、乳酸リンゲル液、リンゲル基礎液、又は酢酸リンゲル液である前項３に記載の薬剤徐放担体。
５．前記リンゲル液は、重炭酸リンゲル液である前項３に記載の薬剤徐放担体。
６．前記リンゲル液は、乳酸リンゲル液である前項３に記載の薬剤徐放担体。
７．前記溶液は、以下のいずれか１から選択される前項１～６のいずれか１に記載の薬剤徐放担体。
（１）塩化カリウム、塩化カルシウム、塩化ナトリウム、炭酸水素ナトリウム、塩化マグネシウム及びクエン酸三ナトリウムを含む溶液
（２）塩化カリウム、塩化カルシウム、塩化ナトリウム及び乳酸ナトリウムを含む溶液
（３）塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む溶液
（４）塩化カリウム、塩化カルシウム、塩化ナトリウム及び酢酸ナトリウム含む溶液
８．前記溶液の各成分の濃度は、以下である前項１～７のいずれか１に記載の薬剤徐放担体。
塩化カリウム：0.001ｍM ～5.00M
塩化カルシウム：0.001ｍM ～10.00M
塩化ナトリウム：0.001ｍM ～10.00M
炭酸水素ナトリウム：0.001ｍM ～2.00M
塩化マグネシウム：0.001ｍM ～5.00M
クエン酸三ナトリウム：0.001ｍM ～2.00M
乳酸ナトリウム：0.001ｍM ～10.00M
酢酸ナトリウム：0.001ｍM ～10.00M
９．前記コラーゲン又はコラーゲン誘導体がアテロコラーゲンである前項１～８のいずれか１に記載の薬剤徐放担体。
１０．前記薬剤徐放担体がゲル形成能を有する前項１～９のいずれか１に記載の薬剤徐放担体。
１１．前記薬剤が、核酸、タンパク質、ペプチド、及び／又は低分子化合物である前項１～１０のいずれか１に記載の薬剤徐放担体。
１２．前記リンゲル液は、重炭酸リンゲル液であり、かつ前記薬剤が核酸である前項４～１１のいずれか１に記載の薬剤徐放担体。
１３．前記リンゲル液は、乳酸リンゲル液であり、かつ前記薬剤が核酸である前項４～１１のいずれか１に記載の薬剤徐放担体。
１４．前項１～１３のいずれか１に記載の薬剤徐放担体が表面に塗布されている医療用具。
１５．前項１～１３のいずれか１に記載の薬剤徐放担体が細胞培養面に塗布されている細胞培養器具。
１６．前項１～１３のいずれか１に記載の薬剤徐放担体を含む薬剤。
１７．前項１～１３のいずれか１に記載の薬剤徐放担体又は前項１３に記載の薬剤を、ヒトを除く哺乳動物に投与する、薬剤徐放担体の使用。
１８．以下の工程を含む薬剤徐放方法、
（１）薬剤と、コラーゲン若しくはコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を混合して、混合溶液を作成する工程、及び
（２）該混合溶液を、該薬剤を必要とする患者に投与する工程。
１９．前記カリウム塩は塩化カリウム、前記カルシウム塩は塩化カルシウム及び前記ナトリウム塩は塩化ナトリウムである前項１８に記載の薬剤徐放方法。
２０．前記溶液は、リンゲル液である前項１８又は１９に記載の薬剤徐放方法。
２１．前記リンゲル液は、重炭酸リンゲル液、乳酸リンゲル液、リンゲル基礎液、又は酢酸リンゲル液である前項２０に記載の薬剤徐放方法。
２２．前記リンゲル液は、重炭酸リンゲル液である前項２０載の薬剤徐放方法。
２３．前記リンゲル液は、乳酸リンゲル液である前項２０載の薬剤徐放方法。
２４．前記溶液は、以下のいずれか１から選択される前項１８～２３のいずれか１に記載の薬剤徐放方法。
（１）塩化カリウム、塩化カルシウム、塩化ナトリウム、炭酸水素ナトリウム、塩化マグネシウム及びクエン酸三ナトリウムを含む溶液
（２）塩化カリウム、塩化カルシウム、塩化ナトリウム及び乳酸ナトリウムを含む溶液
（３）塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む溶液
（４）塩化カリウム、塩化カルシウム、塩化ナトリウム及び酢酸ナトリウム含む溶液
２５．前記溶液の各成分の濃度は、以下である前項１８～２４のいずれか１に記載の薬剤徐放方法。
塩化カリウム：0.001ｍM ～5.00M
塩化カルシウム：0.001ｍM ～10.00M
塩化ナトリウム：0.001ｍM ～10.00M
炭酸水素ナトリウム：0.001ｍM ～2.00M
塩化マグネシウム：0.001ｍM ～5.00M
クエン酸三ナトリウム：0.001ｍM ～2.00M
乳酸ナトリウム：0.001ｍM ～10.00M
酢酸ナトリウム：0.001ｍM ～10.00M
２６．前記コラーゲン又はコラーゲン誘導体がアテロコラーゲンである前項１８～２５のいずれか１に記載の薬剤徐放方法。
２７．前記薬剤徐放担体がゲル形成能を有する前項１８～２６のいずれか１に記載の薬剤徐放方法。
２８．前記薬剤が、核酸、タンパク質、ペプチド、及び／又は低分子化合物である前項１８～２７のいずれか１に記載の薬剤徐放方法。
２９．前記リンゲル液は、重炭酸リンゲル液であり、かつ前記薬剤が核酸である前項２１～２８のいずれか１に記載の薬剤徐放方法。
３０．前記リンゲル液は、乳酸リンゲル液であり、かつ前記薬剤が核酸である前項２１～２８のいずれか１に記載の薬剤徐放方法。」 The present invention is as follows.
“1. Drug sustained-release carrier comprising:
(1) Collagen or collagen derivative (2) Solution containing potassium salt, calcium salt and sodium salt The drug sustained-release carrier according to item 1, wherein the potassium salt is potassium chloride, the calcium salt is calcium chloride, and the sodium salt is sodium chloride.
3. 3. The drug sustained-release carrier according toitem 1 or 2, wherein the solution is a Ringer's solution.
4). 4. The drug sustained-release carrier according to item 3, wherein the Ringer solution is a bicarbonate Ringer solution, a lactated Ringer solution, a Ringer base solution, or an acetate Ringer solution.
5. 4. The drug sustained-release carrier according to item 3, wherein the Ringer's solution is a bicarbonated Ringer's solution.
6). 4. The drug sustained-release carrier according to item 3, wherein the Ringer solution is a lactated Ringer solution.
7). 7. The drug sustained-release carrier according to any one of the preceding items 1 to 6, wherein the solution is selected from any one of the following.
(1) Solution containing potassium chloride, calcium chloride, sodium chloride, sodium bicarbonate, magnesium chloride and trisodium citrate (2) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium lactate (3) Potassium chloride, chloride 7. Solution containing calcium and sodium chloride (4) Solution containing potassium chloride, calcium chloride, sodium chloride andsodium acetate 8. The sustained-release drug carrier according to any one of 1 to 7 above, wherein the concentration of each component of the solution is as follows.
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
9. 9. The drug sustained release carrier according to any one of 1 to 8 above, wherein the collagen or collagen derivative is atelocollagen.
10. 10. The drug sustained-release carrier according to any one of 1 to 9 above, wherein the drug sustained-release carrier has gel forming ability.
11. 11. 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 ofitems 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 ofitems 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. The method for sustained drug release according to 18 above, wherein the potassium salt is potassium chloride, the calcium salt is calcium chloride, and the sodium salt is sodium chloride.
20. 20. The method for sustained drug release according to 18 or 19 above, wherein the solution is a Ringer's solution.
21. 21. The method of sustained drug release as described in 20 above, wherein the Ringer solution is a bicarbonate Ringer solution, a lactated Ringer solution, a Ringer base solution, or an acetate Ringer solution.
22. 20. The method for sustained drug release as described in 20 above, wherein the Ringer's solution is a bicarbonated Ringer's solution.
23. 20. The method for sustained drug release as described in 20 above, wherein the Ringer solution is a lactated Ringer solution.
24. 24. The method for sustained drug release according to any one of items 18 to 23, wherein the solution is selected from any one of the following.
(1) Solution containing potassium chloride, calcium chloride, sodium chloride, sodium bicarbonate, magnesium chloride and trisodium citrate (2) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium lactate (3) Potassium chloride, chloride Solution containing calcium and sodium chloride (4) Solution containing potassium chloride, calcium chloride, sodium chloride andsodium acetate 25. 25. The method for sustained drug release according to any one of 18 to 24 above, wherein the concentration of each component of the solution is as follows.
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 method for sustained drug release according to any one of 18 to 25 above, wherein the collagen or collagen derivative is atelocollagen.
27. 27. The method for sustained drug release according to any one of items 18 to 26 above, wherein the drug sustained-release carrier has gel-forming ability.
28. 28. The method for sustained drug release according to any one of 18 to 27 above, wherein the drug is a nucleic acid, protein, peptide, and / or low molecular weight compound.
29. 29. The method for sustained drug release according to any one of items 21 to 28, wherein the Ringer's solution is a bicarbonated Ringer's solution and the agent is a nucleic acid.
30. 29. The method for sustained drug release according to any one of items 21 to 28, wherein the Ringer solution is a lactated Ringer solution, and the drug is a nucleic acid. "
「１．以下を含む薬剤徐放担体。
（１）コラーゲン又はコラーゲン誘導体
（２）カリウム塩、カルシウム塩及びナトリウム塩を含む溶液
２．前記カリウム塩は塩化カリウム、前記カルシウム塩は塩化カルシウム及び前記ナトリウム塩は塩化ナトリウムである前項１に記載の薬剤徐放担体。
３．前記溶液は、リンゲル液である前項１又は２に記載の薬剤徐放担体。
４．前記リンゲル液は、重炭酸リンゲル液、乳酸リンゲル液、リンゲル基礎液、又は酢酸リンゲル液である前項３に記載の薬剤徐放担体。
５．前記リンゲル液は、重炭酸リンゲル液である前項３に記載の薬剤徐放担体。
６．前記リンゲル液は、乳酸リンゲル液である前項３に記載の薬剤徐放担体。
７．前記溶液は、以下のいずれか１から選択される前項１～６のいずれか１に記載の薬剤徐放担体。
（１）塩化カリウム、塩化カルシウム、塩化ナトリウム、炭酸水素ナトリウム、塩化マグネシウム及びクエン酸三ナトリウムを含む溶液
（２）塩化カリウム、塩化カルシウム、塩化ナトリウム及び乳酸ナトリウムを含む溶液
（３）塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む溶液
（４）塩化カリウム、塩化カルシウム、塩化ナトリウム及び酢酸ナトリウム含む溶液
８．前記溶液の各成分の濃度は、以下である前項１～７のいずれか１に記載の薬剤徐放担体。
塩化カリウム：0.001ｍM ～5.00M
塩化カルシウム：0.001ｍM ～10.00M
塩化ナトリウム：0.001ｍM ～10.00M
炭酸水素ナトリウム：0.001ｍM ～2.00M
塩化マグネシウム：0.001ｍM ～5.00M
クエン酸三ナトリウム：0.001ｍM ～2.00M
乳酸ナトリウム：0.001ｍM ～10.00M
酢酸ナトリウム：0.001ｍM ～10.00M
９．前記コラーゲン又はコラーゲン誘導体がアテロコラーゲンである前項１～８のいずれか１に記載の薬剤徐放担体。
１０．前記薬剤徐放担体がゲル形成能を有する前項１～９のいずれか１に記載の薬剤徐放担体。
１１．前記薬剤が、核酸、タンパク質、ペプチド、及び／又は低分子化合物である前項１～１０のいずれか１に記載の薬剤徐放担体。
１２．前記リンゲル液は、重炭酸リンゲル液であり、かつ前記薬剤が核酸である前項４～１１のいずれか１に記載の薬剤徐放担体。
１３．前記リンゲル液は、乳酸リンゲル液であり、かつ前記薬剤が核酸である前項４～１１のいずれか１に記載の薬剤徐放担体。
１４．前項１～１３のいずれか１に記載の薬剤徐放担体が表面に塗布されている医療用具。
１５．前項１～１３のいずれか１に記載の薬剤徐放担体が細胞培養面に塗布されている細胞培養器具。
１６．前項１～１３のいずれか１に記載の薬剤徐放担体を含む薬剤。
１７．前項１～１３のいずれか１に記載の薬剤徐放担体又は前項１３に記載の薬剤を、ヒトを除く哺乳動物に投与する、薬剤徐放担体の使用。
１８．以下の工程を含む薬剤徐放方法、
（１）薬剤と、コラーゲン若しくはコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を混合して、混合溶液を作成する工程、及び
（２）該混合溶液を、該薬剤を必要とする患者に投与する工程。
１９．前記カリウム塩は塩化カリウム、前記カルシウム塩は塩化カルシウム及び前記ナトリウム塩は塩化ナトリウムである前項１８に記載の薬剤徐放方法。
２０．前記溶液は、リンゲル液である前項１８又は１９に記載の薬剤徐放方法。
２１．前記リンゲル液は、重炭酸リンゲル液、乳酸リンゲル液、リンゲル基礎液、又は酢酸リンゲル液である前項２０に記載の薬剤徐放方法。
２２．前記リンゲル液は、重炭酸リンゲル液である前項２０載の薬剤徐放方法。
２３．前記リンゲル液は、乳酸リンゲル液である前項２０載の薬剤徐放方法。
２４．前記溶液は、以下のいずれか１から選択される前項１８～２３のいずれか１に記載の薬剤徐放方法。
（１）塩化カリウム、塩化カルシウム、塩化ナトリウム、炭酸水素ナトリウム、塩化マグネシウム及びクエン酸三ナトリウムを含む溶液
（２）塩化カリウム、塩化カルシウム、塩化ナトリウム及び乳酸ナトリウムを含む溶液
（３）塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む溶液
（４）塩化カリウム、塩化カルシウム、塩化ナトリウム及び酢酸ナトリウム含む溶液
２５．前記溶液の各成分の濃度は、以下である前項１８～２４のいずれか１に記載の薬剤徐放方法。
塩化カリウム：0.001ｍM ～5.00M
塩化カルシウム：0.001ｍM ～10.00M
塩化ナトリウム：0.001ｍM ～10.00M
炭酸水素ナトリウム：0.001ｍM ～2.00M
塩化マグネシウム：0.001ｍM ～5.00M
クエン酸三ナトリウム：0.001ｍM ～2.00M
乳酸ナトリウム：0.001ｍM ～10.00M
酢酸ナトリウム：0.001ｍM ～10.00M
２６．前記コラーゲン又はコラーゲン誘導体がアテロコラーゲンである前項１８～２５のいずれか１に記載の薬剤徐放方法。
２７．前記薬剤徐放担体がゲル形成能を有する前項１８～２６のいずれか１に記載の薬剤徐放方法。
２８．前記薬剤が、核酸、タンパク質、ペプチド、及び／又は低分子化合物である前項１８～２７のいずれか１に記載の薬剤徐放方法。
２９．前記リンゲル液は、重炭酸リンゲル液であり、かつ前記薬剤が核酸である前項２１～２８のいずれか１に記載の薬剤徐放方法。
３０．前記リンゲル液は、乳酸リンゲル液であり、かつ前記薬剤が核酸である前項２１～２８のいずれか１に記載の薬剤徐放方法。」 The present invention is as follows.
“1. Drug sustained-release carrier comprising:
(1) Collagen or collagen derivative (2) Solution containing potassium salt, calcium salt and sodium salt The drug sustained-release carrier according to item 1, wherein the potassium salt is potassium chloride, the calcium salt is calcium chloride, and the sodium salt is sodium chloride.
3. 3. The drug sustained-release carrier according to
4). 4. The drug sustained-release carrier according to item 3, wherein the Ringer solution is a bicarbonate Ringer solution, a lactated Ringer solution, a Ringer base solution, or an acetate Ringer solution.
5. 4. The drug sustained-release carrier according to item 3, wherein the Ringer's solution is a bicarbonated Ringer's solution.
6). 4. The drug sustained-release carrier according to item 3, wherein the Ringer solution is a lactated Ringer solution.
7). 7. The drug sustained-release carrier according to any one of the preceding items 1 to 6, wherein the solution is selected from any one of the following.
(1) Solution containing potassium chloride, calcium chloride, sodium chloride, sodium bicarbonate, magnesium chloride and trisodium citrate (2) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium lactate (3) Potassium chloride, chloride 7. Solution containing calcium and sodium chloride (4) Solution containing potassium chloride, calcium chloride, sodium chloride and
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
9. 9. The drug sustained release carrier according to any one of 1 to 8 above, wherein the collagen or collagen derivative is atelocollagen.
10. 10. The drug sustained-release carrier according to any one of 1 to 9 above, wherein the drug sustained-release carrier has gel forming ability.
11. 11. 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
13 12. The drug sustained-release carrier according to any one of
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. The method for sustained drug release according to 18 above, wherein the potassium salt is potassium chloride, the calcium salt is calcium chloride, and the sodium salt is sodium chloride.
20. 20. The method for sustained drug release according to 18 or 19 above, wherein the solution is a Ringer's solution.
21. 21. The method of sustained drug release as described in 20 above, wherein the Ringer solution is a bicarbonate Ringer solution, a lactated Ringer solution, a Ringer base solution, or an acetate Ringer solution.
22. 20. The method for sustained drug release as described in 20 above, wherein the Ringer's solution is a bicarbonated Ringer's solution.
23. 20. The method for sustained drug release as described in 20 above, wherein the Ringer solution is a lactated Ringer solution.
24. 24. The method for sustained drug release according to any one of items 18 to 23, wherein the solution is selected from any one of the following.
(1) Solution containing potassium chloride, calcium chloride, sodium chloride, sodium bicarbonate, magnesium chloride and trisodium citrate (2) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium lactate (3) Potassium chloride, chloride Solution containing calcium and sodium chloride (4) Solution containing potassium chloride, calcium chloride, sodium chloride and
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 method for sustained drug release according to any one of 18 to 25 above, wherein the collagen or collagen derivative is atelocollagen.
27. 27. The method for sustained drug release according to any one of items 18 to 26 above, wherein the drug sustained-release carrier has gel-forming ability.
28. 28. The method for sustained drug release according to any one of 18 to 27 above, wherein the drug is a nucleic acid, protein, peptide, and / or low molecular weight compound.
29. 29. The method for sustained drug release according to any one of items 21 to 28, wherein the Ringer's solution is a bicarbonated Ringer's solution and the agent is a nucleic acid.
30. 29. The method for sustained drug release according to any one of items 21 to 28, wherein the Ringer solution is a lactated Ringer solution, and the drug is a nucleic acid. "
本発明の薬剤徐放担体又は薬剤徐放方法は、従来の薬剤担体又は薬剤徐放方法と比較して、下記の効果を有する。
（１）ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができる。
（２）徐放作用能が高いので長時間薬剤を標的細胞に供給することができる。
（３）薬剤の標的細胞内導入効率が高いので薬効効果が高い。
（４）局所に投与した場合の薬剤流出量を抑えることができる。 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.
（１）ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができる。
（２）徐放作用能が高いので長時間薬剤を標的細胞に供給することができる。
（３）薬剤の標的細胞内導入効率が高いので薬効効果が高い。
（４）局所に投与した場合の薬剤流出量を抑えることができる。 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.
（本発明の薬剤徐放担体）
本発明の「薬剤徐放担体」は、コラーゲン又はコラーゲン誘導体並びにリンゲル液を含む溶液を含む運搬体（担体）である。 (Sustained drug release carrier of the present invention)
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.
本発明の「薬剤徐放担体」は、コラーゲン又はコラーゲン誘導体並びにリンゲル液を含む溶液を含む運搬体（担体）である。 (Sustained drug release carrier of the present invention)
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.
本発明の「薬剤徐放担体」は、運搬対象を細胞｛標的細胞（運搬対象を導入する細胞）｝に運搬する作用を有する。さらに、本発明の薬剤徐放担体は、単に運搬対象を細胞に運搬するだけでなく、ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができ、徐放作用能が高いので長時間薬剤を標的細胞に供給することができ、さらに薬剤の標的細胞内導入効率が高いので薬効効果が高い。
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.
（本発明の薬剤徐放方法）
本発明の「薬剤徐放方法」は、コラーゲン又はコラーゲン誘導体並びにリンゲル液を使用して、運搬対象を細胞｛標的細胞（運搬対象を導入する細胞）｝に運搬する方法である。さらに、本発明の薬剤徐放方法は、単に運搬対象を細胞に運搬するだけでなく、ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができ、徐放作用能が高いので長時間薬剤を標的細胞に供給することができ、さらに薬剤の標的細胞内導入効率が高いので薬効効果が高い。 (Method of sustained release of drug of the present invention)
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.
本発明の「薬剤徐放方法」は、コラーゲン又はコラーゲン誘導体並びにリンゲル液を使用して、運搬対象を細胞｛標的細胞（運搬対象を導入する細胞）｝に運搬する方法である。さらに、本発明の薬剤徐放方法は、単に運搬対象を細胞に運搬するだけでなく、ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができ、徐放作用能が高いので長時間薬剤を標的細胞に供給することができ、さらに薬剤の標的細胞内導入効率が高いので薬効効果が高い。 (Method of sustained release of drug of the present invention)
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.
（カリウム塩、カルシウム塩及びナトリウム塩を含む溶液）
本発明の「カリウム塩、カルシウム塩及びナトリウム塩を含む溶液」は、細胞外液補充に用いるために、イオン組成、浸透圧、及び水素イオン濃度を有する生理的電解質溶液を示し、特定の一組成に限定されないが、リンゲル液が好ましい。
カリウム塩としては、塩化カリウム、ヨウ化カリウム、臭化カリウム、炭酸カリウム、炭酸水素カリウム、硫酸カリウム、それらの水和物等を例示することができるが、好ましくは、塩化カリウムである。
カルシウム塩としては、塩化カルシウム、ヨウ化カルシウム、臭化カルシウム、炭酸カルシウム、硫酸カルシウム、水酸化カルシウム、それらの水和物等を例示することができるが、好ましくは、塩化カルシウムである。
ナトリウム塩としては、塩化ナトリウム、ヨウ化ナトリウム、臭化ナトリウム、炭酸ナトリウム、炭酸水素ナトリウム、硫酸ナトリウム、それらの水和物等を例示することができるが、好ましくは、塩化ナトリウムである。
本溶液のｐＨ値は、4.0～10.0、より好ましくは 6.0～8.5、さらに好ましくは6.7～7.8である。さらに、本溶液は、必要に応じて、ｐＨ値を調整するために、リン酸緩衝液、ＨＥＰＥＳ、Ｎａ２ ＨＰＯ４ ／ＮａＨ２ ＰＯ４等 を含んでも良い。 (Solution containing potassium, calcium and sodium salts)
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. Although not limited to this, Ringer's solution is preferable.
Examples of 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. Furthermore, 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.
本発明の「カリウム塩、カルシウム塩及びナトリウム塩を含む溶液」は、細胞外液補充に用いるために、イオン組成、浸透圧、及び水素イオン濃度を有する生理的電解質溶液を示し、特定の一組成に限定されないが、リンゲル液が好ましい。
カリウム塩としては、塩化カリウム、ヨウ化カリウム、臭化カリウム、炭酸カリウム、炭酸水素カリウム、硫酸カリウム、それらの水和物等を例示することができるが、好ましくは、塩化カリウムである。
カルシウム塩としては、塩化カルシウム、ヨウ化カルシウム、臭化カルシウム、炭酸カルシウム、硫酸カルシウム、水酸化カルシウム、それらの水和物等を例示することができるが、好ましくは、塩化カルシウムである。
ナトリウム塩としては、塩化ナトリウム、ヨウ化ナトリウム、臭化ナトリウム、炭酸ナトリウム、炭酸水素ナトリウム、硫酸ナトリウム、それらの水和物等を例示することができるが、好ましくは、塩化ナトリウムである。
本溶液のｐＨ値は、4.0～10.0、より好ましくは 6.0～8.5、さらに好ましくは6.7～7.8である。さらに、本溶液は、必要に応じて、ｐＨ値を調整するために、リン酸緩衝液、ＨＥＰＥＳ、Ｎａ２ ＨＰＯ４ ／ＮａＨ２ ＰＯ４等 を含んでも良い。 (Solution containing potassium, calcium and sodium salts)
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. Although not limited to this, Ringer's solution is preferable.
Examples of 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. Furthermore, 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.
（リンゲル液）
本発明のリンゲル液は、好ましくは、リンゲル基礎液、重炭酸リンゲル液、乳酸リンゲル液、酢酸リンゲル液、又はそれらのリンゲル液に糖質を加えたものを例示することができるが特に限定されない。
糖類としては、キシロース、アラビノース、リボース、グルコース、マンノース、ガラクトース、フルクトース、フコース、イノシトール、グルコサミン、エリスリトールが例示され、二糖類としては、スクロース、マルトース、ラクトース、トレハロース、イソマルトースが例示され、三糖類としては、ラフィノース、及び四糖類としてはスタキオースが例示される。 (Ringer solution)
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.
Examples of sugars include xylose, arabinose, ribose, glucose, mannose, galactose, fructose, fucose, inositol, glucosamine, and erythritol. Examples of disaccharides include sucrose, maltose, lactose, trehalose, and isomaltose. Examples of raffinose and tetrasaccharide include stachyose.
本発明のリンゲル液は、好ましくは、リンゲル基礎液、重炭酸リンゲル液、乳酸リンゲル液、酢酸リンゲル液、又はそれらのリンゲル液に糖質を加えたものを例示することができるが特に限定されない。
糖類としては、キシロース、アラビノース、リボース、グルコース、マンノース、ガラクトース、フルクトース、フコース、イノシトール、グルコサミン、エリスリトールが例示され、二糖類としては、スクロース、マルトース、ラクトース、トレハロース、イソマルトースが例示され、三糖類としては、ラフィノース、及び四糖類としてはスタキオースが例示される。 (Ringer solution)
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.
Examples of sugars include xylose, arabinose, ribose, glucose, mannose, galactose, fructose, fucose, inositol, glucosamine, and erythritol. Examples of disaccharides include sucrose, maltose, lactose, trehalose, and isomaltose. Examples of raffinose and tetrasaccharide include stachyose.
（リンゲル基礎液）
本発明で使用するリンゲル基礎液は、少なくとも、塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む、以下の濃度範囲を例示することができる。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Ringer base solution)
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.
本発明で使用するリンゲル基礎液は、少なくとも、塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む、以下の濃度範囲を例示することができる。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Ringer base solution)
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.
（乳酸リンゲル液）
本発明で使用する「乳酸リンゲル液」は、乳酸ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウムのいずれかから２以上又は３以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
乳酸ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500ｍM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Lactated Ringer's solution)
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 | concentration of each component is not specifically limited, The following can be illustrated.
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.
本発明で使用する「乳酸リンゲル液」は、乳酸ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウムのいずれかから２以上又は３以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
乳酸ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500ｍM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Lactated Ringer's solution)
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 | concentration of each component is not specifically limited, The following can be illustrated.
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.
（重炭酸リンゲル液）
本発明で使用する「重炭酸リンゲル液」は、炭酸水素ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウム、塩化マグネシウム、クエン酸三ナトリウムのいずれかから３以上又は４以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
炭酸水素ナトリウムの濃度は、0.001ｍM～2.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500mM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。
塩化マグネシウムの濃度は、0.001ｍM～5.00M、好ましくは0.005ｍM～1.00M、より好ましくは0.01ｍM～500mM、さらにより好ましくは0.1ｍM～50ｍM、最も好ましくは0.5ｍM～5ｍMである。
クエン酸三ナトリウムの濃度は、0.001ｍM～2.00M、好ましくは0.005ｍM～1.00M、より好ましくは0.01ｍM～500mM、さらにより好ましくは0.1ｍM～50ｍM、最も好ましくは0.5ｍM～5ｍMである。 (Bicarbonate Ringer's solution)
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. Although the density | 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.
本発明で使用する「重炭酸リンゲル液」は、炭酸水素ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウム、塩化マグネシウム、クエン酸三ナトリウムのいずれかから３以上又は４以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
炭酸水素ナトリウムの濃度は、0.001ｍM～2.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500mM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。
塩化マグネシウムの濃度は、0.001ｍM～5.00M、好ましくは0.005ｍM～1.00M、より好ましくは0.01ｍM～500mM、さらにより好ましくは0.1ｍM～50ｍM、最も好ましくは0.5ｍM～5ｍMである。
クエン酸三ナトリウムの濃度は、0.001ｍM～2.00M、好ましくは0.005ｍM～1.00M、より好ましくは0.01ｍM～500mM、さらにより好ましくは0.1ｍM～50ｍM、最も好ましくは0.5ｍM～5ｍMである。 (Bicarbonate Ringer's solution)
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. Although the density | 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.
（酢酸リンゲル液）
本発明で使用する「酢酸リンゲル液」は、酢酸ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウムのいずれかから２以上又は３以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
酢酸ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500ｍM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Acetic Ringer's solution)
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 | concentration of each component is not specifically limited, The following can be illustrated.
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.
本発明で使用する「酢酸リンゲル液」は、酢酸ナトリウムを必須とし、塩化ナトリウム、塩化カリウム、塩化カルシウムのいずれかから２以上又は３以上を含めば良い。各成分の濃度は、特に限定されないが、下記を例示することができる。
酢酸ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～500ｍM、さらにより好ましくは1.0ｍM～100ｍM、最も好ましくは10ｍM～50ｍMである。
塩化カリウムの濃度は、0.001ｍM～5.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化カルシウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～1.00M、より好ましくは0.1ｍM～100ｍM、さらにより好ましくは0.5ｍM～50ｍM、最も好ましくは1.0ｍM～10ｍMである。
塩化ナトリウムの濃度は、0.001ｍM～10.00M、好ましくは0.01ｍM～5.00M、より好ましくは0.1ｍM～1.00M、さらにより好ましくは1.0ｍM～500ｍM、最も好ましくは50ｍM～200ｍMである。 (Acetic Ringer's solution)
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 | concentration of each component is not specifically limited, The following can be illustrated.
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.
（コラーゲン又はコラーゲン誘導体）
本発明の「コラーゲン又はコラーゲン誘導体」とは、通常、医療分野、化粧品分野、工業分野及び食品分野で用いられているあらゆる「コラーゲン又はコラーゲン誘導体」を意味する。コラーゲンは、可溶性又は可溶化コラーゲンを用いることが好ましい。
可溶性コラーゲンとは、酸性又は中性の水や塩溶液に可溶であり、可溶化コラーゲンとは、酵素により可溶化される酵素可溶化コラーゲン、アルカリにより可溶化されるアルカリ可溶化コラーゲンがあり、いずれも孔サイズが１マイクロメートルのメンブレンフィルターを通過できることが好ましい。また、コラーゲンは、いかなる動物種から抽出されたものでも用いることが出来るが、好ましくは脊椎動物から抽出されたもの、さらに好ましくは哺乳類、鳥類、魚類から抽出されたもの、より好ましくは変性温度が高い哺乳類、鳥類から抽出されたコラーゲンが望ましい。コラーゲンのタイプもいかなるタイプのコラーゲンでも良いが、動物体内の存在量からＩ～Ｖ型が好ましい。具体的には例えば、哺乳動物の真皮から酸抽出したＩ型コラーゲンが挙げられ、より好ましくは例えば、仔牛の真皮から酸抽出したＩ型コラーゲン、遺伝子工学的に生産されるＩ型コラーゲンなどが挙げられる。
また、安全性の面から抗原性の高いテロペプチドを酵素的に除去したアテロコラーゲン又は遺伝子工学的に生産されるアテロコラーゲンが望ましく、１０００残基あたりチロシン残基が３以下であるアテロコラーゲンがより好ましい。
なお、本発明の好ましいコラーゲン又はコラーゲン誘導体は、アテロコラーゲンである。 (Collagen or collagen derivative)
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. As the collagen, 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.
本発明の「コラーゲン又はコラーゲン誘導体」とは、通常、医療分野、化粧品分野、工業分野及び食品分野で用いられているあらゆる「コラーゲン又はコラーゲン誘導体」を意味する。コラーゲンは、可溶性又は可溶化コラーゲンを用いることが好ましい。
可溶性コラーゲンとは、酸性又は中性の水や塩溶液に可溶であり、可溶化コラーゲンとは、酵素により可溶化される酵素可溶化コラーゲン、アルカリにより可溶化されるアルカリ可溶化コラーゲンがあり、いずれも孔サイズが１マイクロメートルのメンブレンフィルターを通過できることが好ましい。また、コラーゲンは、いかなる動物種から抽出されたものでも用いることが出来るが、好ましくは脊椎動物から抽出されたもの、さらに好ましくは哺乳類、鳥類、魚類から抽出されたもの、より好ましくは変性温度が高い哺乳類、鳥類から抽出されたコラーゲンが望ましい。コラーゲンのタイプもいかなるタイプのコラーゲンでも良いが、動物体内の存在量からＩ～Ｖ型が好ましい。具体的には例えば、哺乳動物の真皮から酸抽出したＩ型コラーゲンが挙げられ、より好ましくは例えば、仔牛の真皮から酸抽出したＩ型コラーゲン、遺伝子工学的に生産されるＩ型コラーゲンなどが挙げられる。
また、安全性の面から抗原性の高いテロペプチドを酵素的に除去したアテロコラーゲン又は遺伝子工学的に生産されるアテロコラーゲンが望ましく、１０００残基あたりチロシン残基が３以下であるアテロコラーゲンがより好ましい。
なお、本発明の好ましいコラーゲン又はコラーゲン誘導体は、アテロコラーゲンである。 (Collagen or collagen derivative)
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. As the collagen, 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.
さらに、上記コラーゲン又はコラーゲン誘導体は、下記の細胞膜透過性ペプチドを付加しても良い。下記のペプチドを付加することにより、標的細胞の導入率を向上させることができる。
（１）TAT（GRKKRRQRRRPQ：配列番号１）
（２）r8｛rrrrrrrr (D体-R)：配列番号２｝
（３）MPG-8（βAFLGWLGAWGTMGWSPKKKRK：配列番号３） Furthermore, the following cell membrane-permeable peptide may be added to the collagen or collagen derivative. By adding the following peptides, the introduction rate of target cells can be improved.
(1) TAT (GRKKRRQRRRPQ: SEQ ID NO: 1)
(2) r8 {rrrrrrrr (D form-R): SEQ ID NO: 2}
(3) MPG-8 (βAFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 3)
（１）TAT（GRKKRRQRRRPQ：配列番号１）
（２）r8｛rrrrrrrr (D体-R)：配列番号２｝
（３）MPG-8（βAFLGWLGAWGTMGWSPKKKRK：配列番号３） Furthermore, the following cell membrane-permeable peptide may be added to the collagen or collagen derivative. By adding the following peptides, the introduction rate of target cells can be improved.
(1) TAT (GRKKRRQRRRPQ: SEQ ID NO: 1)
(2) r8 {rrrrrrrr (D form-R): SEQ ID NO: 2}
(3) MPG-8 (βAFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 3)
（本発明の薬剤徐放担体の作製方法）
本発明の薬剤徐放担体は、自体公知の方法により、コラーゲン又はコラーゲン誘導体、カリウム塩、カルシウム塩及びナトリウム塩を含む溶液、並びに薬剤を混合することにより作製できる。 (Method for producing sustained-release drug carrier of the present invention)
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.
本発明の薬剤徐放担体は、自体公知の方法により、コラーゲン又はコラーゲン誘導体、カリウム塩、カルシウム塩及びナトリウム塩を含む溶液、並びに薬剤を混合することにより作製できる。 (Method for producing sustained-release drug carrier of the present invention)
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.
（本発明の薬剤徐放担体の組成）
本発明の薬剤徐放担体は、コラーゲン又はコラーゲン誘導体、並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液に加えて、生体親和性材料、添加剤等も含むこともできる。
生体親和性材料としては、例えば、ゼラチン、フィブリン、アルブミン、ヒアルロン酸、ヘパリン、コンドロイチン硫酸、キチン、キトサン、アルギン酸、ペクチン、アガロース、ハイドロキシアパタイト、ポリプロピレン、ポリエチレン、ポリジメチルシロキサン、又はグリコール酸、乳酸もしくはアミノ酸の重合体もしくはこれらの共重合体、又はこれらの生体親和性材料の２種類以上の混合物等が挙げられる。
添加剤としては、注射剤として用いる場合の等張化剤、ｐＨ調節剤、無痛化剤、又は固形剤として用いる場合の賦形剤、崩壊剤、コーティング剤が挙げられる。具体的にはｐＨを６～８に保つため、又は細胞と等張に保つために用いられる塩類や糖類が挙げられる。さらに、本発明の薬剤徐放担体は、固体状であっても溶液状であってもよい。本発明の薬剤徐放担体が固体状である場合、そのままの状態又は精製水、生理的食塩水、生体と等張な緩衝液等を用いて溶液状とし、所望の細胞に導入する。 (Composition of the drug sustained-release carrier of the present invention)
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.
Examples of the biocompatible material 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.
Examples of the additive 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. Furthermore, 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.
本発明の薬剤徐放担体は、コラーゲン又はコラーゲン誘導体、並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液に加えて、生体親和性材料、添加剤等も含むこともできる。
生体親和性材料としては、例えば、ゼラチン、フィブリン、アルブミン、ヒアルロン酸、ヘパリン、コンドロイチン硫酸、キチン、キトサン、アルギン酸、ペクチン、アガロース、ハイドロキシアパタイト、ポリプロピレン、ポリエチレン、ポリジメチルシロキサン、又はグリコール酸、乳酸もしくはアミノ酸の重合体もしくはこれらの共重合体、又はこれらの生体親和性材料の２種類以上の混合物等が挙げられる。
添加剤としては、注射剤として用いる場合の等張化剤、ｐＨ調節剤、無痛化剤、又は固形剤として用いる場合の賦形剤、崩壊剤、コーティング剤が挙げられる。具体的にはｐＨを６～８に保つため、又は細胞と等張に保つために用いられる塩類や糖類が挙げられる。さらに、本発明の薬剤徐放担体は、固体状であっても溶液状であってもよい。本発明の薬剤徐放担体が固体状である場合、そのままの状態又は精製水、生理的食塩水、生体と等張な緩衝液等を用いて溶液状とし、所望の細胞に導入する。 (Composition of the drug sustained-release carrier of the present invention)
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.
Examples of the biocompatible material 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.
Examples of the additive 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. Furthermore, 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.
さらに、本発明の薬剤徐放担体では、さらに、徐放作用能力を向上させるために、単糖類、二糖類、三糖類及び四糖類から選択される１以上の糖を含んでも良い（参照：特許文献３）。
Furthermore, 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).
（本発明の薬剤徐放担体の用途）
本発明の薬剤徐放担体の用途は、特に限定されないが、薬剤、医療器具、細胞培養器具、標識剤等に使用することができる。 (Use of the drug sustained-release carrier of the present invention)
Although 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.
本発明の薬剤徐放担体の用途は、特に限定されないが、薬剤、医療器具、細胞培養器具、標識剤等に使用することができる。 (Use of the drug sustained-release carrier of the present invention)
Although 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.
（本発明の薬剤徐放担体の投与方法）
本発明の薬剤徐放担体を生体（ヒトを含む動物、特に、ヒトを含む哺乳類、例えば、マウス、ラット、ウサギ、イヌ、ネコ、ウマ、ウシ）に投与する方法としては、経口、注射、点眼、点鼻、経肺、皮膚を介した吸収のいずれでも良く、好ましくは注射である。投与部位は疾患に応じて選択できるが、手術時に必要な部位に直接留置することもできる。例えば、本発明の薬剤徐放担体を、静脈注射（点滴等）による全身投与、患部（癌細胞等）に注射することによる局所投与等が挙げられる。 (Method for administering the drug sustained-release carrier of the present invention)
Examples of methods for administering the sustained-release drug carrier of the present invention to living organisms (animals including humans, particularly mammals including humans, such as mice, rats, rabbits, dogs, cats, horses, cows) 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. For example, 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.
本発明の薬剤徐放担体を生体（ヒトを含む動物、特に、ヒトを含む哺乳類、例えば、マウス、ラット、ウサギ、イヌ、ネコ、ウマ、ウシ）に投与する方法としては、経口、注射、点眼、点鼻、経肺、皮膚を介した吸収のいずれでも良く、好ましくは注射である。投与部位は疾患に応じて選択できるが、手術時に必要な部位に直接留置することもできる。例えば、本発明の薬剤徐放担体を、静脈注射（点滴等）による全身投与、患部（癌細胞等）に注射することによる局所投与等が挙げられる。 (Method for administering the drug sustained-release carrier of the present invention)
Examples of methods for administering the sustained-release drug carrier of the present invention to living organisms (animals including humans, particularly mammals including humans, such as mice, rats, rabbits, dogs, cats, horses, cows) 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. For example, 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.
（薬剤徐放方法）
本発明の「薬剤徐放方法」は、少なくとも以下の工程を有する。
（１）薬剤と、コラーゲン若しくはコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を混合して、混合溶液を作成する工程。
（２）該混合溶液を、該薬剤を必要とする患者に投与する工程。
なお、「薬剤」と「該薬剤を必要とする患者」の組合せは、以下を例示することができるが特に限定されない。
・癌治療剤－癌患者
・筋疾患治療剤－筋疾患患者（筋損傷、筋ジストロフィー）
・骨再生誘導剤－骨欠損患者（軟骨）
・線維症治療剤－線維症患者
・抗炎症剤－炎症疾患患者
・黄斑変性症治療剤－黄斑変性症患者
・皮膚疾患治療剤－皮膚疾患患者 (Drug sustained release method)
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.
In addition, although the following can be illustrated as a combination of “medicine” and “patient requiring the drug”, it is not particularly limited.
-Cancer therapeutic agent-Cancer patient-Muscle disease therapeutic agent-Muscle disease patient (muscle injury, muscular dystrophy)
・ Bone regeneration inducer-Bone defect patient (cartilage)
-Fibrosis treatment agent-Fibrosis patient-Anti-inflammatory agent-Inflammatory disease patient-Macular degeneration treatment agent-Macular degeneration patient-Skin disease treatment agent-Skin disease patient
本発明の「薬剤徐放方法」は、少なくとも以下の工程を有する。
（１）薬剤と、コラーゲン若しくはコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を混合して、混合溶液を作成する工程。
（２）該混合溶液を、該薬剤を必要とする患者に投与する工程。
なお、「薬剤」と「該薬剤を必要とする患者」の組合せは、以下を例示することができるが特に限定されない。
・癌治療剤－癌患者
・筋疾患治療剤－筋疾患患者（筋損傷、筋ジストロフィー）
・骨再生誘導剤－骨欠損患者（軟骨）
・線維症治療剤－線維症患者
・抗炎症剤－炎症疾患患者
・黄斑変性症治療剤－黄斑変性症患者
・皮膚疾患治療剤－皮膚疾患患者 (Drug sustained release method)
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.
In addition, although the following can be illustrated as a combination of “medicine” and “patient requiring the drug”, it is not particularly limited.
-Cancer therapeutic agent-Cancer patient-Muscle disease therapeutic agent-Muscle disease patient (muscle injury, muscular dystrophy)
・ Bone regeneration inducer-Bone defect patient (cartilage)
-Fibrosis treatment agent-Fibrosis patient-Anti-inflammatory agent-Inflammatory disease patient-Macular degeneration treatment agent-Macular degeneration patient-Skin disease treatment agent-Skin disease patient
（ゲル形成能）
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例２の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、ゲル形成能が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高いゲル形成能により、高濃度の運搬対象を長期保持することができる。 (Gel forming ability)
According to the results of Example 2 below, 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.
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例２の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、ゲル形成能が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高いゲル形成能により、高濃度の運搬対象を長期保持することができる。 (Gel forming ability)
According to the results of Example 2 below, 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.
（徐放作用能）
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例３の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、徐放作用能が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高い徐放作用能により、長時間薬剤を供給することができる。 (Slow release action ability)
According to the results of Example 3 below, 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.
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例３の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、徐放作用能が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高い徐放作用能により、長時間薬剤を供給することができる。 (Slow release action ability)
According to the results of Example 3 below, 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.
（薬効効果）
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例４の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、標的細胞内導入効率が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高い細胞導入効率により、薬効効果が高い。 (Medicinal effect)
According to the results of Example 4 below, 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.
本発明の薬剤徐放担体又は薬剤徐放方法は、下記実施例４の結果により、公知のコラーゲン（アテロコラーゲン）を含む薬剤担体又は薬剤徐放方法と比較して、標的細胞内導入効率が高い。本発明の薬剤徐放担体又は薬剤徐放方法の高い細胞導入効率により、薬効効果が高い。 (Medicinal effect)
According to the results of Example 4 below, 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.
（薬剤）
本発明における「薬剤」は、特に限定されないが、核酸、タンパク質、ペプチド、低分子化合物等である。 (Drug)
The “drug” in the present invention is not particularly limited, but includes nucleic acids, proteins, peptides, low molecular compounds and the like.
本発明における「薬剤」は、特に限定されないが、核酸、タンパク質、ペプチド、低分子化合物等である。 (Drug)
The “drug” in the present invention is not particularly limited, but includes nucleic acids, proteins, peptides, low molecular compounds and the like.
（タンパク質）
酵素や標的受容体に対するアゴニスト・アンタゴニスト、受容体そのもの、抗体等が挙げられるが、特に限定されるものでは無い。
（ペプチド）
低分子のタンパクであり酵素活性を有するものや機能性タンパク質の一部分を合成したもの、標的受容体に対するアゴニスト・アンタゴニスト等が挙げられるが、特に限定されるものでは無い。
（低分子化合物）
低分子医薬である抗癌剤等の腫瘍細胞の死滅に特異的に効果を示すものや、細胞の生理的活性を亢進又は抑制させるものが挙げられるが、特に限定されるものでは無い。
（核酸）
本発明における「運搬対象である核酸」は、ポリヌクレオチドであってもオリゴヌクレオチドであってもよく、またDNAでもRNA分子でもあり得る。DNA分子の場合、プラスミドDNA、cDNA、ゲノミックDNA又は合成DNAであってもよい。またDNA及びRNAはいずれも２本鎖でも１本鎖でもあり得る。１本鎖の場合、コード鎖又は非コード鎖であり得る。「核酸」にはDNA誘導体又はRNA誘導体が含まれ、該誘導体とはホスホロチオエート結合を有する核酸、又は酵素による分解を避ける為にインターヌクレオチドのリン酸部位、糖部分、塩基部分に化学修飾を施した核酸を意味する。また、「核酸」にはアデノウイルス、レトロウイルス等のウイルスも含まれる。
核酸がプラスミドDNA又はウイルス等の遺伝子治療に用いられるベクターである場合、細胞内に導入されたとき、コードした遺伝情報を細胞内で発現するように構成された形態が好ましく、プロモーター等、目的遺伝子の発現に必要な要素を含有する、又は染色体への組み込みを可能とする要素を含有するベクター等である。 (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.
(Nucleic acid)
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. In the case of 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.
酵素や標的受容体に対するアゴニスト・アンタゴニスト、受容体そのもの、抗体等が挙げられるが、特に限定されるものでは無い。
（ペプチド）
低分子のタンパクであり酵素活性を有するものや機能性タンパク質の一部分を合成したもの、標的受容体に対するアゴニスト・アンタゴニスト等が挙げられるが、特に限定されるものでは無い。
（低分子化合物）
低分子医薬である抗癌剤等の腫瘍細胞の死滅に特異的に効果を示すものや、細胞の生理的活性を亢進又は抑制させるものが挙げられるが、特に限定されるものでは無い。
（核酸）
本発明における「運搬対象である核酸」は、ポリヌクレオチドであってもオリゴヌクレオチドであってもよく、またDNAでもRNA分子でもあり得る。DNA分子の場合、プラスミドDNA、cDNA、ゲノミックDNA又は合成DNAであってもよい。またDNA及びRNAはいずれも２本鎖でも１本鎖でもあり得る。１本鎖の場合、コード鎖又は非コード鎖であり得る。「核酸」にはDNA誘導体又はRNA誘導体が含まれ、該誘導体とはホスホロチオエート結合を有する核酸、又は酵素による分解を避ける為にインターヌクレオチドのリン酸部位、糖部分、塩基部分に化学修飾を施した核酸を意味する。また、「核酸」にはアデノウイルス、レトロウイルス等のウイルスも含まれる。
核酸がプラスミドDNA又はウイルス等の遺伝子治療に用いられるベクターである場合、細胞内に導入されたとき、コードした遺伝情報を細胞内で発現するように構成された形態が好ましく、プロモーター等、目的遺伝子の発現に必要な要素を含有する、又は染色体への組み込みを可能とする要素を含有するベクター等である。 (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.
(Nucleic acid)
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. In the case of 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.
（医療用具又は細胞培養器具）
本発明は、本発明の薬剤徐放担体が表面に塗布されている医療用具又は細胞培養器具も対象とする。
本発明の薬剤徐放担体を固相表面に塗布し、そこへ標的細胞を接触させると、標的細胞の上から本発明の薬剤徐放担体を滴加するよりも、運搬対象の導入効率が向上する。
本発明の医療用具とは、人工臓器、より具体的には、人工血管、血管を補強する医療用具ステント、粘着シート又は人工心臓が含まれる。
本発明の細胞培養器具とは、細胞培養実験に通常用いられているシャーレ、フラスコ、９６穴マイクロプレート、三次元培養担体等が挙げられる。 (Medical device or cell culture device)
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.
When 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. To do.
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.
Examples of 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.
本発明は、本発明の薬剤徐放担体が表面に塗布されている医療用具又は細胞培養器具も対象とする。
本発明の薬剤徐放担体を固相表面に塗布し、そこへ標的細胞を接触させると、標的細胞の上から本発明の薬剤徐放担体を滴加するよりも、運搬対象の導入効率が向上する。
本発明の医療用具とは、人工臓器、より具体的には、人工血管、血管を補強する医療用具ステント、粘着シート又は人工心臓が含まれる。
本発明の細胞培養器具とは、細胞培養実験に通常用いられているシャーレ、フラスコ、９６穴マイクロプレート、三次元培養担体等が挙げられる。 (Medical device or cell culture device)
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.
When 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. To do.
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.
Examples of 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.
（標的細胞における遺伝子又はタンパク質の機能を調べる方法）
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、遺伝子又はタンパク質の標的細胞内での機能を容易に調べることができる。例えば、細胞内に機能を調べたい遺伝子を組み込んだプラスミドDNAを導入して発現させることによってその遺伝子の機能を調べる方法、又は機能を調べたい遺伝子の発現を抑制するsiRNA核酸を細胞内に導入して遺伝子の発現を抑制することによってその遺伝子の機能を調べる方法が有用である。
具体的な測定方法としては、機能を解明したい遺伝子を発現するプラスミドDNA、アデノウイルスベクター又は機能を解明したい遺伝子の発現を抑制するsiRNA核酸と本発明の薬剤徐放担体を混合し、培養プレート固相上に塗布、整列配置する。塗布した薬剤徐放担体を乾燥して固相上に固定した後、細胞を播種し、数日間プレート上で培養する。塗布された薬剤徐放担体は、塗布された部分に接着した細胞に効率的に導入され、機能を調べたい遺伝子を発現又はその発現を長期間抑制する。数日後、細胞の増殖率、形態（表現型）や細胞内での遺伝子発現の状態（遺伝子発現レベル）、又は細胞から産生されたタンパク質の種類や量を調べることによって標的とした遺伝子の機能を明らかにすることができる。 (Method for examining the function of a gene or protein in a target cell)
If the drug sustained-release carrier or drug sustained-release method of the present invention is used, the function of the gene or protein in the target cell can be easily examined. For example, 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. Thus, a method for examining the function of a gene by suppressing the expression of the gene is useful.
As a specific measurement method, 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.
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、遺伝子又はタンパク質の標的細胞内での機能を容易に調べることができる。例えば、細胞内に機能を調べたい遺伝子を組み込んだプラスミドDNAを導入して発現させることによってその遺伝子の機能を調べる方法、又は機能を調べたい遺伝子の発現を抑制するsiRNA核酸を細胞内に導入して遺伝子の発現を抑制することによってその遺伝子の機能を調べる方法が有用である。
具体的な測定方法としては、機能を解明したい遺伝子を発現するプラスミドDNA、アデノウイルスベクター又は機能を解明したい遺伝子の発現を抑制するsiRNA核酸と本発明の薬剤徐放担体を混合し、培養プレート固相上に塗布、整列配置する。塗布した薬剤徐放担体を乾燥して固相上に固定した後、細胞を播種し、数日間プレート上で培養する。塗布された薬剤徐放担体は、塗布された部分に接着した細胞に効率的に導入され、機能を調べたい遺伝子を発現又はその発現を長期間抑制する。数日後、細胞の増殖率、形態（表現型）や細胞内での遺伝子発現の状態（遺伝子発現レベル）、又は細胞から産生されたタンパク質の種類や量を調べることによって標的とした遺伝子の機能を明らかにすることができる。 (Method for examining the function of a gene or protein in a target cell)
If the drug sustained-release carrier or drug sustained-release method of the present invention is used, the function of the gene or protein in the target cell can be easily examined. For example, 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. Thus, a method for examining the function of a gene by suppressing the expression of the gene is useful.
As a specific measurement method, 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.
（疾患を処置できる運搬対象をスクリーニングする方法）
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、遺伝子疾患、癌、AIDS、慢性関節リウマチ、生活習慣病等の様々な疾患を処置し得る候補物質をスクリーニングすることができる。例えば、固相上に疾患の治療効果を調べたい候補物質（例、核酸）と本発明の薬剤徐放担体を混合し、培養プレート固相上に塗布、整列配置する。塗布した薬剤徐放担体を乾燥して固相上に固定した後、細胞を播種し、数日間プレート上で培養する。
候補物質（例、核酸）の効果は、細胞の表現型の変化、細胞死、細胞の増殖、細胞内での遺伝子発現のパターン、産生されるタンパク質の種類や量により解析することができる。 (Method of screening for transportable target that can treat disease)
If the drug sustained-release carrier or drug sustained-release method of the present invention is used, candidate substances capable of treating various diseases such as genetic diseases, cancer, AIDS, rheumatoid arthritis, lifestyle-related diseases and the like can be screened. For example, a candidate substance (eg, nucleic acid) to be examined for the therapeutic effect of a disease on a solid phase and 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) can be analyzed based on changes in cell phenotype, cell death, cell proliferation, gene expression pattern in the cell, and the type and amount of protein produced.
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、遺伝子疾患、癌、AIDS、慢性関節リウマチ、生活習慣病等の様々な疾患を処置し得る候補物質をスクリーニングすることができる。例えば、固相上に疾患の治療効果を調べたい候補物質（例、核酸）と本発明の薬剤徐放担体を混合し、培養プレート固相上に塗布、整列配置する。塗布した薬剤徐放担体を乾燥して固相上に固定した後、細胞を播種し、数日間プレート上で培養する。
候補物質（例、核酸）の効果は、細胞の表現型の変化、細胞死、細胞の増殖、細胞内での遺伝子発現のパターン、産生されるタンパク質の種類や量により解析することができる。 (Method of screening for transportable target that can treat disease)
If the drug sustained-release carrier or drug sustained-release method of the present invention is used, candidate substances capable of treating various diseases such as genetic diseases, cancer, AIDS, rheumatoid arthritis, lifestyle-related diseases and the like can be screened. For example, a candidate substance (eg, nucleic acid) to be examined for the therapeutic effect of a disease on a solid phase and 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) can be analyzed based on changes in cell phenotype, cell death, cell proliferation, gene expression pattern in the cell, and the type and amount of protein produced.
（薬剤徐放担体を使用した標的細胞の性質を変える方法）
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、標的細胞の性質を変えることができる。例えば、特定の細胞内受容体の活性を抑えるアンタゴニストを運搬対象として、標的細胞に投与すれば、該標的細胞は、該アンタゴニストが投与されていない標的細胞と比較して、受容体の活性を低くすることができる（標的細胞の性質を変えることができる）。
加えて、特定のmRNAを分解できるsiRNAを運搬対象として標的細胞に投与すれば、該mRNAを分解し、そのmRNAがコードする機能性タンパク質の発現量を低減させた細胞を得ることができる。 (Method of changing the properties of target cells using a drug sustained-release carrier)
If the drug sustained-release carrier or the drug sustained-release method of the present invention is used, the properties of the target cells can be changed. For example, when 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).
In addition, when 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.
本発明の薬剤徐放担体又は薬剤徐放方法を使用すれば、標的細胞の性質を変えることができる。例えば、特定の細胞内受容体の活性を抑えるアンタゴニストを運搬対象として、標的細胞に投与すれば、該標的細胞は、該アンタゴニストが投与されていない標的細胞と比較して、受容体の活性を低くすることができる（標的細胞の性質を変えることができる）。
加えて、特定のmRNAを分解できるsiRNAを運搬対象として標的細胞に投与すれば、該mRNAを分解し、そのmRNAがコードする機能性タンパク質の発現量を低減させた細胞を得ることができる。 (Method of changing the properties of target cells using a drug sustained-release carrier)
If the drug sustained-release carrier or the drug sustained-release method of the present invention is used, the properties of the target cells can be changed. For example, when 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).
In addition, when 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.
（標識剤）
本発明は、本発明の薬剤徐放担体を含む標識剤も対象とする。例えば、標的細胞等を特異的に認識する物質（標的細胞に特異的に結合するペプチド、タンパク質等）に標識物質を結合させた運搬対象を生体に投与すれば、標的細胞を標識することができる。 (Labeling agent)
The present invention is also directed to a labeling agent comprising the drug sustained-release carrier of the present invention. For example, 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). .
本発明は、本発明の薬剤徐放担体を含む標識剤も対象とする。例えば、標的細胞等を特異的に認識する物質（標的細胞に特異的に結合するペプチド、タンパク質等）に標識物質を結合させた運搬対象を生体に投与すれば、標的細胞を標識することができる。 (Labeling agent)
The present invention is also directed to a labeling agent comprising the drug sustained-release carrier of the present invention. For example, 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). .
（薬剤徐放担体又は薬剤徐放方法の具体例）
本発明の薬剤徐放担体又は薬剤徐放方法の好ましい組み合わせは、以下の通りであるが特に限定されず、さらに、必要に応じて、生体親和性材料及び／又は添加剤を含むこともできる。
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液 (Specific examples of drug sustained release carrier or drug sustained release method)
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.
Nucleic acid (drug), collagen or collagen derivative, bicarbonate Ringer's solution Nucleic acid (drug), collagen or collagen derivative, lactated Ringer's solution Nucleic acid (drug), collagen or collagen derivative, bicarbonated Ringer's solution and lactated Ringer's solution Peptide (drug), collagen or collagen derivative , Bicarbonate Ringer's solution peptide (drug), collagen or collagen derivative, lactated Ringer's solution peptide (drug), collagen or collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution protein (drug), collagen or collagen derivative, bicarbonate Ringer's solution protein (drug), Collagen or collagen derivative, lactated Ringer's solution protein (drug), collagen or collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution low molecular weight compound (drug), co Gen or collagen derivatives, bicarbonate Ringer's solution low molecular compound (drug), collagen or a collagen derivative, lactated Ringer's low molecular compound (drug), collagen or a collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution
本発明の薬剤徐放担体又は薬剤徐放方法の好ましい組み合わせは、以下の通りであるが特に限定されず、さらに、必要に応じて、生体親和性材料及び／又は添加剤を含むこともできる。
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
核酸（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
ペプチド（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
タンパク質（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、乳酸リンゲル液
低分子化合物（薬剤）、コラーゲン又はコラーゲン誘導体、重炭酸リンゲル液及び乳酸リンゲル液 (Specific examples of drug sustained release carrier or drug sustained release method)
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.
Nucleic acid (drug), collagen or collagen derivative, bicarbonate Ringer's solution Nucleic acid (drug), collagen or collagen derivative, lactated Ringer's solution Nucleic acid (drug), collagen or collagen derivative, bicarbonated Ringer's solution and lactated Ringer's solution Peptide (drug), collagen or collagen derivative , Bicarbonate Ringer's solution peptide (drug), collagen or collagen derivative, lactated Ringer's solution peptide (drug), collagen or collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution protein (drug), collagen or collagen derivative, bicarbonate Ringer's solution protein (drug), Collagen or collagen derivative, lactated Ringer's solution protein (drug), collagen or collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution low molecular weight compound (drug), co Gen or collagen derivatives, bicarbonate Ringer's solution low molecular compound (drug), collagen or a collagen derivative, lactated Ringer's low molecular compound (drug), collagen or a collagen derivative, bicarbonate Ringer's solution and lactated Ringer's solution
以下に実施例を挙げて本発明を説明するが、本発明はこれら実施例により何ら限定されるものではない。
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
（コラーゲン又はコラーゲン誘導体並びにリンゲル液を含む薬剤徐放担体の作製）
本実施例では、コラーゲン又はコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を含む薬剤徐放担体の作製をした。詳細は、以下の通りである。 (Preparation of drug sustained-release carrier containing collagen or collagen derivative and Ringer's solution)
In this example, a drug sustained-release carrier containing collagen or a collagen derivative and a solution containing potassium salt, calcium salt and sodium salt was prepared. Details are as follows.
本実施例では、コラーゲン又はコラーゲン誘導体並びにカリウム塩、カルシウム塩及びナトリウム塩を含む溶液を含む薬剤徐放担体の作製をした。詳細は、以下の通りである。 (Preparation of drug sustained-release carrier containing collagen or collagen derivative and Ringer's solution)
In this example, a drug sustained-release carrier containing collagen or a collagen derivative and a solution containing potassium salt, calcium salt and sodium salt was prepared. Details are as follows.
（使用した試薬）
使用した試薬は、以下の通りである。
・siRNA：GL3 siRNA （ ホタルルシフェラーゼに対するsiRNA ）
配列 ( sense側 )：5'-CUUACGCUGAGUACUUCGA-3'（配列番号４）
・アテロコラーゲン（高研社製品、以下ACと記載）
・塩化ナトリウム（Wako）
・塩化カリウム（Wako）
・塩化カルシウム（Wako）
・塩化マグネシウム・六水和物（Wako）
・炭酸水素ナトリウム（Wako）
・クエン酸三ナトリウム・二水和物（Wako）
・乳酸ナトリウム溶液（50%）（Wako）
・PBS（リン酸緩衝生理食塩水、DSファーマ）
・pH 7.2 リン酸緩衝液（以下PBと記載） (Reagent used)
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)
使用した試薬は、以下の通りである。
・siRNA：GL3 siRNA （ ホタルルシフェラーゼに対するsiRNA ）
配列 ( sense側 )：5'-CUUACGCUGAGUACUUCGA-3'（配列番号４）
・アテロコラーゲン（高研社製品、以下ACと記載）
・塩化ナトリウム（Wako）
・塩化カリウム（Wako）
・塩化カルシウム（Wako）
・塩化マグネシウム・六水和物（Wako）
・炭酸水素ナトリウム（Wako）
・クエン酸三ナトリウム・二水和物（Wako）
・乳酸ナトリウム溶液（50%）（Wako）
・PBS（リン酸緩衝生理食塩水、DSファーマ）
・pH 7.2 リン酸緩衝液（以下PBと記載） (Reagent used)
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)
重炭酸リンゲル液の組成は、以下の通りである。
塩化ナトリウム：103 mM
塩化カリウム：4 mM
塩化カルシウム：3 mM
炭酸水素ナトリウム：25 mM
塩化マグネシウム：1 mM
クエン酸三ナトリウム：2 mM The composition of the bicarbonate Ringer's solution is as follows.
Sodium chloride: 103 mM
Potassium chloride: 4 mM
Calcium chloride: 3 mM
Sodium bicarbonate: 25 mM
Magnesium chloride: 1 mM
Trisodium citrate: 2 mM
塩化ナトリウム：103 mM
塩化カリウム：4 mM
塩化カルシウム：3 mM
炭酸水素ナトリウム：25 mM
塩化マグネシウム：1 mM
クエン酸三ナトリウム：2 mM The composition of the bicarbonate Ringer's solution is as follows.
Sodium chloride: 103 mM
Potassium chloride: 4 mM
Calcium chloride: 3 mM
Sodium bicarbonate: 25 mM
Magnesium chloride: 1 mM
Trisodium citrate: 2 mM
乳酸リンゲル液の組成は、以下の通りである。
塩化ナトリウム：103 mM
塩化カリウム：4 mM
塩化カルシウム：3 mM
乳酸ナトリウム：28 mM The 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
塩化ナトリウム：103 mM
塩化カリウム：4 mM
塩化カルシウム：3 mM
乳酸ナトリウム：28 mM The 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
（薬剤徐放担体の作製）
1.1%ACと50 mM GL3 siRNA及び2.2倍濃度の重炭酸又は乳酸リンゲル液を45：10：45の割合で混合した。混合にはローテーターを用い、12rpmで10分間、転倒混和した。
これにより、下記の組成の徐放担体を作製した。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液
コントロールとして、以下も作製した。
○従来バッファー/siRNA画分：：0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS
○乳酸リンゲル液画分：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分：0.5%AC/重炭酸リンゲル液 (Production of drug sustained-release carrier)
1.1% AC and 50 mM GL3 siRNA and 2.2 times concentrated bicarbonate or lactated Ringer's solution were mixed at a ratio of 45:10:45. Mixing was carried out by inversion at 12 rpm for 10 minutes using a rotator.
Thereby, a sustained-release carrier having the following composition was produced.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution The following were also prepared as controls.
○ Conventional buffer / siRNA fraction: 0.5% AC / 5μM GL3 siRNA / 50 mM PB / 0.5 × PBS
○ Lactated Ringer solution fraction: 0.5% AC / Lactated Ringer solution ○ Bicarbonate Ringer solution fraction: 0.5% AC / bicarbonate Ringer solution
1.1%ACと50 mM GL3 siRNA及び2.2倍濃度の重炭酸又は乳酸リンゲル液を45：10：45の割合で混合した。混合にはローテーターを用い、12rpmで10分間、転倒混和した。
これにより、下記の組成の徐放担体を作製した。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液
コントロールとして、以下も作製した。
○従来バッファー/siRNA画分：：0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS
○乳酸リンゲル液画分：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分：0.5%AC/重炭酸リンゲル液 (Production of drug sustained-release carrier)
1.1% AC and 50 mM GL3 siRNA and 2.2 times concentrated bicarbonate or lactated Ringer's solution were mixed at a ratio of 45:10:45. Mixing was carried out by inversion at 12 rpm for 10 minutes using a rotator.
Thereby, a sustained-release carrier having the following composition was produced.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution The following were also prepared as controls.
○ Conventional buffer / siRNA fraction: 0.5% AC / 5μM GL3 siRNA / 50 mM PB / 0.5 × PBS
○ Lactated Ringer solution fraction: 0.5% AC / Lactated Ringer solution ○ Bicarbonate Ringer solution fraction: 0.5% AC / bicarbonate Ringer solution
（本発明の薬剤徐放担体のゲル形成能の確認）
本実施例では、本発明の薬剤徐放担体は、リンゲル液を含むことによりアテロコラーゲンのゲル形成能が変化するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of gel forming ability of drug sustained-release carrier of the present invention)
In this example, it was confirmed whether or not the drug sustained-release carrier of the present invention changes the gel forming ability of atelocollagen by containing Ringer's solution. Details are as follows.
本実施例では、本発明の薬剤徐放担体は、リンゲル液を含むことによりアテロコラーゲンのゲル形成能が変化するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of gel forming ability of drug sustained-release carrier of the present invention)
In this example, it was confirmed whether or not the drug sustained-release carrier of the present invention changes the gel forming ability of atelocollagen by containing Ringer's solution. Details are as follows.
（本発明の薬剤徐放担体のゲル形成能の測定）
実施例１で作製した重炭酸リンゲル液/siRNA画分（0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液）、乳酸リンゲル液/siRNA画分（0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液）及び従来バッファー/siRNA画分（0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS）を撹拌しながら、96wellプレートに全量100μL／wellになるように分注した。
その後、吸光マイクロプレートリーダー（TECAN、サンライズサーモRC-R）を用いて、ゲル化試験を行った（測定温度：37℃、測定波長：400 nm、測定間隔：30秒、測定時間）。測定後、各時間における測定値の平均（n=3）をゲル化試験結果とし、この際の最大勾配時間をゲル形成時間として算出した。 (Measurement of gel-forming ability of drug sustained-release carrier of the present invention)
Bicarbonate Ringer solution / siRNA fraction (0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution), lactated Ringer solution / siRNA fraction (0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution) prepared in Example 1 and conventional buffer / siRNA The fraction (0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × PBS) was dispensed into a 96-well plate at a total volume of 100 μL / well while stirring.
Thereafter, a gelation test was performed using an absorption microplate reader (TECAN, Sunrise Thermo RC-R) (measurement temperature: 37 ° C., measurement wavelength: 400 nm, measurement interval: 30 seconds, measurement time). After the measurement, the average (n = 3) of the measured values at each time was taken as the gelation test result, and the maximum gradient time at this time was calculated as the gel formation time.
実施例１で作製した重炭酸リンゲル液/siRNA画分（0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液）、乳酸リンゲル液/siRNA画分（0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液）及び従来バッファー/siRNA画分（0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS）を撹拌しながら、96wellプレートに全量100μL／wellになるように分注した。
その後、吸光マイクロプレートリーダー（TECAN、サンライズサーモRC-R）を用いて、ゲル化試験を行った（測定温度：37℃、測定波長：400 nm、測定間隔：30秒、測定時間）。測定後、各時間における測定値の平均（n=3）をゲル化試験結果とし、この際の最大勾配時間をゲル形成時間として算出した。 (Measurement of gel-forming ability of drug sustained-release carrier of the present invention)
Bicarbonate Ringer solution / siRNA fraction (0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution), lactated Ringer solution / siRNA fraction (0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution) prepared in Example 1 and conventional buffer / siRNA The fraction (0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × PBS) was dispensed into a 96-well plate at a total volume of 100 μL / well while stirring.
Thereafter, a gelation test was performed using an absorption microplate reader (TECAN, Sunrise Thermo RC-R) (measurement temperature: 37 ° C., measurement wavelength: 400 nm, measurement interval: 30 seconds, measurement time). After the measurement, the average (n = 3) of the measured values at each time was taken as the gelation test result, and the maximum gradient time at this time was calculated as the gel formation time.
（薬剤徐放担体のゲル形成能の測定結果）
薬剤徐放担体のゲル形成能の測定結果を図１に示す。重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分のゲル形成時間は約１０分であった。一方、コントロールである従来バッファー/siRNA画分のゲル形成時間は６０分以上であった。
すなわち、重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分のゲル形成能は、コントロールである従来バッファー/siRNA画分のゲル形成能と比較して、約６倍であることを確認した。
ゲル形成能が高いことは、「迅速にゲル化するので、運搬対象がゲルに封入されている割合が高く、該運搬対象を長期間保持できる」ことを意味する。一方、ゲル形成能が低いことは、「ゲル化が遅いので、ゲル形成時間内に運搬対象が担体から一部抜けていき、該運搬対象を高濃度で保持できない」ことを意味する。
以上により、本発明の薬剤徐放担体は、従来の薬剤担体と比較して、高濃度の運搬対象を長期間保持することができる。 (Measurement result of gel-forming ability of drug sustained-release carrier)
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. On the other hand, 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”. On the other hand, 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”.
As described above, 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.
薬剤徐放担体のゲル形成能の測定結果を図１に示す。重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分のゲル形成時間は約１０分であった。一方、コントロールである従来バッファー/siRNA画分のゲル形成時間は６０分以上であった。
すなわち、重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分のゲル形成能は、コントロールである従来バッファー/siRNA画分のゲル形成能と比較して、約６倍であることを確認した。
ゲル形成能が高いことは、「迅速にゲル化するので、運搬対象がゲルに封入されている割合が高く、該運搬対象を長期間保持できる」ことを意味する。一方、ゲル形成能が低いことは、「ゲル化が遅いので、ゲル形成時間内に運搬対象が担体から一部抜けていき、該運搬対象を高濃度で保持できない」ことを意味する。
以上により、本発明の薬剤徐放担体は、従来の薬剤担体と比較して、高濃度の運搬対象を長期間保持することができる。 (Measurement result of gel-forming ability of drug sustained-release carrier)
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. On the other hand, 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”. On the other hand, 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”.
As described above, 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.
（本発明の薬剤徐放担体の徐放作用能の確認）
本実施例では、本発明の薬剤徐放担体は、リンゲル液を含むことによりアテロコラーゲンの徐放作用能が変化するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of sustained release action ability of drug sustained-release carrier of the present invention)
In this example, it was confirmed whether the sustained release action ability of atelocollagen was changed by including the Ringer's solution in the sustained release carrier of the present invention. Details are as follows.
本実施例では、本発明の薬剤徐放担体は、リンゲル液を含むことによりアテロコラーゲンの徐放作用能が変化するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of sustained release action ability of drug sustained-release carrier of the present invention)
In this example, it was confirmed whether the sustained release action ability of atelocollagen was changed by including the Ringer's solution in the sustained release carrier of the present invention. Details are as follows.
（本発明の薬剤徐放担体の徐放作用能の測定）
徐放作用能を測定するために、フィルターチューブ（Millipore, ultra-free-MC, 0.22μm, PVDF, 参照：図２）を使用した。
4℃下で、該フィルターチューブのフィルター上槽に重炭酸リンゲル液/siRNA画分（0.3%AC/ 50μM GL3 siRNA /重炭酸リンゲル液）又は従来バッファー/siRNA画分（0.3%AC/ 50μM GL3 siRNA /50 mM PB/ 0.5×PBS）を100μl、フィルター下槽にPBS溶液を700μl添加後、37℃下で静置した。上槽のAC中のsiRNAがフィルターを通過して下槽のPBSに放出される様子を調査するために、PBS中のsiRNA濃度を経時的に分光光度計（Thermo scientific, NanoDrop 2000）を用いて測定した。 (Measurement of sustained release action ability of drug sustained-release carrier of the present invention)
A filter tube (Millipore, ultra-free-MC, 0.22 μm, PVDF, see FIG. 2) was used to measure the sustained release action ability.
At 4 ° C, the filter tube of the filter tube is placed in the bicarbonate Ringer solution / siRNA fraction (0.3% AC / 50 μM GL3 siRNA / bicarbonate Ringer solution) or the conventional buffer / siRNA fraction (0.3% AC / 50 μM GL3 siRNA / 50 After adding 100 μl of mM PB / 0.5 × PBS) and 700 μl of PBS solution to the lower filter tank, the plate was allowed to stand at 37 ° C. In order to investigate how siRNA in the AC in the upper tank passes through the filter and is released into the PBS in the lower tank, the concentration of siRNA in PBS is measured over time using a spectrophotometer (Thermo scientific, NanoDrop 2000). It was measured.
徐放作用能を測定するために、フィルターチューブ（Millipore, ultra-free-MC, 0.22μm, PVDF, 参照：図２）を使用した。
4℃下で、該フィルターチューブのフィルター上槽に重炭酸リンゲル液/siRNA画分（0.3%AC/ 50μM GL3 siRNA /重炭酸リンゲル液）又は従来バッファー/siRNA画分（0.3%AC/ 50μM GL3 siRNA /50 mM PB/ 0.5×PBS）を100μl、フィルター下槽にPBS溶液を700μl添加後、37℃下で静置した。上槽のAC中のsiRNAがフィルターを通過して下槽のPBSに放出される様子を調査するために、PBS中のsiRNA濃度を経時的に分光光度計（Thermo scientific, NanoDrop 2000）を用いて測定した。 (Measurement of sustained release action ability of drug sustained-release carrier of the present invention)
A filter tube (Millipore, ultra-free-MC, 0.22 μm, PVDF, see FIG. 2) was used to measure the sustained release action ability.
At 4 ° C, the filter tube of the filter tube is placed in the bicarbonate Ringer solution / siRNA fraction (0.3% AC / 50 μM GL3 siRNA / bicarbonate Ringer solution) or the conventional buffer / siRNA fraction (0.3% AC / 50 μM GL3 siRNA / 50 After adding 100 μl of mM PB / 0.5 × PBS) and 700 μl of PBS solution to the lower filter tank, the plate was allowed to stand at 37 ° C. In order to investigate how siRNA in the AC in the upper tank passes through the filter and is released into the PBS in the lower tank, the concentration of siRNA in PBS is measured over time using a spectrophotometer (Thermo scientific, NanoDrop 2000). It was measured.
（薬剤徐放担体の徐放作用能の測定結果）
薬剤徐放担体の徐放作用能の測定結果を図３に示す。重炭酸リンゲル液/siRNA画分は、コントロールである従来バッファー/siRNA画分と比較して、初期に放出する核酸量が少なく核酸を徐々に放出した。重炭酸リンゲル液/siRNA画分のゲルから放出される核酸量が半分になるまでの時間は、従来バッファー/siRNA画分のゲルから放出される核酸量が半分になるまでの時間と比較して、約２倍であった。
すなわち、本発明の薬剤徐放担体の徐放作用能は、従来の薬剤担体の徐放作用能と比較して、約２倍であることを確認した。 (Measurement result of sustained release action of drug sustained release carrier)
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. Compared to 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.
薬剤徐放担体の徐放作用能の測定結果を図３に示す。重炭酸リンゲル液/siRNA画分は、コントロールである従来バッファー/siRNA画分と比較して、初期に放出する核酸量が少なく核酸を徐々に放出した。重炭酸リンゲル液/siRNA画分のゲルから放出される核酸量が半分になるまでの時間は、従来バッファー/siRNA画分のゲルから放出される核酸量が半分になるまでの時間と比較して、約２倍であった。
すなわち、本発明の薬剤徐放担体の徐放作用能は、従来の薬剤担体の徐放作用能と比較して、約２倍であることを確認した。 (Measurement result of sustained release action of drug sustained release carrier)
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. Compared to 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.
（本発明の薬剤徐放担体の運搬対象の標的細胞内導入効率の確認）
本実施例では、実施例１で作製した重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分が、従来の薬剤担体と比較して、運搬対象の標的細胞内導入効率が向上するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of efficiency of introduction into target cell of target for delivery of sustained-release drug carrier of the present invention)
In this example, whether the bicarbonate Ringer's solution / siRNA fraction and lactate Ringer's solution / siRNA fraction prepared in Example 1 are improved in the efficiency of introduction into the target cell to be transported as compared with conventional drug carriers. confirmed. Details are as follows.
本実施例では、実施例１で作製した重炭酸リンゲル液/siRNA画分及び乳酸リンゲル液/siRNA画分が、従来の薬剤担体と比較して、運搬対象の標的細胞内導入効率が向上するかどうかを確認した。詳細は、以下の通りである。 (Confirmation of efficiency of introduction into target cell of target for delivery of sustained-release drug carrier of the present invention)
In this example, whether the bicarbonate Ringer's solution / siRNA fraction and lactate Ringer's solution / siRNA fraction prepared in Example 1 are improved in the efficiency of introduction into the target cell to be transported as compared with conventional drug carriers. confirmed. Details are as follows.
（本発明の薬剤徐放担体の運搬対象の標的細胞内導入効率の確認方法）
使用したマウス及び試薬は、以下の通りである。
・マウス：C57BL/6NCrSlc、7-week-old、♀、日本SLC
・細胞：B16F10-C2 SV40 Dual Luc ♯8-4（ルシフェラーゼを恒常発現する細胞株）
・培地：D-MEM ＋10％FBS＋1 mg/ml G418 ( Promega )
D-MEM (Invitrogen)
G418 Sulfate Solution(Invitrogen)
・Dual-Luciferase Reporter Assay System ( Promega )
・1 mlシリンジ ( TERUMO )
・26 G注射針 ( TERUMO )
使用した薬剤担体は、以下の通りである。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液
コントロールとして、以下も使用した。
○PBS (従来バッファー)/siRNA画分：0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS
○乳酸リンゲル液画分：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分：0.5%AC/重炭酸リンゲル液
○非投与画分：何も投与しない (Method for confirming the efficiency of introduction into the target cell to be transported of the drug sustained-release carrier of the present invention)
The 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.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution The following were also used as controls.
○ PBS (conventional buffer) / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × PBS
○ Lactated Ringer solution fraction: 0.5% AC / Lactated Ringer solution ○ Bicarbonate Ringer solution fraction: 0.5% AC / bicarbonate Ringer solution ○ Non-administered fraction: No administration
使用したマウス及び試薬は、以下の通りである。
・マウス：C57BL/6NCrSlc、7-week-old、♀、日本SLC
・細胞：B16F10-C2 SV40 Dual Luc ♯8-4（ルシフェラーゼを恒常発現する細胞株）
・培地：D-MEM ＋10％FBS＋1 mg/ml G418 ( Promega )
D-MEM (Invitrogen)
G418 Sulfate Solution(Invitrogen)
・Dual-Luciferase Reporter Assay System ( Promega )
・1 mlシリンジ ( TERUMO )
・26 G注射針 ( TERUMO )
使用した薬剤担体は、以下の通りである。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5μM GL3 siRNA /乳酸リンゲル液
コントロールとして、以下も使用した。
○PBS (従来バッファー)/siRNA画分：0.5%AC/ 5μM GL3 siRNA /50 mM PB/ 0.5×PBS
○乳酸リンゲル液画分：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分：0.5%AC/重炭酸リンゲル液
○非投与画分：何も投与しない (Method for confirming the efficiency of introduction into the target cell to be transported of the drug sustained-release carrier of the present invention)
The 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.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / lactate Ringer solution The following were also used as controls.
○ PBS (conventional buffer) / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × PBS
○ Lactated Ringer solution fraction: 0.5% AC / Lactated Ringer solution ○ Bicarbonate Ringer solution fraction: 0.5% AC / bicarbonate Ringer solution ○ Non-administered fraction: No administration
（局所投与方法）
１．マウスの準備：バリカンでマウスの腰のやや上から後ろ肢の付け根まで毛刈りをした。
２．細胞液の調整：DMEM（+10%FBS）を使用し、6×106 cells/mLに懸濁した。
３．細胞の移植：調整した細胞液（ルシフェラーゼを恒常発現する細胞を含む液）を、腰の辺りの左右2か所に各50 μL/site(3.0×105 cells/site)で移植した。
４．サンプル投与：細胞を移植してから48時間後、以下の濃度に調整した各画分をマウスの左右の腫瘍に26G注射針を用いてラッピング法で（200 μL /site）投与した。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5 μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5 μM GL3 siRNA /乳酸リンゲル液
○PBS/siRNA画分（コントロール）：0.5%AC/ 5 μM GL3 siRNA /50 mM PB/ 0.5×D-PBS
○乳酸リンゲル液画分（コントロール）：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分（コントロール）：0.5%AC/重炭酸リンゲル液
５．摘出：画分を投与してから48時間後、腫瘍を摘出した。
６．ルシフェラーゼアッセイ： Dual-Luciferase Reporter Assay Systemキットを用いてルシフェラーゼ発現抑制率を測定した。具体的には、細胞溶解溶液(該キットに添付)中で腫瘍をホモジナイズし、遠心分離後、上清を採取し、サンプル溶液とした。サンプル溶液20 μLに反応基質試薬(該キットに添付)を100 μLを加え、混合後すぐにマイクロプレートリーダーにて発光光度を測定した。この発光度をホタルの発光光度とした。続いて、反応停止試薬(該キットに添付) を100 μL加え、混合後すぐにマイクロプレートリーダーにて発光光度を測定した。この発光度をウミシイタケの発光光度とした。
測定結果値は、（ホタルの発光光度/ウミシイタケの発光光度）を使用して算出した。 (Topical administration method)
1. Mouse preparation : Hair clipper was used to cut the hair from slightly above the waist of the mouse to the base of the hind limb.
2. Preparation of cell solution : DMEM (+ 10% FBS) was used and suspended in 6 × 10 6 cells / mL.
3. Cell transplantation : The prepared cell solution (a solution containing cells that constantly express luciferase) was transplanted at 50 μL / site (3.0 × 10 5 cells / site) at two locations on the left and right sides of the waist.
4). Sample administration : 48 hours after the cells were transplanted, each fraction adjusted to the following concentration was administered to the left and right tumors of mice by a lapping method (200 μL / site) using 26G injection needles.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / Lactate Ringer solution ○ PBS / siRNA fraction (control): 0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × D-PBS
○ Lactated Ringer's solution fraction (control): 0.5% AC / Lactated Ringer's solution ○ Bicarbonate Ringer's solution fraction (control): 0.5% AC / bicarbonate Ringer's solution Removal : The tumor was removed 48 hours after administration of the fraction.
6). 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 μL of the reaction substrate reagent (attached to the kit) was added to 20 μL 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. Subsequently, 100 μL of a reaction stopping reagent (attached to the kit) was added, and immediately after mixing, the luminescence intensity was measured with a microplate reader. This luminous intensity was taken as the luminous intensity of Renilla.
The measurement result value was calculated using (firefly emission intensity / renilla emission intensity).
１．マウスの準備：バリカンでマウスの腰のやや上から後ろ肢の付け根まで毛刈りをした。
２．細胞液の調整：DMEM（+10%FBS）を使用し、6×106 cells/mLに懸濁した。
３．細胞の移植：調整した細胞液（ルシフェラーゼを恒常発現する細胞を含む液）を、腰の辺りの左右2か所に各50 μL/site(3.0×105 cells/site)で移植した。
４．サンプル投与：細胞を移植してから48時間後、以下の濃度に調整した各画分をマウスの左右の腫瘍に26G注射針を用いてラッピング法で（200 μL /site）投与した。
○重炭酸リンゲル液/siRNA画分：0.5%AC/ 5 μM GL3 siRNA /重炭酸リンゲル液
○乳酸リンゲル液/siRNA画分：0.5%AC/ 5 μM GL3 siRNA /乳酸リンゲル液
○PBS/siRNA画分（コントロール）：0.5%AC/ 5 μM GL3 siRNA /50 mM PB/ 0.5×D-PBS
○乳酸リンゲル液画分（コントロール）：0.5%AC/乳酸リンゲル液
○重炭酸リンゲル液画分（コントロール）：0.5%AC/重炭酸リンゲル液
５．摘出：画分を投与してから48時間後、腫瘍を摘出した。
６．ルシフェラーゼアッセイ： Dual-Luciferase Reporter Assay Systemキットを用いてルシフェラーゼ発現抑制率を測定した。具体的には、細胞溶解溶液(該キットに添付)中で腫瘍をホモジナイズし、遠心分離後、上清を採取し、サンプル溶液とした。サンプル溶液20 μLに反応基質試薬(該キットに添付)を100 μLを加え、混合後すぐにマイクロプレートリーダーにて発光光度を測定した。この発光度をホタルの発光光度とした。続いて、反応停止試薬(該キットに添付) を100 μL加え、混合後すぐにマイクロプレートリーダーにて発光光度を測定した。この発光度をウミシイタケの発光光度とした。
測定結果値は、（ホタルの発光光度/ウミシイタケの発光光度）を使用して算出した。 (Topical administration method)
1. Mouse preparation : Hair clipper was used to cut the hair from slightly above the waist of the mouse to the base of the hind limb.
2. Preparation of cell solution : DMEM (+ 10% FBS) was used and suspended in 6 × 10 6 cells / mL.
3. Cell transplantation : The prepared cell solution (a solution containing cells that constantly express luciferase) was transplanted at 50 μL / site (3.0 × 10 5 cells / site) at two locations on the left and right sides of the waist.
4). Sample administration : 48 hours after the cells were transplanted, each fraction adjusted to the following concentration was administered to the left and right tumors of mice by a lapping method (200 μL / site) using 26G injection needles.
○ Bicarbonate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / bicarbonate Ringer solution ○ Lactate Ringer solution / siRNA fraction: 0.5% AC / 5 μM GL3 siRNA / Lactate Ringer solution ○ PBS / siRNA fraction (control): 0.5% AC / 5 μM GL3 siRNA / 50 mM PB / 0.5 × D-PBS
○ Lactated Ringer's solution fraction (control): 0.5% AC / Lactated Ringer's solution ○ Bicarbonate Ringer's solution fraction (control): 0.5% AC / bicarbonate Ringer's solution Removal : The tumor was removed 48 hours after administration of the fraction.
6). 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 μL of the reaction substrate reagent (attached to the kit) was added to 20 μL 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. Subsequently, 100 μL of a reaction stopping reagent (attached to the kit) was added, and immediately after mixing, the luminescence intensity was measured with a microplate reader. This luminous intensity was taken as the luminous intensity of Renilla.
The measurement result value was calculated using (firefly emission intensity / renilla emission intensity).
（本発明の薬剤徐放担体の運搬対象の標的細胞内導入効率の確認結果）
本発明の薬剤徐放担体の運搬対象の標的細胞内導入効率の確認結果を図４に示す。
図４の結果から明らかなように、重炭酸リンゲル液画分及び乳酸リンゲル液画分の抑制率は、それぞれ、73.2％及び61.2％であった。一方、PBS画分の抑制率は、49.7％であった。
本発明の薬剤徐放担体が、従来の薬剤担体と比較して、高い増殖抑制効果（高い運搬対象の標的細胞内導入効率）を示した。より詳しくは、重炭酸リンゲル液画分及び乳酸リンゲル液画分の運搬対象の標的細胞内導入効率は、従来の薬剤担体の運搬対象の標的細胞内導入効率と比較して、それぞれ、1.47倍及び1.23倍であった。
加えて、重炭酸リンゲル液画分及び乳酸リンゲル液画分を腫瘍（局所）に投与した場合の流出量（腫瘍内部に入らなかった画分）は、PBS画分を腫瘍（局所）に投与した場合の流出量と比較して、抑えることができた。 (Confirmation result of efficiency of introduction into target cell of target for delivery of sustained-release drug carrier of the present invention)
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.
As apparent from the results of FIG. 4, the inhibition rates of the bicarbonate Ringer solution fraction and the lactate Ringer solution fraction were 73.2% and 61.2%, respectively. On the other hand, 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. More specifically, 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.
In addition, 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.
本発明の薬剤徐放担体の運搬対象の標的細胞内導入効率の確認結果を図４に示す。
図４の結果から明らかなように、重炭酸リンゲル液画分及び乳酸リンゲル液画分の抑制率は、それぞれ、73.2％及び61.2％であった。一方、PBS画分の抑制率は、49.7％であった。
本発明の薬剤徐放担体が、従来の薬剤担体と比較して、高い増殖抑制効果（高い運搬対象の標的細胞内導入効率）を示した。より詳しくは、重炭酸リンゲル液画分及び乳酸リンゲル液画分の運搬対象の標的細胞内導入効率は、従来の薬剤担体の運搬対象の標的細胞内導入効率と比較して、それぞれ、1.47倍及び1.23倍であった。
加えて、重炭酸リンゲル液画分及び乳酸リンゲル液画分を腫瘍（局所）に投与した場合の流出量（腫瘍内部に入らなかった画分）は、PBS画分を腫瘍（局所）に投与した場合の流出量と比較して、抑えることができた。 (Confirmation result of efficiency of introduction into target cell of target for delivery of sustained-release drug carrier of the present invention)
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.
As apparent from the results of FIG. 4, the inhibition rates of the bicarbonate Ringer solution fraction and the lactate Ringer solution fraction were 73.2% and 61.2%, respectively. On the other hand, 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. More specifically, 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.
In addition, 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.
（総論）
上記すべての実施例の結果より、本発明の薬剤徐放担体又は薬剤徐放方法は、従来の薬剤担体又は薬剤徐放方法と比較して、以下の効果を有することを確認した。
（１）ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができる。
（２）徐放作用能が高いので長時間薬剤を標的細胞に供給することができる。
（３）薬剤の標的細胞内導入効率が高いので薬効効果が高い。
（４）局所に投与した場合の薬剤流出量を抑えることができる。 (General)
From the results of all the above examples, it was confirmed that 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.
上記すべての実施例の結果より、本発明の薬剤徐放担体又は薬剤徐放方法は、従来の薬剤担体又は薬剤徐放方法と比較して、以下の効果を有することを確認した。
（１）ゲル形成能が高いので高濃度の運搬対象（薬剤）を長時間保持することができる。
（２）徐放作用能が高いので長時間薬剤を標的細胞に供給することができる。
（３）薬剤の標的細胞内導入効率が高いので薬効効果が高い。
（４）局所に投与した場合の薬剤流出量を抑えることができる。 (General)
From the results of all the above examples, it was confirmed that 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.
本発明では、新規かつ有用な薬剤徐放担体及び薬剤徐放方法を提供することができる。
In the present invention, a novel and useful drug sustained release carrier and drug sustained release method can be provided.
Claims (17)
- 以下を含む薬剤徐放担体。
（１）コラーゲン又はコラーゲン誘導体
（２）カリウム塩、カルシウム塩及びナトリウム塩を含む溶液
A drug sustained-release carrier comprising:
(1) Collagen or collagen derivative (2) Solution containing potassium salt, calcium salt and sodium salt
- 前記カリウム塩は塩化カリウム、前記カルシウム塩は塩化カルシウム及び前記ナトリウム塩は塩化ナトリウムである請求項１に記載の薬剤徐放担体。
The sustained-release drug carrier according to claim 1, wherein the potassium salt is potassium chloride, the calcium salt is calcium chloride, and the sodium salt is sodium chloride.
- 前記溶液は、リンゲル液である請求項１又は２に記載の薬剤徐放担体。
The drug sustained-release carrier according to claim 1 or 2, wherein the solution is a Ringer's solution.
- 前記リンゲル液は、重炭酸リンゲル液、乳酸リンゲル、リンゲル基礎液、又は酢酸リンゲル液である請求項３に記載の薬剤徐放担体。
The sustained-release drug carrier according to claim 3, wherein the Ringer's solution is a bicarbonated Ringer's solution, lactated Ringer's, Ringer's basic solution, or acetate Ringer's solution.
- 前記リンゲル液は、重炭酸リンゲル液である請求項３に記載の薬剤徐放担体。
The drug sustained-release carrier according to claim 3, wherein the Ringer's solution is a bicarbonated Ringer's solution.
- 前記リンゲル液は、乳酸リンゲル液である請求項３に記載の薬剤徐放担体。
The drug sustained-release carrier according to claim 3, wherein the Ringer's solution is a lactated Ringer's solution.
- 前記溶液は、以下のいずれか１から選択される請求項１～６のいずれか１に記載の薬剤徐放担体。
（１）塩化カリウム、塩化カルシウム、塩化ナトリウム、炭酸水素ナトリウム、塩化マグネシウム及びクエン酸三ナトリウムを含む溶液
（２）塩化カリウム、塩化カルシウム、塩化ナトリウム及び乳酸ナトリウムを含む溶液
（３）塩化カリウム、塩化カルシウム及び塩化ナトリウムを含む溶液
（４）塩化カリウム、塩化カルシウム、塩化ナトリウム及び酢酸ナトリウム含む溶液
The drug sustained-release carrier according to any one of claims 1 to 6, wherein the solution is selected from any one of the following.
(1) Solution containing potassium chloride, calcium chloride, sodium chloride, sodium bicarbonate, magnesium chloride and trisodium citrate (2) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium lactate (3) Potassium chloride, chloride Solution containing calcium and sodium chloride (4) Solution containing potassium chloride, calcium chloride, sodium chloride and sodium acetate
- 前記溶液の各成分の濃度は、以下である請求項１～７のいずれか１に記載の薬剤徐放担体。
塩化カリウム：0.001ｍM ～5.00M
塩化カルシウム：0.001ｍM ～10.00M
塩化ナトリウム：0.001ｍM ～10.00M
炭酸水素ナトリウム：0.001ｍM ～2.00M
塩化マグネシウム：0.001ｍM ～5.00M
クエン酸三ナトリウム：0.001ｍM ～2.00M
乳酸ナトリウム：0.001ｍM ～10.00M
酢酸ナトリウム：0.001ｍM ～10.00M
The drug sustained-release carrier according to any one of claims 1 to 7, wherein the concentration of each component of the solution is as follows.
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
- 前記コラーゲン又はコラーゲン誘導体がアテロコラーゲンである請求項１～８のいずれか１に記載の薬剤徐放担体。
The drug sustained-release carrier according to any one of claims 1 to 8, wherein the collagen or collagen derivative is atelocollagen.
- 前記薬剤徐放担体がゲル形成能を有する請求項１～９のいずれか１に記載の薬剤徐放担体。
The drug sustained-release carrier according to any one of claims 1 to 9, wherein the drug sustained-release carrier has gel-forming ability.
- 前記薬剤が、核酸、タンパク質、ペプチド、及び／又は低分子化合物である請求項１～１０のいずれか１に記載の薬剤徐放担体。
The sustained-release drug carrier according to any one of claims 1 to 10, wherein the drug is a nucleic acid, protein, peptide, and / or low molecular weight compound.
- 前記リンゲル液は、重炭酸リンゲル液であり、かつ前記薬剤が核酸である請求項４～１１のいずれか１に記載の薬剤徐放担体。
The drug sustained-release carrier according to any one of claims 4 to 11, wherein the Ringer's solution is a bicarbonated Ringer's solution, and the drug is a nucleic acid.
- 前記リンゲル液は、乳酸リンゲル液であり、かつ前記薬剤が核酸である請求項４～１１のいずれか１に記載の薬剤徐放担体。
The drug sustained-release carrier according to any one of claims 4 to 11, wherein the Ringer's solution is a lactated Ringer's solution, and the drug is a nucleic acid.
- 請求項１～１３のいずれか１に記載の薬剤徐放担体が表面に塗布されている医療用具。
A medical device wherein the drug sustained-release carrier according to any one of claims 1 to 13 is applied to a surface.
- 請求項１～１３のいずれか１に記載の薬剤徐放担体が細胞培養面に塗布されている細胞培養器具。
A cell culture device, wherein the drug sustained-release carrier according to any one of claims 1 to 13 is applied to a cell culture surface.
- 請求項１～１３のいずれか１に記載の薬剤徐放担体を含む薬剤。
A drug comprising the drug sustained-release carrier according to any one of claims 1 to 13.
- 請求項１～１３のいずれか１に記載の薬剤徐放担体又は請求項１６に記載の薬剤を、ヒトを除く哺乳動物に投与する、薬剤徐放担体の使用。 Use of a drug sustained-release carrier wherein the drug sustained-release carrier according to any one of claims 1 to 13 or the drug according to claim 16 is administered to a mammal other than a human.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016512715A JP6186572B2 (en) | 2014-10-29 | 2015-10-29 | Drug sustained release carrier and drug sustained release method |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014-220101 | 2014-10-29 | ||
| JP2014220101 | 2014-10-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2016068228A1 true WO2016068228A1 (en) | 2016-05-06 |
Family
ID=55857563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2015/080530 WO2016068228A1 (en) | 2014-10-29 | 2015-10-29 | Carrier for sustained drug release and method for sustained drug release |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP6186572B2 (en) |
| WO (1) | WO2016068228A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITUB20160857A1 (en) * | 2016-02-18 | 2017-08-18 | Bruno Silvestrini | A dosage form comprising calcium sulfate, collagen and a drug and its use in a modified release method having a regulation mechanism inherent in its composition. |
| IT201700093635A1 (en) * | 2017-08-14 | 2019-02-14 | Bruno Silvestrini | A topical dosage form comprising a calcium salt, collagen and a drug and its use in a modified release method having a regulation mechanism inherent in its composition. |
| WO2019064807A1 (en) * | 2017-09-29 | 2019-04-04 | 祐徳薬品工業株式会社 | Method for producing collagen vitrigel, method for producing purified collagen vitrigel, and collagen vitrigel and purified collagen vitrigel produced by said methods |
| WO2023059921A3 (en) * | 2021-10-07 | 2023-05-19 | Intelligent Optical Systems, Inc. | Enhanced lateral flow assays and devices for detecting analytes in blood samples |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59151951A (en) * | 1983-02-17 | 1984-08-30 | 大鳥 盛嗣 | Adhesion of collagen to implant material and tissue activating powder agent used therein |
| JP2006223106A (en) * | 2005-02-15 | 2006-08-31 | Fuji Photo Film Co Ltd | Cell culture carrier |
| JP2008540601A (en) * | 2005-05-19 | 2008-11-20 | クレファク ゲーエムベーハー | Injection solution for RNA |
| WO2013168402A1 (en) * | 2012-05-08 | 2013-11-14 | 株式会社大塚製薬工場 | Dextran-containing mammalian cell suspension for prevention of pulmonary embolism formation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000198743A (en) * | 1999-01-06 | 2000-07-18 | Nissho Corp | Albumin formulation |
-
2015
- 2015-10-29 WO PCT/JP2015/080530 patent/WO2016068228A1/en active Application Filing
- 2015-10-29 JP JP2016512715A patent/JP6186572B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59151951A (en) * | 1983-02-17 | 1984-08-30 | 大鳥 盛嗣 | Adhesion of collagen to implant material and tissue activating powder agent used therein |
| JP2006223106A (en) * | 2005-02-15 | 2006-08-31 | Fuji Photo Film Co Ltd | Cell culture carrier |
| JP2008540601A (en) * | 2005-05-19 | 2008-11-20 | クレファク ゲーエムベーハー | Injection solution for RNA |
| WO2013168402A1 (en) * | 2012-05-08 | 2013-11-14 | 株式会社大塚製薬工場 | Dextran-containing mammalian cell suspension for prevention of pulmonary embolism formation |
Non-Patent Citations (1)
| Title |
|---|
| SANO, A. ET AL.: "Atelocollagen for protein and gene delivery", ADV. DRUG DELIV. REV., vol. 55, no. 12, 2003, pages 1651 - 1677 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITUB20160857A1 (en) * | 2016-02-18 | 2017-08-18 | Bruno Silvestrini | A dosage form comprising calcium sulfate, collagen and a drug and its use in a modified release method having a regulation mechanism inherent in its composition. |
| WO2017141282A1 (en) * | 2016-02-18 | 2017-08-24 | Bruno Silvestrini | A dosage form comprising a calcium salt, collagen and a drug and related use in a method of controlled release endowed with a regulatory mechanism of drug release inside the composition |
| IT201700093635A1 (en) * | 2017-08-14 | 2019-02-14 | Bruno Silvestrini | A topical dosage form comprising a calcium salt, collagen and a drug and its use in a modified release method having a regulation mechanism inherent in its composition. |
| WO2019064807A1 (en) * | 2017-09-29 | 2019-04-04 | 祐徳薬品工業株式会社 | Method for producing collagen vitrigel, method for producing purified collagen vitrigel, and collagen vitrigel and purified collagen vitrigel produced by said methods |
| WO2023059921A3 (en) * | 2021-10-07 | 2023-05-19 | Intelligent Optical Systems, Inc. | Enhanced lateral flow assays and devices for detecting analytes in blood samples |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6186572B2 (en) | 2017-08-30 |
| JPWO2016068228A1 (en) | 2017-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiao et al. | M2-like tumor-associated macrophage-targeted codelivery of STAT6 inhibitor and IKKβ siRNA induces M2-to-M1 repolarization for cancer immunotherapy with low immune side effects | |
| Rufaihah et al. | Enhanced infarct stabilization and neovascularization mediated by VEGF-loaded PEGylated fibrinogen hydrogel in a rodent myocardial infarction model | |
| ES2511791T3 (en) | Mesenchymal stem cells positive for ABCB5 as immunity modulators | |
| JP6186572B2 (en) | Drug sustained release carrier and drug sustained release method | |
| CN107135646A (en) | Method and composition for treating ulcer | |
| JP2012533545A (en) | Methods for increasing liver growth | |
| US20180110832A1 (en) | Clec11a is a bone growth agent | |
| US20190083642A1 (en) | Treatments utilizing a polymer-protein conjugate | |
| WO2009116556A1 (en) | Pharmaceutical composition for injection | |
| Yuan et al. | Platelet-rich plasma gel-loaded collagen/chitosan composite film accelerated rat sciatic nerve injury repair | |
| Kawakami et al. | Atelocollagen‐mediated systemic administration of myostatin‐targeting siRNA improves muscular atrophy in caveolin‐3‐deficient mice | |
| Wegman et al. | Bone morphogenetic protein-2 plasmid DNA as a substitute for bone morphogenetic protein-2 protein in bone tissue engineering | |
| Di-Silvio | A novel application of two biomaterials for the delivery of growth hormone and its effect on osteoblasts | |
| JP2019524753A (en) | Use of IL-12 as a replacement immunotherapeutic | |
| AU2009200996B2 (en) | Method for growing human organs, treating diseases, and increasing longevity | |
| CN106456771A (en) | Compositions and methods for anti-LYST immunomodulation | |
| CN115919804A (en) | Nano-carrier system for inducing Treg cell differentiation and application of nano-carrier system in RA treatment | |
| US20020192198A1 (en) | Method for growing human organs and suborgans | |
| Wang et al. | Locally Delivered Metabolite Derivative Promotes Bone Regeneration in Aged Mice | |
| JP2017538683A (en) | Oligopeptides as anti-angiogenic agents | |
| Trofimova | Molecular Mechanisms of Retina Pathology and Ways of Its Correction | |
| He et al. | Remodeling of Cellular Surfaces via Fast Disulfide–Thiol Exchange To Regulate Cell Behaviors | |
| Hashemi et al. | Extracellular Vesicles and Hydrogels: An Innovative Approach to Tissue Regeneration | |
| JP2024515976A (en) | Biochemical activation of dysfunctional skeletal stem cells for skeletal regeneration | |
| KR20160147055A (en) | Sustained release angiogenesis modulating compositions and methods for induction and modulation of angiogenesis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| ENP | Entry into the national phase |
Ref document number: 2016512715 Country of ref document: JP Kind code of ref document: A |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15854371 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: 15854371 Country of ref document: EP Kind code of ref document: A1 |