WO2006085516A1 - タンパク質導入用担体、前記担体を用いたタンパク質導入剤、タンパク質導入方法、およびタンパク質導入細胞ならびにその製造方法 - Google Patents
タンパク質導入用担体、前記担体を用いたタンパク質導入剤、タンパク質導入方法、およびタンパク質導入細胞ならびにその製造方法 Download PDFInfo
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- WO2006085516A1 WO2006085516A1 PCT/JP2006/302030 JP2006302030W WO2006085516A1 WO 2006085516 A1 WO2006085516 A1 WO 2006085516A1 JP 2006302030 W JP2006302030 W JP 2006302030W WO 2006085516 A1 WO2006085516 A1 WO 2006085516A1
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- protein
- introduction
- carrier
- clay mineral
- agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
-
- 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
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Definitions
- Protein introduction carrier protein introduction agent using the carrier, protein introduction method, protein introduction cell, and production method thereof
- the present invention relates to a protein introduction carrier for introducing a protein into a cell and a protein introduction agent using the carrier. Furthermore, the present invention relates to a protein introduction method using the introduction agent, a cell into which the protein has been introduced, and a production method thereof.
- Non-patent literature l Zelphati, 0. et al. J. Biol. Chem. 276, 35103-35110 (2001) Intracellul ar delivery of proteins with a new lipid—mediated delivery system.
- the present invention aims to provide a new carrier for protein introduction, a protein introduction agent, a protein introduction method, a protein introduction cell, and a production method thereof that are excellent in safety.
- the protein introduction carrier of the present invention is a carrier for introducing a protein into cells, and is characterized by containing a clay mineral.
- the protein introduction agent of the present invention is an introduction agent comprising the protein introduction carrier of the present invention and a protein supported thereon.
- the protein introduction method of the present invention is a method of bringing the protein introduction agent of the present invention into contact with a cell and introducing the protein into the cell.
- the cell of the present invention into which is introduced is produced.
- the clay mineral usually refers to a silicate mineral contained in clay.
- an optical material or a catalyst Conventionally, for example, as a component of cosmetics, as a base material of a foaming agent in the pharmaceutical field, an optical material or a catalyst. Is also widely used.
- the present inventors have discovered for the first time that clay minerals can be used as a carrier for introducing proteins into cells as in the present invention.
- the protein introduction carrier of the present invention for example, the protein can be supported only by mixing the carrier and the protein to be introduced in a solution (the carrier and the protein are separated from each other).
- the protein can be introduced into the cell by bringing the carrier carrying the protein into contact with the target cell. For this reason, the operation of introducing the tank becomes very simple.
- the fact that clay minerals have been widely used in cosmetics and pharmaceuticals as described above has been sufficiently proven to be safe for living bodies, particularly for humans. Therefore, the protein-introducing carrier of the present invention containing such a clay mineral that is extremely excellent in safety is widely applicable and extremely useful in the clinical medicine field and the like.
- the mechanism by which the protein-introducing carrier of the present invention introduces a protein into a cell is not clear. It is considered that the protein introduction carrier is taken up into the cell while carrying the protein and releases the protein in the cell.
- the protein introduction agent of the present invention can be supported on the carrier by mixing the protein-introducing carrier of the present invention and the target protein, for example, and thus can be prepared very easily. is there. And, according to the protein introduction method of the present invention using this protein introduction agent, the protein can be introduced into the cells simply by bringing the introduction agent into contact with the cells. Excellent safety.
- the protein-introduced cell of the present invention can be produced by the introduction method of the present invention, which is extremely safe for cells, for example, a conventional carrier such as polyethyleneimine is used. The bad effects etc. which can be avoided can also be avoided. Therefore, it can be applied with high safety to a treatment method in which cells introduced with foreign proteins are brought into contact with tissue cells in the affected area.
- the “protein” is not limited to a so-called protein, and includes peptides and the like as described later.
- FIG. 1 is a graph showing j8-galatatosidase activity in protein-introduced cells in an example of the present invention.
- FIG. 2 is a graph showing j8-galatatosidase activity in protein-introduced cells in another example of the present invention.
- FIG. 3 is a graph showing the adsorptivity of a protein to a protein-introducing carrier in still another example of the present invention, where FIG. (A) shows the amount of protein in the supernatant fraction, and FIG. The adsorption rate of the protein in the protein introduction carrier is shown respectively.
- FIG. 4 is a graph showing the amount of OVA-specific IgG in mouse serum when OVA is introduced by a protein-introducing carrier in still another example of the present invention.
- FIG. 5 is an electrophoresis photograph of a Western blot showing the presence or absence of intracellular OVA when mitodocain is introduced by a protein introduction carrier in still another example of the present invention.
- FIG. 6 In still another example of the present invention, when a pleiotomach fin is introduced by a protein introduction carrier, an electric wave of a Western plot showing the presence or absence of intracellular PTN. It is an electrophoresis photograph.
- FIG. 7 is a Western blot electrophoresis photograph showing the presence or absence of intracellular OVA when insulin is introduced by a protein introduction carrier in still another example of the present invention.
- the clay mineral is not particularly limited, and the mechanism is unknown, but according to the clay mineral, protein can be supported.
- the clay mineral include layered clay minerals. Such a layered clay mineral usually has a structure in which ions and water are sandwiched between the layers.
- the interlayer material of the layered clay mineral is preferably, for example, an exchangeable cation.
- the interlayer material is an exchangeable cation
- this cation is usually bonded to a hydroxyl group (one OH) on the inner surface of the layer.
- the clay mineral having the exchangeable cation as an interlayer substance is also referred to as “exchangeable cation type” clay mineral.
- Examples of the exchangeable cation include sodium ion, ammonium ion, quaternary ammonium ion, and the like.
- sodium ions are preferred because many natural clay minerals are present, and quaternary ammonium ions, which are large three-dimensional structures, are also preferred.
- Examples of the quaternary ammonium ion include tetramethylammonium chloride, salt benzyltrimethylammonium ion, and the like.
- amino acids such as a amino acid and j8-amino acid, amine compounds such as domino, cations such as amide compounds such as acrylamide
- Surfactants such as alkyl trimethyl ammonium, tetramethyl phosphor, alkyl pyridinium, ruthenium tetra ammonium, tris It can be a cation of a cationic metal complex such as phenanthrolin rhodium.
- a cationic metal complex for example, an ammonia complex such as ruthenium can be used. Further, methyl viologen or the like may be used.
- the layered clay mineral in the present invention is not particularly limited, and examples thereof include force orite, bilophyllite-talc, smectite, vermiculite, mica, brittle mica, chlorite, and sepiolite-
- a total of eight groups of crystalline clay minerals can be used.
- the kaolinite group has “1: 1 type” for the catenate layer type, and the other seven types are “2: 1 type”.
- clay minerals belonging to the kaolinite group without layer charge include, for example, kaolinite, datekite, rhosite, leucite, nacrite, chrysotile, lizardite, as well as clay minerals belonging to the bilophyllite talc group without layer charge.
- examples include neurophyllite and talc.
- Examples of the smectite group having a layer charge include montmorillonite, piderite, nontrolite, sabonite, hectorite, and stevensite.
- examples of the vermiculite group include di-vermiculite and tri-vermiculite.
- the mica group includes, for example, muscovite, noragolite, illite, phlogopite, biotite, and levidrite
- the brittle mica group includes, for example, margarite and clintnite
- the chlorite group includes donpasite, sudowite. Stones, tatsukite, clinochlore and chamosite are examples.
- sepiolite and norgorskite groups having no layer charge include sepiolite and norgorskite.
- a crystalline clay mineral having a layer charge is more preferable, and a smectite group, a vermiculite group, and a mica group are more preferable.
- montmorillonite, vermiculite and illite are preferred, and montmorillonite is particularly preferred.
- amorphous clay minerals such as imogolite, alofen, and hisingerite can also be used.
- the clay mineral is not limited to natural ones, and synthetic products can also be used.
- clay mineral natural clay strength may be collected, but various commercially available products can be used.
- montmorillonite for example, bentonite mainly composed of montmorillonite is preferable.
- Many bentonites are commercially available that comply with the Japanese Pharmacopoeia “Bentonite” and have been proven to be harmless to living organisms. It is extremely useful as a raw material for carriers.
- Examples of such commercially available bentonite include the brand name bentonite and the trade name Kunipia F (both manufactured by Kunimine Industries Co., Ltd.).
- Kunipia F both manufactured by Kunimine Industries Co., Ltd.
- saponite examples include a trade name Smecton SA (manufactured by Kunimine Kogyo Co., Ltd.).
- Various clay minerals can also be obtained from the Japan Clay Society.
- the protein-introducing carrier of the present invention may contain, for example, a single, a mixture of a plurality of clay minerals, or components other than the clay mineral, and the degree of purification of the clay mineral is not limited.
- the clay mineral may be, for example, a purified clay mineral from which various impurities contained or adhered are removed.
- the impurities include soil organic substances (such as various amino acids and lignins), iron, potassium, calcium, and magnesium.
- the purity power by ordinary X-ray diffraction or elemental analysis is, for example, 85% or more, preferably 95% or more, particularly preferably 98% or more.
- the purity by this X-ray diffraction can be determined by comparing the diffraction pattern with a standard sample as is generally done.
- the method for removing the impurities as described above is not particularly limited.
- the following methods can be applied.
- the organic substance removal treatment can be performed, for example, by adding and dispersing a peroxy hydrogen peroxide solution to a clay mineral and then heat-treating the dispersion.
- concentration of aqueous hydrogen peroxide is, for example, 3 to 15% by weight, preferably 3 to 10% by weight, and more preferably 5 to 7% by weight.
- the addition ratio of peroxy hydrogenated water is, for example, 10 to 30 ml, preferably 15 to 30 ml, more preferably 15 to 20 ml with respect to the clay mineral lg.
- the conditions of heat processing are the temperature of 25-50 degreeC, for example, Preferably it is 25-40 degreeC, More preferably, it is 30-40 degreeC.
- the heating time is not particularly limited, for example, it is preferable to carry out until foaming (oxygen) due to heating disappears. After the foaming is completed, further heating treatment is performed in order to remove residual hydrogen peroxide. It is more preferable.
- the conditions are not limited to these.
- a clay mineral having a purity of 85 to 100% can usually be obtained.
- the content of organic matter in the clay mineral used is, for example, 5% or less, preferably 2%, more preferably 1% or less.
- the organically treated clay mineral is used as a protein introduction carrier, for example, the dispersion-strength clay mineral is recovered by filtration, centrifugation, or the like, and then used in a solvent such as water, buffer, or physiological saline. What was washed may be used.
- the interlayer material of the clay mineral subjected to the organic treatment in this manner is the same as the clay mineral before the treatment used as a raw material. For example, in addition to sodium ions, potassium ions, calcium ions, magnesium ions, etc.
- Various exchangeable cations are possible to be used as sodium ions, potassium ions, calcium ions, magnesium ions, etc.
- the clay mineral is subjected to a deironing treatment described below instead of or in addition to the organic matter removal treatment in order to remove, for example, iron adhering to or contained in the clay mineral. Also good.
- This iron removal treatment can be performed, for example, by the following method.
- the clay mineral dispersion treated with the hydrogen peroxide solution is heated, and, for example, an aqueous sodium citrate solution and an aqueous sodium hydrogen carbonate solution are added thereto and sufficiently stirred.
- an aqueous solution of sodium hydrosulfite sodium mouth sodium sulfite
- this mixed solution is allowed to stand.
- the clay mineral after the iron removal treatment is used as a protein introduction carrier, it may be washed with various solvents as in the case of the organic matter treatment.
- the addition ratio of each substance to the clay mineral lg is, for example, in the range of sodium citrate 50 to 70 mmol, sodium bicarbonate 15 to 30 mmol, hydrosulfite 20 to 35 mmol, preferably sodium citrate.
- the range is 55 to 65 mmol, sodium bicarbonate 22 to 28 mmol, hydrosulfite 26 to 29 mmol.
- the clay mineral after the organic substance treatment or the iron removal treatment may be further subjected to induction treatment to various exchangeable cation types by ion exchange reaction. Even when an exchangeable cation such as sodium is included between layers before the treatment, this induction treatment may be performed. As a specific example, an example of the induction process to the exchangeable sodium type is shown below.
- the clay mineral When the clay mineral is induced to an exchangeable sodium type, for example, the clay mineral that has been subjected to the organic substance removal treatment or the deironation treatment is added to a saturated aqueous sodium salt solution such as a saturated aqueous sodium chloride solution. 'Disperse and collect the clay mineral by filtration or centrifugation.
- the addition of the sodium salt (for example, salt sodium salt) and the recovery of clay minerals are: Although it may be performed once, it is preferably performed twice or more, more preferably 3 to 5 times in order to sufficiently induce the sodium type.
- the addition ratio of the saturated aqueous sodium chloride salt solution to the clay mineral is, for example, in the range of 30 to 80 mL, preferably in the range of 40 to 60 mL, and more preferably in the range of 50 to 60 mL with respect to the clay mineral lg.
- the suspension of the clay mineral in the saturated aqueous sodium chloride solution is not particularly limited, but for example, it is preferably 10 seconds or more, more preferably 30 to 60 minutes.
- the solvent used for the induction into the exchangeable sodium type is not limited to the aqueous sodium chloride solution, and in addition, for example, an aqueous solution containing a sodium salt such as sodium bromide or sodium iodide is used. it can.
- the sodium salt concentration in these solutions is not particularly limited, but is preferably a saturated solution as described above.
- the induction treatment may be performed with an aqueous solution of a sodium salt such as sodium chloride sodium, sodium bromide, sodium iodide, or sodium azide, or an aqueous solution containing two or more of the sodium salts.
- a sodium salt such as sodium chloride sodium, sodium bromide, sodium iodide, or sodium azide
- the solution may be treated in the same manner as the above-described method for derivatization into the sodium form with a solution containing an hum-moum salt such as hum.
- a solution containing a quaternary ammonium salt such as salt tetramethyl ammonium or benzyltrimethyl ammonium chloride can be used.
- wear. Mineral function can also be modified by, for example, pillaring using a cationic surfactant as described above, or substitution with an amine compound, amid compound, or cationic metal complex as described above. It is.
- the clay mineral that has been subjected to the exchange cation type in this way may be used as a protein-introducing carrier after dialysis, for example.
- the various clay minerals shown above can be used as a protein-introducing carrier as they are, for example, in a state of being dispersed in a solution, but the dried product obtained by freeze-drying or the like is used as a protein. It may be a carrier for introduction. If it is the dried product, the dispersion described later This is because it is easy to prepare a desired concentration when preparing a protein introduction agent in the form of.
- the protein introduction carrier of the present invention it is not limited to a protein but can be derived by introducing a peptide.
- the protein introduction agent (also referred to as a protein complex) of the present invention includes the protein introduction carrier of the present invention containing a clay mineral and the protein supported thereon.
- the “protein” in the present invention includes not only a protein generally composed of only amino acids but also the above-mentioned peptides, glycoproteins, mucins, and pharmaceutically acceptable salts thereof. Included (in the present invention, referred to as "protein").
- the pharmaceutically acceptable salt include inorganic acid addition salts such as hydrochloride, sulfate, and phosphate, organic acids such as acetate, propionate, kenate, tartrate, and malate.
- Other salts include sodium salts, potassium salts, calcium salts, and the like.
- the (weight ratio A: B) is, for example, in the range of 1: 0.1 to 5, preferably in the range of 1: 0.5 to 4, more preferably in the range of 1: 0.5 to 3, particularly preferably. Is in the range of 1: 1-2.
- the protein to be carried on the protein introduction carrier is not particularly limited, and various proteins can be used, and a single protein or a mixture of a plurality of proteins may be used.
- the characteristics of the protein are not limited at all, and for example, a peptide having a small molecular weight or a protein having a large molecular weight can be sufficiently introduced into a cell.
- Specific examples of the molecular weight are not limited at all, but are, for example, about 300 Da to 500 kDa (as well as larger proteins and small peptides), preferably in the range of 1 kDa to 500 kDa, more preferably 5 kDa to 500 kDa. It can be fully introduced into cells.
- the type of clay mineral to be combined is not limited at all, but may be selected according to the type of protein, for example.
- the type of protein can be appropriately determined according to, for example, the function given to the cell.
- proteins that serve as therapeutic agents, preventive agents, and suppressors of diseases.
- a protein used for desensitization treatment for example, for the purpose of treating hay fever, a pollen allergen or the like is carried, and for the purpose of treating food allergy, a target protein such as ovalbumin (ovalbumin) (allergic
- ovalbumin ovalbumin
- a causative protein or a fragment thereof is supported and introduced into cells.
- it is sufficient to carry a deficient enzyme.
- lactose intolerance ⁇ -galactosidase or the like is It can be illustrated to be introduced in In addition, for diabetes, it can be exemplified that insulin is loaded and introduced into cells.
- the form of the protein introduction agent is not particularly limited as long as it contains the protein introduction carrier and the protein carried on the protein introduction carrier.
- a dispersion liquid in which the protein introduction agent is dispersed may be used. It may be a freeze-dried product. Further, the dispersion may be in a frozen form. In that case, it may be thawed at the time of use.
- pharmaceutical additives such as excipients, binders, disintegrants, stabilizers, preservatives, buffers, or food additives such as sweeteners, fragrances, preservatives, stabilizers, etc. Etc. can also be added.
- the protein concentration in the protein dispersion is not particularly limited, but is, for example, in the range of 0.1 to: LOOO ⁇ g / mL, preferably 25 to 400 ⁇ g / mL, particularly preferably 50. ⁇ 200 / ⁇ 8 ⁇ .
- the dispersion medium of this dispersion is not particularly limited, and examples thereof include distilled water, physiological saline, PBS (phosphate buffered saline) and the like. Further, as described later, when used for protein introduction into cultured cells, a liquid medium or the like may be used in addition to these.
- the dispersion medium is preferably sterilized in order to prevent contamination.
- the pH of the protein dispersion is not particularly limited, and may be, for example, near neutral, and is preferably in the range of 5.5 to 7.5.
- the concentration of the clay mineral in the clay mineral dispersion is not particularly limited.
- ⁇ is in the range of 0.1 to L000 ⁇ g / mL, and preferably ⁇ to 25 to 400 ⁇ gm. [Ma 50-200 / ⁇ 8 ⁇ ⁇ .
- the pH of the clay mineral dispersion is the same as that of the protein dispersion, Not limited.
- a protein introduction agent can be prepared by mixing the protein dispersion and the clay mineral dispersion.
- a complex in which protein is supported on the clay mineral can be formed.
- the ratio (weight ratio A: B) between the protein (A) and the clay mineral (B) is, for example, as described above. It is the same.
- the mixed solution (protein introduction agent) obtained by mixing the protein dispersion and the clay mineral dispersion is usually intended for cells and living bodies, its pH is not particularly limited.
- pH is medium. It may be set near the sex (for example, pH of about 6.5 to 7.5).
- the case where an exchangeable cation is included between clay mineral layers is not limited in the same manner.
- the pH may be adjusted to near neutral.
- the protein can be introduced without being affected by pH even after the protein is retained and after being placed under acidic conditions. . For this reason, for example, it can be said that it is suitable for oral administration as described later, in which the introduction of protein itself is not affected even when it passes through the stomach under strongly acidic conditions.
- the protein dispersion and the clay mineral dispersion can be mixed and used immediately as a protein introduction agent, it is preferable to incubate after mixing in order to sufficiently carry the protein in the clay mineral.
- the temperature conditions for the incubation are not particularly limited, and can usually be treated at room temperature, and the time is not particularly limited.
- Such a protein introduction agent in the form of a dispersion may be used as it is, or may be frozen until use as described above.
- methods such as freeze-drying and spray-drying can be applied, and it may be dispersed in the above-mentioned dispersion medium at the time of use.
- the clay mineral in the present invention is known to be taken internally as a natural remedy, for example, and may be contained in an internal preparation.
- the protein introduction agent of the present invention is extremely useful as, for example, a pharmaceutical, a functional food, and a food additive.
- the present invention introduces a protein by bringing the protein introduction agent into contact with a cell.
- Specific examples of the protein introduction method in vitro and in vivo are shown in the following Embodiments 3 and 4, respectively. According to this method, cells into which a foreign protein has been introduced can be produced. In addition, this invention is not restrict
- the protein introduction agent may be added to a cultured cell into which the target protein is introduced and incubated.
- the cultured cells are preincubated for a certain period of time in a liquid medium according to the type, and then the protein introduction agent is added and further incubated.
- the protein introduction agent is added and further incubated.
- the protein can be smoothly introduced into the cultured cells by the introduction agent, and incubation is performed for a certain period of time after the addition. Can introduce proteins more efficiently.
- the type of the liquid medium can be appropriately determined depending on the cells as described above, and is not particularly limited.
- the addition ratio of the protein introduction agent to the cultured cells is not particularly limited!
- the addition ratio of the clay mineral to 6 cells IX 10 6 is in the range of 0.01 to: LOO / zg.
- the protein introduction agent more preferably, the addition rate of the clay mineral is 0.1 to 50 g, particularly preferably 0.2 to 25 / zg.
- the protein introduction agent such that the ratio of the protein to the cell 1 X 10 6 is in the range of 0.01 to: LOO / zg, more preferably the addition ratio of the protein is 0.1. ⁇ 50 8 , particularly preferably ⁇ . 0.2 to 25 / zg.
- the cultured cells into which the target protein is introduced are not limited. Applicable.
- human cells are non-human animal cells (mammalian cells), these In addition, it can be applied to cells of all species such as microorganisms, fish, reptiles, amphibians, birds and insects.
- the cell culture is not particularly limited as described above, and can be performed according to known culture conditions using a known liquid medium according to the cell type.
- the culture time in which cells are cultured (pre-incubated) in advance is preferably, for example, 18-30 hours, more preferably 18 to 24 hours, particularly preferably 22 to 24 hours.
- the incubation time after the addition of the protein introduction agent is not particularly limited as long as, for example, the protein is introduced into the cell, but is, for example, about 3 hours, and the upper limit is not particularly limited.
- the culture temperature is usually 37 ° C, and it is preferable to culture in the presence of 5% carbon dioxide.
- Cells into which the protein obtained by the above method has been introduced can be used for the following treatments. For example, when a certain disease has developed due to a gene deficiency, a protein encoded by the deficient gene is introduced into cultured cells by the introduction method of the present invention. Then, the obtained protein-introduced cell is administered to the affected area of the disease. In this way, the transferred protein in the administered cells can alleviate or cure the symptoms of the disease.
- the administration method include injection into the affected area, and surgical treatment in which the protein-introduced cell is carried on a carrier and implanted in the affected area, subcutaneously or abdominally.
- the protein introducing agent is administered to a living body, and the protein is introduced into cells of an organ or tissue by the introducing agent.
- the protein-introducing agent can be introduced into the target cells in the living body, for example, by oral administration or simply administered to the target organ or tissue by surgical treatment or injection. .
- the protein introduction agent of the present invention when used, the target protein can be selectively and reliably introduced into small intestinal cells. in this way It is considered that the protein can be selectively introduced into the small intestine for the following reasons.
- the protein is supported on the clay mineral, for example, by mixing the clay mineral and protein.
- the carrier for introduction of proteins thus carried supports the protein tightly by increasing the charge when the pH is acidic, while the charge is charged under neutral or alkaline conditions. By reducing the size, protein is released and released. Since the clay mineral has such a mechanism, when the protein introduction agent is orally administered, it passes through the stomach under acidic conditions and reaches the small intestine without releasing the protein. In addition, because it is a clay mineral, it is not digested in the stomach. And since the moon cake has a neutral pH and a smaller charge than the stomach, protein is released from the clay mineral of the protein introduction agent.
- the protein introduction carrier (protein introduction agent) of the present invention is orally administered, the protein will not be digested in the stomach, and it will enter various cells and be selected into the small intestine like intravenous injection. There is no problem that makes it difficult to administer. That is, according to the present invention, selective administration to the small intestine can be easily performed by simple oral administration.
- allergy treatment desensitization treatment
- intestinal immunity By such selective protein introduction agent contact with the small intestine, allergy treatment (desensitization treatment) utilizing intestinal immunity can also be realized by simple oral administration.
- a protein-introducing agent is prepared by supporting a protein that causes various allergies on a protein-introducing carrier, and this is regularly orally administered to allergic patients. Then, since the protein introduction agent is carried to the small intestine and selectively releases the target protein in the small intestinal cells, immune tolerance occurs in intestinal immunity, and allergic symptoms are reduced.
- Such a method can be said to be an excellent therapy with less burden on patients who do not need frequent administration by injection, for example, as compared with conventional desensitization treatment.
- Intestinal cells usually peel off in about two weeks, so that cells into which proteins are introduced do not remain in the body for a long period of time. Therefore, if the present invention is applied, it can be said that it is possible to provide a preventive method and a therapeutic method that are further excellent in safety.
- the protein introduction method in vivo can be applied to, for example, a human living body, and can also be applied to a living body of a mammal other than human.
- the dose of the protein introduction agent can be appropriately determined according to the purpose, type of living body, etc. For example, cells and clay minerals or The ratio with the protein may be the same ratio as the in vitro introduction described above.
- protein can be selectively introduced into the small intestine, it can be said to be a useful method for clinical treatment.
- the pharmaceutical composition of the present invention is a pharmaceutical composition containing a protein as a therapeutic agent, preventive agent or inhibitor of a disease, and further comprises the protein introduction carrier of the present invention (that is, the present invention) A protein composition comprising a protein transducing agent).
- the active ingredient that serves as a therapeutic agent, preventive agent, or suppressor for a disease is a protein
- administration of the pharmaceutical composition of the present invention containing the protein-introducing carrier allows easy and safe human safety.
- the prevention and treatment of mammals other than can be performed.
- the type of protein is not limited at all, and can be selected according to the disease. For example, in the case of desensitization treatment, protein such as pollen or albumin, etc., and in the case of diabetes, insulin or the like can be selected.
- the method for administering the pharmaceutical composition is not particularly limited, and oral administration and parenteral administration such as injection can be selected depending on the disease.
- the pharmaceutical composition of the present invention may be administered by being implanted into a living body by a surgical treatment such as a protein-introduced cell as described above.
- ⁇ -galatatosidase was introduced into small intestinal epithelial cells in vitro using clay minerals.
- DMEM Dullbecco'sModified E agle's Medium: serum-free
- Rat small intestinal epithelial cells IEC-6 (ATCC-CRL1592) distributed by ATCC were used as cells.
- 2.5 ml of the following liquid medium was placed in a 12 well plate (Falcon), and 1 ⁇ 10 5 small intestinal epithelial cells were seeded on each well and cultured at 37 ° C. for 24 hours.
- the carbon dioxide concentration was adjusted to 5% with a carbon dioxide incubator.
- DMEM Dulbecco's modified Eagle medium: Nissui Pharmaceutical Co., Ltd.
- FBS fetal bovine serum: manufactured by Dainippon Pharmaceutical
- the cultured cells were washed twice with the PBS (-), and the DEME (serum-free) lml was added to each well. Further, the protein introduction agent 1001 was added to each well so that j8-galactosidase was: L g (905 mU) Z well. After incubating at 37 ° C. for 3 hours, serum (FBS) 111 ⁇ 1 was added to adjust the FBS concentration in the medium to 10% by weight and incubated at 37 ° C. for 24 hours.
- FBS serum
- Z buffer 90 ⁇ 1 of the following composition recovered cell extract 10 ⁇ 1 and 4 mg / ml ONPG ( 0- Nitrophenyl / 3-D-galatatopyranoside) solution 20 ⁇ 1 after 1 hour at 3 7 ° C and ⁇ Ka ⁇ , 100 i ul of reaction stopping solution (LMNA CO) and ⁇ Ka ⁇ , The absorbance of this reaction solution at a wavelength of 415 nm was measured. Then, ⁇ -galatatosidase activity (mU) in each cell was calculated from the obtained absorbance and a calibration curve prepared in advance. These results are shown in Fig. 1. Note that “j8-galonly” in the figure indicates that only galactosidase was introduced in Comparative Example 1 (same in FIG. 2).
- ⁇ -galatatosidase was introduced into small intestinal epithelial cells in vivo using clay minerals.
- the same trade name Smecton SA and the trade name bentonite as in Example 1 were used.
- the carrier dispersion (lmgZml) 50 ⁇ 1 and the protein dispersion (1 ⁇ 8 ⁇ 1) 50 / ⁇ 1 prepared in the same manner as in Example 1 were each diluted with 1001 of mili-Q water. Then, both dilutions were mixed and incubated at 25 ° C for 1 hour to prepare a protein introduction agent.
- the protein introduction agent was forcibly orally administered to a 6-week-old ddY mouse (OS: Japan SLC) using an oral sonde so that the concentration of 13-galatatosidase was 50 ⁇ g (45.25 U) / mouse.
- the mice were fasted from the day before the administration of the protein introduction agent.
- mili-Q water 300 1 was administered instead of the protein introduction agent, and Comparative Example 2 Instead of the protein introduction agent, a diluted solution obtained by diluting the protein dispersion (lmgZml) 50 1 with mili-Q water 250 1 was orally administered.
- mice were anesthetized with ether and dislocated the cervical spine. Then, the mouse small intestine was taken out, cut out by 4 cm from the stomach side, and a total of 3 sections were each immersed in 200 ⁇ 1 of PBS ( ⁇ ) containing 5 mM E GTA.
- the small intestine was homogenized (Fiscotron homogenizer: manufactured by Sakai-ON), and the supernatant collected by centrifugation (14,500 rpm, 5 minutes, 4 ° C.) was used as a cell extract. For these cell extracts, j8-galatatosidase activity was measured in the same manner as in Example 1.
- Fig. 2 shows the results of cell extracts from a section of a section close to the large intestine.
- Example 2 and Comparative Example 2 can be judged to be activity values by the protein into which the difference from the control is introduced. Then, as shown in FIG. 2, compared with Comparative Example 2, Examples 2-1 (Kunipia) and 2-2 (Bentonite) showed extremely high activity. In other words, it can be said that j8-galactosidase can be introduced into small intestinal cells extremely efficiently even in vivo by using these protein introduction carriers.
- j8-galactosidase is introduced in vivo in this way and shows activity in vivo
- an introduction agent carrying j8-galactosidase is, for example, for lactose intolerance. It can be said that it can be used as a medicine or a functional food.
- OVA ovalbumin
- bentonite trade name Bengel Fw; manufactured by Houjiyun Co., Ltd.
- FIG. 3 (A) is a graph showing the protein concentration in the supernatant
- Fig. 3 (B) shows the ratio of the amount of protein decrease from OVA alone, that is, the ratio to the bentonite, where OVA alone is 100%. Indicates adsorption rate (%) It is a graph.
- a protein transduction agent was prepared using a protein containing ovalbumin (OVA), a major allergen of egg allergy, and a clay mineral, and the introduction of OVA into mouse blood in vivo was confirmed.
- OVA ovalbumin
- Example 3 OVA and bentonite (trade name Bengel Fw) were suspended in the PBS () of Example 1 to prepare an OVA dispersion (lOOmgZml) and a carrier dispersion (lOOmgZml). These dispersions were mixed in the following ratio in an Ebendful tube and incubated at room temperature for 1 hour to obtain a protein introduction agent.
- PBS (—) 300 / zl of Example 1 was administered instead of the protein introduction agent, and in Comparative Example 4, a solution of OVA alone shown in Table 2 instead of the protein introduction agent 300 ⁇ 1 Were orally administered.
- the OVA-specific Ig G antibody contained in the serum sample was allowed to act on the OVA-coated well plate, and the HRP-labeled antibody was used as the secondary antibody to detect the OVA-specific IgG antibody contained in the sample.
- the following Coating Buffer was added to the well plate to 100 ⁇ l Zwell, sealed, and incubated at 4 ° C for 1 hour. The next day, the well plate was washed three times with the following Wash buffer, and the following blocking buffer was added to 300 ⁇ l Zwell. After incubation for 1 hour, the well plate was washed 3 times with the wash buffer. The serum sample was diluted 500-fold with the following sample diluent, and this diluted sample was added to the well plate so as to be 100 1 / well and incubated for 2 hours. After incubation, the well plate was washed 5 times with the wash buffer.
- Coating buffer Dissolved in lOOmM carbonate buffer (pH 9.6) so that the OVA concentration is 10 ⁇ g / ml.
- Wash buffer Mix 50 mM Tris, O. 14 M NaCl, and O. 05% Tween20 (trade name) (pH 8.0).
- Blocking buffer Blocking one (trade name) and 50 mM Tris—HC1 are mixed so that the volume ratio is 1: 3.
- Sample diluent Blocking one (trade name) and the above Wash solution are mixed so that the volume ratio is 1:19.
- TMB solution TMB solution (trade name) and Peroxidase solution (trade name) are mixed immediately before use so that the volume ratio is 1: 1.
- the protein-introduced carrier of the present invention is useful as a carrier for protein delivery, for example, in oral desensitization treatment for food allergies and the like.
- MK mitodocaine
- MK for example, a recombinant in which Escherichia coli is used as a host is prepared, and a preparation obtained by refining the expressed MK can be used in the experiment.
- human MKcDNA is routinely obtained by PCR using the Wilms tumor-derived culture cell line G-401 according to the method of Take et al. (J. Biochem.ll6, ppl063- 1068 (1994)).
- the human MKcDN A is transduced into E. coli to prepare a recombinant.
- a preparation can be prepared by purifying human MK protein from inclusion bodies of recombinant E. coli grown in culture according to the method of Take et al. It can also be prepared by purchasing a commercial product from Peptide Research Institute (Osaka), R & D Systems (MN, USA), or the like.
- the carrier dispersion 15 ⁇ 1 and the protein dispersion 151 were added to and mixed with DMEM (Dullbecco's Modified Eagle's Medium: serum-free) 270 ⁇ 1.
- DMEM Dullbecco's Modified Eagle's Medium: serum-free
- the mixture was allowed to stand at room temperature for 1 hour, and the DMEMlml was further added and mixed to obtain a protein introduction agent.
- Rat small intestinal epithelial cells IEC-6 (ECACC-88071 401) distributed by ATCC were used as cells. First, place 5 ml of the following liquid medium in a 12-well plate (Corning) and inoculate 5 x 10 4 rat rat small intestinal epithelial cells IEC-6 on each well for 4 days at 37 ° C. did. All cells were cultured under conditions of 5% carbon dioxide. [0098] (Composition of liquid medium)
- FBS fetal bovine serum: made by Hyclone
- the cultured cells were washed twice with the PBS (-) solution, and 1.3 ml of the protein introduction agent was added to each well. Each well was then incubated at 37 ° C for 2 hours to introduce these proteins (MK) into cultured cells.
- each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli et al. PAG mini “Daiichi” 15Z25 (Daiichi Chemical Co., Ltd.) was used as the gel for electrophoresis, and the electrophoresis conditions were about 90 minutes at a constant current of 20 mA.
- Each well includes marker protein solution, mitocaine + smetato A total of 7 samples were sampled: N, SA, Mitodocaine + Kunipia F, Mitodocaine + Bengel FW, Mitsodocaine + Bengenore Bright 25, Comparative Example 5 (Middocaine only), and Control.
- the amount of sample per lane was 2 X 10 5 cells.
- chemically synthesized middocaine human (Peptide Institute, Inc.) was prepared and run at 200 to 500 ng per lane (not shown in FIG. 5).
- molecular weight marker the trade name Kaleidoscope Prestained Standards (BIO—RAD) was used.
- the membrane was reacted with donkey anti-goat IgG peroxidase-labeled antibody (SantaCruz Biotechnology, In; CA, U.S.A.) for 1 hour at room temperature. After completion of the reaction, the membrane was washed 4 times (each for 10 minutes) with a PBS solution containing 0.05% Tween 20, and then once (10 minutes) with a PBS solution.
- donkey anti-goat IgG peroxidase-labeled antibody SantaCruz Biotechnology, In; CA, U.S.A.
- lanes 1 and 8 are the results of using the marker protein solution
- lanes 2 to 5 are the results of using the protein transducing agent
- lane 2 is the midocaine + smecton SA
- lane 3 is the mitodo force in + Kunipia F
- lane 4 Mitodocaine + Bengenore FW
- Lane 5 is the result of Mitodocaine + Bengelbright 25
- Lane 6 is the result of Comparative Example 5 (Mitsudocaine only)
- Lane 7 is the result of control (one).
- Example 6 A protein having a molecular weight of about 18 kD was introduced into small intestinal epithelial cells IEC-6 (ECAC C 88071401) in vitro using various carriers for protein introduction.
- PTN can also be produced as a recombinant, and a recombinant PTN protein can be prepared and used as a standard.
- Peptide Laboratories Osaka
- R & DSystems MN, USA
- Etc. may be used.
- the carrier dispersion 15 ⁇ 1 and the protein dispersion 151 were added to DMEM (Dullbecco's Modified Eagle's Medium: serum-free) 270 ⁇ 1 and mixed. The mixture was allowed to stand at room temperature for 1 hour, and the DMEMlml was further added and mixed to obtain a protein introduction agent.
- DMEM Dullbecco's Modified Eagle's Medium: serum-free
- Rat small intestinal epithelial cells IEC-6 (ECACC-88071 401) distributed by ATCC were used as cells. First, in a 12 well plate (manufactured by Corning Co., Ltd.), store 5 ml of the same liquid medium as in Example 5 and seed each well with 5 x 10 4 rat small intestinal epithelial cells IEC-6. The cells were cultured at 37 ° C for 4 days. All cells were cultured under conditions of 5% carbon dioxide.
- lanes 1 and 8 are the results of using the marker protein solution
- lanes 2 to 5 are the protein introduction agents
- lane 2 is the pleotite fin + smecton SA
- lane 3 is the pleat mouth.
- Lane 4 is Pleoto Mouth Fin + Wenger FW
- Lane 5 is Pleoto Mouth Fin + Wenger Bright 25
- Lane 6 is Comparative Example 6 (pre-trophin only)
- Lane 7 is control (one) Is the result of As shown in the figure, even when any of the above four types of protein introduction carriers was used, a band was confirmed that was darker than the one introduced alone (Comparative Example 6).
- Polypeptides having a molecular weight of about 5 kD were introduced into small intestinal epithelial cells IEC-6 (ECAC C 88071401) in vitro using various carriers for protein introduction.
- the same carrier dispersion (2 mgZml) was prepared using the same clay mineral of the trade name Sumetaton SA containing saponite as the carrier for protein introduction as in Example 6.
- Sumetaton SA containing saponite
- insulin SIGMA-A LDRICH; MO, U.S.A.
- INS insulin
- This INS was dispersed in 1 mgZml with 5 mmol Zl hydrochloric acid aqueous solution to prepare a polypeptide dispersion.
- the carrier dispersion 15 ⁇ 1 and the polypeptide dispersion 151 were added to DMEM (Dullbecco's Modified Eagle's Medium: serum-free) 270 ⁇ 1 and mixed.
- DMEM Dullbecco's Modified Eagle's Medium: serum-free
- the blend The mixture was allowed to stand at room temperature for 1 hour, and the above DMEMlml was further added and mixed to obtain a polypeptide introducing agent.
- Rat small intestinal epithelial cells IEC-6 (ECACC-88071 401) distributed by ATCC were used as cells. First, in a 12 well plate (manufactured by Corning Co., Ltd.), store 5 ml of the same liquid medium as in Example 5 and seed each well with 5 x 10 4 rat small intestinal epithelial cells IEC-6. The cells were cultured at 37 ° C for 4 days. All cells were cultured under conditions of 5% carbon dioxide.
- the cultured cells were washed twice with the PBS (-) solution, and 1.3 ml of the above-mentioned polypeptide introduction agent was added to each tool. Each well was then incubated at 37 ° C for 2 hours to introduce INS into the cultured cells.
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Isocratic Gel Invitrogen; CA, USA
- the sample amount per lane was 2 ⁇ 10 5 ce lis protein.
- insulin SIGMA-ALDRICH; MO, USA
- As the molecular weight marker the trade name Kaleidoscope Prestained Standards (BIO-RAD) was used.
- lane 1 is the marker protein solution
- lane 2 is the result of using the polypeptide transfection agent (insulin + smecton SA)
- lane 3 is the control (one)
- lane 4 is the standard solution (positive control insulin). )
- Result As shown in the figure, as a result of using smecton SA containing saponate, a dark band indicating the introduction of insulin was confirmed.
- the protein introduction carrier of the present invention when used, a target protein can be introduced into cells with high safety. Therefore, the protein introduction method of the present invention using the protein introduction carrier is very useful in the field of clinical medicine.
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EP06713173A EP1854472A1 (en) | 2005-02-09 | 2006-02-07 | Support for protein transfer, protein transfer agent using the support, protein transfer method, cell having protein transferred thereinto and method of producing the same |
CA002597583A CA2597583A1 (en) | 2005-02-09 | 2006-02-07 | Support for protein transfer, protein transfer agent using the support, protein transfer method, cell having protein transferred thereinto and method of producing the same |
US12/827,648 US20100267627A1 (en) | 2005-02-09 | 2010-06-30 | Support for protein transfer, protein transfer agent using the support, protein transfer method, cell having protein transferred thereinto and method of producing the same |
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