WO2020045162A1 - Drug delivery carrier - Google Patents
Drug delivery carrier Download PDFInfo
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- WO2020045162A1 WO2020045162A1 PCT/JP2019/032411 JP2019032411W WO2020045162A1 WO 2020045162 A1 WO2020045162 A1 WO 2020045162A1 JP 2019032411 W JP2019032411 W JP 2019032411W WO 2020045162 A1 WO2020045162 A1 WO 2020045162A1
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- WO
- WIPO (PCT)
- Prior art keywords
- drug delivery
- base sequence
- hydrogel
- delivery system
- aav
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
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- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
Definitions
- the present invention relates to a drug delivery carrier. More specifically, the present invention relates to a drug delivery carrier and a drug delivery system.
- the cochlea which exists in the inner ear and converts sound into neural activity, is buried in the compact bone called the temporal bone, and contacts the middle ear cavity through a structure called a round window and an oval window.
- the former is a membrane-like structure located at the bottom of a recess called a round window fossa, and is expected as a route for drug administration to the inner ear.
- AAV is used as a drug
- AAV passes through the round window membrane and diffuses into the cochlea, while the middle ear surgery raises the eardrum and clearly approaches the round window fossa by visualization.
- DDS drug delivery system
- Non-Patent Document 1 is mainly applied to a body surface such as a wound, and is difficult to apply to a narrow and complicated tissue. Also, control at any timing of sustained release has not been realized.
- the present invention has been made in view of the above circumstances, and provides a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.
- the purpose is to do.
- a drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
- a drug delivery system comprising the drug delivery carrier according to any one of [1] to [6] and a drug.
- the drug delivery system according to [9] wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
- the nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell: 11] or the drug delivery system according to [12]: (A) the base sequence represented by SEQ ID NO: 1, (B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1, (C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1, (D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above-mentioned (A) to ( Degenerate isomer of the base sequence of D).
- a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.
- FIG. 4 is a graph showing the results of a stability test of AAV-containing calcium alginate gel beads expressing GFP stored at 4 ° C. or ⁇ 80 ° C. It is a graph which shows the result of the time-dependent change test of the infectivity of AAV containing calcium alginate gel beads. It is a graph which shows the test result of the virus infectivity after subjecting AAV containing collagen gel beads (Low Density and High Density) to alginate lyase treatment.
- the present invention provides a carrier for drug delivery comprising a hydrogel comprising a polysaccharide having a crosslinkable functional group.
- the polysaccharide having a crosslinkable functional group is crosslinked by a crosslinking agent to form a hydrogel.
- the hydrogel means a gel containing water or an aqueous solution.
- the method for crosslinking the polysaccharide is not limited as long as the hydrogel forms a three-dimensional network structure, and includes chemical crosslinking or physical crosslinking. Specifically, crosslinking by a covalent bond, a hydrogen bond, or an ionic bond can be mentioned, and crosslinking by an ionic bond is preferable. As the crosslinking by ionic bonding, crosslinking by divalent metal ions is preferable.
- an acidic functional group is preferable, and examples thereof include a carboxyl group, an alcoholic hydroxyl group, and a phenolic hydroxyl group, and a carboxyl group is more preferable.
- polysaccharides having a crosslinkable functional group include alginic acid, pectic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethyl cellulose, carboxymethyl chitosan, carboxymethyl dextran, Examples include carboxymethyl starch, xanthan, karaya gum, gum gutti, gellan, agar, heparin sulfate, chondroitin sulfate, or salts or derivatives thereof. Among them, alginic acid, pectic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid, pectic
- those containing a divalent metal ion are preferable, and Pb 2+ , Cu 2+ , Cd 2+ , Ba 2+ , Sr 2+ , Ca 2+ , Co 2+ , Ni 2+ , Zn 2+ , Mn 2+ , Mg 2+ , those containing Ca 2+ and Ba 2+ are preferable, and those containing Ca 2+ are more preferable.
- the crosslinking agent can be used in the form of an aqueous solution by dissolving in water, a salt containing the above divalent metal ion is preferable, and a halide, hydroxide, carbonate, nitrate containing the above cation, Acetates are more preferred, halogen salts are even more preferred, and calcium chloride is particularly preferred.
- the hydrogel is obtained, for example, by extruding a solution containing a polysaccharide having a crosslinkable functional group into a solution containing a crosslinking agent.
- concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1 mM to 10 M, more preferably 10 mM to 5 M, and particularly preferably 100 mM to 1000 mM.
- concentration of the polysaccharide in the solution containing the polysaccharide having a crosslinkable functional group is preferably 0.1% by weight to 50% by weight, more preferably 0.5% by weight to 20% by weight, and more preferably 1% by weight to 10% by weight. Is particularly preferred.
- the hydrogel can take any shape depending on the site to be delivered and the purpose. For example, a shape such as a bead, a sheet, and a fiber may be used, but is not limited thereto.
- the hydrogel can be in the form of beads.
- Hydrogel beads can be produced, for example, by a microgel bead production method using centrifugal force (see Non-Patent Document 2).
- the pore size (diameter) of the hydrogel beads is preferably 1 nm to 10 ⁇ m, more preferably 5 nm to 1 ⁇ m, and particularly preferably 10 nm to 200 nm.
- the size (diameter) of the hydrogel beads is preferably 1 ⁇ m to 1000 ⁇ m, more preferably 10 ⁇ m to 500 ⁇ m, and particularly preferably 50 ⁇ m to 200 ⁇ m.
- the carrier for drug delivery of the present invention preferably contains a fibrous extracellular matrix containing a hydrogel, in which case the hydrogel is preferably in the form of beads.
- the drug delivery carrier of the present invention can be stably placed at a desired location in a living body.
- the drug delivery carrier of the present invention can be appropriately placed in an auricular space having a specific shape such as a gap in a space where stapes are located. Can be detained.
- the diameter of the fibrous extracellular matrix is preferably 1 ⁇ m to 1000 ⁇ m, more preferably 10 ⁇ m to 500 ⁇ m, and particularly preferably 50 ⁇ m to 200 ⁇ m.
- the length of the fibrous extracellular matrix is preferably from 10 ⁇ m to 100 cm, more preferably from 100 ⁇ m to 10 cm, and particularly preferably from 1 mm to 1 cm.
- a method for producing a fibrous extracellular matrix containing a hydrogel can be produced, for example, by pouring the extracellular matrix into a fiber-shaped mold.
- basal reconstituted from collagen type I, II, III, V, XI, etc.
- mouse EHS tumor extract including type IV collagen, laminin, heparan sulfate proteoglycan, etc.
- membrane components trade name: Matrigel
- glycosaminoglycans hyaluronic acid
- proteoglycans gelatin, and the like.
- the hydrogel itself may be in the form of a fiber.
- the diameter of the fibrous hydrogel is preferably 1 ⁇ m to 1000 ⁇ m, more preferably 10 ⁇ m to 500 ⁇ m, and particularly preferably 50 ⁇ m to 200 ⁇ m.
- the length of the fibrous hydrogel is preferably from 10 ⁇ m to 100 cm, more preferably from 100 ⁇ m to 10 cm, and particularly preferably from 1 mm to 1 cm.
- the present invention provides a drug delivery system including the above-described drug delivery carrier and a drug.
- the drug delivery system according to the present embodiment can deliver a drug to a desired organ without controlled cytotoxicity, with controlled sustained release.
- the drug delivery system of the present embodiment can be used in a form containing a therapeutic agent for a disease, a contrast agent, and the like as a drug.
- Examples of the active ingredient of the therapeutic agent for a disease include low molecular weight compounds, nucleic acids, proteins, and peptides.
- the low molecular weight compound a compound whose size is adjusted by micelle formation or the like so as to be supported on the pores of the hydrogel beads is preferable.
- the nucleic acid include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA and the like.
- proteins include enzymes and antibodies, and examples of peptides include ligands and vaccines.
- a nucleic acid is preferable as the disease therapeutic agent.
- a cDNA of the wild-type gene is preferable to use as a therapeutic agent for a disease.
- the therapeutic agent for a disease is a nucleic acid
- the viral vector has a form containing the cDNA as the therapeutic agent for a disease, from the viewpoint of delivery and expression efficiency.
- the virus vector used here includes a retrovirus vector, an adenovirus vector, an adeno-associated virus vector (AAV), a herpes virus vector, and the like. From the viewpoint of low toxicity, an adeno-associated virus vector (AAV) is preferable.
- Atonal-related factors are transcription factors belonging to the basic helix-loop-helix family.
- Examples of the gene encoding an atonal-related factor include a gene having a base sequence of any one of the following (A) to (D) and encoding a protein capable of differentiating a supporting cell into a sensory hair cell.
- a gene having a base sequence of any one of the following (A) to (D) and encoding a protein capable of differentiating a supporting cell into a sensory hair cell Listed: (A) the base sequence represented by SEQ ID NO: 1, (B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1, (C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1, (D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).
- the gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 is cDNA of Hath1 (Human @ atonal @ homolog1).
- the number of bases which may be deleted, substituted, inserted, or added is preferably 1 to 320, more preferably 1 to 160, still more preferably 1 to 80, and 1 to 80.
- the number is more preferably 40, particularly preferably 1 to 20, and most preferably 1 to 10.
- the identity is preferably at least 75%, more preferably at least 80%, further preferably at least 85%, particularly preferably at least 90%.
- “under stringent conditions” means, for example, 5 ⁇ SSC (composition of 20 ⁇ SSC: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 0.1% by weight N Incubation at 55-70 ° C. for several hours to overnight in a hybridization buffer consisting of lauroyl sarcosine, 0.02% by weight SDS, 2% by weight nucleic acid hydridation blocking reagent and 50% formamide Thus, conditions for hybridization can be given.
- the washing buffer used for washing after incubation is preferably a 1 ⁇ SSC solution containing 0.1% by weight SDS, more preferably a 0.1 ⁇ SSC solution containing 0.1% by weight SDS.
- the degenerate isomer of the base sequence means another base sequence corresponding to an amino acid encoded by a certain base sequence.
- the drug delivery system of the present embodiment preferably further includes a solubilizing agent for the hydrogel.
- a solubilizing agent for the hydrogel include a chelating agent for removing divalent metal ions when crosslinked by ionic bonding.
- an enzyme that decomposes a polysaccharide having a crosslinkable functional group itself is also included.
- Examples of the chelating agent include citric acid, ethylenediamine, ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, and glycol ether diaminetetraacetic acid.
- Examples of the enzyme include alginate lyase.
- the drug delivery system of the present embodiment can be reliably placed inside a complex tissue by adjusting the size or shape of the hydrogel, or by enclosing the hydrogel in a polymer structure such as a gel fiber. can do. Furthermore, by subjecting the hydrogel beads carrying the AAV to degradation by an enzyme such as alginate lyase, or irradiation of ultrasonic waves or the like, the AAV can be inactivated at desired timing without deactivation of AAV or damage to surrounding tissues. Can be sustained released. The sustained-release AAV can introduce a desired gene into a target tissue.
- contrast agent as a drug in the drug delivery system of the present embodiment, barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass Contrast agents for computed tomography (CT) such as iodine contrast agents; contrast agents for MRI such as gadolinium preparations, superparamagnetic iron oxide preparations (Super Paramagnetic Iron Oxide, SPIO); technetium 99m (99m Tc), molybdenum 99 (99 Mo) Single photon emission computed tomography (SPECT) for radioactive isotopes such like good It can be cited as Shii drug.
- CT computed tomography
- contrast agents for MRI such as gadolinium preparations, superparamagnetic iron oxide preparations (Super Paramagnetic Iron Oxide, SPIO); technetium 99m (99m Tc), molybdenum 99
- the present invention provides a method of treating a disease caused by a defect or loss of sensory hair cells, comprising administering a drug delivery system as described above to a patient in need of treatment.
- the hydrogel carrying AAV containing the nucleic acid encoding the atonal-related factor described above was used as a drug delivery system.
- gene therapy is performed.
- the carrier for drug delivery preferably contains a fibrous hydrogel or a fibrous extracellular matrix containing a beaded hydrogel.
- alginate lyase may be administered to the inner ear several times periodically.
- the sustained release of AAV containing the nucleic acid encoding the atonal-related factor carried on the hydrogel can be controlled.
- the dose of AAV supported on the hydrogel is 10 5 gc / mL to 10 12 gc / mL, and the lower limit is 10 5 gc / mL, 10 6 gc / mL, 10 7 gc / mL and 10 7 gc / mL.
- the dose of alginate lyase is 1 ⁇ g to 10 mg per administration, the lower limit is 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 1 mg, and the upper limit is 10 mg, 1 mg, 100 ⁇ g, 10 ⁇ g. No.
- ultrasound may be applied to a predetermined part of the ear to physically dissolve the hydrogel placed in the inner ear.
- the present invention provides a drug delivery system for the treatment of a disease caused by a defect or loss of sensory hair cells.
- the same drug delivery system as described above can be used.
- the present invention provides a pharmaceutical composition for the treatment of a disease caused by defect or loss of sensory hair cells, comprising a drug delivery carrier and a drug.
- a drug delivery carrier and drug those similar to those described above can be used.
- the present invention provides a drug delivery system for use as a treatment for a disease caused by a defect or loss of sensory hair cells.
- a drug delivery system for use as a treatment for a disease caused by a defect or loss of sensory hair cells.
- the same drug delivery system as described above can be used.
- a solution was prepared so that the final concentration was 2% by weight of sodium alginate and 10% by weight of fluorescent silica, and microgel beads were prepared using centrifugal force (K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340-1346 (2012)).
- the stability of the microbeads after 3 days in a calcium chloride solution was evaluated.
- FIG. 1A is a fluorescence image of microbeads immediately after preparation
- FIG. 1B is a fluorescence image of microbeads after storage in a calcium chloride solution for 3 days. As shown in FIG. 1B, it was confirmed that the silica beads were stably encapsulated in the microgel beads.
- Example 2 (Sustained release test of microbeads) Next, a sustained release test of the encapsulated silica beads was performed. An aqueous solution of 3% by weight of sodium alginate was injected into a 100 mM calcium chloride solution in the same manner as in Experimental Example 1 to produce calcium alginate gel microbeads (about 80 ⁇ m in diameter). After the microbeads were dropped on the dish in which the NIH / 3T3 cells were cultured, alginate lyase was dropped on the dish such that the final concentration became 50 ⁇ g / mL, and the state until the beads were dissolved was observed. Successfully decomposed.
- Example 3 (Sustained release test of AAV-containing calcium alginate gel beads) A verification test of DDS using AAV instead of silica beads was performed. Specifically, AAV prepared to express GFP in cells by infection was encapsulated in micro-beads of calcium alginate gel, and the same sustained release test as in Example 2 was performed. Example 3 demonstrates whether a DDS carrier can maintain the infectious function of AAV. In the same manner as in Experimental Examples 1 and 2, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 ⁇ 10 10 gc / mL of GFP was placed in a 100 mM, 500 mM, or 1000 mM calcium chloride solution.
- Example 2 In the same manner as in Example 1, injection was performed to produce calcium alginate gel microbeads (about 80 ⁇ m in diameter).
- the prepared AAV-containing calcium alginate gel beads expressing GFP and alginate lyase at final concentrations of 10 ⁇ g / mL and 30 ⁇ g / mL were dropped on HeLa cells, and the proportion of GFP-expressing cells 72 hours later was measured.
- the results are shown in FIG. In FIG. 3, from the left lane, Control indicates AAV-containing calcium alginate gel beads expressing GFP and HeLa cells without addition of alginate lyase, and AAV + Lyase_0 indicates HeLa cells with only AAV expressing GFP added.
- AAV + Lyase_10 indicates AAV expressing GFP and HeLa cells to which alginate lyase at a final concentration of 10 ⁇ g / mL was added
- AAV + Lyase_30 indicates AAV expressing GFP and an alginate lyase at a final concentration of 30 ⁇ g / mL.
- AAV-Beads + Lyase_0 indicates HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP prepared in a 100 mM calcium chloride solution were added
- AAV-Beads + Lyase_30 indicates AAV-containing calcium alginate gel beads expressing GFP prepared in 100 mM calcium chloride solution, and HeLa cells to which alginate lyase at a final concentration of 30 ⁇ g / mL was added.
- AAV-containing calcium alginate gel beads expressing GFP prepared in a calcium solution, and HeLa cells to which alginate lyase was added at a final concentration of 30 ⁇ g / mL are shown.
- AAV-Beads + Lyase_30 + CaCl2_1000 shows GFP prepared in a 1000 mM calcium chloride solution. Expressed AAV-containing calcium alginate gel beads and alginate lyase at a final concentration of 30 ⁇ g / mL Show the added HeLa cells.
- FIG. 4 shows the results. From the left lane in FIG. 4, Fresh B (-) L (-) indicates HeLa cells to which only AAV expressing fresh GFP without preservation was added, and Fresh B (-) L (+) indicates AAV expressing fresh GFP without preservation and HeLa cells supplemented with alginate lyase at a final concentration of 30 ⁇ g / mL. Fresh B (+) L (+) shows fresh AAV-containing calcium alginate gel beads containing GFP expressing and HeLa cells supplemented with alginate lyase at a final concentration of 30 ⁇ g / mL are shown. 4B (+) L (+) shows AAV expressing GFP stored at 4 ° C.
- Example 5 (Evaluation test of infectivity of virus after elapse of time after AAV encapsulation in hydrogel microbeads) AAV prepared to express GFP in cells by infection was encapsulated in calcium alginate gel microbeads, (i) a sample retained for up to 72 hours after encapsulation, and (ii) alginate lyase immediately after encapsulation in microbeads. Samples that were degraded and retained for up to 72 hours after encapsulation, and (iii) samples that were retained for 24 hours after encapsulation in microbeads and then degraded by alginate lyase to retain for up to 72 hours after encapsulation were prepared.
- the lyase concentration was 33 ⁇ g / mL.
- the samples (i) to (iii) were used at a MOI of 1 ⁇ 10 6 for 5 ⁇ 10 3 cells per well.
- the cell infection rate was determined for samples (i) to data (iii) and controls (positive and negative).
- FIG. 5 shows the results.
- Example 6 Evaluation of the infectivity of samples that have been subjected to encapsulation of AAV-containing beads in a collagen gel and subsequent degradation treatment
- beads containing AAV prepared to express GFP in cells by infection Low Density, High Density
- a sample in which Low Density beads were encapsulated in a collagen gel and held for 72 hours (ii) Low Density Immediately after encapsulating the beads in the collagen gel, the sample was degraded by alginate lyase and retained for up to 72 hours after encapsulation
- High Density beads were encapsulated in the collagen gel and retained for 72 hours
- the density beads were digested with alginate lyase to prepare a sample that was retained for up to 72 hours after the encapsulation.
- High Density beads have 1/10 the number of beads and 10 times the virus concentration of Low density.
- the lyase concentration was 33 ⁇ g / mL.
- the samples (i) to (iv) were used at a MOI of 1 ⁇ 10 6 for 5 ⁇ 10 3 cells per well.
- the infection rate of the cells was determined for the samples (i) to (iv) and the controls (positive and negative).
- FIG. 6 shows the results.
- a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.
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Abstract
The purpose of the present invention is to provide a drug delivery carrier which can be stably placed in a narrow tissue having a complex structure such as inner ear and enables the control of the sustained release of a drug, and a drug delivery system comprising the drug delivery carrier. The drug delivery carrier according to the present invention comprises a hydrogel which contains a polysaccharide having a crosslinkable functional group.
Description
本発明は、薬物送達用担体に関する。より詳細には、本発明は、薬物送達用担体、及び薬物送達システムに関する。
The present invention relates to a drug delivery carrier. More specifically, the present invention relates to a drug delivery carrier and a drug delivery system.
この数年、難聴に対する新規治療法開発が国内外で活発化している。特に、内耳性難聴については、老人性難聴、突発性難聴、メニエール病等、罹患数も多く、６５歳以上の日本人口の３０～４０％が罹患している。しかも哺乳類蝸牛は、自発的再生能に乏しいため、慢性化した感音難聴は難治性である。この現状ゆえに海外の数々の大小企業が内耳性難聴をアンメット・メディカルニーズとして捉え、特に根治治療を目指した内耳への遺伝子治療の開発研究にしのぎを削っている。例えば、大手製薬メーカーは、アデノウイルスでの遺伝子導入において既に臨床試験を進めている。遺伝子導入においては、安全性を鑑み、アデノ随伴ウイルス（以下、ＡＡＶともいう。）による遺伝子導入媒体が主流となりつつある。
開 発 In recent years, the development of new treatments for hearing loss has been active in Japan and overseas. In particular, with regard to inner ear deafness, there are many cases of presbycusis, sudden deafness, Meniere's disease, etc., and 30 to 40% of the Japanese population aged 65 or older is affected. Moreover, since the mammalian cochlea is poor in spontaneous regenerative ability, chronic sensorineural hearing loss is intractable. Because of this situation, a number of large and small companies abroad regard inner ear hearing loss as an unmet medical need, and have been competing in research into the development of gene therapy for the inner ear, especially for the curative treatment. For example, major pharmaceutical manufacturers are already conducting clinical trials for gene transfer with adenovirus. In gene transfer, in view of safety, a gene transfer medium using an adeno-associated virus (hereinafter, also referred to as AAV) is becoming mainstream.
内耳に存在し、音を神経活動に変換する臓器「蝸牛」は、側頭骨という緻密骨の中に埋もれて存在し、正円窓および卵円窓という構造を通して中耳腔と接している。特に前者は正円窓窩と呼ばれる凹みの底に位置する膜様構造であり、内耳への薬剤投与経路として期待されている。
例えば、薬剤としてＡＡＶを用いた場合、ＡＡＶが正円窓膜を通過して蝸牛内へ拡散するが、他方中耳手術によって鼓膜を挙上し明視化で正円窓窩に確実にアプローチしても、投与した薬剤と正円窓膜の接触面積や滞留時間、さらには徐放化といったドラッグデリバリーシステム（以下、ＤＤＳともいう。）の課題が残る。内耳への局所投与に関するＤＤＳ開発研究は世界的にもまだ端を発した段階であり、現在まさにシーズ探索～開発研究をはじめる萌芽期にある。 The cochlea, which exists in the inner ear and converts sound into neural activity, is buried in the compact bone called the temporal bone, and contacts the middle ear cavity through a structure called a round window and an oval window. In particular, the former is a membrane-like structure located at the bottom of a recess called a round window fossa, and is expected as a route for drug administration to the inner ear.
For example, when AAV is used as a drug, AAV passes through the round window membrane and diffuses into the cochlea, while the middle ear surgery raises the eardrum and clearly approaches the round window fossa by visualization. However, there still remain problems of a drug delivery system (hereinafter, also referred to as DDS) such as a contact area and a residence time between the administered drug and the round window membrane and a sustained release. Research on DDS for topical administration to the inner ear has just begun in the world, and it is in the early stages of seeds exploration and development research.
例えば、薬剤としてＡＡＶを用いた場合、ＡＡＶが正円窓膜を通過して蝸牛内へ拡散するが、他方中耳手術によって鼓膜を挙上し明視化で正円窓窩に確実にアプローチしても、投与した薬剤と正円窓膜の接触面積や滞留時間、さらには徐放化といったドラッグデリバリーシステム（以下、ＤＤＳともいう。）の課題が残る。内耳への局所投与に関するＤＤＳ開発研究は世界的にもまだ端を発した段階であり、現在まさにシーズ探索～開発研究をはじめる萌芽期にある。 The cochlea, which exists in the inner ear and converts sound into neural activity, is buried in the compact bone called the temporal bone, and contacts the middle ear cavity through a structure called a round window and an oval window. In particular, the former is a membrane-like structure located at the bottom of a recess called a round window fossa, and is expected as a route for drug administration to the inner ear.
For example, when AAV is used as a drug, AAV passes through the round window membrane and diffuses into the cochlea, while the middle ear surgery raises the eardrum and clearly approaches the round window fossa by visualization. However, there still remain problems of a drug delivery system (hereinafter, also referred to as DDS) such as a contact area and a residence time between the administered drug and the round window membrane and a sustained release. Research on DDS for topical administration to the inner ear has just begun in the world, and it is in the early stages of seeds exploration and development research.
遺伝子治療を目的としたＤＤＳには、コラーゲンによるＤＮＡベクターの傷口への徐放などが報告されている（例えば、非特許文献１参照。）。
徐 As for the DDS for the purpose of gene therapy, it has been reported that the collagen vector is slowly released to the wound by collagen (for example, see Non-Patent Document 1).
しかしながら、非特許文献１の技術は、傷口など体表面への塗布などが主であり、狭い複雑な組織に適用は困難である。また、徐放の任意のタイミングでの制御も実現されてはいない。
However, the technique of Non-Patent Document 1 is mainly applied to a body surface such as a wound, and is difficult to apply to a narrow and complicated tissue. Also, control at any timing of sustained release has not been realized.
本発明は上記事情を鑑みてなされたものであり、内耳などの狭く複雑な構造をしている組織に安定して留置可能であり、薬剤の徐放制御を可能とする薬物送達用担体を提供することを目的とする。
The present invention has been made in view of the above circumstances, and provides a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug. The purpose is to do.
本発明は以下の態様を含む。
［１］架橋性官能基を有する多糖を含むハイドロゲルを含む、薬物送達用担体。
［２］前記架橋性官能基を有する多糖は、アルギン酸、又はその塩である、［１］に記載の薬物送達用担体。
［３］前記ハイドロゲルは、シート状、ビーズ状、又はファイバ状である、［１］又は［２］に記載の薬物送達用担体。
［４］前記ハイドロゲルは、ビーズ状である、［３］に記載の薬物送達用担体。
［５］前記ハイドロゲルは、ファイバ状である、［３］に記載の薬物送達用担体。
［６］ファイバ状の細胞外マトリクスを更に含み、前記細胞外マトリクスは、前記ハイドロゲルを含む、［１］～［４］のいずれか一つに記載の薬物送達用担体。
［７］［１］～［６］のいずれか一つに記載の薬物送達用担体と、薬物とを含む、薬物送達システム。
［８］前記ハイドロゲルの可溶化剤を更に含む、［７］に記載の薬物送達システム。
［９］疾患治療剤である、［７］又は［８］に記載の薬物送達システム。
［１０］前記疾患治療剤は、難聴治療剤である、［９］に記載の薬物送達システム。
［１１］前記薬物は、核酸を含む、［７］～［１０］のいずれかに記載の薬物送達システム。
［１２］ウイルスベクターを更に含み、当該ウイルスベクターが前記核酸を含む、［１１］に記載の薬物送達システム。
［１３］前記核酸は、以下の（Ａ）～（Ｄ）のいずれか一つの塩基配列からなり、且つ、支持細胞を感覚有毛細胞に分化させる能力を有するタンパク質をコードする遺伝子を含む、［１１］又は［１２］に記載の薬物送達システム：
（Ａ）配列番号１で表される塩基配列、
（Ｂ）配列番号１で表される塩基配列において、１又は数個の塩基が欠失、置換、挿入、又は付加されている塩基配列、
（Ｃ）配列番号１で表される塩基配列において、同一性が７０％以上である塩基配列、
（Ｄ）配列番号１で表される塩基配列からなる遺伝子と相補的な塩基配列からなる遺伝子とストリンジェントな条件下でハイブリダイズすることができる塩基配列、及び
（Ｅ）前記（Ａ）～（Ｄ）の塩基配列の縮重異性体。 The present invention includes the following aspects.
[1] A drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
[2] The carrier for drug delivery according to [1], wherein the polysaccharide having a crosslinkable functional group is alginic acid or a salt thereof.
[3] The carrier for drug delivery according to [1] or [2], wherein the hydrogel is in a sheet shape, a bead shape, or a fiber shape.
[4] The drug delivery carrier according to [3], wherein the hydrogel is in the form of beads.
[5] The drug delivery carrier according to [3], wherein the hydrogel is in the form of a fiber.
[6] The drug delivery carrier according to any one of [1] to [4], further comprising a fibrous extracellular matrix, wherein the extracellular matrix contains the hydrogel.
[7] A drug delivery system comprising the drug delivery carrier according to any one of [1] to [6] and a drug.
[8] The drug delivery system according to [7], further comprising a solubilizing agent for the hydrogel.
[9] The drug delivery system according to [7] or [8], which is a therapeutic agent for a disease.
[10] The drug delivery system according to [9], wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
[11] The drug delivery system according to any one of [7] to [10], wherein the drug includes a nucleic acid.
[12] The drug delivery system according to [11], further comprising a viral vector, wherein the viral vector comprises the nucleic acid.
[13] The nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell: 11] or the drug delivery system according to [12]:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above-mentioned (A) to ( Degenerate isomer of the base sequence of D).
［１］架橋性官能基を有する多糖を含むハイドロゲルを含む、薬物送達用担体。
［２］前記架橋性官能基を有する多糖は、アルギン酸、又はその塩である、［１］に記載の薬物送達用担体。
［３］前記ハイドロゲルは、シート状、ビーズ状、又はファイバ状である、［１］又は［２］に記載の薬物送達用担体。
［４］前記ハイドロゲルは、ビーズ状である、［３］に記載の薬物送達用担体。
［５］前記ハイドロゲルは、ファイバ状である、［３］に記載の薬物送達用担体。
［６］ファイバ状の細胞外マトリクスを更に含み、前記細胞外マトリクスは、前記ハイドロゲルを含む、［１］～［４］のいずれか一つに記載の薬物送達用担体。
［７］［１］～［６］のいずれか一つに記載の薬物送達用担体と、薬物とを含む、薬物送達システム。
［８］前記ハイドロゲルの可溶化剤を更に含む、［７］に記載の薬物送達システム。
［９］疾患治療剤である、［７］又は［８］に記載の薬物送達システム。
［１０］前記疾患治療剤は、難聴治療剤である、［９］に記載の薬物送達システム。
［１１］前記薬物は、核酸を含む、［７］～［１０］のいずれかに記載の薬物送達システム。
［１２］ウイルスベクターを更に含み、当該ウイルスベクターが前記核酸を含む、［１１］に記載の薬物送達システム。
［１３］前記核酸は、以下の（Ａ）～（Ｄ）のいずれか一つの塩基配列からなり、且つ、支持細胞を感覚有毛細胞に分化させる能力を有するタンパク質をコードする遺伝子を含む、［１１］又は［１２］に記載の薬物送達システム：
（Ａ）配列番号１で表される塩基配列、
（Ｂ）配列番号１で表される塩基配列において、１又は数個の塩基が欠失、置換、挿入、又は付加されている塩基配列、
（Ｃ）配列番号１で表される塩基配列において、同一性が７０％以上である塩基配列、
（Ｄ）配列番号１で表される塩基配列からなる遺伝子と相補的な塩基配列からなる遺伝子とストリンジェントな条件下でハイブリダイズすることができる塩基配列、及び
（Ｅ）前記（Ａ）～（Ｄ）の塩基配列の縮重異性体。 The present invention includes the following aspects.
[1] A drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
[2] The carrier for drug delivery according to [1], wherein the polysaccharide having a crosslinkable functional group is alginic acid or a salt thereof.
[3] The carrier for drug delivery according to [1] or [2], wherein the hydrogel is in a sheet shape, a bead shape, or a fiber shape.
[4] The drug delivery carrier according to [3], wherein the hydrogel is in the form of beads.
[5] The drug delivery carrier according to [3], wherein the hydrogel is in the form of a fiber.
[6] The drug delivery carrier according to any one of [1] to [4], further comprising a fibrous extracellular matrix, wherein the extracellular matrix contains the hydrogel.
[7] A drug delivery system comprising the drug delivery carrier according to any one of [1] to [6] and a drug.
[8] The drug delivery system according to [7], further comprising a solubilizing agent for the hydrogel.
[9] The drug delivery system according to [7] or [8], which is a therapeutic agent for a disease.
[10] The drug delivery system according to [9], wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
[11] The drug delivery system according to any one of [7] to [10], wherein the drug includes a nucleic acid.
[12] The drug delivery system according to [11], further comprising a viral vector, wherein the viral vector comprises the nucleic acid.
[13] The nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell: 11] or the drug delivery system according to [12]:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above-mentioned (A) to ( Degenerate isomer of the base sequence of D).
本発明によれば、内耳などの狭く複雑な構造をしている組織に安定して留置可能であり、薬剤の徐放制御を可能とする薬物送達用担体を提供することができる。
According to the present invention, it is possible to provide a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.
［薬物送達用担体］
一の実施形態において、本発明は、架橋性官能基を有する多糖を含むハイドロゲルを含む、薬物送達用担体を提供する。 [Drug Delivery Carrier]
In one embodiment, the present invention provides a carrier for drug delivery comprising a hydrogel comprising a polysaccharide having a crosslinkable functional group.
一の実施形態において、本発明は、架橋性官能基を有する多糖を含むハイドロゲルを含む、薬物送達用担体を提供する。 [Drug Delivery Carrier]
In one embodiment, the present invention provides a carrier for drug delivery comprising a hydrogel comprising a polysaccharide having a crosslinkable functional group.
架橋性官能基を有する多糖は、架橋剤により架橋されハイドロゲルを形成する。本発明において、ハイドロゲルとは、水又は水系の溶液を含むゲルを意味する。多糖の架橋方法としては、ハイドロゲルが３次元網目構造を形成する方法であれば限定されず、化学的架橋又は物理的架橋が挙げられる。具体的には、共有結合、水素結合、イオン結合による架橋が挙げられ、イオン結合による架橋が好ましい。イオン結合による架橋としては、２価の金属イオンによる架橋が好ましい。
(4) The polysaccharide having a crosslinkable functional group is crosslinked by a crosslinking agent to form a hydrogel. In the present invention, the hydrogel means a gel containing water or an aqueous solution. The method for crosslinking the polysaccharide is not limited as long as the hydrogel forms a three-dimensional network structure, and includes chemical crosslinking or physical crosslinking. Specifically, crosslinking by a covalent bond, a hydrogen bond, or an ionic bond can be mentioned, and crosslinking by an ionic bond is preferable. As the crosslinking by ionic bonding, crosslinking by divalent metal ions is preferable.
架橋性官能基としては、酸性官能基が好ましく、カルボキシル基、アルコール性水酸基、フェノール性水酸基が挙げられ、カルボキシル基がより好ましい。
架橋性官能基を有する多糖としては、アルギン酸、ペクチン酸、ヒアルロン酸、セルロース、キトサン、キチン、澱粉、デキストラン、ヘパリン、コンドロイチン、陽イオングアー、陽イオン澱粉、カルボキシメチルセルロース、カルボキシメチルキトサン、カルボキシメチルデキストラン、カルボキシメチル澱粉、キサンタン、カラヤゴム、ガッティゴム、ジェラン、寒天、ヘパリンスルフェート、コンドロイチンスルフェート、又はこれらの塩若しくは誘導体が挙げられる。
中でも、アルギン酸、ペクチン酸、ヒアルロン酸、又はこれらの塩若しくは誘導体が好ましく、アルギン酸、又はアルギン酸塩がより好ましい。アルギン酸塩としては、アルギン酸ナトリウム、アルギン酸カリウムが挙げられる。 As the crosslinkable functional group, an acidic functional group is preferable, and examples thereof include a carboxyl group, an alcoholic hydroxyl group, and a phenolic hydroxyl group, and a carboxyl group is more preferable.
Examples of polysaccharides having a crosslinkable functional group include alginic acid, pectic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethyl cellulose, carboxymethyl chitosan, carboxymethyl dextran, Examples include carboxymethyl starch, xanthan, karaya gum, gum gutti, gellan, agar, heparin sulfate, chondroitin sulfate, or salts or derivatives thereof.
Among them, alginic acid, pectic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid or alginates are more preferred. Alginates include sodium alginate and potassium alginate.
架橋性官能基を有する多糖としては、アルギン酸、ペクチン酸、ヒアルロン酸、セルロース、キトサン、キチン、澱粉、デキストラン、ヘパリン、コンドロイチン、陽イオングアー、陽イオン澱粉、カルボキシメチルセルロース、カルボキシメチルキトサン、カルボキシメチルデキストラン、カルボキシメチル澱粉、キサンタン、カラヤゴム、ガッティゴム、ジェラン、寒天、ヘパリンスルフェート、コンドロイチンスルフェート、又はこれらの塩若しくは誘導体が挙げられる。
中でも、アルギン酸、ペクチン酸、ヒアルロン酸、又はこれらの塩若しくは誘導体が好ましく、アルギン酸、又はアルギン酸塩がより好ましい。アルギン酸塩としては、アルギン酸ナトリウム、アルギン酸カリウムが挙げられる。 As the crosslinkable functional group, an acidic functional group is preferable, and examples thereof include a carboxyl group, an alcoholic hydroxyl group, and a phenolic hydroxyl group, and a carboxyl group is more preferable.
Examples of polysaccharides having a crosslinkable functional group include alginic acid, pectic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethyl cellulose, carboxymethyl chitosan, carboxymethyl dextran, Examples include carboxymethyl starch, xanthan, karaya gum, gum gutti, gellan, agar, heparin sulfate, chondroitin sulfate, or salts or derivatives thereof.
Among them, alginic acid, pectic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid or alginates are more preferred. Alginates include sodium alginate and potassium alginate.
架橋剤としては、２価の金属イオンを含むものが好ましく、Ｐｂ２+、Ｃｕ２+、Ｃｄ２+、Ｂａ２+、Ｓｒ２+、Ｃａ２＋、Ｃｏ２＋、Ｎｉ２＋、Ｚｎ２＋、Ｍｎ２＋、Ｍｇ２＋を含むものが挙げられ、Ｃａ２＋、Ｂａ２＋を含むものが好ましく、Ｃａ２＋を含むものがより好ましい。
As the cross-linking agent, those containing a divalent metal ion are preferable, and Pb 2+ , Cu 2+ , Cd 2+ , Ba 2+ , Sr 2+ , Ca 2+ , Co 2+ , Ni 2+ , Zn 2+ , Mn 2+ , Mg 2+ , those containing Ca 2+ and Ba 2+ are preferable, and those containing Ca 2+ are more preferable.
更に架橋剤は、水に溶かして水溶液の形態で用いることができることから、上記の２価の金属イオンを含む塩が好ましく、上記の陽イオンを含むハロゲン化物、水酸化物、炭酸塩、硝酸塩、酢酸塩がより好ましく、ハロゲン塩が更に好ましく、塩化カルシウムが特に好ましい。
Further, since the crosslinking agent can be used in the form of an aqueous solution by dissolving in water, a salt containing the above divalent metal ion is preferable, and a halide, hydroxide, carbonate, nitrate containing the above cation, Acetates are more preferred, halogen salts are even more preferred, and calcium chloride is particularly preferred.
ハイドロゲルは、例えば、架橋剤を含む溶液中に、架橋性官能基を有する多糖を含む溶液を押し出すことにより得られる。
架橋剤を含む溶液中の架橋剤の濃度としては、１ｍＭ～１０Ｍが好ましく、１０ｍＭ～５Ｍがより好ましく、１００ｍＭ～１０００ｍＭが特に好ましい。
架橋性官能基を有する多糖を含む溶液中の多糖の濃度としては、０．１重量％～５０重量％が好ましく、０．５重量％～２０重量％がより好ましく、１重量％～１０重量％が特に好ましい。ハイドロゲルは送達する部位、目的に合わせていかなる形状もとることができる。例えばビーズ上、シート状、ファイバー状などの形状があげられるが、これに限るものではない。 The hydrogel is obtained, for example, by extruding a solution containing a polysaccharide having a crosslinkable functional group into a solution containing a crosslinking agent.
The concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1 mM to 10 M, more preferably 10 mM to 5 M, and particularly preferably 100 mM to 1000 mM.
The concentration of the polysaccharide in the solution containing the polysaccharide having a crosslinkable functional group is preferably 0.1% by weight to 50% by weight, more preferably 0.5% by weight to 20% by weight, and more preferably 1% by weight to 10% by weight. Is particularly preferred. The hydrogel can take any shape depending on the site to be delivered and the purpose. For example, a shape such as a bead, a sheet, and a fiber may be used, but is not limited thereto.
架橋剤を含む溶液中の架橋剤の濃度としては、１ｍＭ～１０Ｍが好ましく、１０ｍＭ～５Ｍがより好ましく、１００ｍＭ～１０００ｍＭが特に好ましい。
架橋性官能基を有する多糖を含む溶液中の多糖の濃度としては、０．１重量％～５０重量％が好ましく、０．５重量％～２０重量％がより好ましく、１重量％～１０重量％が特に好ましい。ハイドロゲルは送達する部位、目的に合わせていかなる形状もとることができる。例えばビーズ上、シート状、ファイバー状などの形状があげられるが、これに限るものではない。 The hydrogel is obtained, for example, by extruding a solution containing a polysaccharide having a crosslinkable functional group into a solution containing a crosslinking agent.
The concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1 mM to 10 M, more preferably 10 mM to 5 M, and particularly preferably 100 mM to 1000 mM.
The concentration of the polysaccharide in the solution containing the polysaccharide having a crosslinkable functional group is preferably 0.1% by weight to 50% by weight, more preferably 0.5% by weight to 20% by weight, and more preferably 1% by weight to 10% by weight. Is particularly preferred. The hydrogel can take any shape depending on the site to be delivered and the purpose. For example, a shape such as a bead, a sheet, and a fiber may be used, but is not limited thereto.
ハイドロゲルは、ビーズ状とすることができる。ハイドロゲルビーズは、例えば、遠心力を用いたマイクロゲルビーズ作製法(非特許文献２参照。)により作製することができる。
ハイドロゲルビーズの有するポアサイズ（直径）としては、１ｎｍ～１０μｍが好ましく、５ｎｍ～１μｍがより好ましく、１０ｎｍ～２００ｎｍが特に好ましい。
ハイドロゲルビーズのサイズ（直径）としては、１μｍ～１０００μｍが好ましく、１０μｍ～５００μｍがより好ましく、５０μｍ～２００μｍが特に好ましい。 The hydrogel can be in the form of beads. Hydrogel beads can be produced, for example, by a microgel bead production method using centrifugal force (see Non-Patent Document 2).
The pore size (diameter) of the hydrogel beads is preferably 1 nm to 10 μm, more preferably 5 nm to 1 μm, and particularly preferably 10 nm to 200 nm.
The size (diameter) of the hydrogel beads is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
ハイドロゲルビーズの有するポアサイズ（直径）としては、１ｎｍ～１０μｍが好ましく、５ｎｍ～１μｍがより好ましく、１０ｎｍ～２００ｎｍが特に好ましい。
ハイドロゲルビーズのサイズ（直径）としては、１μｍ～１０００μｍが好ましく、１０μｍ～５００μｍがより好ましく、５０μｍ～２００μｍが特に好ましい。 The hydrogel can be in the form of beads. Hydrogel beads can be produced, for example, by a microgel bead production method using centrifugal force (see Non-Patent Document 2).
The pore size (diameter) of the hydrogel beads is preferably 1 nm to 10 μm, more preferably 5 nm to 1 μm, and particularly preferably 10 nm to 200 nm.
The size (diameter) of the hydrogel beads is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
本発明の薬物送達用担体は、ハイドロゲルを含むファイバ状の細胞外マトリクスを含むことが好ましく、その場合、前記ハイドロゲルは、ビーズ状であることが好ましい。本発明の薬物送達用担体を所望の形状を有するものとすることにより、生体内の所望の箇所に、係る薬物送達用担体を安定に留置することができる。例えば、本発明の薬物送達用担体をファイバ状の形状に調製することにより、アブミ骨が位置する空間の隙間といった、特異的形状を有する耳内空間に、本発明の薬物送達用担体を適切に留置することができる。
担 体 The carrier for drug delivery of the present invention preferably contains a fibrous extracellular matrix containing a hydrogel, in which case the hydrogel is preferably in the form of beads. By making the drug delivery carrier of the present invention have a desired shape, the drug delivery carrier can be stably placed at a desired location in a living body. For example, by preparing the drug delivery carrier of the present invention in a fiber-like shape, the drug delivery carrier of the present invention can be appropriately placed in an auricular space having a specific shape such as a gap in a space where stapes are located. Can be detained.
ファイバ状の細胞外マトリクスの直径としては、１μｍ～１０００μｍが好ましく、１０μｍ～５００μｍがより好ましく、５０μｍ～２００μｍが特に好ましい。
ファイバ状の細胞外マトリクスの長さとしては、１０μｍ～１００ｃｍが好ましく、１００μｍ～１０ｃｍがより好ましく、１ｍｍ～１ｃｍが特に好ましい。
ハイドロゲルを含むファイバ状の細胞外マトリクスの製造方法としては、例えば、ファイバ形状の型に、細胞外マトリクスを流し込むことにより作製することができる。 The diameter of the fibrous extracellular matrix is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous extracellular matrix is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.
A method for producing a fibrous extracellular matrix containing a hydrogel can be produced, for example, by pouring the extracellular matrix into a fiber-shaped mold.
ファイバ状の細胞外マトリクスの長さとしては、１０μｍ～１００ｃｍが好ましく、１００μｍ～１０ｃｍがより好ましく、１ｍｍ～１ｃｍが特に好ましい。
ハイドロゲルを含むファイバ状の細胞外マトリクスの製造方法としては、例えば、ファイバ形状の型に、細胞外マトリクスを流し込むことにより作製することができる。 The diameter of the fibrous extracellular matrix is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous extracellular matrix is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.
A method for producing a fibrous extracellular matrix containing a hydrogel can be produced, for example, by pouring the extracellular matrix into a fiber-shaped mold.
細胞外マトリクスとしては、コラーゲン（Ｉ型、ＩＩ型、ＩＩＩ型、Ｖ型、ＸＩ型等）、マウスＥＨＳ腫瘍抽出物（ＩＶ型コラーゲン、ラミニン、ヘパラン硫酸プロテオグリカン等を含む）より再構成された基底膜成分（商品名：マトリゲル）、グリコサミノグリカン、ヒアルロン酸、プロテオグリカン、ゼラチン等が挙げられる。
As extracellular matrix, basal reconstituted from collagen (type I, II, III, V, XI, etc.), mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.) Examples include membrane components (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin, and the like.
また、ハイドロゲル自体は、ファイバ状とすることもできる。
ファイバ状のハイドロゲルの直径としては、１μｍ～１０００μｍが好ましく、１０μｍ～５００μｍがより好ましく、５０μｍ～２００μｍが特に好ましい。
ファイバ状のハイドロゲルの長さとしては、１０μｍ～１００ｃｍが好ましく、１００μｍ～１０ｃｍがより好ましく、１ｍｍ～１ｃｍが特に好ましい。 Further, the hydrogel itself may be in the form of a fiber.
The diameter of the fibrous hydrogel is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous hydrogel is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.
ファイバ状のハイドロゲルの直径としては、１μｍ～１０００μｍが好ましく、１０μｍ～５００μｍがより好ましく、５０μｍ～２００μｍが特に好ましい。
ファイバ状のハイドロゲルの長さとしては、１０μｍ～１００ｃｍが好ましく、１００μｍ～１０ｃｍがより好ましく、１ｍｍ～１ｃｍが特に好ましい。 Further, the hydrogel itself may be in the form of a fiber.
The diameter of the fibrous hydrogel is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous hydrogel is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.
［薬物送達システム］
一の実施形態において、本発明は、上述した薬物送達用担体と、薬物とを含む、薬物送達システムを提供する。実施例において後述するように、本実施形態の薬物送達システムにより、細胞毒性を有さず、薬物を所望の器官へ、徐放性を制御した状態で送達することができる。 [Drug delivery system]
In one embodiment, the present invention provides a drug delivery system including the above-described drug delivery carrier and a drug. As described later in Examples, the drug delivery system according to the present embodiment can deliver a drug to a desired organ without controlled cytotoxicity, with controlled sustained release.
一の実施形態において、本発明は、上述した薬物送達用担体と、薬物とを含む、薬物送達システムを提供する。実施例において後述するように、本実施形態の薬物送達システムにより、細胞毒性を有さず、薬物を所望の器官へ、徐放性を制御した状態で送達することができる。 [Drug delivery system]
In one embodiment, the present invention provides a drug delivery system including the above-described drug delivery carrier and a drug. As described later in Examples, the drug delivery system according to the present embodiment can deliver a drug to a desired organ without controlled cytotoxicity, with controlled sustained release.
本実施形態の薬物送達システムは、疾患治療剤、造影剤等を薬剤として含む形態で利用することができる。
薬 物 The drug delivery system of the present embodiment can be used in a form containing a therapeutic agent for a disease, a contrast agent, and the like as a drug.
疾患治療剤の有効成分としては、低分子化合物、核酸、タンパク質、ペプチド等が挙げられる。低分子化合物としては、ハイドロゲルビーズの有するポアに担持されるように、ミセル化等によりサイズを調整したものが好ましい。核酸としては、ｃＤＮＡ、ｍＲＮＡ、ｓｉＲＮＡ、ｓｈＲＮＡ、ｍｉＲＮＡ、アンチセンスＲＮＡ等が挙げられる。タンパク質としては、酵素、抗体等が挙げられ、ペプチドとしては、リガンド、ワクチン等が挙げられる。
有効 Examples of the active ingredient of the therapeutic agent for a disease include low molecular weight compounds, nucleic acids, proteins, and peptides. As the low molecular weight compound, a compound whose size is adjusted by micelle formation or the like so as to be supported on the pores of the hydrogel beads is preferable. Examples of the nucleic acid include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA and the like. Examples of proteins include enzymes and antibodies, and examples of peptides include ligands and vaccines.
本実施形態の薬物送達システムを遺伝子治療に用いる場合、疾患治療剤としては、核酸が好ましい。遺伝子配列の欠損、挿入又は変異等により、患者において野生型遺伝子の発現が喪失又は抑制されている場合には、疾患治療剤として野生型遺伝子のｃＤＮＡを用いることが好ましい。また、疾患治療剤が核酸である場合には、デリバリー及び発現効率の観点から、ウイルスベクターが、疾患治療剤としての係るｃＤＮＡを含む形態をとることが好ましい。
用 い る When the drug delivery system of the present embodiment is used for gene therapy, a nucleic acid is preferable as the disease therapeutic agent. When the expression of a wild-type gene is lost or suppressed in a patient due to a deletion, insertion or mutation of the gene sequence, it is preferable to use a cDNA of the wild-type gene as a therapeutic agent for a disease. When the therapeutic agent for a disease is a nucleic acid, it is preferable that the viral vector has a form containing the cDNA as the therapeutic agent for a disease, from the viewpoint of delivery and expression efficiency.
ここで使用するウイルスベクターとしては、レトロウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター（ＡＡＶ）、ヘルペスウイルスベクター等が挙げられ、毒性が少ないという観点から、アデノ随伴ウイルスベクター（ＡＡＶ）が好ましい。
ウ イ ル ス The virus vector used here includes a retrovirus vector, an adenovirus vector, an adeno-associated virus vector (AAV), a herpes virus vector, and the like. From the viewpoint of low toxicity, an adeno-associated virus vector (AAV) is preferable.
本実施形態の薬物送達システムを、難聴に対する遺伝子治療に用いる場合、ａｔｏｎａｌ関連因子をコードする核酸を含むＡＡＶを用いることが好ましい。ａｔｏｎａｌ関連因子は、塩基性へリックス－ループ－へリックスファミリーに属する転写因子である。
用 い る When the drug delivery system of the present embodiment is used for gene therapy for hearing loss, it is preferable to use an AAV containing a nucleic acid encoding an atonal-related factor. Atonal-related factors are transcription factors belonging to the basic helix-loop-helix family.
ａｔｏｎａｌ関連因子をコードする遺伝子としては、以下の（Ａ）～（Ｄ）のいずれか一つの塩基配列からなり、且つ、支持細胞を感覚有毛細胞に分化させる能力を有するタンパク質をコードする遺伝子が挙げられる：
（Ａ）配列番号１で表される塩基配列、
（Ｂ）配列番号１で表される塩基配列において、１又は数個の塩基が欠失、置換、挿入、又は付加されている塩基配列、
（Ｃ）配列番号１で表される塩基配列において、同一性が７０％以上である塩基配列、
（Ｄ）配列番号１で表される塩基配列からなる遺伝子と相補的な塩基配列からなる遺伝子とストリンジェントな条件下でハイブリダイズすることができる塩基配列、及び
（Ｅ）前記（Ａ）～（Ｄ）の塩基配列の縮重異性体。 Examples of the gene encoding an atonal-related factor include a gene having a base sequence of any one of the following (A) to (D) and encoding a protein capable of differentiating a supporting cell into a sensory hair cell. Listed:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).
（Ａ）配列番号１で表される塩基配列、
（Ｂ）配列番号１で表される塩基配列において、１又は数個の塩基が欠失、置換、挿入、又は付加されている塩基配列、
（Ｃ）配列番号１で表される塩基配列において、同一性が７０％以上である塩基配列、
（Ｄ）配列番号１で表される塩基配列からなる遺伝子と相補的な塩基配列からなる遺伝子とストリンジェントな条件下でハイブリダイズすることができる塩基配列、及び
（Ｅ）前記（Ａ）～（Ｄ）の塩基配列の縮重異性体。 Examples of the gene encoding an atonal-related factor include a gene having a base sequence of any one of the following (A) to (D) and encoding a protein capable of differentiating a supporting cell into a sensory hair cell. Listed:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).
上記（Ａ）において配列番号１で表される塩基配列からなる遺伝子は、Ｈａｔｈ１（Ｈｕｍａｎ ａｔｏｎａｌ ｈｏｍｏｌｏｇ１）のｃＤＮＡである。
遺 伝 子 In the above (A), the gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 is cDNA of Hath1 (Human @ atonal @ homolog1).
上記（Ｂ）において、欠失、置換、挿入、又は付加されてもよい塩基の数としては、１ ～３２０個が好ましく、１～１６０個がより好ましく、１～８０個が更に好ましく、１～４０個が更に好ましく、１～２０個が特に好ましく、１～１０個が最も好ましい。
In the above (B), the number of bases which may be deleted, substituted, inserted, or added is preferably 1 to 320, more preferably 1 to 160, still more preferably 1 to 80, and 1 to 80. The number is more preferably 40, particularly preferably 1 to 20, and most preferably 1 to 10.
上記（Ｃ）において、同一性は、好ましくは７５％以上、より好ましくは８０％以上、さらに好ましくは８５％以上、特に好ましくは、９０％以上である。
に お い て In the above (C), the identity is preferably at least 75%, more preferably at least 80%, further preferably at least 85%, particularly preferably at least 90%.
上記（Ｄ）において、「ストリンジェントな条件下」とは、例えば、５×ＳＳＣ（２０×ＳＳＣの組成：３Ｍ 塩化ナトリウム，０．３Ｍ クエン酸溶液，ｐＨ７．０）、０．１重量％ Ｎ－ラウロイルサルコシン、０．０２重量％のＳＤＳ、２重量％の核酸ハイブルダイゼーション用ブロッキング試薬、及び５０％フォルムアミドから成るハイブリダイゼーションバッファー中で、５５～７０℃で数時間から一晩インキュベーションを行うことによりハイブリダイズさせる条件を挙げることができる。なお、インキュベーション後の洗浄の際に用いる洗浄バッファーとしては、好ましくは０．１重量％ＳＤＳ含有１×ＳＳＣ溶液、より好ましくは０．１重量％ＳＤＳ含有０．１×ＳＳＣ溶液である。
In the above (D), “under stringent conditions” means, for example, 5 × SSC (composition of 20 × SSC: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 0.1% by weight N Incubation at 55-70 ° C. for several hours to overnight in a hybridization buffer consisting of lauroyl sarcosine, 0.02% by weight SDS, 2% by weight nucleic acid hydridation blocking reagent and 50% formamide Thus, conditions for hybridization can be given. The washing buffer used for washing after incubation is preferably a 1 × SSC solution containing 0.1% by weight SDS, more preferably a 0.1 × SSC solution containing 0.1% by weight SDS.
メチオニンとトリプトファン以外のアミノ酸は、１つのアミノ酸に対して複数のコドンが対応する。このことを遺伝暗号の縮重という。上記（Ｅ）において、塩基配列の縮重異性体とは、ある塩基配列がコードするアミノ酸に対応する他の塩基配列を意味する。
ア ミ ノ 酸 For amino acids other than methionine and tryptophan, multiple codons correspond to one amino acid. This is called the degeneracy of the genetic code. In the above (E), the degenerate isomer of the base sequence means another base sequence corresponding to an amino acid encoded by a certain base sequence.
また、徐放性を制御する観点から、本実施形態の薬物送達システムは、前記ハイドロゲルの可溶化剤を更に含むことが好ましい。
ハイドロゲルの可溶化剤としては、イオン結合により架橋されてなる場合には、二価の金属イオンが取り除くキレート剤が挙げられる。また、架橋性官能基を有する多糖自体を分解する酵素も挙げられる。 From the viewpoint of controlling the sustained release, the drug delivery system of the present embodiment preferably further includes a solubilizing agent for the hydrogel.
Examples of the solubilizing agent for the hydrogel include a chelating agent for removing divalent metal ions when crosslinked by ionic bonding. In addition, an enzyme that decomposes a polysaccharide having a crosslinkable functional group itself is also included.
ハイドロゲルの可溶化剤としては、イオン結合により架橋されてなる場合には、二価の金属イオンが取り除くキレート剤が挙げられる。また、架橋性官能基を有する多糖自体を分解する酵素も挙げられる。 From the viewpoint of controlling the sustained release, the drug delivery system of the present embodiment preferably further includes a solubilizing agent for the hydrogel.
Examples of the solubilizing agent for the hydrogel include a chelating agent for removing divalent metal ions when crosslinked by ionic bonding. In addition, an enzyme that decomposes a polysaccharide having a crosslinkable functional group itself is also included.
キレート剤としては、クエン酸、エチレンジアミン、エチレンジアミン四酢酸、ニトリロ三酢酸、ジエチレントリアミン五酢酸、グリコールエーテルジアミン四酢酸等が挙げられる。
酵素としては、例えば、アルギン酸リアーゼ等が挙げられる。 Examples of the chelating agent include citric acid, ethylenediamine, ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, and glycol ether diaminetetraacetic acid.
Examples of the enzyme include alginate lyase.
酵素としては、例えば、アルギン酸リアーゼ等が挙げられる。 Examples of the chelating agent include citric acid, ethylenediamine, ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, and glycol ether diaminetetraacetic acid.
Examples of the enzyme include alginate lyase.
本実施形態の薬物送達システムは、ハイドロゲルのサイズや形状を調節することにより、またはこのハイドロゲルをゲルファイバなどの高分子構造物内に封入することにより、複雑な組織の内部に確実に留置することができる。
更に、上記ＡＡＶが担持されたハイドロゲルビーズを、アルギン酸リアーゼなどの酵素による分解、又は超音波などの照射にかけることにより、ＡＡＶの失活や周辺組織へのダメージなく、且つ意図したタイミングでのＡＡＶの徐放が可能となる。徐放されたＡＡＶは、ターゲットとする組織に所望の遺伝子を導入できる。 The drug delivery system of the present embodiment can be reliably placed inside a complex tissue by adjusting the size or shape of the hydrogel, or by enclosing the hydrogel in a polymer structure such as a gel fiber. can do.
Furthermore, by subjecting the hydrogel beads carrying the AAV to degradation by an enzyme such as alginate lyase, or irradiation of ultrasonic waves or the like, the AAV can be inactivated at desired timing without deactivation of AAV or damage to surrounding tissues. Can be sustained released. The sustained-release AAV can introduce a desired gene into a target tissue.
更に、上記ＡＡＶが担持されたハイドロゲルビーズを、アルギン酸リアーゼなどの酵素による分解、又は超音波などの照射にかけることにより、ＡＡＶの失活や周辺組織へのダメージなく、且つ意図したタイミングでのＡＡＶの徐放が可能となる。徐放されたＡＡＶは、ターゲットとする組織に所望の遺伝子を導入できる。 The drug delivery system of the present embodiment can be reliably placed inside a complex tissue by adjusting the size or shape of the hydrogel, or by enclosing the hydrogel in a polymer structure such as a gel fiber. can do.
Furthermore, by subjecting the hydrogel beads carrying the AAV to degradation by an enzyme such as alginate lyase, or irradiation of ultrasonic waves or the like, the AAV can be inactivated at desired timing without deactivation of AAV or damage to surrounding tissues. Can be sustained released. The sustained-release AAV can introduce a desired gene into a target tissue.
また、本実施形態の薬物送達システムにおいて薬剤として造影剤を用いる場合、当該薬剤として、硫酸バリウム、次炭酸ビスマス、酸化ビスマス、酸化ジルコニウム、フッ化イッテルビウム、ヨードホルム、バリウムアパタイト、チタン酸バリウム、ランタンガラス、バリウムガラス、ストロンチウムガラス等のＸ線造影剤；ヨード造影剤等のＣｏｍｐｕｔｅｄ Ｔｏｍｏｇｒａｐｈｙ（ＣＴ）用造影剤；ガドリニウム製剤、超常磁性酸化鉄製剤（Ｓｕｐｅｒ Ｐａｒａｍａｇｎｅｔｉｃ Ｉｒｏｎ Ｏｘｉｄｅ、ＳＰＩＯ）等のＭＲＩ用造影剤；テクネチウム９９ｍ（９９ｍＴｃ）、モリブデン９９（９９Ｍｏ）等のＳｉｎｇｌｅ ｐｈｏｔｏｎ ｅｍｉｓｓｉｏｎ ｃｏｍｐｕｔｅｄ ｔｏｍｏｇｒａｐｈｙ（ＳＰＥＣＴ）用放射性同位体等を好ましい薬剤として挙げることができる。
Further, when using a contrast agent as a drug in the drug delivery system of the present embodiment, as the drug, barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass Contrast agents for computed tomography (CT) such as iodine contrast agents; contrast agents for MRI such as gadolinium preparations, superparamagnetic iron oxide preparations (Super Paramagnetic Iron Oxide, SPIO); technetium 99m (99m Tc), molybdenum 99 (99 Mo) Single photon emission computed tomography (SPECT) for radioactive isotopes such like good It can be cited as Shii drug.
［治療方法］
一の実施形態において、本発明は、上述した薬物送達システムを、治療を必要とする患者に投与することを含む、感覚有毛細胞の欠陥又は喪失によって生じる疾患の治療方法を提供する。 [Method of treatment]
In one embodiment, the present invention provides a method of treating a disease caused by a defect or loss of sensory hair cells, comprising administering a drug delivery system as described above to a patient in need of treatment.
一の実施形態において、本発明は、上述した薬物送達システムを、治療を必要とする患者に投与することを含む、感覚有毛細胞の欠陥又は喪失によって生じる疾患の治療方法を提供する。 [Method of treatment]
In one embodiment, the present invention provides a method of treating a disease caused by a defect or loss of sensory hair cells, comprising administering a drug delivery system as described above to a patient in need of treatment.
感覚有毛細胞の欠陥又は喪失によって生じる疾患としては、難聴又は平衡障害が挙げられ、難聴が好ましい。内耳有毛細胞は、長期にわたって大きな音にさらされると破壊され、自然に再生することはないため、上述したａｔｏｎａｌ関連因子をコードする核酸を含むＡＡＶを担持したハイドロゲルを薬物送達システムとして用いた遺伝子治療を行うことが好ましい。
薬物送達用担体としては、ファイバ状のハイドロゲルを含むか、ビーズ状のハイドロゲルを含むファイバ状の細胞外マトリクスを含むことが好ましい。係る形状を有することにより、アブミ骨が位置する空間の隙間といった、内耳における空間に、内視鏡で確認しながら、本発明の薬物送達用担体を安定に留置することができる。 Diseases caused by sensory hair cell defect or loss include hearing loss or impaired balance, with hearing loss being preferred. Since the inner ear hair cells are destroyed when exposed to loud sounds for a long period of time and do not regenerate spontaneously, the hydrogel carrying AAV containing the nucleic acid encoding the atonal-related factor described above was used as a drug delivery system. Preferably, gene therapy is performed.
The carrier for drug delivery preferably contains a fibrous hydrogel or a fibrous extracellular matrix containing a beaded hydrogel. By having such a shape, the drug delivery carrier of the present invention can be stably placed in a space in the inner ear, such as a gap in the space where the stapes are located, while confirming the endoscope.
薬物送達用担体としては、ファイバ状のハイドロゲルを含むか、ビーズ状のハイドロゲルを含むファイバ状の細胞外マトリクスを含むことが好ましい。係る形状を有することにより、アブミ骨が位置する空間の隙間といった、内耳における空間に、内視鏡で確認しながら、本発明の薬物送達用担体を安定に留置することができる。 Diseases caused by sensory hair cell defect or loss include hearing loss or impaired balance, with hearing loss being preferred. Since the inner ear hair cells are destroyed when exposed to loud sounds for a long period of time and do not regenerate spontaneously, the hydrogel carrying AAV containing the nucleic acid encoding the atonal-related factor described above was used as a drug delivery system. Preferably, gene therapy is performed.
The carrier for drug delivery preferably contains a fibrous hydrogel or a fibrous extracellular matrix containing a beaded hydrogel. By having such a shape, the drug delivery carrier of the present invention can be stably placed in a space in the inner ear, such as a gap in the space where the stapes are located, while confirming the endoscope.
留置した後は、アルギン酸リアーゼを複数回、定期的に内耳に投与してもよい。係る投与により、ハイドロゲルに担持されたａｔｏｎａｌ関連因子をコードする核酸を含むＡＡＶの徐放を制御することができる。
し た After placement, alginate lyase may be administered to the inner ear several times periodically. By such administration, the sustained release of AAV containing the nucleic acid encoding the atonal-related factor carried on the hydrogel can be controlled.
ハイドロゲルに担持させるＡＡＶの用量としては、１０５ｇｃ／ｍＬ～１０１２ｇｃ／ｍＬが挙げられ、下限値としては、１０５ｇｃ／ｍＬ、１０６ｇｃ／ｍＬ、１０７ｇｃ／ｍＬ、１０８ｇｃ／ｍＬ、１０９ｇｃ／ｍＬ、１０１０ｇｃ／ｍＬ、１０１１ｇｃ／ｍＬが挙げられ、上限値としては、１０１２ｇｃ／ｍＬ、１０１１ｇｃ／ｍＬ、１０１０ｇｃ／ｍＬ、１０９ｇｃ／ｍＬ、１０８ｇｃ／ｍＬ、１０７ｇｃ／ｍＬ、１０６ｇｃ／ｍＬが挙げられる。
The dose of AAV supported on the hydrogel is 10 5 gc / mL to 10 12 gc / mL, and the lower limit is 10 5 gc / mL, 10 6 gc / mL, 10 7 gc / mL and 10 7 gc / mL. 8 gc / mL, 10 9 gc / mL, 10 10 gc / mL, 10 11 gc / mL, and the upper limit is 10 12 gc / mL, 10 11 gc / mL, 10 10 gc / mL, 10 9 gc / mL, 10 8 gc / mL, 10 7 gc / mL, and 10 6 gc / mL.
アルギン酸リアーゼの投与量としては、１回の投与につき、１μｇ～１０ｍｇが挙げられ、下限値としては、１μｇ、１０μｇ、１００μｇ、１ｍｇが挙げられ、上限値としては、１０ｍｇ、１ｍｇ、１００μｇ、１０μｇが挙げられる。
The dose of alginate lyase is 1 μg to 10 mg per administration, the lower limit is 1 μg, 10 μg, 100 μg, 1 mg, and the upper limit is 10 mg, 1 mg, 100 μg, 10 μg. No.
アルギン酸リアーゼの投与の代わりに、耳の所定の箇所に超音波を当てて、内耳に留置されたハイドロゲルを物理的に溶解させてもよい。
代 わ り Instead of administration of alginate lyase, ultrasound may be applied to a predetermined part of the ear to physically dissolve the hydrogel placed in the inner ear.
［その他の実施形態］
[Other Embodiments]
一の実施形態において、本発明は、感覚有毛細胞の欠陥又は喪失によって生じる疾患の治療のための薬物送達システムを提供する。薬物送達システムとしては、上述したものと同様のものを使用することができる。
In one embodiment, the present invention provides a drug delivery system for the treatment of a disease caused by a defect or loss of sensory hair cells. The same drug delivery system as described above can be used.
一の実施形態において、本発明は、感覚有毛細胞の欠陥又は喪失によって生じる疾患の治療のための医薬組成物であって、薬物送達用担体と、薬物とを含む、医薬組成物を提供する。薬物送達用担体、薬物としては、上述したものと同様のものを使用することができる。
In one embodiment, the present invention provides a pharmaceutical composition for the treatment of a disease caused by defect or loss of sensory hair cells, comprising a drug delivery carrier and a drug. . As the drug delivery carrier and drug, those similar to those described above can be used.
一の実施形態において、本発明は、感覚有毛細胞の欠陥又は喪失によって生じる疾患の治療として使用ための薬物送達システムを提供する。薬物送達システムとしては、上述したものと同様のものを使用することができる。
In one embodiment, the present invention provides a drug delivery system for use as a treatment for a disease caused by a defect or loss of sensory hair cells. The same drug delivery system as described above can be used.
次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
［実験例１］
（シリカビーズ含有マイクロビーズの安定性試験）
ＡＡＶが担持された直径約１００μｍのアルギン酸カルシウムゲルビーズを作製するための検証として、ＡＡＶと同程度の直径をもつ２０ｎｍ径の蛍光シリカビーズをＡＡＶと見立て、アルギン酸カルシウムゲルのマイクロビーズに封入した。
最終濃度が、アルギン酸ナトリウム２重量％、蛍光シリカ１０重量％となるように溶液を調製し、遠心力を用いたマイクロゲルビーズ作製法(K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340－1346 (2012)参照。)により、５００ｍＭ塩化カルシウム溶液中に射出した。
このマイクロビーズの塩化カルシウム溶液内での３日後の安定性を評価した。 [Experimental example 1]
(Stability test of microbeads containing silica beads)
As a test for producing calcium alginate gel beads having a diameter of about 100 μm carrying AAV, fluorescent silica beads having a diameter similar to that of AAV and having a diameter of 20 nm were regarded as AAV, and encapsulated in calcium alginate gel microbeads.
A solution was prepared so that the final concentration was 2% by weight of sodium alginate and 10% by weight of fluorescent silica, and microgel beads were prepared using centrifugal force (K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340-1346 (2012)).
The stability of the microbeads after 3 days in a calcium chloride solution was evaluated.
（シリカビーズ含有マイクロビーズの安定性試験）
ＡＡＶが担持された直径約１００μｍのアルギン酸カルシウムゲルビーズを作製するための検証として、ＡＡＶと同程度の直径をもつ２０ｎｍ径の蛍光シリカビーズをＡＡＶと見立て、アルギン酸カルシウムゲルのマイクロビーズに封入した。
最終濃度が、アルギン酸ナトリウム２重量％、蛍光シリカ１０重量％となるように溶液を調製し、遠心力を用いたマイクロゲルビーズ作製法(K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340－1346 (2012)参照。)により、５００ｍＭ塩化カルシウム溶液中に射出した。
このマイクロビーズの塩化カルシウム溶液内での３日後の安定性を評価した。 [Experimental example 1]
(Stability test of microbeads containing silica beads)
As a test for producing calcium alginate gel beads having a diameter of about 100 μm carrying AAV, fluorescent silica beads having a diameter similar to that of AAV and having a diameter of 20 nm were regarded as AAV, and encapsulated in calcium alginate gel microbeads.
A solution was prepared so that the final concentration was 2% by weight of sodium alginate and 10% by weight of fluorescent silica, and microgel beads were prepared using centrifugal force (K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340-1346 (2012)).
The stability of the microbeads after 3 days in a calcium chloride solution was evaluated.
図１Ａは、作製直後のマイクロビーズの蛍光像であり、図１Ｂは、塩化カルシウム溶液内で３日保存後のマイクロビーズの蛍光像である。図１Ｂに示されるように、マイクロゲルビーズ内にシリカビーズが安定して封入されることが確認された。
FIG. 1A is a fluorescence image of microbeads immediately after preparation, and FIG. 1B is a fluorescence image of microbeads after storage in a calcium chloride solution for 3 days. As shown in FIG. 1B, it was confirmed that the silica beads were stably encapsulated in the microgel beads.
［実験例２］
（マイクロビーズの徐放試験）
次に封入されたシリカビーズの徐放試験を行った。アルギン酸ナトリウム３重量％の水溶液を、１００ｍＭ塩化カルシウム溶液中に、実験例１と同様の手法で、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
このマイクロビーズを、ＮＩＨ／３Ｔ３細胞を培養したディシュに滴下した後、アルギン酸リアーゼを最終濃度が５０μｇ／ｍＬとなるようにディシュに滴下し、ビーズが溶けるまでの様子を観察したところ、数分で分解することに成功した。その後、アルギン酸リアーゼを滴下したこの細胞を３日間培養した後、生死判定試験により評価した。生細胞の染色に、カルセイン-ＡＭを用い（図２Ａ参照。）、死細胞の染色にエチジウムホモダイマーを用いた（図２Ｂ参照。）。図２Ａ及び図２Ｂの結果からわかるように、培養したほぼ全ての細胞が生存しており、徐放のプロセスにより細胞がダメージを受けないことが確認された。 [Experimental example 2]
(Sustained release test of microbeads)
Next, a sustained release test of the encapsulated silica beads was performed. An aqueous solution of 3% by weight of sodium alginate was injected into a 100 mM calcium chloride solution in the same manner as in Experimental Example 1 to produce calcium alginate gel microbeads (about 80 μm in diameter).
After the microbeads were dropped on the dish in which the NIH / 3T3 cells were cultured, alginate lyase was dropped on the dish such that the final concentration became 50 μg / mL, and the state until the beads were dissolved was observed. Successfully decomposed. Thereafter, the cells to which the alginate lyase had been dropped were cultured for 3 days, and then evaluated by a viability test. Calcein-AM was used to stain live cells (see FIG. 2A), and ethidium homodimer was used to stain dead cells (see FIG. 2B). As can be seen from the results of FIGS. 2A and 2B, almost all of the cultured cells were alive, and it was confirmed that the cells were not damaged by the sustained release process.
（マイクロビーズの徐放試験）
次に封入されたシリカビーズの徐放試験を行った。アルギン酸ナトリウム３重量％の水溶液を、１００ｍＭ塩化カルシウム溶液中に、実験例１と同様の手法で、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
このマイクロビーズを、ＮＩＨ／３Ｔ３細胞を培養したディシュに滴下した後、アルギン酸リアーゼを最終濃度が５０μｇ／ｍＬとなるようにディシュに滴下し、ビーズが溶けるまでの様子を観察したところ、数分で分解することに成功した。その後、アルギン酸リアーゼを滴下したこの細胞を３日間培養した後、生死判定試験により評価した。生細胞の染色に、カルセイン-ＡＭを用い（図２Ａ参照。）、死細胞の染色にエチジウムホモダイマーを用いた（図２Ｂ参照。）。図２Ａ及び図２Ｂの結果からわかるように、培養したほぼ全ての細胞が生存しており、徐放のプロセスにより細胞がダメージを受けないことが確認された。 [Experimental example 2]
(Sustained release test of microbeads)
Next, a sustained release test of the encapsulated silica beads was performed. An aqueous solution of 3% by weight of sodium alginate was injected into a 100 mM calcium chloride solution in the same manner as in Experimental Example 1 to produce calcium alginate gel microbeads (about 80 μm in diameter).
After the microbeads were dropped on the dish in which the NIH / 3T3 cells were cultured, alginate lyase was dropped on the dish such that the final concentration became 50 μg / mL, and the state until the beads were dissolved was observed. Successfully decomposed. Thereafter, the cells to which the alginate lyase had been dropped were cultured for 3 days, and then evaluated by a viability test. Calcein-AM was used to stain live cells (see FIG. 2A), and ethidium homodimer was used to stain dead cells (see FIG. 2B). As can be seen from the results of FIGS. 2A and 2B, almost all of the cultured cells were alive, and it was confirmed that the cells were not damaged by the sustained release process.
［実験例３］
（ＡＡＶ含有アルギン酸カルシウムゲルビーズの徐放試験）
シリカビーズの替わりにＡＡＶを用いたＤＤＳの検証試験を実施した。具体的には、感染により細胞にＧＦＰを発現するよう調製されたＡＡＶをアルギン酸カルシウムゲルのマイクロビーズに封入し、実施例２と同様の徐放試験を実施した。実施例３は、ＤＤＳ担体がＡＡＶの感染機能を維持できるかを実証するものである。
実験例１～２と同様の手法で、１．７×１０１０ｇｃ／ｍＬのＧＦＰを発現するＡＡＶを含むアルギン酸ナトリウム３重量％の水溶液を、１００ｍＭ、５００ｍＭ、１０００ｍＭの塩化カルシウム溶液中に、実験例１と同様の手法で、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度１０μｇ／ｍＬ、３０μｇ／ｍＬのアルギン酸リアーゼを、ＨｅＬａ細胞に滴下し、７２時間後のＧＦＰ発現細胞の割合を計測した。結果を図３に示す。図３中、左のレーンから、Ｃｏｎｔｒｏｌは、ＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、アルギン酸リアーゼ共に無添加のＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿０は、ＧＦＰを発現するＡＡＶのみを添加したＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿１０は、ＧＦＰを発現するＡＡＶ、及び、最終濃度１０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿３０は、ＧＦＰを発現するＡＡＶ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿０は、１００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズのみを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０は、１００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０＋ＣａＣｌ２＿５００は、５００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０＋ＣａＣｌ２＿１０００は、１０００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示す。 [Experimental example 3]
(Sustained release test of AAV-containing calcium alginate gel beads)
A verification test of DDS using AAV instead of silica beads was performed. Specifically, AAV prepared to express GFP in cells by infection was encapsulated in micro-beads of calcium alginate gel, and the same sustained release test as in Example 2 was performed. Example 3 demonstrates whether a DDS carrier can maintain the infectious function of AAV.
In the same manner as in Experimental Examples 1 and 2, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 10 gc / mL of GFP was placed in a 100 mM, 500 mM, or 1000 mM calcium chloride solution. In the same manner as in Example 1, injection was performed to produce calcium alginate gel microbeads (about 80 μm in diameter).
The prepared AAV-containing calcium alginate gel beads expressing GFP and alginate lyase at final concentrations of 10 μg / mL and 30 μg / mL were dropped on HeLa cells, and the proportion of GFP-expressing cells 72 hours later was measured. The results are shown in FIG. In FIG. 3, from the left lane, Control indicates AAV-containing calcium alginate gel beads expressing GFP and HeLa cells without addition of alginate lyase, and AAV + Lyase_0 indicates HeLa cells with only AAV expressing GFP added. AAV + Lyase_10 indicates AAV expressing GFP and HeLa cells to which alginate lyase at a final concentration of 10 μg / mL was added, and AAV + Lyase_30 indicates AAV expressing GFP and an alginate lyase at a final concentration of 30 μg / mL. AAV-Beads + Lyase_0 indicates HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP prepared in a 100 mM calcium chloride solution were added, AAV-Beads + Lyase_30 indicates AAV-containing calcium alginate gel beads expressing GFP prepared in 100 mM calcium chloride solution, and HeLa cells to which alginate lyase at a final concentration of 30 μg / mL was added. AAV-containing calcium alginate gel beads expressing GFP prepared in a calcium solution, and HeLa cells to which alginate lyase was added at a final concentration of 30 μg / mL are shown. AAV-Beads + Lyase_30 + CaCl2_1000 shows GFP prepared in a 1000 mM calcium chloride solution. Expressed AAV-containing calcium alginate gel beads and alginate lyase at a final concentration of 30 μg / mL Show the added HeLa cells.
（ＡＡＶ含有アルギン酸カルシウムゲルビーズの徐放試験）
シリカビーズの替わりにＡＡＶを用いたＤＤＳの検証試験を実施した。具体的には、感染により細胞にＧＦＰを発現するよう調製されたＡＡＶをアルギン酸カルシウムゲルのマイクロビーズに封入し、実施例２と同様の徐放試験を実施した。実施例３は、ＤＤＳ担体がＡＡＶの感染機能を維持できるかを実証するものである。
実験例１～２と同様の手法で、１．７×１０１０ｇｃ／ｍＬのＧＦＰを発現するＡＡＶを含むアルギン酸ナトリウム３重量％の水溶液を、１００ｍＭ、５００ｍＭ、１０００ｍＭの塩化カルシウム溶液中に、実験例１と同様の手法で、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度１０μｇ／ｍＬ、３０μｇ／ｍＬのアルギン酸リアーゼを、ＨｅＬａ細胞に滴下し、７２時間後のＧＦＰ発現細胞の割合を計測した。結果を図３に示す。図３中、左のレーンから、Ｃｏｎｔｒｏｌは、ＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、アルギン酸リアーゼ共に無添加のＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿０は、ＧＦＰを発現するＡＡＶのみを添加したＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿１０は、ＧＦＰを発現するＡＡＶ、及び、最終濃度１０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ＋Ｌｙａｓｅ＿３０は、ＧＦＰを発現するＡＡＶ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿０は、１００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズのみを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０は、１００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０＋ＣａＣｌ２＿５００は、５００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、ＡＡＶ－Ｂｅａｄｓ＋Ｌｙａｓｅ＿３０＋ＣａＣｌ２＿１０００は、１０００ｍＭの塩化カルシウム溶液中で作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示す。 [Experimental example 3]
(Sustained release test of AAV-containing calcium alginate gel beads)
A verification test of DDS using AAV instead of silica beads was performed. Specifically, AAV prepared to express GFP in cells by infection was encapsulated in micro-beads of calcium alginate gel, and the same sustained release test as in Example 2 was performed. Example 3 demonstrates whether a DDS carrier can maintain the infectious function of AAV.
In the same manner as in Experimental Examples 1 and 2, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 10 gc / mL of GFP was placed in a 100 mM, 500 mM, or 1000 mM calcium chloride solution. In the same manner as in Example 1, injection was performed to produce calcium alginate gel microbeads (about 80 μm in diameter).
The prepared AAV-containing calcium alginate gel beads expressing GFP and alginate lyase at final concentrations of 10 μg / mL and 30 μg / mL were dropped on HeLa cells, and the proportion of GFP-expressing cells 72 hours later was measured. The results are shown in FIG. In FIG. 3, from the left lane, Control indicates AAV-containing calcium alginate gel beads expressing GFP and HeLa cells without addition of alginate lyase, and AAV + Lyase_0 indicates HeLa cells with only AAV expressing GFP added. AAV + Lyase_10 indicates AAV expressing GFP and HeLa cells to which alginate lyase at a final concentration of 10 μg / mL was added, and AAV + Lyase_30 indicates AAV expressing GFP and an alginate lyase at a final concentration of 30 μg / mL. AAV-Beads + Lyase_0 indicates HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP prepared in a 100 mM calcium chloride solution were added, AAV-Beads + Lyase_30 indicates AAV-containing calcium alginate gel beads expressing GFP prepared in 100 mM calcium chloride solution, and HeLa cells to which alginate lyase at a final concentration of 30 μg / mL was added. AAV-containing calcium alginate gel beads expressing GFP prepared in a calcium solution, and HeLa cells to which alginate lyase was added at a final concentration of 30 μg / mL are shown. AAV-Beads + Lyase_30 + CaCl2_1000 shows GFP prepared in a 1000 mM calcium chloride solution. Expressed AAV-containing calcium alginate gel beads and alginate lyase at a final concentration of 30 μg / mL Show the added HeLa cells.
図３に示すように、ＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズのみを添加したＨｅＬａ細胞では、ＡＡＶの感染が抑えられ、アルギン酸カルシウムゲルが、ＡＡＶを担持することができることが確認された。更に、アルギン酸リアーゼを添加することにより、ＧＦＰを発現するＡＡＶの徐放が制御され、培養細胞におけるＧＦＰ遺伝子の発現が確認された。
示 す As shown in FIG. 3, in HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP were added, AAV infection was suppressed, and it was confirmed that the calcium alginate gel could carry AAV. Furthermore, by adding alginate lyase, the sustained release of AAV expressing GFP was controlled, and the expression of the GFP gene in cultured cells was confirmed.
［実験例４］
（４℃又は－８０℃で保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズの安定性試験）
実験例３と同様の手法で、１．７×１０１０ｇｃ／ｍＬのＧＦＰを発現するＡＡＶを含むアルギン酸ナトリウム３重量％の水溶液を、１００ｍＭの塩化カルシウム溶液中に、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズをＰＢＳに懸濁したビーズ懸濁液を、４℃又は－８０℃で７２時間保存した後、実験例３と同様の感染実験を行った。結果を図４に示す。図４中左のレーンから、Ｆｒｅｓｈ Ｂ（－）Ｌ（－）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶのみを添加したＨｅＬａ細胞を示し、Ｆｒｅｓｈ Ｂ（－）Ｌ（＋）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、Ｆｒｅｓｈ Ｂ（＋）Ｌ（＋）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、４Ｂ（＋）Ｌ（＋）は、４℃で７２時間保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、－８０Ｂ（＋）Ｌ（＋）は、－８０℃で７２時間保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示す。
図４に示すように、コントロールと比較して、４℃又は－８０℃で保存した後でも感染効率に違いがいないことから、ＡＡＶ含有アルギン酸カルシウムゲルビーズを４℃又は－８０℃で保存可能なことが確認された。 [Experimental example 4]
(Stability test of AAV-containing calcium alginate gel beads expressing GFP stored at 4 ° C. or −80 ° C.)
In the same manner as in Experimental Example 3, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 10 gc / mL of GFP was injected into a 100 mM calcium chloride solution, and a calcium alginate gel was obtained. Microbeads (about 80 μm in diameter) were prepared.
A bead suspension obtained by suspending the prepared GFP-expressing AAV-containing calcium alginate gel beads in PBS was stored at 4 ° C. or −80 ° C. for 72 hours, and the same infection experiment as in Experimental Example 3 was performed. FIG. 4 shows the results. From the left lane in FIG. 4, Fresh B (-) L (-) indicates HeLa cells to which only AAV expressing fresh GFP without preservation was added, and Fresh B (-) L (+) indicates AAV expressing fresh GFP without preservation and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL. Fresh B (+) L (+) shows fresh AAV-containing calcium alginate gel beads containing GFP expressing and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL are shown. 4B (+) L (+) shows AAV expressing GFP stored at 4 ° C. for 72 hours. Containing calcium alginate gel beads and HeLa microspheres added with a final concentration of 30 μg / mL alginate lyase. Are shown, -80B (+) L (+) shows AAV-containing calcium alginate gel beads expressing GFP stored at -80 ° C. 72 h, and HeLa cells was added alginate lyase a final concentration of 30 [mu] g / mL.
As shown in FIG. 4, the AAV-containing calcium alginate gel beads can be stored at 4 ° C. or −80 ° C. since there is no difference in the infection efficiency even after storage at 4 ° C. or −80 ° C. as compared with the control. Was confirmed.
（４℃又は－８０℃で保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズの安定性試験）
実験例３と同様の手法で、１．７×１０１０ｇｃ／ｍＬのＧＦＰを発現するＡＡＶを含むアルギン酸ナトリウム３重量％の水溶液を、１００ｍＭの塩化カルシウム溶液中に、射出し、アルギン酸カルシウムゲルのマイクロビーズ（直径約８０μｍ）を作製した。
作製したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズをＰＢＳに懸濁したビーズ懸濁液を、４℃又は－８０℃で７２時間保存した後、実験例３と同様の感染実験を行った。結果を図４に示す。図４中左のレーンから、Ｆｒｅｓｈ Ｂ（－）Ｌ（－）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶのみを添加したＨｅＬａ細胞を示し、Ｆｒｅｓｈ Ｂ（－）Ｌ（＋）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、Ｆｒｅｓｈ Ｂ（＋）Ｌ（＋）は、保存を行っていないフレッシュなＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、４Ｂ（＋）Ｌ（＋）は、４℃で７２時間保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示し、－８０Ｂ（＋）Ｌ（＋）は、－８０℃で７２時間保存したＧＦＰを発現するＡＡＶ含有アルギン酸カルシウムゲルビーズ、及び、最終濃度３０μｇ／ｍＬのアルギン酸リアーゼを添加したＨｅＬａ細胞を示す。
図４に示すように、コントロールと比較して、４℃又は－８０℃で保存した後でも感染効率に違いがいないことから、ＡＡＶ含有アルギン酸カルシウムゲルビーズを４℃又は－８０℃で保存可能なことが確認された。 [Experimental example 4]
(Stability test of AAV-containing calcium alginate gel beads expressing GFP stored at 4 ° C. or −80 ° C.)
In the same manner as in Experimental Example 3, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 10 gc / mL of GFP was injected into a 100 mM calcium chloride solution, and a calcium alginate gel was obtained. Microbeads (about 80 μm in diameter) were prepared.
A bead suspension obtained by suspending the prepared GFP-expressing AAV-containing calcium alginate gel beads in PBS was stored at 4 ° C. or −80 ° C. for 72 hours, and the same infection experiment as in Experimental Example 3 was performed. FIG. 4 shows the results. From the left lane in FIG. 4, Fresh B (-) L (-) indicates HeLa cells to which only AAV expressing fresh GFP without preservation was added, and Fresh B (-) L (+) indicates AAV expressing fresh GFP without preservation and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL. Fresh B (+) L (+) shows fresh AAV-containing calcium alginate gel beads containing GFP expressing and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL are shown. 4B (+) L (+) shows AAV expressing GFP stored at 4 ° C. for 72 hours. Containing calcium alginate gel beads and HeLa microspheres added with a final concentration of 30 μg / mL alginate lyase. Are shown, -80B (+) L (+) shows AAV-containing calcium alginate gel beads expressing GFP stored at -80 ° C. 72 h, and HeLa cells was added alginate lyase a final concentration of 30 [mu] g / mL.
As shown in FIG. 4, the AAV-containing calcium alginate gel beads can be stored at 4 ° C. or −80 ° C. since there is no difference in the infection efficiency even after storage at 4 ° C. or −80 ° C. as compared with the control. Was confirmed.
[実施例５]
（ハイドロゲルマイクロビーズへのＡＡＶ封入後、時間経過した後のウイルスの感染力評価試験）
感染により細胞にＧＦＰを発現するよう調製されたＡＡＶをアルギン酸カルシウムゲルのマイクロビーズに封入し、(i)封入から７２時間後まで保持した試料、(ii)マイクロビーズに封入後すぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料、及び(iii)マイクロビーズに封入後２４時間保持し、その後アルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料を調製した。リアーゼ濃度は、３３μｇ/ｍLとした。１well当たり５×１０3個の細胞に対して、MOI=1×１０6で上記(i)～(iii)の試料を使用した。細胞の感染率を試料(i)～資料(iii)、コントロール（ポジティブ、ネガティブ）について求めた。結果を図５に示す。 [Example 5]
(Evaluation test of infectivity of virus after elapse of time after AAV encapsulation in hydrogel microbeads)
AAV prepared to express GFP in cells by infection was encapsulated in calcium alginate gel microbeads, (i) a sample retained for up to 72 hours after encapsulation, and (ii) alginate lyase immediately after encapsulation in microbeads. Samples that were degraded and retained for up to 72 hours after encapsulation, and (iii) samples that were retained for 24 hours after encapsulation in microbeads and then degraded by alginate lyase to retain for up to 72 hours after encapsulation were prepared. The lyase concentration was 33 μg / mL. The samples (i) to (iii) were used at a MOI of 1 × 10 6 for 5 × 10 3 cells per well. The cell infection rate was determined for samples (i) to data (iii) and controls (positive and negative). FIG. 5 shows the results.
（ハイドロゲルマイクロビーズへのＡＡＶ封入後、時間経過した後のウイルスの感染力評価試験）
感染により細胞にＧＦＰを発現するよう調製されたＡＡＶをアルギン酸カルシウムゲルのマイクロビーズに封入し、(i)封入から７２時間後まで保持した試料、(ii)マイクロビーズに封入後すぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料、及び(iii)マイクロビーズに封入後２４時間保持し、その後アルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料を調製した。リアーゼ濃度は、３３μｇ/ｍLとした。１well当たり５×１０3個の細胞に対して、MOI=1×１０6で上記(i)～(iii)の試料を使用した。細胞の感染率を試料(i)～資料(iii)、コントロール（ポジティブ、ネガティブ）について求めた。結果を図５に示す。 [Example 5]
(Evaluation test of infectivity of virus after elapse of time after AAV encapsulation in hydrogel microbeads)
AAV prepared to express GFP in cells by infection was encapsulated in calcium alginate gel microbeads, (i) a sample retained for up to 72 hours after encapsulation, and (ii) alginate lyase immediately after encapsulation in microbeads. Samples that were degraded and retained for up to 72 hours after encapsulation, and (iii) samples that were retained for 24 hours after encapsulation in microbeads and then degraded by alginate lyase to retain for up to 72 hours after encapsulation were prepared. The lyase concentration was 33 μg / mL. The samples (i) to (iii) were used at a MOI of 1 × 10 6 for 5 × 10 3 cells per well. The cell infection rate was determined for samples (i) to data (iii) and controls (positive and negative). FIG. 5 shows the results.
図５に示されるように、試料(i)～試料(iii)の全てにおいて感染力が認められた。試料(i)における感染力は、試料(ii)及び試料(iii)における感染力と比較して有為に低かった。この結果は、AAVの感染がビーズによって抑制されたことを示すものである。
感染 As shown in FIG. 5, infectivity was observed in all of the samples (i) to (iii). Infectivity in sample (i) was significantly lower than infectivity in samples (ii) and (iii). This result indicates that AAV infection was suppressed by the beads.
[実施例６]
（コラーゲンゲルへのＡＡＶ含有ビーズの封入と、その後に分解処理にかけた試料の感染力評価試験）
感染により細胞にＧＦＰを発現するよう調製されたＡＡＶを含有するビーズ（Low Density, High Density）に関して、(i)Low Densityビーズをコラーゲンゲルに封入して７２時間保持した試料、 (ii)Low Densityビーズをコラーゲンゲルに封入してからすぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料、(iii)High Densityビーズをコラーゲンゲルに封入して７２時間保持した試料、(iv)High Densityビーズをコラーゲンゲルに封入してからすぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料を調製した。High Densityビーズは、ビーズ数がLow densityの１/１０であり、ウイルス濃度が１０倍のものとなっている。
リアーゼ濃度は、３３μｇ/ｍLとした。１well当たり５×１０3個の細胞に対して、MOI=1×１０6で上記(i)～(iv)の試料を使用した。細胞の感染率を試料(i)～試料(iv)、コントロール（ポジティブ、ネガティブ）について求めた。結果を図６に示す。 [Example 6]
(Evaluation of the infectivity of samples that have been subjected to encapsulation of AAV-containing beads in a collagen gel and subsequent degradation treatment)
Regarding beads containing AAV prepared to express GFP in cells by infection (Low Density, High Density), (i) a sample in which Low Density beads were encapsulated in a collagen gel and held for 72 hours, (ii) Low Density Immediately after encapsulating the beads in the collagen gel, the sample was degraded by alginate lyase and retained for up to 72 hours after encapsulation, (iii) High Density beads were encapsulated in the collagen gel and retained for 72 hours, Immediately after the Density beads were encapsulated in the collagen gel, the density beads were digested with alginate lyase to prepare a sample that was retained for up to 72 hours after the encapsulation. High Density beads have 1/10 the number of beads and 10 times the virus concentration of Low density.
The lyase concentration was 33 μg / mL. The samples (i) to (iv) were used at a MOI of 1 × 10 6 for 5 × 10 3 cells per well. The infection rate of the cells was determined for the samples (i) to (iv) and the controls (positive and negative). FIG. 6 shows the results.
（コラーゲンゲルへのＡＡＶ含有ビーズの封入と、その後に分解処理にかけた試料の感染力評価試験）
感染により細胞にＧＦＰを発現するよう調製されたＡＡＶを含有するビーズ（Low Density, High Density）に関して、(i)Low Densityビーズをコラーゲンゲルに封入して７２時間保持した試料、 (ii)Low Densityビーズをコラーゲンゲルに封入してからすぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料、(iii)High Densityビーズをコラーゲンゲルに封入して７２時間保持した試料、(iv)High Densityビーズをコラーゲンゲルに封入してからすぐにアルギン酸リアーゼにかけて分解し、封入から７２時間後まで保持した試料を調製した。High Densityビーズは、ビーズ数がLow densityの１/１０であり、ウイルス濃度が１０倍のものとなっている。
リアーゼ濃度は、３３μｇ/ｍLとした。１well当たり５×１０3個の細胞に対して、MOI=1×１０6で上記(i)～(iv)の試料を使用した。細胞の感染率を試料(i)～試料(iv)、コントロール（ポジティブ、ネガティブ）について求めた。結果を図６に示す。 [Example 6]
(Evaluation of the infectivity of samples that have been subjected to encapsulation of AAV-containing beads in a collagen gel and subsequent degradation treatment)
Regarding beads containing AAV prepared to express GFP in cells by infection (Low Density, High Density), (i) a sample in which Low Density beads were encapsulated in a collagen gel and held for 72 hours, (ii) Low Density Immediately after encapsulating the beads in the collagen gel, the sample was degraded by alginate lyase and retained for up to 72 hours after encapsulation, (iii) High Density beads were encapsulated in the collagen gel and retained for 72 hours, Immediately after the Density beads were encapsulated in the collagen gel, the density beads were digested with alginate lyase to prepare a sample that was retained for up to 72 hours after the encapsulation. High Density beads have 1/10 the number of beads and 10 times the virus concentration of Low density.
The lyase concentration was 33 μg / mL. The samples (i) to (iv) were used at a MOI of 1 × 10 6 for 5 × 10 3 cells per well. The infection rate of the cells was determined for the samples (i) to (iv) and the controls (positive and negative). FIG. 6 shows the results.
図６に示されるように、試料(iii)を除く、試料(i)、試料(ii)、及び試料(iv)において感染力が認められた。
感染 As shown in FIG. 6, infectivity was observed in sample (i), sample (ii), and sample (iv) except sample (iii).
本発明によれば、内耳などの狭く複雑な構造をしている組織に安定して留置可能であり、薬剤の徐放制御を可能とする薬物送達用担体を提供することができる。
According to the present invention, it is possible to provide a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.
Claims (13)
- 架橋性官能基を有する多糖を含むハイドロゲルを含む、薬物送達用担体。 (4) A drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
- 前記架橋性官能基を有する多糖は、アルギン酸、又はその塩である、請求項１に記載の薬物送達用担体。 The drug delivery carrier according to claim 1, wherein the polysaccharide having a crosslinkable functional group is alginic acid or a salt thereof.
- 前記ハイドロゲルは、シート状、ビーズ状、又はファイバ状である、請求項１又は２に記載の薬物送達用担体。 薬 物 The drug delivery carrier according to claim 1 or 2, wherein the hydrogel is in the form of a sheet, a bead, or a fiber.
- 前記ハイドロゲルは、ビーズ状である、請求項３に記載の薬物送達用担体。 薬 物 The drug delivery carrier according to claim 3, wherein the hydrogel is in the form of beads.
- 前記ハイドロゲルは、ファイバ状である、請求項３に記載の薬物送達用担体。 担 体 The drug delivery carrier according to claim 3, wherein the hydrogel is in the form of a fiber.
- ファイバ状の細胞外マトリクスを更に含み、前記細胞外マトリクスは、前記ハイドロゲルを含む、請求項１～４のいずれか一項に記載の薬物送達用担体。 The drug delivery carrier according to any one of claims 1 to 4, further comprising a fibrous extracellular matrix, wherein the extracellular matrix comprises the hydrogel.
- 請求項１～６のいずれか一項に記載の薬物送達用担体と、薬物とを含む、薬物送達システム。 (8) A drug delivery system comprising the drug delivery carrier according to any one of (1) to (6) and a drug.
- 前記ハイドロゲルの可溶化剤を更に含む、請求項７に記載の薬物送達システム。 The drug delivery system according to claim 7, further comprising a solubilizing agent for the hydrogel.
- 疾患治療剤である、請求項７又は８に記載の薬物送達システム。 The drug delivery system according to claim 7 or 8, which is an agent for treating a disease.
- 前記疾患治療剤は、難聴治療剤である、請求項９に記載の薬物送達システム。 The drug delivery system according to claim 9, wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
- 前記薬物は、核酸を含む、請求項７～１０のいずれか一項に記載の薬物送達システム。 薬 物 The drug delivery system according to any one of claims 7 to 10, wherein the drug includes a nucleic acid.
- ウイルスベクターを更に含み、当該ウイルスベクターが前記核酸を含む、請求項１１に記載の薬物送達システム。 12. The drug delivery system of claim 11, further comprising a viral vector, wherein the viral vector comprises the nucleic acid.
- 前記核酸は、以下の（Ａ）～（Ｄ）のいずれか一つの塩基配列からなり、且つ、支持細胞を感覚有毛細胞に分化させる能力を有するタンパク質をコードする遺伝子を含む、請求項１１又は１２に記載の薬物送達システム：
（Ａ）配列番号１で表される塩基配列、
（Ｂ）配列番号１で表される塩基配列において、１又は数個の塩基が欠失、置換、挿入、又は付加されている塩基配列、
（Ｃ）配列番号１で表される塩基配列において、同一性が７０％以上である塩基配列、
（Ｄ）配列番号１で表される塩基配列からなる遺伝子と相補的な塩基配列からなる遺伝子とストリンジェントな条件下でハイブリダイズすることができる塩基配列、及び
（Ｅ）前記（Ａ）～（Ｄ）の塩基配列の縮重異性体。 12. The nucleic acid according to claim 11, wherein the nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell. A drug delivery system according to item 12,
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114392361A (en) * | 2022-01-23 | 2022-04-26 | 重庆医科大学附属儿童医院 | A kind of carboxymethyl chitosan-adenovirus mixture and its application |
| EP4252790A1 (en) * | 2022-03-30 | 2023-10-04 | Nordovo Biosciences As | Use of polymer-based microcarriers in the production of tissue scaffolds with complex geometry |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0971542A (en) * | 1995-07-03 | 1997-03-18 | Sumitomo Pharmaceut Co Ltd | Gene product |
| JP2001524084A (en) * | 1997-04-17 | 2001-11-27 | アムジエン・インコーポレーテツド | Sustained release alginate gel |
| JP2005536513A (en) * | 2002-07-18 | 2005-12-02 | ネオックス、インコーポレイテッド | Oral administration of therapeutic agents conjugated to transport agents |
| JP2011503157A (en) * | 2007-11-14 | 2011-01-27 | エムアー．イ．アー ワウンドケアー | Biomaterials for controlled delivery of components |
| JP2011224378A (en) * | 2007-02-21 | 2011-11-10 | Mochida Pharmaceut Co Ltd | Composition for treatment of cartilage disease |
| JP2012513394A (en) * | 2008-12-22 | 2012-06-14 | オトノミ―，インク． | Controlled release of an ear sensory cell modulator composition for the treatment of otic disorders and methods thereof |
| WO2017153947A1 (en) * | 2016-03-09 | 2017-09-14 | Norwegian University Of Science And Technology | Methods of forming ionically cross-linked gels |
| JP2018059248A (en) * | 2016-10-07 | 2018-04-12 | 学校法人慶應義塾 | Stimulation-responsive fiber, stimulation-responsive fiber production method, and manufacturing apparatus of stimulation-responsive fiber |
-
2019
- 2019-08-20 WO PCT/JP2019/032411 patent/WO2020045162A1/en active Application Filing
- 2019-08-20 JP JP2020539368A patent/JPWO2020045162A1/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0971542A (en) * | 1995-07-03 | 1997-03-18 | Sumitomo Pharmaceut Co Ltd | Gene product |
| JP2001524084A (en) * | 1997-04-17 | 2001-11-27 | アムジエン・インコーポレーテツド | Sustained release alginate gel |
| JP2005536513A (en) * | 2002-07-18 | 2005-12-02 | ネオックス、インコーポレイテッド | Oral administration of therapeutic agents conjugated to transport agents |
| JP2011224378A (en) * | 2007-02-21 | 2011-11-10 | Mochida Pharmaceut Co Ltd | Composition for treatment of cartilage disease |
| JP2011503157A (en) * | 2007-11-14 | 2011-01-27 | エムアー．イ．アー ワウンドケアー | Biomaterials for controlled delivery of components |
| JP2012513394A (en) * | 2008-12-22 | 2012-06-14 | オトノミ―，インク． | Controlled release of an ear sensory cell modulator composition for the treatment of otic disorders and methods thereof |
| WO2017153947A1 (en) * | 2016-03-09 | 2017-09-14 | Norwegian University Of Science And Technology | Methods of forming ionically cross-linked gels |
| JP2018059248A (en) * | 2016-10-07 | 2018-04-12 | 学校法人慶應義塾 | Stimulation-responsive fiber, stimulation-responsive fiber production method, and manufacturing apparatus of stimulation-responsive fiber |
Non-Patent Citations (2)
| Title |
|---|
| MIYAHARA, HIDEO ET AL.: "Non-official translation: Controlled release triggered by enzymatic degradation for DDS using alginate hydrogel microparticles", THE PROCEEDINGS OF 2018 ANNUAL MEETING OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS, September 2018 (2018-09-01) * |
| MIYAHARA, HIDEO ET AL.: "Non-official translation: Drug delivery system using composite hydrogel microstructures controlled by enzymatic degradation", THE PROCEEDINGS OF THE 9TH SYMPOSIUM ON MICRO-NANO SCIENCE AND TECHNOLOGY, THE JAPAN SOCIETY OF MECHANICAL ENGINEERS, 30 October 2018 (2018-10-30) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114392361A (en) * | 2022-01-23 | 2022-04-26 | 重庆医科大学附属儿童医院 | A kind of carboxymethyl chitosan-adenovirus mixture and its application |
| EP4252790A1 (en) * | 2022-03-30 | 2023-10-04 | Nordovo Biosciences As | Use of polymer-based microcarriers in the production of tissue scaffolds with complex geometry |
| WO2023187018A1 (en) | 2022-03-30 | 2023-10-05 | Nordovo Biosciences As | Use of polymer-based microcarriers in the production of tissue scaffolds with complex geometry |
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