Drug delivery carrier and pharmaceutical composition

Abstract

To provide drug delivery carriers capable of releasing a drug by ultrasonic irradiation at such a low output that would not give damage to biological tissues and capable of stable sustained release of a drug over a longer period of time as well as pharmaceutical compositions using the drug delivery carrier.SOLUTION: The drug delivery carrier comprises microbeads comprising a hydrogel made by gelation of a polysaccharide having a cross-linkable functional group and particles of ultrasonic reflection material, and a coating layer comprising a hydrophilic polymer where the coating layer is coating the surface of the microbeads. The drug delivery carrier comprises microbeads comprising a hydrogel made by gelation of a polysaccharide having a cross-linkable functional group, and particles of inorganic phosphate embedded in the microbeads. The pharmaceutical composition comprises the drug delivery carrier and a drug, where the drug is embedded in the microbeads.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a drug delivery carrier and a pharmaceutical composition. [Background technology]

[0002] Drug delivery systems (DDS) have been proposed as a technology to efficiently exert drug effects while suppressing side effects by precisely controlling the pharmacokinetics of drugs in the body and ensuring they act at desired concentrations and time patterns. In particular, "on-demand DDS," which allows for more precise control of drug release from drug delivery carriers using external stimuli such as magnetic fields, electric fields, light, and ultrasound, has attracted attention in recent years. Ultrasound-based release control, in particular, is being actively researched because ultrasound has high spatial resolution, can easily deliver stimuli deep into biological tissues, and can achieve high output with simple equipment.

[0003] For example, Non-Patent Document 1 reports that by irradiating calcium alginate gel beads containing osteomorphic proteins with ultrasound, the osteomorphic proteins were released from the calcium alginate gel beads at a desired time, thereby promoting cell growth. The calcium alginate gel beads referred to here correspond to the drug delivery carriers mentioned above. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kearney CJ et al., “Switchable Release of Entrapped Nanoparticles from Alginate Hydrogels.”, Adv. Healthcare Mater., Vol. 4, pp. 1634-1639, 2015. [Overview of the project] [Problems that the invention aims to solve]

[0005] However, in the calcium alginate gel beads described in Non-Patent Document 1, drug release control depends on air bubbles (cavitation) contained in the drug delivery carrier, and there is room for improvement in terms of stable and efficient drug release control. In addition, in some cases, strong ultrasonic waves need to be irradiated to achieve drug release, which poses a risk of damaging living tissues. For these reasons, it is required to establish a method capable of safely and efficiently releasing drugs in a sustained manner when irradiated with sound waves.

[0006] The present invention has been made in view of the above circumstances, and provides a drug delivery carrier capable of releasing drugs by irradiating low-output ultrasonic waves that do not damage living tissues, and capable of controlling the sustained release of drugs more stably over a longer period than before, and a pharmaceutical composition using the drug delivery carrier.

Means for Solving the Problems

[0007] That is, the present invention includes the following aspects. (1) Microbeads composed of a hydrogel formed by gelating a polysaccharide having a crosslinkable functional group, Particles composed of an ultrasonic reflector embedded in the microbeads, A coating layer composed of a hydrophilic polymer covering the surface of the microbeads, A drug delivery carrier comprising: (2) The drug delivery carrier according to (1), wherein the polysaccharide having a crosslinkable functional group is alginic acid or a monovalent metal salt thereof. (3) The drug delivery carrier according to (2), wherein the hydrophilic polymer is a cationic hydrophilic polymer. (4) The drug delivery carrier according to (3), wherein the cationic hydrophilic polymer is one or more polyamino acids selected from the group consisting of poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly-L-ornithine. (5) The drug delivery carrier according to any one of (1) to (4), wherein the ultrasonic reflector is hydroxyapatite. Microbeads composed of a hydrogel formed by gelling a polysaccharide having a crosslinkable functional group, and particles composed of an inorganic phosphate embedded in the microbeads, and A carrier for drug delivery, comprising: (7) The carrier for drug delivery according to (6), wherein the inorganic phosphate is hydroxyapatite. (8) A pharmaceutical composition comprising the carrier for drug delivery according to any one of (1) to (7) and a drug, wherein the drug is embedded in the microbeads. (9) The pharmaceutical composition according to (8), which is for treating a disease. [Advantages of the Invention]

[0008] [[ID=ID=18]] According to the carrier for drug delivery of the above aspect, it is possible to release a drug by irradiating ultrasonic waves with a low output that does not damage living tissues, and a carrier for drug delivery capable of controlling the sustained release of a drug stably over a longer period than before can be provided. <00001)05>[Brief Description of the Drawings]

[0009] [Figure 1] It is a schematic configuration diagram of a carrier for drug delivery according to a first embodiment of the present invention. [Figure 2] It is a schematic configuration diagram of a carrier for drug delivery according to a second embodiment of the present invention. [Figure 3] It is a schematic configuration diagram of a pharmaceutical composition according to a third embodiment of the present invention. [Figure 4] It is a schematic configuration diagram of a pharmaceutical composition according to a fourth embodiment of the present invention. [Figure 5] It is a graph showing the relationship between the ultrasonic intensity and irradiation time in Experimental Example 1 and the release rate of fluorescent nanosilica. [Figure 6] It is a graph showing the relationship between the content of tungsten particles and the release rate of fluorescent nanosilica in Experimental Example 2. [Figure 7]This graph shows the results of confirming the stepwise control of fluorescent nanosilica emission using ultrasound in calcium alginate beads embedded with tungsten particles and fluorescent nanosilica in Experimental Example 3. [Figure 8A] This graph shows the results of confirming the stepwise emission control of fluorescent nanosilica by ultrasound for beads without poly-L-lysine (PLL) coating in Example 1. [Figure 8B] This graph shows the results of confirming the stepwise emission control of fluorescent nanosilica by ultrasound from beads with PLL coating in Example 1. [Figure 9A] These are fluorescence images of calcium alginate beads embedded with tungsten particles and fluorescently labeled bovine serum albumin in Experimental Example 4. The left image shows the fluorescence image of the beads before ultrasonic irradiation, and the right image shows the fluorescence image of the beads after ultrasonic irradiation. The scale bar is 100 μm. [Figure 9B] This graph shows the results of confirming the stepwise control of the release of fluorescently labeled bovine serum albumin by ultrasound using tungsten particles and calcium alginate beads embedded with fluorescently labeled bovine serum albumin in Experimental Example 4. [Figure 10] This graph shows the relationship between the hydroxyapatite particle content and the emission rate of fluorescent nanosilica in Example 2. [Modes for carrying out the invention]

[0010] <Drug delivery carrier> [First Embodiment] Figure 1 is a schematic diagram of a drug delivery carrier according to a first embodiment of the present invention. The drug delivery carrier 10 includes microbeads 1, particles 2 made of an ultrasonic reflector embedded in the microbeads 1, and a coating layer 3 made of a hydrophilic polymer covering the surface of the microbeads 1. The microbeads 1 are made of a hydrogel formed by gelling a polysaccharide having a crosslinkable functional group. The particles 2 made of the ultrasonic reflector can also be said to be discretely localized inside the microbeads 1.

[0011] By using a drug delivery carrier in which particles 2 made of an ultrasonic reflector are embedded in microbeads 1, the particles 2 made of the ultrasonic reflector vibrate when irradiated with ultrasound, expanding the pore size of the hydrogel network constituting the microbeads 1, and allowing for the sustained release of the drug. However, as shown in the examples described later, a problem with microbeads 1 embedded with particles 2 made of an ultrasonic reflector is that the drug gradually leaks out even before ultrasound irradiation, although the detailed mechanism is unknown. In contrast, the drug delivery carrier 10 according to the first embodiment of the present invention has a coating layer 3 made of a hydrophilic polymer on the surface of the microbeads 1, which effectively suppresses drug leakage before ultrasonic irradiation. In other words, the drug delivery carrier 10 can more precisely control the sustained release of the drug compared to one that does not have a coating layer 3 made of a hydrophilic polymer.

[0012] The average particle diameter of the drug delivery carrier 10 can be, for example, greater than 1 μm and less than or equal to 1000 μm, preferably between 10 μm and 500 μm, and more preferably between 50 μm and 400 μm. The average particle diameter of the drug delivery carrier 10 can be obtained, for example, by measuring the particle diameters of multiple drug delivery carriers 10 (approximately 3 to 1000) using a phase-contrast microscope and calculating their average value.

[0013] Next, the individual components of the drug delivery carrier 10 will be described in detail below.

[0014] [Microbeads] Microbeads 1 are spherical particles with a particle size on the order of micrometers. Microbeads are made of hydrogel.

[0015] Hydrogels are obtained by crosslinking polysaccharides having crosslinkable functional groups with a crosslinking agent to form a gel. In this specification, "hydrogel" means a gel containing water or an aqueous solution.

[0016] Hydrogels have an acoustic impedance close to that of water, which makes up the majority of biological tissue. Specifically, the acoustic impedance of hydrogels is 1.5 × 10⁻⁶. 6 N·S / m 3 In contrast, the acoustic impedance of water is 1.44 × 10⁻⁶. 6 N·S / m 3 Therefore, ultrasound irradiated from outside the body penetrates biological tissue and hydrogel.

[0017] The method for crosslinking polysaccharides is not limited to any method that forms a three-dimensional network structure in the hydrogel, and includes chemical crosslinking or physical crosslinking. Specifically, this includes crosslinking by covalent bonds, hydrogen bonds, and ionic bonds, with ionic crosslinking being preferred. Among ionic crosslinking methods, crosslinking by divalent metal ions is preferred.

[0018] Examples of crosslinkable functional groups include carboxyl groups, alcoholic hydroxyl groups, and phenolic hydroxyl groups, with the carboxyl group, which is an acidic functional group, being preferred. Examples of polysaccharides having crosslinkable functional groups include alginic acid, pectic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethylcellulose, carboxymethyl chitosan, carboxymethyl dextran, carboxymethyl starch, xanthan gum, karaya gum, gatti gum, gellan, agar, heparin sulfate, chondroitin sulfate, and salts or derivatives thereof. These polysaccharides may be used individually or in combination of two or more. Among these, alginic acid, pectic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid or a monovalent metal salt of alginic acid is more preferred. Examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate.

[0019] The crosslinking agent is preferably one containing a divalent metal ion. Examples of divalent metal ions include Pb. 2+ Cu 2+, Cd 2+ , Ba 2+ , Sr 2+ , Ca 2+ , Co 2+ , Ni 2+ , Zn 2+ , Mn 2+ , or Mg 2+ etc. Among these, Ca 2+ , or Ba 2+ is preferable, and those containing Ca 2+ are more preferable.

[0020] Furthermore, since the crosslinking agent can be dissolved in water and used in the form of an aqueous solution, salts containing the above divalent metal ions are preferable, halides, hydroxides, carbonates, nitrates, or acetates containing the above cations are more preferable, halide salts are even more preferable, and calcium chloride is particularly preferable.

[0021] The average particle diameter of the microbeads is preferably 1 μm or more and 1000 μm or less, more preferably 10 μm or more and 500 μm or less, and even more preferably 50 μm or more and 300 μm or less. The average particle diameter of the microbeads can be obtained, for example, by measuring the particle diameters of a plurality (about 3 or more and 1000 or less) of microbeads using a phase-contrast microscope and calculating their average value.

[0022] As the pore size of the hydrogel in the microbeads (the pore diameter of the network structure), 1 nm or more and 1 μm or less is preferable, 3 nm or more and 500 nm or less is more preferable, and 5 nm or more and 100 nm or less is even more preferable. The pore size of the hydrogel can be obtained, for example, by measuring the pore sizes of a plurality (about 3 or more and 1000 or less) of hydrogels using the dynamic light scattering method and calculating their average value.

[0023] [Particles made of ultrasonic reflectors] The particles made of the ultrasonic reflector are preferably made of a material whose difference in acoustic impedance is greater than the difference between the acoustic impedance of the hydrogel and the acoustic impedance of water, in order to efficiently reflect the irradiated ultrasonic waves. Specifically, the acoustic impedance of a hydrogel made of calcium alginate, etc., is 1.5 × 10⁻⁶. 6 N·S / m 3 The acoustic impedance of water is 1.44 × 10⁻⁶. 6 N·S / m 3 The difference is 0.06 × 10 6 N·S / m 3 Therefore, the difference between the acoustic impedance of the ultrasonic reflector and the acoustic impedance of water is, for example, 0.5 × 10⁻⁶. 6 N·S / m 3 It can be done as 1.0 × 10 6 N·S / m 3 It is preferable that this be the case. In other words, the specific range of the acoustic impedance of the ultrasonic reflector is 0.5 × 10⁻⁶. 6 N·S / m 3 The following, or 2.5 × 10 6 N·S / m 3 It is preferable that the above conditions are met. Examples of materials that fall within this range of acoustic impedance include copper (44.6 × 10 6 N·S / m 3 ), iron (46.4 x 10 6 N·S / m 3 ), tungsten (106 x 10 6 N·S / m 3 ) and other metals; magnesium (10.0 × 10 6 N·S / m 3 ), calcium (3.9 × 10 6 N·S / m 3 Examples include inorganic compounds such as salts of inorganic phosphoric acid or other acids or bases thereof. These materials may be used individually or in combination of two or more. In particular, it is preferable that the material be made of a biocompatible material, and inorganic phosphate is more preferable. Here, "biocompatibility" means the property of being able to coexist with biological components, biological tissues, and biologically derived substances while performing its original function without causing adverse effects or strong irritation to the living body over a long period of time. Examples of inorganic phosphates include tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), octacalcium phosphate (OCP), calcium pyrophosphate, amorphous calcium phosphate (ACP), hydroxyapatite (HAp), carbonate apatite, and wittrockite. Among these, hydroxyapatite (7.8 × 10) 6 N·S / m 3 ) is preferable.

[0024] The shape of the particles made of the ultrasonic reflector is not particularly limited, but it is preferably spherical. This allows for more efficient reflection of ultrasonic waves. When the particles made of ultrasonic reflector are spherical, their average particle diameter should be large enough to be embedded in the microbeads and sufficiently larger than the pore size of the hydrogel. For example, it can be 500 nm to 500 μm, preferably 600 nm to 300 μm, more preferably 700 nm to 100 μm, and even more preferably 800 nm to 10 μm. The average particle diameter of particles made of ultrasonic reflecting material can be obtained, for example, by measuring the particle diameters of multiple particles (approximately 3 to 1000) made of ultrasonic reflecting material using dynamic light scattering and calculating their average value.

[0025] [Coating layer] The coating layer consists of a hydrophilic polymer. The hydrophilic polymer should be capable of coating the surface of the microbeads and dissolving or dispersing the drug delivery carrier in an aqueous solution. Furthermore, it is preferable that the hydrophilic polymer is biocompatible. Specifically, cationic, anionic, or nonionic hydrophilic polymers can be used. For example, in the case of microbeads made of anionic hydrogels such as alginic acid or its monovalent metal salts, it is preferable to use a cationic hydrophilic polymer because the microbead surface can be easily coated by ionic bonding. On the other hand, in the case of microbeads made of cationic hydrogels, it is preferable to use an anionic hydrophilic polymer. Alternatively, in the case of microbeads made of nonionic hydrogels, any of cationic, anionic, or nonionic hydrophilic polymers may be used. In particular, it is preferable to use a combination of an anionic hydrogel and a cationic hydrophilic polymer.

[0026] Examples of such hydrophilic polymers include cationic polyamino acids such as poly-L-ornithine, poly-L-lysine, poly-L-arginine, and poly-L-histidine; anionic polyamino acids such as poly-L-glutamic acid and poly-L-aspartic acid; and nonionic polyamino acids such as polysarcosine. These hydrophilic polymers may be used individually or in combination of two or more. Furthermore, these polyamino acids are suitable materials for sustained-release formulations because, in addition to their biocompatibility, they are biodegradable and have low immunogenicity. In particular, in the case of microbeads made of anionic hydrogels, it is preferable to use one or more cationic polyamino acids selected from the group consisting of poly-L-ornithine, poly-L-lysine, poly-L-arginine, and poly-L-histidine.

[0027] The thickness of the coating layer can be, for example, 1 μm to 20 μm, or 5 μm to 10 μm.

[0028] [Second Embodiment] Figure 2 is a schematic diagram of a drug delivery carrier according to a second embodiment of the present invention. In Figures 2 and beyond, components common to the drug delivery carrier 10 shown in Figure 1 are denoted by the same reference numerals, and their detailed descriptions are omitted. The drug delivery carrier 20 differs from the drug delivery carrier 10 shown in Figure 1 in that, as particles 2 made of ultrasonic reflecting material, inorganic phosphate particles 2a are embedded in microbeads 1, and the surface of the microbeads 1 is not covered with a coating layer 3. Otherwise, it is the same as the drug delivery carrier 10. That is, the drug delivery carrier 20 includes microbeads 1 and inorganic phosphate particles 2a embedded in the microbeads 1.

[0029] The drug delivery carrier 20 can be made more biocompatible by embedding inorganic phosphate particles 2a as particles 2 made of an ultrasonic reflector. The inorganic phosphate constituting the inorganic phosphate particles 2a is the same as that exemplified in "Ultrasonic Reflector" above. The average particle diameter of the inorganic phosphate particles and the method of measuring it are as described in "Ultrasonic Reflector" above.

[0030] <Method for manufacturing drug delivery carriers> The drug delivery carrier of this embodiment can be produced, for example, by placing a solution containing a polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflector into a liquid gelling apparatus described in Japanese Patent Application Publication No. 2012-176374 (Reference 1) or "Maeda K et al., “Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barreled Capillary.”, Adv. Mater., Vol. 24, pp. 1340-1346, 2012." (Reference 2) and subjecting it to a microgel bead production method using centrifugal force (see References 1 and 2). Specifically, first, a solution containing a polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflector is added to the liquid gelling apparatus. Next, droplets controlled to have a predetermined particle size are released from the solution in the liquid gelling apparatus by centrifugal force. Next, the droplet is brought into contact with a solution containing a crosslinking agent. This allows the polysaccharide to gel, and a drug delivery carrier with a predetermined particle size is obtained.

[0031] Examples of crosslinking agents include those similar to those exemplified in the "hydrogel" section above.

[0032] 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 even more preferably 100 mM to 1000 mM. Here, the unit "M" is an abbreviation for mol / L.

[0033] The concentration of the polysaccharide in the solution containing the polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflector is preferably 0.1% by mass or more and 50% by mass or less, more preferably 0.5% by mass or more and 20% by mass or less, and even more preferably 1% by mass or more and 10% by mass or less.

[0034] The concentration of the ultrasonic reflector particles in a solution containing a polysaccharide having a crosslinkable functional group and ultrasonic reflector particles is sufficient if one or more ultrasonic reflector particles are present in one drug delivery carrier. For example, 0.1% by mass or more and 50% by mass or less is preferred, 0.5% by mass or more and 20% by mass or less is more preferred, and 1% by mass or more and 15% by mass or less is even more preferred.

[0035] Water or an aqueous solvent can be used as the solvent for dissolving or dispersing the crosslinking agent, or the polysaccharide and ultrasonic reflector. Specific examples of aqueous solvents include buffers known in this field, such as phosphate buffer, Tris-HCl buffer, citrate buffer, carbonate buffer, borate buffer, succinate buffer, and acetate buffer.

[0036] The buffer solution may contain NaCl, a surfactant (e.g., Tween 20, Triton X-100, etc.), or a preservative (e.g., sodium azide, etc.).

[0037] Specific examples of known buffer solutions include, but are not limited to, phosphate-buffered saline (PBS), PBS-T (PBS-Tween 20), Tris-buffered saline (TBS), and TBS-T (TBS-Tween 20).

[0038] In this case, the drug delivery carrier 20 can be prepared by using particles 2a made of inorganic phosphate as particles 2 made of ultrasonic reflector material.

[0039] Next, the microbeads embedded with the obtained ultrasonic reflector particles 2 are brought into contact with, for example, a solution containing a hydrophilic polymer. This causes the hydrophilic polymer to physically or chemically bond to the surface of the microbeads. As a result, the hydrophilic polymer coats the surface of the microbeads, forming a coating layer 3, thereby obtaining the drug delivery carrier 10. Examples of methods for contacting the microbeads with the solution containing the hydrophilic polymer include immersing the microbeads in the solution containing the hydrophilic polymer or dropping the solution containing the hydrophilic polymer onto the microbeads.

[0040] The concentration of the hydrophilic polymer in the solution containing the hydrophilic polymer can be, for example, 0.001 w / v% or more and 0.01 w / v% or less, for example, 0.0025 w / v% or more and 0.0075 w / v% or less.

[0041] Water or an aqueous solvent can be used as the solvent for dissolving or dispersing the hydrophilic polymer. The aqueous solvents mentioned above can be used.

[0042] <Pharmaceutical composition> [Third Embodiment] Figure 3 is a schematic diagram of a pharmaceutical composition according to a third embodiment of the present invention. The pharmaceutical composition 100 shown in Figure 3 differs from the drug delivery carrier 10 shown in Figure 1 in that it further contains a drug 11 inside the microbeads 1 of the drug delivery carrier 10, but is otherwise the same as the drug delivery carrier 10. That is, the pharmaceutical composition 100 comprises the drug delivery carrier 10 and the drug 11, with the drug 11 embedded in the microbeads 1.

[0043] The pharmaceutical composition 100, having the above configuration, allows for the sustained release of the drug 11 by vibrating particles 2 made of ultrasonic reflector material upon irradiation with ultrasound, thereby expanding the pore size of the hydrogel network constituting the microbeads 1. Furthermore, as described above, the pharmaceutical composition 100 effectively suppresses drug leakage before ultrasonic irradiation because the surface of the microbeads 1 is covered with a coating layer 3 made of a hydrophilic polymer. In other words, the pharmaceutical composition 100 allows for more precise control of the sustained release of the drug 11 compared to those without a coating layer. Moreover, the pharmaceutical composition 100 is cytotoxic and can deliver the drug 11 to the desired organ with controlled sustained release.

[0044] The pharmaceutical composition 100 can be prepared using the same method as the method for producing the drug delivery carrier 10 described in the above-mentioned "Method for Producing a Drug Delivery Carrier," except that it is added to a solution containing a polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflector, so that the drug 11 reaches a desired concentration.

[0045] [Drugs] The pharmaceutical composition 100 can be used in a form containing a disease treatment agent, a contrast agent, etc., as the drug 11. That is, the pharmaceutical composition 100 is suitably used for disease treatment and contrast imaging.

[0046] Examples of active ingredients in disease treatment agents include small molecule compounds, nucleic acids, proteins, and peptides. For small molecule compounds, it is preferable to adjust their size by micellization or other means so that they are supported on the pores of microbeads made of hydrogel. Examples of nucleic acids include cDNA, mRNA, siRNA, shRNA, miRNA, and antisense RNA. Examples of proteins include enzymes and antibodies, and examples of peptides include ligands and vaccines.

[0047] When the pharmaceutical composition of this embodiment is used in gene therapy, nucleic acids are preferred as the disease treatment agent. When the expression of the wild-type gene is lost or suppressed in the patient due to deletions, insertions, or mutations in the gene sequence, it is preferable to use the cDNA of the wild-type gene as the disease treatment agent. Furthermore, when the disease treatment agent is nucleic acid, from the viewpoint of delivery and expression efficiency, it is preferable that the viral vector takes the form of containing the cDNA as the disease treatment agent.

[0048] Examples of viral vectors used here include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (AAVs), and herpesvirus vectors. Adeno-associated virus vectors (AAVs) are preferred from the standpoint of low toxicity.

[0049] Examples of contrast agents include X-ray contrast agents such as barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass, barium glass, and strontium glass; contrast agents for computed tomography (CT) such as iodine contrast agents; contrast agents for MRI such as gadolinium preparations and super paramagnetic iron oxide preparations (SPIO); and radioactive isotopes for single-photon emission computed tomography (SPECT) such as technetium-99m (99mTc) and molybdenum-99 (99Mo).

[0050] Depending on the type of drug desired, the type and amount of hydrogel and hydrophilic polymer used in the coating layer, as well as the particle size of the microbeads, can be appropriately selected.

[0051] [Fourth Embodiment] Figure 4 is a schematic diagram of a pharmaceutical composition according to a fourth embodiment of the present invention. The pharmaceutical composition 200 shown in Figure 4 differs from the drug delivery carrier 20 shown in Figure 2 in that it further contains the drug 11 inside the microbeads 1 of the drug delivery carrier 20, but is otherwise the same as the drug delivery carrier 20. That is, the pharmaceutical composition 200 comprises the drug delivery carrier 20 and the drug 11, with the drug 11 embedded in the microbeads 1.

[0052] The pharmaceutical composition 200 can be made more biocompatible by embedding inorganic phosphate particles 2a as ultrasonic reflector particles 2.

[0053] The pharmaceutical composition 200 can be prepared using the same method as the method for producing the drug delivery carrier 20 described in the above-mentioned "Method for Producing a Drug Delivery Carrier," except that it is added to a solution containing a polysaccharide having a crosslinkable functional group and particles 2a consisting of an inorganic phosphate, so that the drug 11 reaches a desired concentration.

[0054] [Other ingredients] The pharmaceutical composition of this embodiment can be used in combination with a pharmaceutically acceptable carrier.

[0055] Pharmaceutically acceptable carriers can be those commonly used in the formulation of pharmaceutical compositions, without any particular limitations. More specifically, examples include binders such as gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as starch and crystalline cellulose; leavening agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives.

[0056] The pharmaceutical composition of this embodiment may further contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red ginger oil; stabilizers such as benzyl alcohol and phenol; buffering agents such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

[0057] The pharmaceutical composition of this embodiment can be formulated by mixing it with the above-mentioned pharmaceutically acceptable carriers and additives in a unit dose form generally accepted for pharmaceutical production.

[0058] The pharmaceutical composition of this embodiment may be in an orally administered dosage form or a parenterally administered dosage form, but an orally administered dosage form is preferred. Examples of orally administered dosage forms include tablets, capsules, elixirs, and microcapsules. Examples of parenterally administered dosage forms include injections, ointments, and patches.

[0059] <Treatment method> In one embodiment, the present invention provides a method for treating a disease, comprising administering the above-described pharmaceutical composition to a patient or animal in need of treatment, and irradiating the affected area of ​​the patient or animal in need of treatment with ultrasound.

[0060] The patient or animal is preferably a mammal with a specific disease. Examples of mammals include humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters, with humans being the most preferred.

[0061] Administration to patients or animals can be carried out by methods known to those skilled in the art, such as intrathecal injection, intra-arterial injection, intravenous injection, subcutaneous injection, local injection, intranasal injection, transbronchial injection, intramuscular injection, transcutaneous injection, or oral injection. The dosage and frequency of administration will vary depending on the type of disease, the type of drug, the weight and age of the patient or animal, the patient's symptoms, the method of administration, etc., but those skilled in the art can appropriately select the appropriate dosage and frequency of administration.

[0062] For the device that emits ultrasound, known devices used in medical applications can be used. The ultrasonic frequency can be, for example, between 20 kHz and 10 MHz, and the output intensity is 5.0 W / cm² in the case of continuous ultrasonic irradiation. 2 More than 50W / cm 2 The following can be used, and in the case of pulsed ultrasound irradiation, 25 W / cm². 2 More than 250W / cm 2 The following is possible: According to the pharmaceutical composition of this embodiment, the sustained release of the drug can be controlled safely and efficiently at a lower ultrasonic output intensity than in conventional methods. Furthermore, the ultrasonic frequency and output intensity can be set appropriately after confirming the sustained release properties through preliminary tests using the pharmaceutical composition to be used.

[0063] The ultrasound irradiation time can be, for example, between 10 seconds and 1 hour, or between 30 seconds and 30 minutes.

[0064] The number of ultrasound irradiations can be one time only on the affected area after a predetermined time has elapsed since administering the pharmaceutical composition, or multiple times at intervals of one day, three days, or one week, once or several times a day.

[0065] Furthermore, in the treatment method of this embodiment, in addition to ultrasonic irradiation, the sustained release of the drug can also be controlled by locally administering a hydrogel solubilizer to the affected area.

[0066] As solubilizing agents for hydrogels, when they are crosslinked by ionic bonds, chelating agents that remove divalent metal ions are available. Enzymes that decompose the polysaccharides themselves that have crosslinkable functional groups are also available.

[0067] Examples of chelating agents include citric acid, ethylenediamine, ethylenediamine tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriamine pentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), glycol ether diamine tetraacetic acid (GEDTA, EGTA), triethylene tetramine-N,N,N',N'',N''',N'''-hexaacetic acid (TTHA), N-(2-hydroxyethyl)iminodiacetic acid (HIDA), and N,N-di(2-hydroxyethyl)glycine (Dihydroxyethyl Examples include, but are not limited to, glycine (DHEG). Furthermore, citric acid may also be in the form of citrate salts such as sodium citrate or potassium citrate.

[0068] Examples of enzymes include, but are not limited to, λ-carraginase, pectinase, gellan gum-degrading enzyme, xyloglucan-degrading enzyme, hyaluronidase, and alginate lyase. [Examples]

[0069] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0070] [Experimental Example 1] (Changes in drug release rate in drug delivery models based on ultrasound intensity and irradiation time) A solution was prepared to have a final concentration of 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm (simulating small molecule drugs), and 3% by mass of tungsten particles with an average particle size of 1 μm. This solution was then injected into a 100 mmol / L calcium chloride solution using a microgel bead fabrication method with a centrifugal force of 200 × g (see References 1 and 2). The average particle size of the resulting calcium alginate gel beads, which embedded the fluorescent nanosilica and tungsten particles, was 220 μm. The average particle size of the calcium alginate gel beads was obtained by measuring the particle sizes of multiple beads using an inverted microscope (Olympus, IX73P1-22FL / PH) and calculating their average value.

[0071] Next, the obtained calcium alginate gel beads were dispersed in pure water and irradiated with ultrasound (20 kHz, 0, 5, 7.5, or 10 W) for 30 minutes using a homogenizer. The amount of fluorescent nanosilica emitted was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. The results are shown in Figure 5.

[0072] As shown in Figure 5, the amount of fluorescent nanosilica released tended to increase as the ultrasound irradiation intensity and irradiation time increased. This suggests that the amount of drug released can be controlled by changing the ultrasound irradiation intensity and irradiation time.

[0073] [Experimental Example 2] (Changes in drug release rate in drug delivery models depending on tungsten particle content) Solutions were prepared so that the final concentration was 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm, which simulates small molecules of drugs, and 0, 5, or 10% by mass of tungsten particles with an average particle size of 1 μm. Three types of beads with different tungsten particle contents were obtained using the same method as in Experimental Example 1.

[0074] Next, each of the obtained calcium alginate gel beads was dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 30 minutes. The amount of fluorescent nanosilica emitted was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. The results are shown in Figure 6.

[0075] As shown in Figure 6, the amount of fluorescent nanosilica emitted tended to increase with increasing tungsten particle content. Furthermore, beads with a tungsten particle content of 10% by mass showed 1.7 times more fluorescent nanosilica emission after 10 minutes of irradiation compared to beads without tungsten particles. These results suggest that the amount of drug released can be controlled by changing the content of particles made of ultrasonic reflective materials, such as tungsten particles.

[0076] [Experimental Example 3] (Confirmation of long-term sustained-release control in drug delivery models) Solutions were prepared so that the final concentration was 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm, which simulates small molecules of drugs, and 0, 1, or 3% by mass of tungsten particles with an average particle size of 1 μm. Three types of beads with different tungsten particle contents were obtained using the same method as in Experimental Example 1.

[0077] Next, each of the obtained calcium alginate gel beads was dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 3 minutes every other day, for a total of 3 times. The amount of fluorescent nanosilica emitted was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. The results are shown in Figure 7. In Figure 7, "0% NO US" indicates the group that did not undergo ultrasound irradiation using beads that did not contain tungsten particles.

[0078] As shown in Figure 7, ultrasonic irradiation triggered the release of fluorescent nanosilica and improved the sustained release rate of fluorescent nanosilica from the beads. Furthermore, by combining ultrasonic irradiation with the sustained release ability of the beads themselves, it was confirmed that 65% of the fluorescent nanosilica was released gradually over three days. These results suggest that the long-term sustained release of drugs can be controlled by changing the content of particles made of ultrasonic reflectors, such as tungsten particles.

[0079] [Example 1] (Confirmation of sustained-release control in drug delivery models using poly-L-lysine (PLL) coating) A solution was prepared to have a final concentration of 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm (simulating small molecule drugs), and 0 or 3% by mass of tungsten particles with an average particle size of 1 μm. Three types of beads with different tungsten particle contents were obtained using the same method as in Experimental Example 1. Next, some of the obtained beads containing tungsten particles and some of the beads without tungsten particles were added to a solution containing 0.005 w / v% poly-L-lysine (PLL) and stirred. This ionically bonded the positively charged PLL to the negatively charged surface of the beads, coating the bead surface with poly-L-lysine and obtaining PLL-coated beads. The average particle size of the PLL-coated beads was 248.7 μm. The average particle size of the beads was obtained by measuring the particle size of multiple beads using an inverted microscope (Olympus, IX73P1-22FL / PH) and calculating their average value.

[0080] Next, each of the obtained beads was dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 3 minutes every other day, for a total of 3 times. The amount of fluorescent nanosilica emitted was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. The results are shown in Figure 8A (beads without PLL coating) and Figure 8B (beads with PLL coating). In Figures 8A and 8B, "US" indicates ultrasound irradiation, "without W" indicates beads without tungsten particles, and "with W" indicates particles containing tungsten particles.

[0081] As shown in Figure 8A, it became clear that in beads without PLL coating, leakage of fluorescent nanosilica gradually occurs even before ultrasonic irradiation. In contrast, as shown in Figure 8B, no emission of fluorescent nanosilica was observed in the beads with the PLL coating until ultrasonic irradiation was performed, confirming that the leakage of the fluorescent nanosilica could be effectively suppressed. Therefore, it was suggested that applying a coating layer such as a PLL coating to the beads can achieve more precise control of drug sustained release.

[0082] [Experimental Example 4] (Confirmation of sustained release in beads embedded with fluorescently labeled bovine serum albumin) A solution was prepared to have a final concentration of 2% by mass of sodium alginate, 12% by mass of tetramethylrhodamine-labeled bovine serum albumin (BSA), and 3% by mass of tungsten particles with an average particle size of 1 μm. Calcium alginate beads embedded with tetramethylrhodamine-labeled BSA and tungsten particles were obtained using the same method as in Experimental Example 1.

[0083] Next, the obtained beads were dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 3 minutes every other day, for a total of 3 times. The amount of tetramethylrhodamine-labeled BSA released was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. Figure 9A shows the fluorescence images of the gel beads before and after ultrasound irradiation. Figure 9B is a graph showing the relationship between the number of ultrasound irradiations and the release rate of tetramethylrhodamine-labeled BSA.

[0084] As shown in Figures 9A and 9B, it was confirmed that tetramethylrhodamine-labeled BSA was released from the gel beads in a sustained manner by ultrasound irradiation. Furthermore, the release rate of tetramethylrhodamine-labeled BSA tended to increase with increasing ultrasound irradiation frequency. These results suggest that sustained release can be controlled by ultrasound irradiation even when a protein is used as a drug.

[0085] [Example 2] (Confirmation of sustained release properties in beads embedded with hydroxyapatite) A solution was prepared so that the final concentration consisted of 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm, which simulates small molecule drugs, and 0, 1, or 3% by mass of hydroxyapatite particles with a particle size of 1 μm to 5 μm, which serve as biocompatible vibration nuclei. Each bead was then obtained using the same method as in Experimental Example 1.

[0086] Next, each of the obtained calcium alginate gel beads was dispersed in pure water and irradiated with ultrasound (20 kHz, 2.5 W) using a homogenizer for 30 minutes. The amount of fluorescent nanosilica emitted was evaluated by measuring the fluorescence intensity of the solution with a fluorescence spectrophotometer. The results are shown in Figure 10. In Figure 10, "no US" indicates the group that was not irradiated with ultrasound, and "US 2.5W" indicates the group that was irradiated with ultrasound (20 kHz, 2.5 W).

[0087] As shown in Figure 10, similar to the case using tungsten particles, the amount of fluorescent nanosilica released tended to increase with increasing hydroxyapatite particle content. This result suggests that even with particles made of biocompatible ultrasonic reflective materials, such as hydroxyapatite particles, the amount of drug released can be controlled by changing their content. [Industrial applicability]

[0088] According to the drug delivery carrier of this embodiment, it is possible to release drugs by irradiation with low-power ultrasound that does not damage biological tissue, and a drug delivery carrier that enables stable sustained drug release control over a longer period than conventional carriers can be provided. [Explanation of Symbols]

[0089] 1...Microbeads, 2...Particles made of ultrasonic reflector, 2a...Particles made of inorganic phosphate, 3...Coating layer, 10, 20...Drug delivery carrier, 11...Drug, 100, 200...Pharmaceutical composition

Claims

1. Microbeads made of a hydrogel obtained by gelling a polysaccharide having a crosslinkable functional group; particles of an ultrasound reflecting material embedded in the microbeads; a coating layer made of a hydrophilic polymer that covers the surface of the microbeads; A drug delivery carrier comprising:

2. The drug delivery carrier according to claim 1 , wherein the polysaccharide having a crosslinkable functional group is alginic acid or a monovalent metal salt thereof.

3. The drug delivery carrier according to claim 2 , wherein the hydrophilic polymer is a cationic hydrophilic polymer.

4. 4. The drug delivery carrier according to claim 3, wherein the cationic hydrophilic polymer is one or more polyamino acids selected from the group consisting of poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly-L-ornithine.

5. The drug delivery carrier according to any one of claims 1 to 4, wherein the ultrasonic reflecting material is hydroxyapatite.

6. Microbeads made of a hydrogel obtained by gelling a polysaccharide having a crosslinkable functional group; particles comprising inorganic phosphate embedded in the microbeads; A drug delivery carrier comprising:

7. The drug delivery carrier according to claim 6, wherein the inorganic phosphate is hydroxyapatite.

8. A drug delivery carrier according to any one of claims 1 to 7, and a drug, A pharmaceutical composition wherein the drug is embedded in the microbeads.

9. The pharmaceutical composition according to claim 8, which is for treating a disease.

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