JP7668481B2 - Drug delivery carrier and pharmaceutical composition - Google Patents

Description

The present invention relates to a drug delivery carrier and a pharmaceutical composition.

Drug delivery systems (DDS) have been proposed as a technology for precisely controlling the pharmacokinetics of drugs and for efficiently exerting their efficacy by acting at the desired concentration and time pattern while suppressing side effects. In particular, in recent years, "on-demand DDS" has attracted attention, 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. In particular, release control using ultrasound has been actively researched, since ultrasound has high spatial resolution, can easily deliver stimuli deep into biological tissues, and can obtain high output with simple equipment.

For example, Non-Patent Document 1 reports that by irradiating calcium alginate gel beads containing bone morphogenetic protein with ultrasound, the bone morphogenetic protein is released from the calcium alginate gel beads at a desired time, promoting cell growth. The calcium alginate gel beads referred to here correspond to the above-mentioned drug delivery carrier.

Kearney CJ et al., “Switchable Release of Entrapped Nanoparticles from Alginate Hydrogels.”, Adv. Healthcare Mater., Vol. 4, pp. 1634-1639, 2015.

However, the calcium alginate gel beads described in Non-Patent Document 1 rely on air bubbles (cavitation) contained within the drug delivery carrier to control drug release, and there is room for improvement in terms of stable and efficient drug release control. In addition, there are cases where it is necessary to irradiate strong ultrasound to achieve drug release, which carries the risk of damaging biological tissue. For these reasons, there is a need to establish a method that can safely and efficiently release drugs sustainedly when irradiated with sound waves.

The present invention has been made in consideration of the above circumstances, and provides a drug delivery carrier that can release a drug by irradiating it with low-output ultrasound that does not damage biological tissue, and that can stably control the sustained release of the drug for a longer period of time than conventional methods, and a pharmaceutical composition that uses the drug delivery carrier.

That is, the present invention includes the following aspects.
(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 (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 reflecting material is hydroxyapatite.
(6) Microbeads made of a hydrogel obtained by gelling a polysaccharide having a crosslinkable functional group;
Particles comprised of inorganic phosphate embedded in the microbeads;
A drug delivery carrier comprising:
(7) The drug delivery carrier according to (6), wherein the inorganic phosphate is hydroxyapatite.
(8) A drug delivery carrier according to any one of (1) to (7) and a drug,
A pharmaceutical composition, wherein the drug is embedded in the microbeads.
(9) The pharmaceutical composition according to (8), which is for treating a disease.

The drug delivery carrier of the above embodiment can release drugs by irradiating them with low-power ultrasound that does not damage biological tissue, and can provide a drug delivery carrier that can stably control the sustained release of drugs for a longer period of time than conventional carriers.

1 is a schematic diagram of a drug delivery carrier according to a first embodiment of the present invention. FIG. FIG. 2 is a schematic diagram of a drug delivery carrier according to a second embodiment of the present invention. FIG. 1 is a schematic diagram of a pharmaceutical composition according to a third embodiment of the present invention. FIG. 1 is a schematic diagram of a pharmaceutical composition according to a fourth embodiment of the present invention. 1 is a graph showing the relationship between ultrasonic intensity and irradiation time and the emission rate of fluorescent nanosilica in Experimental Example 1. 1 is a graph showing the relationship between the content of tungsten particles and the emission rate of fluorescent nanosilica in Experimental Example 2. 13 is a graph showing the results of confirming the stepwise control of fluorescent nanosilica release by ultrasound from calcium alginate beads in which tungsten particles and fluorescent nanosilica are embedded in Experimental Example 3. 1 is a graph showing the results of confirming the stepwise controlled release of fluorescent nanosilica by ultrasound from beads not having a poly-L-lysine (PLL) coating in Example 1. 1 is a graph showing the results of confirming the stepwise controlled release of fluorescent nanosilica from beads having a PLL coating in Example 1 by ultrasound. Fluorescence images of calcium alginate beads in which tungsten particles and fluorescently labeled bovine serum albumin are embedded in Experimental Example 4. The left image is a fluorescence image of the beads before ultrasonic irradiation, and the right image is a fluorescence image of the beads after ultrasonic irradiation. The scale bar is 100 μm. 13 is a graph showing the results of confirming the stepwise controlled release of fluorescently labeled bovine serum albumin by ultrasound from tungsten particles and calcium alginate beads in which fluorescently labeled bovine serum albumin is embedded in Experimental Example 4. 1 is a graph showing the relationship between the content of hydroxyapatite particles and the release rate of fluorescent nanosilica in Example 2.

<Drug delivery carrier>
[First embodiment]
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 a microbead 1, particles 2 made of an ultrasound reflecting material embedded in the microbead 1, and a coating layer 3 made of a hydrophilic polymer that covers the surface of the microbead 1. The microbead 1 is made of a hydrogel obtained by gelling a polysaccharide having a crosslinkable functional group. It can also be said that the particles 2 made of the ultrasound reflecting material are discretely localized inside the microbead 1.

By using a drug delivery carrier in which particles 2 made of an ultrasound reflecting material are embedded in microbeads 1, the particles 2 made of ultrasound reflecting material vibrate upon application of ultrasound, expanding the pore size of the hydrogel mesh that constitutes the microbeads 1, enabling the drug to be released gradually. However, as shown in the examples described below, microbeads 1 in which particles 2 made of an ultrasound reflecting material are embedded have an issue in that the drug gradually leaks out even before application of ultrasound, although the detailed mechanism is unclear.
In contrast, the drug delivery carrier 10 according to the first embodiment of the present invention can effectively prevent the drug from leaking out before ultrasonic irradiation because the surfaces of the microbeads 1 are covered with a coating layer 3 made of a hydrophilic polymer. That is, the drug delivery carrier 10 can more strictly control the sustained release of the drug compared to a carrier that does not have a coating layer 3 made of a hydrophilic polymer.

The average particle diameter of the drug delivery carrier 10 can be, for example, more than 1 μm and not more than 1000 μm, preferably 10 μm or more and not more than 500 μm, and more preferably 50 μm or more and not more than 400 μm. The average particle diameter of the drug delivery carrier 10 can be obtained, for example, by using a phase contrast microscope to measure the particle diameters of multiple drug delivery carriers 10 (approximately 3 to 1000 particles) and calculating the average value.

Next, each component of the drug delivery carrier 10 will be described in detail below.

[Microbeads]
The microbeads 1 are spherical particles having a particle size on the order of microns and are made of hydrogel.

Hydrogel is obtained by gelling polysaccharides having crosslinkable functional groups through crosslinking with a crosslinking agent. In this specification, "hydrogel" means a gel containing water or an aqueous solution.

The acoustic impedance of hydrogel is close to that of water, which constitutes most of biological tissues. Specifically, the acoustic impedance of hydrogel is 1.5×10 ⁶ N·S/m ³ , while the acoustic impedance of water is 1.44×10 ⁶ N·S/m ^3. Therefore, ultrasound irradiated from outside the body passes through biological tissues and hydrogel.

The method of crosslinking polysaccharides is not limited as long as it is a method that allows the hydrogel to form a three-dimensional network structure, and examples of such methods include chemical crosslinking and physical crosslinking. Specific examples of such methods include crosslinking by covalent bonds, hydrogen bonds, and ionic bonds, with crosslinking by ionic bonds being preferred. As crosslinking by ionic bonds, crosslinking by divalent metal ions is preferred.

Examples of the crosslinkable functional group include a carboxy group, an alcoholic hydroxyl group, and a phenolic hydroxyl group. Among these, the carboxy group, which is an acidic functional group, is preferred.
Examples of polysaccharides having crosslinkable functional groups include alginic acid, pectinic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethylcellulose, carboxymethylchitosan, carboxymethyldextran, carboxymethylstarch, xanthan, karaya gum, ghatti gum, gellan, agar, heparin sulfate, chondroitin sulfate, and salts or derivatives thereof. These polysaccharides may be used alone or in combination of two or more.
Among them, alginic acid, pectinic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid or monovalent metal salts of alginic acid are more preferred. Examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate.

The crosslinking agent preferably contains a divalent metal ion. Examples of the divalent metal ion include Pb ²⁺ , Cu ²⁺ , Cd 2+ , Ba ²⁺ , Sr ²⁺ , Ca ²⁺ , Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Mn ²⁺ , and Mg ²⁺ . Among them, the crosslinking agent containing Ca ²⁺ or Ba ²⁺ is preferable, and the ^crosslinking agent containing Ca ²⁺ is more preferable.

Furthermore, since the crosslinking agent can be dissolved in water and used in the form of an aqueous solution, a salt containing the above-mentioned divalent metal ion is preferable, a halide, hydroxide, carbonate, nitrate, or acetate containing the above-mentioned cation is more preferable, a halogen salt is even more preferable, and calcium chloride is particularly preferable.

The average particle size 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 size of the microbeads can be obtained, for example, by using a phase contrast microscope to measure the particle sizes of multiple microbeads (approximately 3 to 1000 microbeads) and calculating the average value.

The pore size (diameter of the mesh structure) of the hydrogel in the microbeads is preferably 1 nm to 1 μm, more preferably 3 nm to 500 nm, and even more preferably 5 nm to 100 nm. The pore size of the hydrogel can be obtained, for example, by measuring the pore sizes of multiple hydrogels (approximately 3 to 1000) using dynamic light scattering and calculating the average value.

[Particles made of ultrasonic reflecting material]
In order to efficiently reflect the irradiated ultrasonic waves, the particles made of the ultrasonic reflecting material are preferably made of a material whose acoustic impedance difference with water is larger than the difference between the acoustic impedance of the hydrogel and the acoustic impedance of water. Specifically, the acoustic impedance of the hydrogel made of calcium alginate or the like is 1.5×10 ⁶ NS/m ³ , and the acoustic impedance of water is 1.44×10 ⁶ NS/m ³ , and the difference is 0.06×10 ⁶ NS/m ^3. Therefore, the difference between the acoustic impedance of the ultrasonic reflecting material and the acoustic impedance of water can be, for example, 0.5×10 ⁶ NS/m ³ , and is preferably 1.0×10 ⁶ NS/m ³ .
Specifically, the acoustic impedance of the ultrasonic reflecting material is preferably in the range of 0.5×10 ⁶ N·S/m ³ or less, or 2.5×10 ⁶ N·S/m ³ or more.
Examples of materials that have an acoustic impedance in this range include metals such as copper (44.6×10 ⁶ N·S/m ³ ), iron (46.4×10 ⁶ N·S/m ³ ), and tungsten (106×10 ⁶ N·S/m ³ ), and inorganic compounds such as magnesium (10.0×10 ⁶ N·S/m ³ ), calcium (3.9×10 ⁶ N·S/m ³ ), and salts of these with acids or bases such as inorganic phosphoric acid. These materials may be used alone or in combination of two or more.
Among them, it is preferable to use a material having biocompatibility, and inorganic phosphate is more preferable. "Biocompatibility" means a property that can coexist with biological components, biological tissues, and biological substances while performing its original function without adversely affecting or strongly irritating the living body for a long period of time. Examples of inorganic phosphate include tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), octacalcium phosphate (OCP), calcium pyrophosphate, amorphous calcium phosphate (ACP), hydroxyapatite (HAp), carbonate apatite, and Whitlockite. Among them, hydroxyapatite (7.8 x ¹⁰⁶ N.S/ ^m3 ) is preferable.

The shape of the particles made of the ultrasonic reflecting material is not particularly limited, but it is preferable that they are spherical, which allows them to reflect ultrasonic waves more efficiently.
When the particles made of the ultrasound reflecting material are spherical, the average particle size can be embedded in the microbeads and can be sufficiently larger than the pore size of the hydrogel, and can be, for example, 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 size of the particles made of ultrasonic reflecting material can be obtained, for example, by measuring the particle sizes of multiple particles made of ultrasonic reflecting material (approximately 3 to 1,000 particles) using a dynamic light scattering method and calculating the average value of the particle sizes.

[Coating layer]
The coating layer is made of a hydrophilic polymer. The hydrophilic polymer may be any polymer capable of coating the surface of the microbeads and dissolving or dispersing the drug delivery carrier in an aqueous solution. In addition, the hydrophilic polymer is preferably biocompatible. Specifically, cationic, anionic or nonionic hydrophilic polymers can be used as the hydrophilic polymer. For example, in the case of microbeads made of an anionic hydrogel such as alginic acid or a monovalent metal salt thereof, it is preferable to use a cationic hydrophilic polymer since the microbead surface can be easily coated by ionic bonds. On the other hand, in the case of microbeads made of a cationic hydrogel, it is preferable to use an anionic hydrophilic polymer. Alternatively, in the case of microbeads made of a nonionic hydrogel, any of cationic, anionic and nonionic hydrophilic polymers may be used.
Among these, it is preferable to use a combination of an anionic hydrogel and a cationic hydrophilic polymer.

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 alone or in combination of two or more. Furthermore, these polyamino acids are suitable materials for sustained-release preparations because they are biocompatible, biodegradable, and have low immunogenicity.
Among these, in the case of microbeads made of anionic hydrogel, 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.

The thickness of the coating layer can be, for example, 1 μm or more and 20 μm or less, for example, 5 μm or more and 10 μm or less.

[Second embodiment]
Fig. 2 is a schematic diagram of a drug delivery carrier according to a second embodiment of the present invention. In Fig. 2 and subsequent figures, components common to the drug delivery carrier 10 shown in Fig. 1 are denoted by the same reference numerals, and detailed descriptions thereof will be omitted.
1 in that particles 2a made of inorganic phosphate are embedded in microbeads 1 as particles 2 made of ultrasound reflecting material, and the surfaces of the microbeads 1 are not covered with a coating layer 3, but otherwise the drug delivery carrier 20 is the same as the drug delivery carrier 10. That is, the drug delivery carrier 20 includes microbeads 1 and particles 2a made of inorganic phosphate embedded in the microbeads 1.

The drug delivery carrier 20 can be made more biocompatible by embedding particles 2a made of inorganic phosphate as particles 2 made of ultrasound reflecting material. Examples of inorganic phosphate constituting particles 2a made of inorganic phosphate include the same as those exemplified in the above "ultrasound reflecting material". The average particle size of particles made of inorganic phosphate and the method for measuring the same are as described in the above "ultrasound reflecting material".

<Method of manufacturing drug delivery carrier>
The drug delivery carrier of this embodiment can be prepared by, for example, placing a solution containing a polysaccharide having a crosslinkable functional group and particles made of an ultrasound reflecting material in a liquid gelation device described in JP 2012-176374 A (Reference 1) or "Maeda K et al., "Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary.", Adv. Mater., Vol. 24, pp. 1340-1346, 2012." (Reference 2) and subjecting it to a microgel bead preparation 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 ultrasound reflecting material is added to a liquid gelation device. Next, in the liquid gelation device, droplets controlled to have a predetermined particle size are released from the solution by centrifugal force. The droplets are then contacted with a solution containing a crosslinking agent, which causes the polysaccharide to gel and gives a drug delivery carrier with a desired particle size.

Examples of crosslinking agents include those exemplified above for "hydrogel."

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. The unit "M" here is an abbreviation for mol/L.

The concentration of the polysaccharide in a solution containing the polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflecting material 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.

The concentration of the particles made of ultrasound reflecting material in a solution containing a polysaccharide having a crosslinkable functional group and particles made of ultrasound reflecting material may be such that one or more particles made of ultrasound reflecting material are contained in one drug delivery carrier, and 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 15% by mass or less.

As a solvent for dissolving or dispersing the crosslinking agent, or the polysaccharide and the ultrasonic reflecting material, water or an aqueous solvent can be used. Specific examples of aqueous solvents include buffers known in the art, such as phosphate buffer, Tris-HCl buffer, citrate buffer, carbonate buffer, borate buffer, succinate buffer, acetate buffer, etc.

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

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).

In this case, the above-mentioned drug delivery carrier 20 can be produced by using particles 2a made of inorganic phosphate as particles 2 made of ultrasonic reflecting material.

The microbeads with the embedded particles 2 made of the ultrasound reflecting material are then brought into contact with, for example, a solution containing a hydrophilic polymer. This causes the hydrophilic polymer to bond physically or chemically to the surface of the microbeads. As a result, the hydrophilic polymer covers the surface of the microbeads to form a coating layer 3, thereby obtaining the drug delivery carrier 10. Examples of methods for bringing the microbeads into contact with the solution containing the hydrophilic polymer include a method of immersing the microbeads in a solution containing the hydrophilic polymer and a method of dripping the solution containing the hydrophilic polymer onto the microbeads.

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.

Water or an aqueous solvent can be used as a solvent for dissolving or dispersing the hydrophilic polymer. As the aqueous solvent, the above-mentioned solvents can be used.

Pharmaceutical Compositions
[Third embodiment]
Fig. 3 is a schematic diagram of a pharmaceutical composition according to a third embodiment of the present invention. The pharmaceutical composition 100 shown in Fig. 3 differs from the drug delivery carrier 10 shown in Fig. 1 in that a drug 11 is further contained 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 contains the drug delivery carrier 10 and the drug 11, and the drug 11 is embedded in the microbeads 1.

The pharmaceutical composition 100 has the above-mentioned configuration, and when exposed to ultrasound, the particles 2 made of an ultrasound reflecting material vibrate, expanding the pore size of the hydrogel mesh that constitutes the microbeads 1, allowing the drug 11 to be released in a sustained manner. As described above, the pharmaceutical composition 100 has the surface of the microbeads 1 covered with a coating layer 3 made of a hydrophilic polymer, which effectively prevents the drug from leaking out before the application of ultrasound. In other words, the pharmaceutical composition 100 allows for more strict control of the sustained release of the drug 11 compared to a pharmaceutical composition without a coating layer. Furthermore, the pharmaceutical composition 100 does not have cytotoxicity, and allows the drug 11 to be delivered to a desired organ in a sustained release controlled state.

The pharmaceutical composition 100 can be produced using the same method as the method for producing the drug delivery carrier 10 described in the above "Method for producing a drug delivery carrier" except that in the above "Method for producing a drug delivery carrier", the drug 11 is added to a solution containing a polysaccharide having a crosslinkable functional group and particles made of an ultrasonic reflecting material so as to achieve a desired concentration.

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

Examples of active ingredients of disease treatment agents include low molecular weight compounds, nucleic acids, proteins, peptides, etc. Low molecular weight compounds are preferably those whose size is adjusted by micellization or the like so that they can be supported in the pores of microbeads made of hydrogel. Examples of nucleic acids include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA, etc. Examples of proteins include enzymes, antibodies, etc., and examples of peptides include ligands, vaccines, etc.

When the pharmaceutical composition of this embodiment is used for gene therapy, the disease therapeutic agent is preferably a nucleic acid. When expression of a wild-type gene is lost or suppressed in a patient due to deletion, insertion, mutation, or the like of a gene sequence, it is preferable to use a cDNA of the wild-type gene as the disease therapeutic agent. Furthermore, when the disease therapeutic agent is a nucleic acid, it is preferable from the viewpoint of delivery and expression efficiency that the viral vector takes a form containing the relevant cDNA as the disease therapeutic agent.

The viral vectors used here include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), herpes viral vectors, etc., with adeno-associated viral vectors (AAV) being preferred from the viewpoint of low toxicity.

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; computed tomography (CT) contrast agents such as iodine contrast agents; MRI contrast agents such as gadolinium preparations and superparamagnetic iron oxide preparations (SPIO); and single photon emission computed tomography (SPECT) radioisotopes such as technetium 99m (99mTc) and molybdenum 99 (99Mo).

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

[Fourth embodiment]
Fig. 4 is a schematic diagram of a pharmaceutical composition according to a fourth embodiment of the present invention. The pharmaceutical composition 200 shown in Fig. 4 differs from the drug delivery carrier 20 shown in Fig. 2 in that a drug 11 is further contained 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 contains the drug delivery carrier 20 and the drug 11, and the drug 11 is embedded in the microbeads 1.

The pharmaceutical composition 200 can be made more biocompatible by embedding particles 2a made of inorganic phosphate as particles 2 made of an ultrasound reflecting material.

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 "Method for producing a drug delivery carrier", except that in the above "Method for producing a drug delivery carrier", the drug 11 is added to a solution containing a polysaccharide having a crosslinkable functional group and particles 2a made of inorganic phosphate so as to achieve a desired concentration.

[Other ingredients]
The pharmaceutical composition of the present embodiment can be used in combination with a pharma- ceutically acceptable carrier.

As the pharma- ceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular restrictions. More specifically, examples include binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; swelling 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.

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; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc.

The pharmaceutical composition of this embodiment can be formulated by appropriately combining with the above-mentioned pharma- ceutically acceptable carriers and additives and mixing in a unit dose form required for generally accepted pharmaceutical practice.

The pharmaceutical composition of this embodiment may be in a dosage form for oral use or a dosage form for parenteral use, but is preferably in a dosage form for oral use. Examples of dosage forms for oral use include tablets, capsules, elixirs, microcapsules, etc. Examples of dosage forms for parenteral use include injections, ointments, patches, etc.

<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 an affected area of the patient or animal in need of treatment with ultrasound.

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

Administration to patients or animals can be performed by methods known to those skilled in the art, such as intrathecal injection, intraarterial injection, intravenous injection, subcutaneous injection, local injection, intranasal injection, transbronchial injection, intramuscular injection, transdermal injection, or oral injection. The dosage and frequency of administration 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 select an appropriate dosage and frequency of administration.

As a device for irradiating ultrasonic waves, a known device used for medical purposes can be used.
The frequency of the ultrasonic wave can be, for example, 20 kHz or more and 10 MHz or less, and the output intensity can be 5.0 W/cm ² or more and 50 W/cm ² or less in the case of continuous ultrasonic irradiation, and 25 W/cm ² or more and 250 W/cm ² or less in the case of pulsed ultrasonic irradiation. According to the pharmaceutical composition of this embodiment, the sustained release of the drug can be safely and efficiently controlled with an ultrasonic output intensity lower than that of the conventional one. In addition, the frequency and output intensity of the ultrasonic wave can be appropriately set after confirming the sustained release property by a preliminary test using the pharmaceutical composition to be used.

The ultrasonic irradiation time can be, for example, from 10 seconds to 1 hour, or from 30 seconds to 30 minutes.

The frequency of ultrasound irradiation can be one time only after a certain time has elapsed since administration of the pharmaceutical composition, or it can be multiple times once or several times a day, every day, every three days, or every week.

In addition to irradiating the affected area with ultrasound, the treatment method of this embodiment can also control sustained release of the drug by locally administering a hydrogel solubilizer to the affected area.

Solubilizing agents for hydrogels include chelating agents that remove divalent metal ions when the hydrogel is crosslinked by ionic bonds. Also included are enzymes that degrade the polysaccharides themselves that have crosslinkable functional groups.

Examples of chelating agents include citric acid, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), glycol ether diaminetetraacetic acid (Glycol Ether Diamine Tetraacetic Acid Examples of citric acid include, but are not limited to, N,N,N',N'',N''',N'''-hexaacetic acid (TTHA), N-(2-hydroxyethyl)iminodiacetic acid (HIDA), and N,N-di(2-hydroxyethyl)glycine (DHEG). Citric acid may also be in the form of a citrate salt, such as sodium citrate or potassium citrate.

Examples of enzymes include, but are not limited to, lambda-carrageenase, pectinase, gellan gum decomposing enzyme, xyloglucan decomposing enzyme, hyaluronidase, alginate lyase, etc.

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

[Experimental Example 1]
(Changes in drug release rate of drug delivery model depending on ultrasound intensity and irradiation time)
A solution was 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 simulating a small molecule drug, and 3% by mass of tungsten particles with an average particle size of 1 μm, and injected into a 100 mmol/L calcium chloride solution by a microgel bead preparation method (see References 1 and 2) using a centrifugal force of 200×g. The average particle size of the resulting calcium alginate gel beads in which fluorescent nanosilica and tungsten particles were embedded 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 the average value.

The resulting calcium alginate gel beads were then 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 released was evaluated by measuring the fluorescence intensity of the solution using a fluorescence spectrophotometer. The results are shown in Figure 5.

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

[Experimental Example 2]
(Changes in drug release rate in drug delivery models depending on tungsten particle content)
Solutions were prepared to have final concentrations of 2% by mass of sodium alginate, 15% by mass of fluorescent nanosilica with an average particle size of 20 nm to simulate a small molecule drug, and 0, 5, or 10% by mass of tungsten particles with an average particle size of 1 μm, and three types of beads with different tungsten particle contents were obtained using a method similar to that of Experimental Example 1.

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

As shown in Figure 6, there was a tendency for the amount of fluorescent nanosilica released to increase with an increase in the content of tungsten particles. In addition, beads containing 10% tungsten particles by mass were found to release 1.7 times as much fluorescent nanosilica after 10 minutes of irradiation compared to beads containing no tungsten particles. These results suggest that the amount of drug released can be controlled by changing the content of particles made of ultrasound-reflecting material, such as tungsten particles.

[Experimental Example 3]
(Confirmation of long-term sustained release control of drug delivery model)
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 to simulate a small molecule drug, and 0, 1, or 3% by mass of tungsten particles with an average particle size of 1 μm. Using a method similar to that of Experimental Example 1, three types of beads with different tungsten particle contents were obtained.

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 released was evaluated by measuring the fluorescence intensity of the solution using a fluorescence spectrophotometer. The results are shown in Figure 7. In Figure 7, "0% NO US" indicates a group that was not irradiated with ultrasound using beads that did not contain tungsten particles.

As shown in Figure 7, it was confirmed that ultrasound irradiation was used as a trigger to release fluorescent nanosilica and improve the rate of sustained release of fluorescent nanosilica from the beads. It was also confirmed that by combining ultrasound irradiation with the sustained release ability of the beads themselves, 65% of the fluorescent nanosilica was released gradually over a period of three days. These results suggest that it is possible to control the long-term sustained release of drugs by changing the content of particles made of ultrasound-reflecting material, such as tungsten particles.

[Example 1]
(Confirmation of sustained release control of drug delivery model by poly-L-lysine (PLL) coating)
A solution was 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 simulating a small molecule drug, and 0 or 3% by mass of tungsten particles with an average particle size of 1 μm, and three types of beads with different tungsten particle contents were obtained using the same method as in Experimental Example 1. Next, a part of the obtained beads containing tungsten particles and beads not containing tungsten particles was added to a solution containing 0.005 w/v% poly-L-lysine (PLL) and stirred to ionically bond the positively charged PLL to the negatively charged bead surface, and the bead surface was coated with poly-L-lysine to obtain 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 sizes of multiple beads using an inverted microscope (Olympus, IX73P1-22FL/PH) and calculating the average value.

Next, each of the beads obtained was dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 3 minutes every other day, a total of three times. The amount of fluorescent nanosilica released was evaluated by measuring the fluorescence intensity of the solution using 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 that do not contain tungsten particles, and "with W" indicates particles that contain tungsten particles.

As shown in FIG. 8A, it was clear that in beads without PLL coating, fluorescent nanosilica gradually leaked out even before ultrasonic irradiation.
In contrast, as shown in FIG. 8B, in the case of beads having a PLL coating, no release of fluorescent nanosilica was observed until ultrasonic irradiation was performed, confirming that leakage of the fluorescent nanosilica could be effectively suppressed.
This suggests that more precise control of sustained drug release can be achieved by providing a coating layer such as a PLL coating on the beads.

[Experimental Example 4]
(Confirmation of sustained release from beads embedded with fluorescently labeled bovine serum albumin)
A solution was prepared so that the final concentrations were 2% by mass of sodium alginate, 12% by mass of bovine serum albumin (BSA) labeled with tetramethylrhodamine, and 3% by mass of tungsten particles having an average particle size of 1 μm. Using the same method as in Experimental Example 1, calcium alginate beads in which tetramethylrhodamine-labeled BSA and tungsten particles were embedded were obtained.

The resulting beads were then dispersed in pure water and irradiated with ultrasound (20 kHz, 10 W) using a homogenizer for 3 minutes every other day, a total of three times. The amount of BSA labeled with tetramethylrhodamine released was evaluated by measuring the fluorescence intensity of the solution using 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 BSA labeled with tetramethylrhodamine.

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

[Example 2]
(Confirmation of sustained release from beads with embedded hydroxyapatite)
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 to simulate a small molecule drug, and 0, 1, or 3% by mass of hydroxyapatite particles with a particle size of 1 μm or more and 5 μm or less as a biocompatible vibrating nucleus, and each bead was obtained using a method similar to that in Experimental Example 1.

Next, each of the obtained calcium alginate gel beads was dispersed in pure water and irradiated with ultrasound (20 khz, 2.5 W) for 30 minutes using a homogenizer. The amount of fluorescent nanosilica released 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 2.5 W" indicates the group that was irradiated with ultrasound (20 khz, 2.5 W).

As shown in Figure 10, similar to the case of using tungsten particles, the amount of fluorescent nanosilica released tended to increase with an increase in the content of hydroxyapatite particles. These results suggest that the amount of drug released can be controlled by changing the content of particles made of biocompatible ultrasound reflecting materials, such as hydroxyapatite particles.

The drug delivery carrier of this embodiment can release drugs by irradiating them with low-power ultrasound that does not damage biological tissue, and can provide a drug delivery carrier that can stably control the sustained release of drugs for a longer period of time than conventional carriers.

1...Microbeads, 2...Particles made of ultrasound reflecting material, 2a...Particles made of inorganic phosphate, 3...Coating layer, 10, 20...Drug delivery carrier, 11...Drug, 100, 200...Pharmaceutical composition

Claims (6)

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;
Including,
A drug delivery carrier, wherein the ultrasonic reflecting material is hydroxyapatite . 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. The drug delivery carrier according to claim 2, wherein the hydrophilic polymer is a cationic hydrophilic polymer. 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. A drug delivery carrier according to any one of claims 1 to 4 , and a drug,
A pharmaceutical composition, wherein the drug is embedded in the microbeads. The pharmaceutical composition according to claim 5 , which is for treating a disease.

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