Particles and method for producing particles, as well as drug, drug production method, and anticancer drug

Abstract

Provided are: particles each comprising a polymer and having a particle diameter suitable for use in a drug delivery system (DDS) or the like; and a method for producing the particles. Also provided are a drug and an anti-cancer agent each utilizing the particles.　Particles each of which comprises a polymer having a structural unit derived from a sugar compound having a hydroxyl group and having a inclusion property and a structural unit derived from a monomer having a functional group capable of reacting with a hydroxyl group, and which have an average hydrodynamic radius (Rhav) of 5 to 100 nm. A method for producing particles, the method comprising a step of agitating a mixed composition comprising a sugar compound having a hydroxyl group and having an inclusion property, a monomer having a functional group capable of reacting with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH value of 12 or higher, wherein each of the particles comprises a polymer having a structural unit derived from the sugar compound and a structural unit derived from the monomer in which the structural units are formed in the mixed composition. A drug which comprises a hydrophobic physiologically active substance, such as α-mangostin, contained in the particles; and an anti-cancer agent comprising the drug.

Description

[Technical field]

[0001] The present invention relates to particles that can be used for drugs encapsulating hydrophobic bioactive agents and a method for producing the particles, to drugs and a method for producing the drugs using the particles, and to anticancer drugs using them. [Background technology]

[0002] It is known that some organic compounds called xanthones have anti-cancer properties. For example, alpha-mangosteen and gamma-mangosteen, which are derivatives of mangosteen, a fruit native to Southeast Asia, can be extracted as xanthones that have anti-cancer properties.

[0003] There is a demand for the development of new drugs that utilize the strong anti-cancer effects of mangosteen, but it is difficult to deliver the drug only to the target site while suppressing side effects. As a method to solve this problem, a technology called drug delivery system (DDS) that uses a polymer carrier to deliver the drug only to the target site has attracted attention.

[0004] On the other hand, hyperbranched polymers consisting of cyclodextrin and epichlorohydrin are known. The structure of this hyperbranched polymer is a large spherical polymer containing many branched structures. In addition, the cyclodextrin contained in this polymer has a hydrophobic cavity inside the molecule, which can encapsulate molecules of appropriate size, and has attracted attention due to its property of supporting various molecules.

[0005] For example, Patent Document 1 discloses a crosslinked cyclodextrin obtained by crosslinking cyclodextrin with epihalohydrin under alkaline conditions, and discloses that the crosslinked cyclodextrin is used as an agent for removing endocrine disrupters.

[0006] Patent Document 2 discloses a composition comprising an aqueous dispersion of particles (p) having an average hydrodynamic diameter of 50 to 5,000 nm, characterized in that the particles contain (A) a polymer based on cyclodextrin units having an average content of at least 4 cyclodextrin units in its structure, and (B) a polysaccharide polymer having an average number of groups G of at least 3 per polysaccharide polymer and containing a group G capable of forming an inclusion compound with the cyclodextrin present in the structure of the polymer (A), in association with each other, and that the compounds (A) and (B) are water-soluble in isolated form.

[0007] Furthermore, studies on the properties of compounds using cyclodextrin and epichlorohydrin have been disclosed in Non-Patent Documents 1 and 2. For example, Non-Patent Document 1 mainly studies the synthesis of ionic cyclodextrin-based polymers and the properties of their ability to encapsulate an antibacterial agent called triclosan. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2003-226737 A [Patent Document 2] Special Publication No. 2005-536587 [Non-patent literature]

[0009] [Non-Patent Document 1] Carbohydrate Polymers 142 (2016) 149-157, Preparation and characterization of soluble branched ionicβ-cyclodextrins and their inclusion complexes with triclosan. [Non-Patent Document 2] European Polymer Journal 46 (2010) 1915-1922, Water-soluble c-cyclodextrin polymers with high molecular weight and their complex forming properties. Summary of the Invention [Problem to be solved by the invention]

[0010] In DDS technology, the particle size of the polymer carrier must be such that it is not easily cleared (excreted) from the body, that is, it must have a diameter of 10 nm or more (radius of 5 nm or more), and it is also necessary to efficiently introduce the target drug into the polymer carrier.

[0011] As disclosed in Patent Document 1 and Patent Document 2, a crosslinked body can be synthesized using cyclodextrin (hereinafter sometimes abbreviated as "CD") and epichlorohydrin (hereinafter sometimes abbreviated as "ECH"). However, these synthesis methods proceed by vigorously stirring an aqueous CD solution and hydrophobic ECH, but because the reaction is non-uniform, CD and ECH do not react efficiently. Therefore, the particle size of the obtained product varies, and the particle size is very small, with a diameter of less than 10 nm (radius less than 5 nm), and is easily cleared in the body, or the particle size is large and is captured in the body and cannot move.

[0012] In addition, if an excessive amount of ECH is added to increase the particle size, only the ECH reacts with other molecules, resulting in an insoluble product. In order to increase the particle size to a certain degree while still being soluble in water, it is necessary to precisely control the reaction between CD and ECH, but the ring-opening reaction of ECH tends to proceed at an accelerated rate, making this control extremely difficult. In addition, in Patent Document 2, polysaccharide polymers are used for association, and the particle size tends to become too large. In addition, there have been no attempts to introduce xanthone drugs such as α-mangostin into this crosslinked material. It is considered difficult to introduce highly hydrophobic drugs such as α-mangostin into hydrophilic polymer carriers.

[0013] An object of the present invention is to provide particles using a polymer having a particle size suitable for DDS and the like, and a method for producing the same. [Means for solving the problem]

[0014] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the following invention meets the above object, thereby completing the present invention.

[0015] <a1>The polymer has a structural unit derived from a saccharide compound having a hydroxyl group and clathrate property, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group, and has an average hydrodynamic radius (R hav ) is 5 to 100 nm. <a2>The saccharide compound includes any one of saccharide compounds selected from the group consisting of cyclodextone, β-1,3-glucan, and amylose. <a1>The particle according to claim 1. <a3>The monomer includes a multifunctional epoxy compound. <a1>or <a2>The particle according to claim 1. <a4>The mean hydrodynamic radius (R hav ) is the median, the standard deviation (σ) of the particle size distribution is the mean hydrodynamic radius (R hav ) divided by (σ / R hav ) is 0.25 or less. <a1> ~ <a3>2. The particle according to any one of claims 1 to 11. <a5>The above <a1> ~ <a4>2. A drug having a hydrophobic bioactive agent encapsulated in the particle according to claim 1. <a6>The hydrophobic bioactive agent includes a hydrophobic bioactive agent dispersed in one or more dispersion media selected from the group consisting of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol. <a5>The drug described in. <a7>The above <a5>or <a6>The anti-cancer agent according to claim 1, wherein the hydrophobic bioactive agent comprises any one selected from the group consisting of alpha-mangostin, curcumin, and doxorubicin, and further comprises a pharma- ceutically acceptable carrier. <a8>The cancer is a solid cancer. <a7>The anticancer agent described in

[0016] <b1>The method includes a step of mixing a mixed composition including a saccharide compound having a hydroxyl group and having inclusion properties, a monomer having a functional group reactive with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH of 12 or more; A method for producing particles comprising a polymer having a structural unit derived from the saccharide compound and a structural unit derived from the monomer, the polymer being formed in the mixed composition. <b2>The surfactant includes one or more surfactants selected from the group consisting of quaternary ammonium salt surfactants, sulfonate surfactants, and oligoethylene glycol surfactants. <b1>A method for producing the particles according to claim 1. <b3>The quaternary ammonium salt surfactant is hexadecyltrimethylammonium bromide, the sulfonate surfactant is sodium dodecyl sulfate, and the oligoethylene glycol salt surfactant is Triton-X100. <b2>A method for producing the particles according to claim 1. <b4>The concentration of the surfactant in the mixed composition (mass of the surfactant / total mass of the mixed composition) is 4 times or less the critical micelle concentration of the surfactant in neutral water. <b1> ~ <b3>13. A method for producing particles according to any one of the preceding claims. <b5>The above <b1> ~ <b4>2. A method for producing a drug, comprising mixing a first composition containing particles obtained by any one of the production methods described above with a second composition containing a hydrophobic bioactive agent, thereby encapsulating the hydrophobic bioactive agent in the particles. <b6>The second composition contains one or more dispersion media selected from the group consisting of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol. <b5>A method for producing the drug described in Effect of the Invention

[0017] The particles of the present invention have a particle size suitable for DDS, etc. Moreover, according to the method for producing particles of the present invention, such particles can be obtained efficiently. [Brief description of the drawings]

[0018] [Figure 1] FIG. 2 is a flow chart for explaining a process for producing particles in the examples. [Diagram 2] 1 is a graph showing particle size distribution of particles with and without SDS according to Example 1. [Diagram 3] 2 is a graph showing the particle size distribution of particles according to Production Example 1. [Figure 4] 4 is a graph showing the particle size distribution of particles according to Production Example 2. [Diagram 5] 1 is a graph showing the results of a test for encapsulating α-mangostin according to Production Example 3. [Figure 6] 1 is a graph showing the particle size distribution of particles according to Production Example 5. [Figure 7] 1 is a graph showing the results of a drug inclusion test in Production Example 7. [Figure 8] 1 is a graph showing the results of evaluating the cell viability of colon cancer cells in vitro according to Example 2. [Figure 9] 13 is a graph showing the results of evaluating the effect of inhibiting the proliferation of colon cancer cells in mice in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following is a detailed description of the embodiments of the present invention, but the following description of the constituent elements is one example (representative example) of the embodiment of the present invention, and the present invention is not limited to the following content as long as the gist of the present invention is not changed. Note that in this specification, when the expression "~" is used, it is used as an expression including the numerical values ​​before and after it.

[0020] [Particles of the present invention] The particles of the present invention contain a polymer having a structural unit derived from a saccharide compound having a hydroxyl group and having inclusion properties, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group, and have an average hydrodynamic radius (R hav ) is 5 to 100 nm. The particles of the present invention can encapsulate hydrophobic compounds and the like, and have such a particle size, which is suitable for DDS and the like.

[0021] [Method of producing particles of the present invention] The method for producing particles of the present invention includes a step of mixing a mixed composition containing a saccharide compound having a hydroxyl group and clathrate property, a monomer having a functional group reactive with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH of 12 or more, and is a method for producing particles containing a polymer having a structural unit derived from the saccharide compound and a structural unit derived from the monomer formed in the mixed composition. By producing particles by such a production method, particles with a hydrodynamic radius controlled to about 5 to 100 nm can be efficiently produced. The method for producing particles of the present invention is a preferred method for producing particles of the present invention, and the corresponding configurations in the present application can be used interchangeably.

[0022] [Sugar compound] The method for producing particles of the present invention uses a saccharide compound having a hydroxyl group and clathrate properties as one of the raw materials. The particles of the present invention have a structural unit derived from the saccharide compound having a hydroxyl group and clathrate properties. Such saccharide compounds are polymerized using the monomers used in the production of the particles of the present invention to form large particles. These particles can encapsulate hydrophobic substances, etc.

[0023] The sugar compounds include sugars and sugar derivatives. The sugars may be monosaccharides, oligosaccharides, polysaccharides, etc. For example, cyclic oligosaccharides in which glucose is linked by α-1,4 glucoside bonds or α-1,6 glucoside bonds and a glucoside bond is formed between the reducing end and the final end are preferably used. A representative example of such cyclic oligosaccharides is cyclodextrin (CD). In addition, the cyclic oligosaccharides are not limited to these, and for example, cyclic amylose described in Applied Glycoscience, Vol. 1, No. 1, pp. 39-46 (2011) and references thereto may also be used.

[0024] Also, the derivatives of sugars contained in sugar compounds are compounds in which some of these sugars have been modified or substituted. As the derivatives, sugars or polysaccharides in which some of the hydroxyl groups have been methylated, aminated, or sulfonated can be used. Specifically, carboxycellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, etc. can be used. Also, similarly modified polysaccharides other than cellulose can be used. Sugar compounds have hydroxyl groups, and these hydroxyl groups are crosslinked via monomers to form crosslinked bodies. The molecular structure of the sugar compound preferably has two or more hydroxyl groups. Alternatively, it may have three or more hydroxyl groups.

[0025] The saccharide compound used in the particles of the present invention is preferably any saccharide compound selected from the group consisting of CD, β-1,3-glucan, and amylose. CD is a cyclic oligosaccharide having a cyclic structure in which multiple glucose units are bonded by glycosidic bonds. CD encapsulates hydrophobic molecules.

[0026] Any CD may be used, such as α-cyclodextrin (cyclohexaamylose) (αCD) with six glucose molecules bound, β-cyclodextrin (cycloheptaamylose) (βCD) with seven glucose molecules bound, or γ-cyclodextrin (cyclooctaamylose) (γCD) with eight glucose molecules bound.

[0027] β-1,3-glucan is a polysaccharide in which D-glucose is linked by glycosidic bonds, and is found in seaweed and mushrooms, such as laminaran. Amylose is a polysaccharide in which α-glucose is linked by α1→4 glycosidic bonds, and is found in starch.

[0028] These sugars may be used alone or in appropriate combination. These sugars are suitable for controlling the particle size of the polymer used in the particles of the present invention, and are also excellent in encapsulating hydrophobic compounds, etc. In addition, these sugars have low toxicity and are easy to be formulated and used as part of the raw material to be ingested by humans or animals.

[0029] [monomer] The method for producing particles of the present invention uses a monomer having a functional group that reacts with a hydroxyl group. The monomer is preferably a polyfunctional epoxy compound. Here, a monomer having two or more functional groups is called polyfunctional. In particular, it is preferable to use a monomer having a functional group that reacts with a hydroxyl group at multiple points, such as epihalohydrin, ethylene glycol diglycidyl ether, or butanediol diglycidyl ether, as one of the raw materials. The number of functional groups that react with a hydroxyl group that the monomer has is preferably two or more, and may be three or more, or four or more. In addition, the particles of the present invention have a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group. This monomer reacts with the hydroxyl group of the above-mentioned saccharide compound to form a crosslinked body.

[0030] As such a monomer, epihalohydrin is preferably used. In particular, epichlorohydrin (ECH) and / or epibromohydrin are preferably used, and ECH is more preferably used. Epihalohydrin is suitable for forming a polymer having a crosslinked structure by a reaction of crosslinking the hydroxyl groups of the saccharide compound used in the present invention.

[0031] [Polymer] The particles of the present invention have a polymer having a structural unit derived from a saccharide compound having a hydroxyl group and clathrate property, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group. This polymer has, for example, a structural unit derived from a saccharide compound and a structural unit derived from a monomer. For example, when CD is used as the saccharide compound and ECH is used as the monomer, a crosslinked polymer (hyperbranched polymer) having a very large branch can be obtained by reacting them. Formula 1 is an assumed example of a chemical structural formula related to such a polymer by CD and ECH. In Formula 1, n is the number of glucose contained in cyclodextrin (CD), and when n is 6, it corresponds to αCD, when n is 7, it corresponds to βCD, and when n is 8, it corresponds to γCD.

[0032] [ka]

[0033] The particles of the present invention can be made of a polymer substantially composed of a structural unit derived from a saccharide compound having a hydroxyl group and clathrate property and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group. By making the polymer of such a constitution, it is easy to manage and control the components from which the structural units are derived without requiring other components for enlarging the particle size. Here, "substantially composed of these structural units" means that the proportion of the structural units derived from the above-mentioned saccharide compound and the structural units derived from the above-mentioned monomer in the structure of the polymer is very large. However, within a range that does not impair the purpose of the particles of the present invention, impurities and structural units that may be contained in the manufacturing process, etc., are also included in the end or side chain, etc., in a small amount. For example, when the structure of the polymer is analyzed, the proportion of the structural units derived from the above-mentioned saccharide compound and the structural units derived from the above-mentioned monomer may be 80% by mass or more or 90% by mass or more, which may be used as an index that the polymer is substantially composed of these structural units. This proportion may be 95% by mass or more, 98% by mass or more.

[0034] [Particle shape, etc.] The particles of the present invention contain such a polymer. The polymer particles produced by controlling the particle size by the particle production method of the present invention can selectively obtain particles with a hydrodynamic radius of 5 to 100 nm. When the hydrodynamic radius is less than 5 nm, the size is easily eliminated by various physiological phenomena such as urine and sweat due to metabolism of the living body, or by movement between cells. By making the hydrodynamic radius 5 nm or more, these eliminations are less likely to occur, and by encapsulating an active ingredient such as a drug in the particles, the active ingredient can be released gradually by reaching a specific tissue or cell of the living body. When the hydrodynamic radius is more than 100 nm, the particles may be captured in the living body and may not be able to move, or may be difficult to apply to formulations such as powders and tablets.

[0035] This average particle size and its distribution are obtained by particle size distribution analysis using dynamic light scattering. Regarding the measurement method, for example, the standard method described in "Basics and Applications of Light Scattering Method" (Kodansha) can be used. Specifically, a laser beam is irradiated onto particles dispersed in a solution, and the scattered light is measured by a photon detector. Since the particles are constantly moving due to Brownian motion, the intensity distribution due to the interference of the scattered light also constantly fluctuates. The state of this Brownian motion is observed as the fluctuation of the scattered light intensity. Usually, the autocorrelation function is obtained by the photon correlation method, and the diffusion coefficient indicating the Brownian motion speed, as well as the particle size and particle size distribution, can be obtained by using the cumulant method, histogram method analysis, and contin analysis. In the present invention, contin analysis is used. For contin analysis, a program attached to the particle size measuring device can be used.

[0036] The particles of the present invention have an average hydrodynamic radius (R hav ) (hereinafter abbreviated as "average particle size") is 5 to 100 nm. The lower limit of the average particle size of the particles of the present invention is preferably 10 nm or more, more preferably 12 nm or more, even more preferably 15 nm or more, and particularly preferably 20 nm or more. The upper limit of the average particle size of the particles of the present invention is preferably 80 nm or less, more preferably 50 nm or less, even more preferably less than 50 nm, and particularly preferably 30 nm or less. The particle size distribution is preferably unimodal with one peak.

[0037] In addition, the particle size distribution is normal, and the average particle size (R hav ) is the median, the standard deviation (σ) divided by the average particle size (σ / R hav ) is preferably 0.25 or less. The method of determining this is to plot the obtained scattering intensity (Intensity) versus the hydrodynamic radius Rh, with the intensity on the vertical axis and the logarithm of Rh on the horizontal axis, and to obtain the standard deviation σ from peak fitting on this graph, assuming a Gaussian distribution. Then, σ / R hav Calculate the σ / R hav is the value calculated in this way. The standard deviation (σ) divided by the average particle size (σ / R hav ) is more preferably 0.23 or less, and even more preferably 0.20 or less.

[0038] [Drugs] A drug can be obtained by encapsulating a hydrophobic bioactive agent in the particles of the present invention. Any hydrophobic compound that can be dissolved in an organic solvent that can be mixed with water in any ratio can be used as the drug. Examples of such compounds include those described in Chem. Pharm. Bull. 66, 493-505 (2018).

[0039] [Hydrophobic bioactive agents] The particles of the present invention can encapsulate a hydrophobic bioactive agent. A hydrophobic bioactive agent is a compound having hydrophobic bioactivity. Such compounds are difficult to disperse in water, and even if they are expected to exhibit various bioactivities such as for use in medicines and functional foods, there are cases where there is no suitable mixing method for them as components of drugs, or where it is difficult to control them using DDS. According to the present invention, such a hydrophobic bioactive agent can be encapsulated to control the tissue of the body into which it is released and the time during which it exhibits sustained release. Examples of hydrophobic bioactive agents include xanthone derivatives, polyphenols, glucosides, and flavanols. These include xanthone derivatives such as α-mangostin, polyphenols such as curcumin, stilbene derivative polyphenols such as resveratrol, bioactive substances such as 2,3,5,4-tetrahydroxy-diphenylethylene-2-O-glucoside (THSG), and flavanols such as dihydromyristicene and astilbin.

[0040] [α-Mangosteen] An example of a hydrophobic bioactive agent is α-mangosteen. α-mangosteen is a type of xanthone contained in the outer skin of mangosteen, an evergreen tree of the genus Garcinia. The chemical structure of α-mangosteen is shown in Formula 2. α-mangosteen has a cancer-suppressing effect.

[0041] [ka]

[0042] The particles of the present invention may be used as a drug by encapsulating a physiologically active agent or the like as particles, or may be used as particles for drugs to be encapsulated with a physiologically active agent or the like. Although it depends on the physiologically active agent and the encapsulation conditions, the particle size can be maintained at the same level as the particles of the present invention even after the physiologically active agent is encapsulated. The particles of the present invention may be used as the particles recovered by filtration, drying, centrifugation, etc., or may be stored or used in any form that is easy to use, such as a dispersion or suspension in which the particles are dispersed or suspended in a liquid, or a powder, tablet, or capsule formed by mixing with various excipients, etc.

[0043] [Anti-cancer drugs] The anticancer agent of the present invention may contain a hydrophobic bioactive agent selected from the group consisting of alpha-mangostin, curcumin, and doxorubicin as an active ingredient. The anticancer agent may further contain a pharma- ceutically acceptable carrier as appropriate. The agent may be used to manufacture an anticancer agent. The agent may be used for anticancer treatment. The method of treating cancer may include administering a therapeutically effective amount of the agent to a patient in need of treatment.

[0044] Since the active ingredient of an anticancer drug such as α-mangosteen is a low molecular weight, it is quickly removed from the body by metabolism, such as excretion from the kidney into the urine. However, when the particles of the present invention are used, the particles have a hydrodynamic radius of 5 nm or more and are not easily removed from the body by renal excretion. Therefore, the active ingredient can be retained in the blood for a long time. In addition, since the vascular permeability of tumor tissue is enhanced compared to normal tissue, particles with a hydrodynamic radius of about 5 to 100 nm are likely to flow out of the blood vessels. Furthermore, since the lymphatic system of tumor tissue is underdeveloped, particles are difficult to disappear and tend to accumulate. This phenomenon is known as the enhanced permeability and retention (EPR) effect. The particles of the present invention can avoid renal excretion and have a size suitable for this EPR effect. Therefore, a remarkable antitumor effect (anticancer effect) can be obtained.

[0045] The anticancer agent of the present invention can be used for humans, mammals, animals, etc. When the anticancer agent of the present invention is used for humans, the daily dose of the hydrophobic bioactive agent and active ingredient α-mangostin, curcumin, and doxorubicin can be as follows. The daily dose can be 0.5 mg / (kg·day) to 20 mg / (kg·day) per kg of body weight. That is, this dose can be 30 mg / day to 1200 mg / day when converted to an adult weighing 60 kg. The lower limit of this dose can be preferably 1.0 mg / (kg·day) or more, or 1.5 mg / (kg·day) or more, per kg of body weight per day. The upper limit of this dose can be preferably 15.0 mg / (kg·day) or less, or 10.0 mg / (kg·day) or less, per kg of body weight per day.

[0046] The anticancer agent of the present invention can be administered by injection, transdermal administration, oral administration, etc., depending on the target cancer. In particular, it is preferable to administer it by intravenous injection, since it can remain in the blood for a sufficient time and exert its effect. The anticancer agent of the present invention can be appropriately prepared as a solid preparation or liquid preparation containing a pharma- ceutically acceptable carrier that is commonly used depending on these administration methods.

[0047] Specific examples of the dosage form of the anticancer agent include solid preparations such as powders, granules, tablets, capsules, troches, etc. Also, examples of liquid preparations include internal liquids, external liquids, suspensions, emulsions, syrups, injections, infusions, etc. These dosage forms or other dosage forms are appropriately selected depending on the purpose.

[0048] In solid preparations, auxiliary agents such as excipients, binders, disintegrants, lubricants, flavoring agents, and stabilizers may be used in addition to the main agent. The ratio of the main agent to the auxiliary agent is appropriately selected according to the purpose. Examples of excipients in solid preparations include lactose, D-mannitol, and starch. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, and hydroxypropyl cellulose. Examples of disintegrants include starch, carboxymethylcellulose, and carboxymethylcellulose calcium. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners may be used as necessary.

[0049] When used as a liquid preparation, the solvent is selected from those that have the ability to disperse the active ingredient or drug and are biologically safe. Suitable examples of the solvent include water for injection, ethanol, and propylene glycol.

[0050] The liquid preparation may contain auxiliary components such as a solubilizing agent, a suspending agent, an isotonicity agent, a buffer, and an antioxidant, together with the main agent. Examples of the solubilizing agent include ethanol, polyethylene glycol, propylene glycol, benzyl benzoate, sodium carbonate, and sodium citrate. Examples of the suspending agent include sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, and hydroxymethylcellulose. Examples of the buffer include buffer solutions such as phosphates, acetates, and carbonates. Examples of the antioxidant include sulfites and ascorbic acid salts.

[0051] The anticancer agent of the present invention can be used to treat various types of cancer. In particular, since the agent remains in the blood and exerts a long-term effect on tissues and organs, it is preferable to use the agent to treat solid cancer. Examples of solid cancers include cancers (epithelial tumors) that develop from epithelial cells and sarcomas that develop from non-epithelial cells. Representative examples include lung cancer, breast cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma.

[0052] [Method of producing particles of the present invention] The particle production method of the present invention includes a step of mixing a mixed composition containing a saccharide compound having a hydroxyl group and inclusion property, a monomer having a functional group that reacts with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH of 12 or higher, and is a method for producing particles containing a polymer having structural units derived from the saccharide compound and structural units derived from the monomer formed in the mixed composition.

[0053] [Mixed composition] The method for producing particles of the present invention includes a step of mixing a mixed composition containing a saccharide compound having a hydroxyl group and clathrate property, a monomer having a functional group reactive with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH of 12 or more. By reacting this mixed composition under predetermined conditions, particles that are polymers having structural units derived from the saccharide compound and structural units derived from the monomer can be obtained.

[0054] The mixed composition can be prepared by adding the saccharide compound and the surfactant used in the particles of the present invention to an alkaline aqueous solution adjusted to pH 12 or more, and then adding the monomer used in the particles of the present invention. The order and combination may be changed as appropriate. The saccharide compound and the monomer are appropriately selected from those described above.

[0055] [Surfactants] The surfactant may be anionic, cationic, neutral, or other surfactants, either alone or in appropriate mixture. By reacting CD with ECH in an alkaline aqueous solution, extremely small particles are obtained, but the size is very small, with a hydrodynamic radius of 0.5 nm to 2.5 nm, and particles with a stable hydrodynamic radius of 5 nm or more have not been obtained in the past. The present inventors have found that by using a surfactant in combination, the average particle size can be controlled to 5 nm or more. This is because the surfactant contains a hydrophilic part that dissolves in water and a hydrophobic part that dissolves in oil, and is expected to function as a phase transfer catalyst that transfers ECH, etc. to the aqueous phase, and it is believed that this makes it easier for monomers such as ECH to come into contact with CD, making it easier for crosslinked bodies to grow.

[0056] The mixed composition preferably contains one or more surfactants selected from the group consisting of quaternary ammonium salt surfactants, sulfonate surfactants, and oligoethylene glycol surfactants. These surfactants may be used alone or in combination. In the method for producing particles of the present invention, it is considered preferable that the surfactant has a high critical micelle concentration, and the above-mentioned surfactants have physical properties such as critical micelle concentration suitable for the present invention.

[0057] Regardless of which surfactant is used, the particle size of the resulting hyperbranched polymer is larger than 5 nm on average, which is unattainable by conventional synthesis methods. Moreover, the particle size can be controlled by the molar ratio of ECH and CD added.

[0058] [Quaternary ammonium salt surfactants] As the surfactant, a quaternary ammonium salt surfactant can be preferably used. As the quaternary ammonium salt surfactant, for example, hexadecyltrimethylammonium bromide (hereinafter, "CTAB") can be preferably used. Tetradecyltrimethylammonium bromide and octadecyltrimethylammonium bromide having different alkyl chain lengths, and hexadecyltrimethylammonium chloride having different counter ions can also be used.

[0059] [Sulfonate surfactants] As the surfactant, a sulfonate surfactant can be preferably used. As the sulfonate surfactant, for example, sodium dodecyl sulfate (hereinafter, referred to as "SDS") can be preferably used. Sodium decyl sulfate and sodium tetradecyl sulfate, which have different alkyl chain lengths, and potassium dodecyl sulfate, which has different counter ions, can also be used.

[0060] [Oligoethylene glycol salt surfactants] As the surfactant, an oligoethylene glycol salt surfactant can be used. As the oligoethylene glycol surfactant, for example, Triton X-100 (hereinafter, "TX100") can be suitably used. Triton X-114, which has a different polyoxyethylene chain length, and Tween 20, a similar nonionic surfactant, can be used.

[0061] [Surfactant concentration] The concentration of the surfactant can be an extremely important factor in adjusting the particle size. The concentration varies depending on the type of surfactant, but it is preferable that the concentration is four times or less than the critical micelle concentration (CMC) in neutral water, and it is particularly preferable to use it at or below the CMC to freely control the particle size. If no surfactant is added, the ECH separates from the solvent, and the reaction progresses unevenly. However, at a moderate surfactant concentration, it can be seen that even when ECH is added, the solution becomes translucent and is uniformly dispersed. Note that below a certain concentration, surfactants are dissolved as molecules or ionized ions, but above a certain concentration, they associate to form micelles and exhibit the properties of a colloidal solution. This boundary concentration is called the CMC of the surfactant.

[0062] The CMC is determined based on the literature value or, if necessary, the measured value for the conditions of use for each surfactant used. For example, the CMC of hexadecyltrimethylammonium bromide is about 1.0 mM. The CMC of SDS is about 8.0 mM. The CMC of TX100 is about 0.23 mM.

[0063] [Alkaline aqueous solution] The mixed composition includes an alkaline aqueous solution having a pH of 12 or more. In the presence of the alkaline aqueous solution, the sugar compound and the monomer undergo a crosslinking reaction to form a polymer by reacting. The pH of the alkaline aqueous solution is preferably 13 or more, more preferably 13.5 or more. For example, a mixture of an alkali metal hydroxide such as sodium hydroxide (NaOH), lithium hydroxide (LiOH), or potassium hydroxide (KOH) and a hydroxide of tetraalkylammonium in water can be used. In particular, sodium hydroxide (NaOH) can be preferably used from the viewpoint of availability. The concentration of the hydroxide in the alkaline aqueous solution can be as high as 1% by mass or more, 5% by mass or more, 10% by mass or more, or 20% by mass or more.

[0064] [Mixed composition] The concentration of the saccharide compound in the mixed composition (mass of the saccharide compound / total mass of the mixed composition) is about 5 to 20% by mass. If the concentration of the saccharide compound is too low, the particles may not grow to a sufficient size. If the concentration of the saccharide compound is too high, the particles may become too large, or the ratio with other components may be difficult to crosslink, causing the particles to not grow, or the particles may not dissolve and cause precipitation.

[0065] The monomer concentration in the mixed composition (mass of monomer / total mass of mixed composition) is about 40 to 90 mass %. If the monomer concentration is too low, the particles may not grow to a sufficient size. If the monomer concentration is too high, the particles may become too large, the ratio with other components may be such that crosslinking is difficult, the particles may not grow, or the monomer may not be sufficiently dispersed or dissolved in the mixed composition, causing it to separate on the surface and not react.

[0066] The concentration of the surfactant in the mixed composition (mass of surfactant / total mass of the mixed composition) varies depending on the type of surfactant, but is preferably 4 times or less than the critical micelle concentration (CMC) of the surfactant in neutral water, more preferably 2 times or less than the CMC, and even more preferably, the particle size can be freely controlled by using the surfactant at or below the CMC. When a lower limit is set for the concentration, it is preferably 1 / 20 or more of the CMC, more preferably 1 / 15 or more of the CMC, or 1 / 10 or more of the CMC, or 1 / 5 or more of the CMC.

[0067] If the surfactant concentration is too low, the monomer may not be able to disperse or dissolve in the mixed composition, and the particles may not grow to a sufficient size. If the surfactant concentration is too high, the surfactant may form micelles on its own, resulting in the surfactant's effects not being achieved and the particles may not grow.

[0068] The weight ratio of the saccharide compound to the monomer in the mixed composition (saccharide compound:monomer) is appropriately set within the range of about 10:90 to 20:80. It is preferably 13:87 to 17:83. If either the saccharide compound or the monomer is present in a large amount, the crosslinking reaction is difficult to occur, and the particles may not grow to a sufficient size.

[0069] In order to increase the particle size, it is possible to increase the weight ratio of the monomer added or adjust the reaction temperature, but the most important point in the present invention is that the amount of surfactant added is extremely important. As a guideline, it is particularly effective to use a surfactant at a concentration of about 4 times the CMC or less, based on the CMC measured in a neutral salt-free aqueous solution. At a concentration much higher than the CMC, the number of monomers per emulsion particle dispersed by the surfactant becomes small, and it may not be possible to form large particles after polymerization.

[0070] On the other hand, below the CMC, the monomers act as nuclei to self-assemble the surfactant, so the number of monomers per emulsion particle increases, and as a result, the particles after polymerization become stable and large. As estimated from this reaction mechanism, the optimal surfactant concentration and the corresponding concentrations and composition ratios of monomers and sugar compounds vary depending on the size of the target particles, but it is usually preferable to use a concentration no greater than about four times the CMC, based on the CMC measured in a neutral salt-free aqueous solution.

[0071] The alkaline aqueous solution concentration in the mixed composition (mass of alkaline aqueous solution / total mass of mixed composition) is preferably 35 mass% or less, and the alkaline aqueous solution is included as a reaction site, and is the remainder excluding other sugar compounds, monomers, surfactants, and other additional components. If the alkaline aqueous solution concentration is too low, the hydroxyl groups of the sugar compounds and monomers are not sufficiently activated to promote the reaction, and particles may not be generated. On the other hand, if the alkaline aqueous solution concentration is too high, the hydroxyl groups of the reactants are overactivated, making it difficult to control the reaction, and the target particles may not be obtained.

[0072] [Reaction conditions] After preparing the mixed composition, the particles of the present invention can be obtained by reacting the mixed composition under predetermined conditions. During the reaction, it is preferable to carry out the reaction while appropriately stirring the mixed composition using a stirrer, stirring blades, or the like.

[0073] This reaction can be carried out at room temperature, or under appropriate heating or cooling conditions. For example, the reaction can be carried out at 0°C to 100°C, which is easy to operate even at normal pressure, using an alkaline aqueous solution. This temperature is preferably 10°C or higher, more preferably 20°C or higher, and more preferably 25°C or higher. If the temperature is too low, the reaction may be slow, or the surfactant or the like may not function sufficiently. The upper limit of the temperature may be 80°C or lower, 60°C or lower, or 50°C or lower, so as not to overheat the reaction.

[0074] The reaction time can be appropriately adjusted depending on the combination of the saccharide compound and the monomer for forming the particles, the particle size, the reaction temperature, etc. The reaction time of the particle production method of the present invention is preferably carried out for a relatively long time in order to grow the particles, and the reaction can be carried out for 1 hour or more, 2 hours or more, 3 hours or more, or 6 hours or more. Since the reaction time is considered to be completed within a certain range and saturated in a state where no particle growth occurs, the upper limit of the reaction time may be set to 100 hours or less, 50 hours or less, 30 hours or less, or less.

[0075] After the reaction, a neutralizing solution (such as an acidic aqueous solution) is mixed into the mixed composition in which the particles have been formed to neutralize the reaction solution and completely stop the reaction. Then, the mixture is mixed with a non-polar solvent such as acetone and reprecipitated to isolate only the particles. The molecular weight and particle size of the obtained particles are appropriately evaluated by static and dynamic light scattering measurements.

[0076] [Encapsulation of hydrophobic bioactive agents] A first composition containing the particles of the present invention and a second composition containing a hydrophobic bioactive agent are mixed to carry out an encapsulation step in which the hydrophobic bioactive agent is encapsulated in the particles of the present invention, thereby making it possible to produce a drug in which the hydrophobic bioactive agent is encapsulated in the particles of the present invention.

[0077] [First composition] The first composition includes the particles of the present invention. The first composition may be the particles of the present invention alone or may be a mixture with other dispersion media, but the concept of including both is referred to as the first composition in the present application. Since the particles of the present invention are dispersed in water, when a dispersion medium is used, it is preferable to use a polar solvent such as water or a lower alcohol having about 1 to 4 carbon atoms.

[0078] [Second Composition] The second composition contains a hydrophobic bioactive agent. The second composition may contain the hydrophobic bioactive agent alone or may be a mixture with other dispersion media, but the term "second composition" in this application includes both of these. It is preferable to use a dispersion medium such as a liquid for mixing the first composition and the second composition, and it is preferable that at least the second composition contains a dispersion medium.

[0079] The dispersion medium of the second composition is preferably an organic solvent that can be mixed with water at any ratio. The dispersion medium of the second composition preferably contains one or more dispersion media selected from the group consisting of dimethyl sulfoxide (hereinafter, "DMSO"), tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol. These dispersion media are amphipathic and suitable for dispersing hydrophobic bioactive agents. They are also suitable for mixing and dispersing with the first composition containing the particles of the present invention.

[0080] In this way, the first composition and the second composition are mixed, and the particles of the present invention come into contact with the hydrophobic bioactive agent in the mixed dispersion medium, so that the hydrophobic bioactive agent is encapsulated in the particles of the present invention. The mixing for encapsulation can be performed at about 10 to 50°C, or may be performed at about 20 to 40°C. The reaction time can be about 0.5 to 24 hours, or may be about 1 to 12 hours.

[0081] Equation 3 shows the reaction scheme for synthesizing particles as hyperbranched polymers using CD and ECH, and the scheme for introducing α-mangostin into the particles. A polymer can be obtained by reacting CD and ECH in an alkaline aqueous solution using a 33% by mass NaOH solution containing a surfactant at 30°C for 6 hours, and this polymer becomes particles of a specified particle size. This particle is mixed with α-mangostin in the presence of a solvent such as DMSO, and contacted with stirring at room temperature for 2 hours, to obtain a mixture in which α-mangostin is encapsulated in particles of a specified particle size.

[0082] [ka]

[0083] The reaction according to formula 3 will be taken as an example to provide a more detailed explanation. As a method for introducing (encapsulating) α-mangostin into particles, a preparation method is adopted in which α-mangostin / DMSO solution is added, which is dissolved in DMSO, an organic solvent that shows high affinity to polymerized particles and is soluble in water. CD is insoluble in DMSO, and even if α-mangostin / DMSO solution is simply added to CD aqueous solution, the encapsulation rate remains low. However, if the particles of the present invention are used, the dispersibility in DMSO is significantly improved even for particles having the structural unit of CD, so it is expected that α-mangostin will be efficiently introduced into CD in the polymer via DMSO.

[0084] For example, this mixing is performed by adding a small amount of 10 mg / mL α-mangostin DMSO solution to the aqueous solution of the particles of the present invention using CD. At this time, α-mangostin that has not been incorporated into the particles of the present invention precipitates, and is removed by centrifugation. The fact that α-mangostin is encapsulated in the particles of the present invention can be confirmed by FFF-UV / RI measurement using a flow field fractionation method and a UV-visible spectrophotometer / differential refractometer. The loading rate of α-mangostin in the particles of the present invention can be calculated from UV absorbance.

[0085] By introducing (encapsulating) α-mangostin into the particles of the present invention using the method of the present invention, a high loading rate can be achieved, which is about 10 times higher than that of polymeric micelles, which are often used as other polymeric carriers. EXAMPLES

[0086] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples unless the gist of the present invention is changed.

[0087] [Evaluation items] [Particle size (average particle size, particle size distribution)] Particle size was measured using "DelsaMax Pro" manufactured by Beckman Coulter, Inc. The particle size was determined by contin analysis of the autocorrelation function obtained by dynamic light scattering measurement using the attached software. The main measurement conditions and analysis conditions for particle size measurement are as follows: sample concentration: 5.0 mg / mL, solvent: water, number of measurements: 5, temperature: 25°C, scattering angle: 172°, wavelength: 532 nm. In the following, the mean hydrodynamic radius (R hav ) is the "average particle size." In addition, the average hydrodynamic radius (R hav ) is the median, the standard deviation (σ) is the average hydrodynamic radius (R hav ) divided by (σ / R hav ) is used as an index to represent the degree of distribution.

[0088] [Inclusiveness] The inclusion of hydrophobic bioactive agents in particles was evaluated using a Wyatt "Asymmetric Flow Field Fractionation Device (FFF) connected to an ultraviolet-visible spectrophotometer (UV) / differential refractometer (RI) FFF-UV / RI measurement."

[0089] [Loading rate] The loading rate of the hydrophobic bioactive agent on the particles was measured using a JASCO V-600.

[0090] [Reagents, etc.] Cyclodextrin (CD): Tokyo Chemical Industry Co., Ltd. Epichlorohydrin (ECH): Tokyo Chemical Industry Co., Ltd. Sodium hydroxide (NaOH): Fujifilm Wako Pure Chemical Industries, Ltd. Hexadecyltrimethylammonium bromide: Tokyo Chemical Industry Co., Ltd. Sodium dodecyl sulfate (SDS): Fujifilm Wako Pure Chemical Industries, Ltd. Triton-X100 (TX100): Nacalai Tesque, Inc. Alpha-mangosteen: Tokyo Chemical Industry Co., Ltd. ·Water: Ultra pure water Dimethyl sulfoxide (DMSO): Fujifilm Wako Pure Chemical Industries, Ltd.

[0091] [Example 1] Production of particles The method for producing the particles was carried out according to the flow chart shown in Figure 1. The experimental procedure was as follows. (1): Prepare a 33% by weight NaOH aqueous solution (water: 3.2 g, NaOH: 1.6 g). (2): Dissolve 500 mg of βCD in (1). (3): Next, disperse the mixture using ultrasound. (4): Add 100 μL of SDS solution (stock solution of 98.5 mM (abbreviated simply as "mM" from "mmol / L")) to the solution (3) (surfactant). Since 3.2 g of water is already present, the concentration of SDS is 3 mM. From now on, this will be referred to as X (first variable parameter). (5): Stir at room temperature for 5 minutes using a magnetic stirrer. (6): Add 2.4 mL of ECH while stirring the solution. This value will be referred to as Y (second variable parameter) hereafter. Then, stir at room temperature for 24 hours. (7): After stirring for 24 hours, the reaction solution is neutralized. (8): Reprecipitation is performed (good solvent: water, poor solvent: acetone). (9): The reprecipitated material was collected and dialyzed. Spectra / Por (registered trademark) Standard Regenerated Cellulose (RC) Membrane with a cut-off molecular weight of 3.5 kD was used for dialysis. After dialysis, the particles were freeze-dried for 3 to 4 days to obtain the particles of the present invention.

[0092] Figure 2 shows the particle size distribution of the obtained particles. The distribution of the peak to the variance σ / R hav The variance was calculated to be 0.226, which was a single peak. The average particle size calculated from the maximum of the peak was 59 nm. Hereafter, the cases where the variance is 0.25 or less, which indicates monodispersion, and the average particle size is in the range of 10 nm to 100 nm are shown as double circles.

[0093] As shown in FIG. 2, in the absence of SDS, particles with an average particle size of about 2 nm are produced, and even larger particles exist at around 20 nm. In this way, a multimodal distribution with multiple peaks is usually produced. This is because epoxy monomers have multiple reaction sites, and the reaction increases in a chain reaction, making it difficult to control. On the other hand, in the system in which the surfactant discovered in this invention is added, the surfactant acts as a phase transfer catalyst, controlling the reaction and giving a uniform, unimodal distribution. An example is shown below, where the average particle size obtained when SDS is added to 3 mM is very large, at about 60 nm.

[0094] As seen above, it is possible to produce polymers using CDs with controlled particle size by adding a surfactant.

[0095] [Production Example 1] The composition of SDS and ECH was changed in exactly the same manner as in Example 1, except that X and Y were changed. The combinations at that time are shown in Table 1. The dispersion was measured in the same manner as in Example 1. A single circle indicates a case where the condition of either unimodality or a dispersion of 0.25 or less is not met, but the main peak is in the range of 10 nm to 100 nm. An "x" is indicated when the sample is bimodal and the dispersion exceeds 0.25, and an "x" is also indicated when the average particle size is less than 10 nm or exceeds 100 nm. The results are shown in Table 1.

[0096] As shown in Table 1, ideal particles can be obtained with a specific combination of X and Y. Figure 3 shows the particle size distribution of the obtained particles. When SDS was 0 mM, the average particle size was less than 10 nm, resulting in an ×. Depending on the balance between the variable parameters X and Y, the results could show multiple peaks or the variance could become large.

[0097] As shown in FIG. 3, when the amount of ECH is 2.2 mL, the average particle size is about 28 nm, and when the amount of ECH is 2.3 mL, particles with an average particle size of about 76 nm are obtained.

[0098] [Table 1]

[0099] [Production Example 2] In Example 1, the concentration of SDS was set to 5 times the CMC. The particle size was evaluated using the same method as in Example 1, and the results are shown in Figure 4. The average particle size of the particles obtained under these conditions was about 6 nm, which was a small average particle size.

[0100] [Production Example 3] Using the particles obtained in Example 1, α-mangostin was encapsulated in the particles. 50 μL of α-mangostin DMSO solution (100 mM: this value is the third variable parameter. In FIG. 5, it is shown as Feeding cocn. of αMGS) was added to the microparticle aqueous solution (10 mg / mL, 450 μL) and then left at room temperature for 2 hours. Then, α-mangostin that was not encapsulated in the microparticles and became a precipitate was removed by centrifugation, and DMSO in the aqueous solution was removed by performing ultrafiltration three times. The resulting aqueous solution was subjected to FFF-UV / RI measurement and UV-vis measurement to confirm the encapsulation of α-mangostin in the microparticles and to determine the loading rate. In the same manner, the concentration of the α-mangostin DMSO solution was changed from 10 mM to 150 mM to perform encapsulation. The results are shown in FIG. 5.

[0101] The top graph in Figure 5 shows UV absorption data. Because there is no absorption before α-mangostin is encapsulated, and because α-mangostin absorbs UV at this wavelength (323 nm), the encapsulation rate per unit weight can be calculated from the absorbance. The results of the calculation are shown in the bottom graph in Figure 5. A loading rate of approximately 14 wt% was achieved. This is an extremely high value compared to Production Example 4 below.

[0102] [Production Example 4] 450μL of 10mg / mL βCD was prepared, 50μL of α-mangostin DMSO solution (concentration 100mM) was added, and the precipitated α-mangostin was removed by centrifugation. After removing the DMSO by dialysis, the loading rate was measured in the same manner as in Production Example 3, and was found to be 2-3%.

[0103] [Production Example 5] Particles were produced in the same manner as in Example 1, except that the value of the first variable parameter and the type of surfactant were changed to TX100. The evaluation results are shown in Table 2. An example of the particle size distribution measurement is shown in Figure 6. As shown in Table 2, with TX100, ideal particles can be obtained with a specific combination of X and Y, just like with SDS.

[0104] As shown in Figure 6, the average particle size of the particles obtained increases from about 15 nm to about 60 nm as the concentration of TX100 decreases from 0.75 mM to 0.05 mM. When TX100 was 0 mM, the average particle size was less than 10 nm, resulting in an X. Depending on the balance between the variable parameters X and Y, the results may show multiple peaks or the variance may become large.

[0105] [Table 2]

[0106] [Production Example 6] Particles were prepared in the same manner as in Example 1 except that the type of surfactant was changed to hexadecyltrimethylammonium bromide (CTAB). hav ) was calculated to be 0.2, which was a single peak. The average particle size calculated from the maximum of the peak was 40 nm. This can be judged to be equivalent to the double circles in Tables 1 and 2.

[0107] [Production Example 7] An attempt was made to encapsulate a drug other than α-mangostin in the particles obtained in Example 1. In this case, the drug was encapsulated in the same manner as in Production Example 3, and the loading rate was determined.

[0108] "Curcumin I" is curcumin, a polyphenol compound found in turmeric and other plants. "Resveratorol" is resveratrol, a type of stilbene derivative polyphenol. "THSG" is 2,3,5,4-tetrahydroxy-diphenylethylene-2-O-glucoside, a physiologically active substance extracted from Houttuynia cordata. "Dihydromyricetin" is dihydromyristicene, a type of flavanol. "Astilbin" is astilbin, a type of flavanol. "αMangostin" is α-mangostin (Production Example 3). "Dox" is doxorubicin, a type of anticancer drug.

[0109] A comparison of the loading rates of each drug is shown in Figure 7. All drugs could be encapsulated in the particles of the present invention, and the amount of encapsulation depended on the structure of the drug molecule.

[0110] [Production Example 8] Particles were synthesized using αCD and γCD instead of βCD in the same manner as in Example 1, and the particle size evaluation results are summarized in Table 3. As shown in Table 3, in the case of any CD, the average particle size of the obtained particles could be controlled within the range of 10 to 100 nm.

[0111] [Table 3]

[0112] [Example 2] In vitro experiment using cell lines Mouse colon cancer cells CT26WT were seeded onto a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. Cytotoxicity assays were performed using the highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] provided by Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.). WST-8 is reduced by intracellular dehydrogenases to produce formazan (orange), and the amount of this formazan dye is directly proportional to the number of viable cells. After adding the samples to the cells, the cells were cultured for 48 hours, and the amount of formazan dye was measured using a Multiscan FC spectrophotometer (Thermo Scientific) to determine the cell viability. The concentration of each drug was 50 μM.

[0113] The following samples were used: Nanoparticles used in animal experiments: average particle size 6 nm (produced under the conditions of Production Example 2) Drugs: Doxorubicin (Dox), Curcumin (Cur), Alpha-Mangosteen (MGS) Final drug concentration: 50μM Final drug-containing nanoparticle concentration: 0.45 mg / mL (drug concentration is 50 μM) Doxorubicin hydrochloride (Dox) purity 95% or more: Tokyo Chemical Industry Co., Ltd. Curcumin (Cur): Wako Pure Chemical Industries, Ltd. Alpha Mangosteen (MGS): Tokyo Chemical Industry Co., Ltd. Tween 80: Tokyo Chemical Industry Co., Ltd.

[0114] The evaluation results are shown in Figure 8. The nanoparticles (CDNP) themselves have almost no toxicity, and it can be seen that the toxicity of the nanoparticles encapsulating doxorubicin (Dox), curcumin (Cur), and α-mangostin (MGS) is almost the same as the toxicity of the original drugs. From these results, it was found that the nanoparticles (CDNP) used in the present invention do not impair the physiological activity of the drug even when encapsulated. In other words, it can be expected that the physiological activity of the drug itself will be expressed at the affected area to which it is delivered.

[0115] [Example 3] Animal experiments using mice Anticancer activity was tested using mice. All animal experiments described below were approved by the University of Kitakyushu and the Animal Ethics Committee of the University of Kitakyushu.

[0116] The antitumor effect of the particles of the present invention containing α-mangostin was examined using mice (BALB / c, 7 weeks old, male) bearing the mouse colon cancer cell CT26. 3 After confirming that the tumor volume had reached 10 mg / kg (7 days later), the sample was administered intravenously. The dose of α-mangostin was standardized to 10 mg / kg. After administration, the change in tumor volume was observed.

[0117] The following samples were used: Nanoparticles: average particle size 20 nm (prepared under the conditions of X (TX100): 0.15 mM, Y (ECH): 2.1 mL in Manufacturing Example 5) Alpha-mangosteen: Tokyo Chemical Industry Co., Ltd. Buffer solution: Phosphate buffer (pH 7.5) ·Non-α-mangostin nanoparticles: Nanoparticles only (50mg / mL) α-Mangosteen solution: α-Mangosteen solution (1.0 mM) containing 0.4 vol% DMSO, 2 vol% ethanol, and 2 vol% Tween 80 (surfactant) Alpha-mangostin-containing nanoparticles: A solution containing nanoparticles containing alpha-mangostin (nanoparticle concentration: 50 mg / mL, alpha-mangostin concentration: 1.0 mM)

[0118] Experimental data (animal experiments using mice) FIG. 9 shows the results of observing the transition of tumor volume after administration of the sample. Buffer (Control), α-mangostin solution (MGS), and nanoparticles not containing α-mangostin (Polymer) did not show any effect of suppressing cancer growth, whereas α-mangostin-containing nanoparticles (P / MGS) were able to suppress cancer growth. Furthermore, a significant antitumor effect was observed when compared with buffer and α-mangostin solution. This is believed to be because the particles of the present invention have an average particle size of 5 nm or more, making them less likely to be removed from the body by renal excretion, etc., and can remain in the blood, etc., for a long time by using the particles of the present invention. Furthermore, since the particles of the present invention can avoid renal excretion and are of a size suitable for the EPR effect, it is believed that a remarkable anticancer effect was obtained. [Industrial Applicability]

[0119] The particles of the present invention can be used as medicines containing hydrophobic bioactive agents and are therefore industrially useful. < / b1> < / b1> < / a1> < / a1>

Claims

1. The polymer includes a structural unit derived from a saccharide compound having a hydroxyl group and having clathrate properties, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group, and has an average hydrodynamic radius (R hav ) is 5 to 100 nm; The mean hydrodynamic radius (R hav ) is the median, the standard deviation (σ) of the particle size distribution is the mean hydrodynamic radius (R hav ) divided by (σ / R hav ) is 0.25 or less.

2. 2. The particle according to claim 1, wherein the saccharide compound comprises any saccharide compound selected from the group consisting of cyclodextone, β-1,3-glucan, and amylose.

3. 3. The particle according to claim 1, wherein the monomer comprises a multifunctional epoxy compound.

4. A drug comprising a hydrophobic bioactive agent encapsulated in the particle according to any one of claims 1 to 3.

5. The polymer includes a structural unit derived from a saccharide compound having a hydroxyl group and having clathrate properties, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group, and has an average hydrodynamic radius (R hav ) is a drug having a hydrophobic bioactive agent encapsulated in particles having a diameter of 5 to 100 nm, A drug comprising a hydrophobic bioactive agent, the hydrophobic bioactive agent being dispersed in one or more dispersion media selected from the group consisting of dimethylsulfoxide, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol.

6. 5. The method of claim 4, wherein the hydrophobic bioactive agent comprises a hydrophobic bioactive agent dispersed in one or more dispersion media selected from the group consisting of dimethylsulfoxide, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol.

7. The polymer includes a structural unit derived from a saccharide compound having a hydroxyl group and having clathrate properties, and a structural unit derived from a monomer having a functional group that reacts with a hydroxyl group, and has an average hydrodynamic radius (R hav ) is a drug having a hydrophobic bioactive agent encapsulated in particles having a diameter of 5 to 100 nm, The anticancer agent comprises a hydrophobic bioactive agent selected from the group consisting of alpha-mangostin, curcumin, and doxorubicin, and further comprises a pharma- ceutically acceptable carrier.

8. 7. An anti-cancer agent comprising the hydrophobic bioactive agent of claim 5 or 6, which comprises any one selected from the group consisting of alpha-mangostin, curcumin, and doxorubicin, and further comprises a pharma- ceutically acceptable carrier.

9. The anticancer agent according to claim 7 or 8, wherein the cancer is a solid cancer.

10. The method includes a step of mixing a mixed composition including a saccharide compound having a hydroxyl group and having inclusion properties, a monomer having a functional group reactive with a hydroxyl group, a surfactant, and an alkaline aqueous solution having a pH of 12 or more; A method for producing particles comprising a polymer having a structural unit derived from the saccharide compound and a structural unit derived from the monomer, the polymer being formed in the mixed composition.

11. The method for producing particles according to claim 10, wherein the surfactant comprises one or more surfactants selected from the group consisting of quaternary ammonium salt-based surfactants, sulfonate-based surfactants, and oligoethylene glycol-based surfactants.

12. The method for producing particles described in claim 10 or 11, wherein the concentration of the surfactant in the mixed composition (mass of surfactant / total mass of the mixed composition) is 4 times or less the critical micelle concentration of the surfactant in neutral water.

13. A method for producing a drug, comprising mixing a first composition containing particles obtained by the production method according to any one of claims 10 to 12 with a second composition containing a hydrophobic bioactive agent, thereby encapsulating the hydrophobic bioactive agent in the particles.

14. The method for producing a drug according to claim 13, wherein the second composition contains one or more dispersion media selected from the group consisting of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, propanol, and butanol.

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