Drug carrier and drug release-controlling formulation

Abstract

Provided are a novel drug carrier and a drug release-controlling formulation using the same. A drug carrier according to an embodiment comprises an aggregate of cellooligosaccharides having, at an anomeric position of a reducing terminal, a substituent including an alkyl group. A drug release-controlling formulation according to an embodiment comprises said drug carrier and a hydrophobic drug.

Description

Drug carriers and controlled release drug formulations

[0001] The present invention relates to a drug carrier and a controlled release drug formulation using the same.

[0002] In drug delivery systems (DDS), which are technologies for delivering drugs to required sites in the body, the vehicles that carry the drugs are called drug carriers or drug delivery carriers, and attempts have been made to use polymeric micelles, liposomes, etc. (See, for example, Patent Document 1.) Drug carriers release drugs in response to specific environments in the affected area, such as endogenous stimuli such as pH changes or overexpression of proteases.

[0003] It is known that crystalline cellulose can be artificially synthesized. The cellulose obtained by artificial synthesis is generally an oligomer called cellooligosaccharide. Patent Document 2 discloses cellooligosaccharides in which an alkyl group is introduced as a substituent at the anomeric position of the reducing end, and proposes that aggregates of such cellooligosaccharides be used as scaffolds for growing animal cells, films, battery separators, and the like.

[0004] JP 2022-075622 A International Publication No. 2016 / 113933

[0005] While investigating new uses for the above-mentioned cellooligosaccharides, the present inventors thought that it might be possible to construct a drug delivery system that releases drugs in response to bacteria expressing cellulase, for example.

[0006] The embodiments of the present invention have been made in view of the above points, and have an object to provide a novel drug carrier using cellooligosaccharide and a controlled-release drug formulation using the same.

[0007] The present invention includes the following embodiments: [1] A drug carrier comprising an aggregate of cellooligosaccharides having a substituent containing an alkyl group at the anomeric position of the reducing end. [2] The drug carrier according to [1], wherein the alkyl group is an alkyl group having 4 or more carbon atoms. [3] The cellooligosaccharide is represented by the following general formula (1): In the formula, n is the average degree of polymerization and represents a number from 5 to 20, and m is an integer of 4 or more. [4] The drug carrier according to any one of [1] to [3], wherein the aggregate of cellooligosaccharides has a cellulose type I crystalline structure. [5] The drug carrier according to any one of [1] to [3], wherein the aggregate of cellooligosaccharides has a cellulose type II crystalline structure. [6] A controlled drug release formulation comprising the drug carrier according to any one of [1] to [5] and a hydrophobic drug. [7] The controlled drug release formulation according to [6], wherein the hydrophobic drug is an antibacterial agent.

[0008] According to an embodiment of the present invention, it is possible to provide a novel drug carrier capable of releasing a hydrophobic drug in the presence of cellulase, and a novel controlled-release drug formulation using the same.

[0009] A conceptual diagram showing the preparation process of a drug carrier according to one embodiment. A conceptual diagram for explaining the mechanism of preparation and drug release of a controlled drug release formulation using a drug carrier according to one embodiment. Ultraviolet-visible absorption spectrum in an antibacterial agent loading test according to an example. A graph showing the antibacterial agent loading rate in an antibacterial agent loading test according to an example. A graph showing the results of an antibacterial agent release test for a controlled drug release formulation according to an example. A graph showing the results of an antibacterial agent test for a controlled drug release formulation according to an example.

[0010] The drug carrier according to this embodiment comprises an aggregate of cellooligosaccharides having a substituent containing an alkyl group at the anomeric position of the reducing end.

[0011] Cellooligosaccharides are oligosaccharides with a structure in which glucose units are linked by β-1,4-glycosidic bonds, and are also called cellulose oligomers. In this embodiment, a cellooligosaccharide in which a substituent containing an alkyl group is introduced at the anomeric position of the reducing end (hereinafter referred to as "alkylated cellooligosaccharide") is used.

[0012] The alkyl group of the alkylated cellooligosaccharide preferably has 4 or more carbon atoms in order to enhance hydrophobicity. There is no particular upper limit to the number of carbon atoms in the alkyl group, but from the viewpoint of ease of availability of the primer described below, the upper limit is preferably 30. The number of carbon atoms in the alkyl group is preferably 4 to 30, more preferably 4 to 20, more preferably 5 to 12, more preferably 5 to 10, and even more preferably 6 to 8. The alkyl group may be linear or branched. The alkyl group is preferably a linear alkyl group.

[0013] The average degree of polymerization (DP) of the alkylated cellooligosaccharide (the average number of glucose units present in one molecule) is not particularly limited, but is preferably 5 to 20. The average degree of polymerization (DP) is more preferably 6 to 16, more preferably 6 to 10, and even more preferably 7 to 9. The average degree of polymerization of the alkylated cellooligosaccharide is a weighted average of the degrees of polymerization according to the mass ratio of the alkylated cellooligosaccharides. The alkylated cellooligosaccharide is usually a mixture of compounds with different degrees of polymerization, and may include, for example, those with a degree of polymerization of 4 to 20, or those with a degree of polymerization of 5 to 18.

[0014] As the alkylated cellooligosaccharide, it is preferable to use a compound represented by the following general formula (1).

[0015] In formula (1), an alkyl group is bonded as a substituent to the carbon at position 1 (anomeric carbon) of the reducing end via an oxygen atom. The wavy line in the bond between the anomeric carbon and the alkoxy group in formula (1) indicates that the alkoxy group has an α-configuration, a β-configuration, or a mixture of α- and β-configurations. The β-configuration of the alkoxy group is preferred.

[0016] In formula (1), n ​​represents the average degree of polymerization of cellooligosaccharides, and as described above, it is preferably 5 to 20, more preferably 6 to 16, more preferably 6 to 10, and even more preferably 7 to 9.

[0017] In formula (1), m represents the number of carbon atoms in the alkyl group, and as described above, is preferably an integer of 4 or more, more preferably 4 to 30, more preferably 4 to 20, more preferably 5 to 12, more preferably 5 to 10, and even more preferably 6 to 8. The alkyl group may be linear or branched, as described above. Preferred specific examples of the alkyl group include linear alkyl groups such as n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and n-tetradecyl groups.

[0018] The aggregate of alkylated cellooligosaccharides is a crystallization of alkylated cellooligosaccharides, and may have a crystalline structure of cellulose type I (type 1) or a crystalline structure of cellulose type II (type 2).

[0019] The crystalline structure of cellulose type I is similar to that of naturally occurring cellulose, in that adjacent molecular chains have the same orientation, whereas the crystalline structure of cellulose type II is such that adjacent molecular chains have opposite orientations.

[0020] Specifically, as shown below in formula (1), the alkylated cellooligosaccharide is represented by an arrow pointing from the reducing end to the non-reducing end of the cellulose chain, and the alkyl group-containing substituent is represented by a wavy line. In the crystalline structure of cellulose type I, as shown in the cross-sectional structure in Figure 1, the alkylated cellooligosaccharides are arranged so that the arrows point in the same direction between adjacent molecular chains. Because the alkyl group-containing substituents are hydrophobic, an assembly of alkylated cellooligosaccharides having the cellulose type I crystalline structure forms a bilayer membrane-like structure in water, with the reducing end containing the substituent on the inside and the non-reducing end on the outside. Hereinafter, this is referred to as a bilayer membrane-like assembly. The bilayer membrane-like assembly allows hydrophobic drugs to be trapped in the inner hydrophobic portion, thereby enhancing the hydrophobic drug loading effect.

[0021] In contrast, in the crystalline structure of cellulose type II, as shown in the cross-sectional structure in Figure 1, alkylated cellooligosaccharides are arranged so that the arrows point alternately between adjacent molecular chains. Therefore, an aggregate of alkylated cellooligosaccharides having the crystalline structure of cellulose type II has a sheet-like structure consisting of a monolayer, with substituents having hydrophobic alkyl groups exposed on both the front and back sides of the sheet. Hereinafter, this is referred to as a monolayer nanosheet-like aggregate. Herein, the sheet-like structure is a concept that encompasses ribbon-like structures such as those shown in Figure 1, and therefore, a monolayer nanosheet-like aggregate is also referred to as a monolayer nanoribbon-like aggregate. In a monolayer nanosheet-like aggregate, the hydrophobic drug is not confined as in a bilayer membrane-like aggregate, and therefore the hydrophobic drug can be released relatively quickly during decomposition by cellulase.

[0022] The method for preparing the alkylated cellooligosaccharide aggregate is not particularly limited. For example, alkylated cellooligosaccharides may be prepared by an enzymatic synthesis reaction utilizing the reverse reaction of cellodextrin phosphorylase (CDP) (see Y. Yataka et al., Langmuir, 2016, 32, 10120-10125), or the alkylated cellooligosaccharides may be self-assembled to prepare the alkylated cellooligosaccharide aggregate (see T. Serizawa et al., J. Colloid Interface Sci., 2021, 601, 505).

[0023] Specifically, in the enzymatic synthesis reaction, α-glucose-1-phosphate (αG1P) and alkyl-β-D-glucopyranoside are reacted with CDP, whereby αG1P is sequentially polymerized as a monomer with the alkyl-β-D-glucopyranoside as a primer to obtain the alkylated cellooligosaccharide represented by formula (1).

[0024] CDP is known to be produced by microorganisms such as Clostridium thermocellum and Cellulomonas, and can be obtained by known methods using these microorganisms. For example, CDP derived from Clostridium thermocellum YM4 can be prepared in an Escherichia coli expression system according to the method described by M. Krishnareddy et al., J. Appl. Glycosci., 2002, 49, 1-8, but is not limited thereto.

[0025] The concentration of CDP is not particularly limited and may be, for example, 0.1 U / mL or more, or 0.2 U / mL or more. Here, the amount of CDP enzyme can be determined, for example, based on the enzyme activity. For example, αG1P, D-(+)-cellobiose, and CDP are incubated, the amount of phosphate produced by CDP is quantified, and the amount of enzyme that liberates 1 μmol of phosphate per minute can be defined as 1 U.

[0026] For example, 10 to 1000 mM αG1P, 10 to 200 mM alkyl-β-D-glucoside, and 0.1 U / mL or more of CDP are mixed in 100 to 1000 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.0 to 8.0), and the mixture is incubated at 10 to 80°C for 30 minutes to 30 days to allow the reaction to proceed, thereby synthesizing the alkylated cellooligosaccharide of formula (1).

[0027] In the self-assembly step, for example, alkylated cellooligosaccharides obtained by enzymatic synthesis may be incubated to self-assemble, thereby obtaining aggregates of alkylated cellooligosaccharides. Alternatively, the alkylated cellooligosaccharides obtained by enzymatic synthesis may be dissolved in alkali and then neutralized with acid to self-assemble (hereinafter, this self-assembly is referred to as neutralization-induced self-assembly). Alternatively, the alkylated cellooligosaccharides obtained by enzymatic synthesis may be dissolved in phosphoric acid, and then water may be added to cause self-assembly.

[0028] In one embodiment of neutralization-induced self-assembly, for example, in the case of cellooligosaccharides having alkyl groups with 6 or more carbon atoms, the crystalline structure of cellulose type I and the crystalline structure of cellulose type II can be selectively produced depending on the temperature conditions during neutralization, as shown in Figure 1. For example, an aqueous solution of alkylated cellooligosaccharides dissolved in sodium hydroxide is neutralized by adding hydrochloric acid, and the solution is incubated at 25°C to obtain an aggregate of the crystalline structure of cellulose type II, i.e., a monolayer nanosheet-like aggregate. On the other hand, an aqueous solution of alkylated cellooligosaccharides dissolved in sodium hydroxide is neutralized by adding hydrochloric acid, and the solution is incubated at 60°C to obtain an aggregate of the crystalline structure of cellulose type I, i.e., a bilayer membrane-like aggregate.

[0029] The alkylated cellooligosaccharide aggregate obtained in this manner has a portion where hydrophobic alkyl groups are aggregated, and therefore can carry hydrophobic drugs. Furthermore, the aggregate can be decomposed by the action of cellulase, and the hydrophobic drug can be released by the decomposition. Therefore, the alkylated cellooligosaccharide aggregate can be used as a drug carrier for hydrophobic drugs. The drug carrier of this embodiment contains the alkylated cellooligosaccharide aggregate, and may consist solely of the aggregate, or may contain optional components such as buffer components and serum components in addition to the aggregate.

[0030] The controlled release formulation of this embodiment includes the drug carrier and a hydrophobic drug supported on the drug carrier. Here, "supported" means that the hydrophobic drug is immobilized or supported so as not to fall off from the drug carrier, and the form of support is not particularly limited.

[0031] Regarding the loading form, for example, when the assembly of alkylated cellooligosaccharides has a cellulose type I crystal structure, the hydrophobic drug (in the figure, the hydrophobic antibacterial drug) may be loaded in a state of being confined in the hydrophobic portion inside the bilayer membrane-like assembly, as shown in Figure 2. On the other hand, when the assembly of alkylated cellooligosaccharides has a cellulose type II crystal structure, the hydrophobic drug may be loaded in the hydrophobic portion on the front and / or back side of the monolayer nanosheet-like assembly, as shown in Figure 2.

[0032] The hydrophobic drug is not particularly limited as long as it has hydrophobicity to such an extent that it can be stably supported by the aggregate of alkylated cellooligosaccharides having hydrophobic groups, but it is preferable to use a drug that is poorly soluble in water (including cases where it is insoluble). The solubility of the hydrophobic drug in water at 25°C may be less than 1 mg / mL or less than about 100 μg / mL.

[0033] Specific examples of hydrophobic drugs include antibiotics, anticancer drugs, antiviral drugs, etc., which may be used alone or in combination of two or more. Antibacterial drugs are preferably used as the hydrophobic drug. Specific examples of hydrophobic antibiotics include rifampicin, macrolide drugs, fluoroquinolones, tetracyclines, linezolid, chloramphenicol, etc., which may be used alone or in combination of two or more.

[0034] In the controlled release drug formulation, the amount of the hydrophobic drug is not particularly limited, and may be, for example, 0.01 to 10% by mass, or 0.1 to 1% by mass, based on the total mass of the assembly of alkylated cellooligosaccharides.

[0035] The method for loading a hydrophobic drug onto a drug carrier is not particularly limited. In one embodiment, as shown in Figure 2, the hydrophobic drug can be loaded onto the alkylated cellooligosaccharide aggregate by adding the hydrophobic drug dissolved in a water-miscible organic solvent to an aqueous dispersion of the alkylated cellooligosaccharide aggregate and mixing them.

[0036] The dispersion medium for the aqueous dispersion of alkylated cellooligosaccharide aggregates may be water alone, a buffer solution such as PBS, or an aqueous solvent consisting primarily of water mixed with a hydrophilic organic solvent. The concentration of alkylated cellooligosaccharide in the aqueous dispersion is not particularly limited and may be, for example, 0.01 to 5% (w / v) or 0.1 to 1% (w / v). The concentration of the hydrophobic drug is also not particularly limited and may be, for example, 1 to 500 μM or 10 to 100 μM.

[0037] In this specification, "% (w / v)" means a mass / volume percent concentration, which is the mass (g) of a substance of interest contained in a volume of 100 mL. Also, "M" means a molar concentration (mol / L), so "μM" means "μmol / L."

[0038] When adding a hydrophobic drug to an aqueous dispersion of alkylated cellooligosaccharides, it is preferable to add the hydrophobic drug as a solution in a water-miscible organic solvent, such as, but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, N-methylpyrrolidone, or dioxane.

[0039] The controlled release drug formulation may consist of only a drug carrier and a hydrophobic drug, or may contain optional components such as a buffer component, serum component, and the like.

[0040] Because the controlled-release preparation is an aggregate of cellooligosaccharides that carries the hydrophobic drug, it can be decomposed by cellulase, a cellulose hydrolase enzyme, to release the hydrophobic drug, as shown in Figure 2. This allows the release behavior of the hydrophobic drug to be controlled, making it possible to construct a drug delivery system that delivers the hydrophobic drug to the necessary site in the body. Furthermore, the controlled-release preparation can be decomposed by the action of the enzyme cellulase, thereby gradually releasing the hydrophobic drug, thereby providing a sustained-release effect.

[0041] Specifically, for example, a controlled-release drug formulation containing an antibacterial agent against a pathogenic bacterium that expresses cellulase to decompose cellulose can be administered to a patient infected with the pathogenic bacterium. In this case, the controlled-release drug formulation is decomposed in the patient's body by cellulase produced by the pathogenic bacterium, releasing the antibacterial agent, thereby suppressing the growth of the pathogenic bacterium or killing it. Specifically, for example, when orally administered to a human, the controlled-release drug formulation is not decomposed until it reaches the site where the pathogenic bacterium that expresses cellulase is present, and is decomposed by cellulase at the site where the pathogenic bacterium is present in the digestive tract (e.g., stomach, small intestine, large intestine), allowing the antibacterial effect of the antibacterial agent to be exerted.

[0042] Examples of pathogenic bacteria that express such cellulases include Enterococcus faecalis, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Bacillus cereus.

[0043] The cellulase used to degrade the controlled release drug formulation is not limited to that expressed by pathogenic bacteria, and may be administered to a specific site in the body by a means separate from the controlled release drug formulation.

[0044] The present invention will be further explained below with reference to examples, but is not limited to these.

[0045] [Synthesis of Hexylated Cellooligosaccharides] Hexylated cellooligosaccharides with an average degree of polymerization of 7 (n = 7) were synthesized by enzyme-catalyzed polymerization according to the following reaction formula, according to the method described in Y. Yataka et al., Langmuir, 2016, 32, 10120-10125.

[0046] Specifically, 200 mmol / L of αG1P, 50 mmol / L of hexyl-β-D-glucopyranoside, and 0.2 U / mL of cellodextrin phosphorylase (CDP) were mixed in 500 mmol / L of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.5) and incubated at 60°C for 3 days. The reaction solution containing the product was centrifuged (15,000 rpm, 10 minutes or more, 4°C), the supernatant was removed, and then ultrapure water was added to redisperse the product. This was followed by centrifugation (under the same conditions) repeatedly to purify the product until the supernatant replacement rate reached 99.999% or more, yielding hexylated cellooligosaccharide (CEL-C). ６ ) was obtained.

[0047] [Synthesis of other cellooligosaccharides] Butyl-β-D-glucopyranoside, octyl-β-D-glucopyranoside, and cellobiose were used instead of hexyl-β-D-glucopyranoside, and the other procedures were the same as for hexylated cellooligosaccharides to prepare butylated cellooligosaccharides (CEL-C). ４ ), octylated cellooligosaccharides (CEL-C ８The average degree of polymerization was 7 (n=7) for both the oligosaccharides.

[0048] The average degree of polymerization of cellooligosaccharides was measured using a proton nuclear magnetic resonance (NMR) spectrometer. Specifically, 12 mg or more of the freeze-dried product was dissolved in 600 μL of a 4% by weight sodium deuterium hydroxide heavy water solution to prepare a measurement sample. The NMR spectrometer used was an ADVANCE III HD500 (Bruker Biospin, magnetic field strength: 500 MHz, number of accumulations: 16). The average degree of polymerization was calculated based on the integral values ​​of the protons at the anomeric position of the reducing end of the cellooligosaccharide and at other anomeric positions.

[0049] [Preparation of cellooligosaccharide aggregates] Cellooligosaccharide aggregates were prepared by neutralization-induced self-assembly according to the method described in T. Serizawa et al., J. Colloid Interface Sci., 2021, 601, 505. The details are as follows.

[0050] Example 1: CEL Ｉ -C ６ Preparation of CEL-C in 1 mol / L NaOH aqueous solution ６ The resulting aqueous solution was neutralized with an equal volume of 1 mol / L HCl aqueous solution containing 100 mmol / L of phosphate buffer (adjusted so that the pH after neutralization was 6.8), and allowed to stand at 60°C for 24 hours to obtain bilayer membrane aggregates (CEL) with a cellulose I type crystalline structure. Ｉ -C ６ The fact that it has a cellulose type I crystal structure was confirmed by infrared absorption spectroscopy.

[0051] Example 2: CEL ＩＩ -C ６ Preparation of CEL-C in 1 mol / L NaOH aqueous solution ６The resulting aqueous solution was neutralized with an equal volume of 1 mol / L HCl aqueous solution containing 100 mmol / L of phosphate buffer component (adjusted so that the pH after neutralization was 6.8), and allowed to stand at 25°C for 24 hours to obtain a single-layer nanosheet-like aggregate CEL having a cellulose type II crystal structure. ＩＩ -C ６ The fact that it has a cellulose type II crystal structure was confirmed by infrared absorption spectroscopy.

[0052] Example 3: CEL ＩＩ -C ４ ) The butylated cellooligosaccharide (CEL-C) obtained by the above enzyme-catalyzed polymerization ４ ) is a single-layer nanosheet-like assembly with a cellulose type II crystalline structure, CEL ＩＩ -C ４ (confirmed by infrared absorption spectroscopy), and this was used as it was as the cellooligosaccharide assembly in Example 3.

[0053] Example 4: CEL Ｉ -C ８ Octylated cellooligosaccharide (CEL-C) obtained by the above enzyme-catalyzed polymerization ８ ) is a bilayer membrane-like assembly with the crystalline structure of cellulose type I. Ｉ -C ８ (confirmed by infrared absorption spectrum), and this was used as it was as the cellooligosaccharide assembly in Example 4.

[0054] (Comparative example 1: CEL ＩＩ The cellooligosaccharide (CEL-OH) obtained by the above enzyme-catalyzed polymerization is a single-layer nanosheet-like aggregate with a cellulose II type crystalline structure. ＩＩ This was used as it was as the cellooligosaccharide assembly of Comparative Example 1.

[0055] [Antibacterial Drug Loading Test] A loading test of the antibacterial drug rifampicin was carried out on the cellooligosaccharide assemblies of Examples 1 to 4 and Comparative Example 1. Specifically, a dispersion of cellooligosaccharide assemblies at a concentration of 0.2% (w / v) was prepared using PBS (phosphate buffered saline) as the dispersion medium. A DMSO solution of rifampicin was added to the dispersion so that the rifampicin concentration was 20 μM and the DMSO concentration was 0.2% (v / v), and the dispersion was allowed to stand at 25°C for 24 hours. Here, "% (v / v)" refers to the volume percentage concentration.

[0056] After standing, the mixture was centrifuged (15,000 rpm, 10 minutes or more, 4°C), and the supernatant was dispensed and subjected to UV-visible absorption spectroscopy. UV-visible absorption spectroscopy was also performed on a control prepared in the same manner as in the Examples, except that no cellooligosaccharide assembly was added. The antibacterial loading rate (%) was calculated from the ratio of the absorbance at a wavelength of 475 nm, which is the absorbance derived from rifampicin, for each Example and Comparative Example to the absorbance of the control.

[0057] The ultraviolet-visible absorption spectrum was measured using a V-670 (manufactured by JASCO Corporation) in the wavelength range of 300-600 nm.

[0058] FIG. 3 shows the results of Example 1 (CEL Ｉ -C ６ ), Example 2 (CEL ＩＩ -C ６ 3 is a graph showing the ultraviolet-visible absorption spectra of the alkylated cellooligosaccharides (A) and the control (-). As shown in FIG. 3, the absorbance at a wavelength of 475 nm derived from the antimicrobial agent decreased in the Examples, indicating that the alkylated cellooligosaccharides carried the antimicrobial agent.

[0059] The antimicrobial loading rates for each Example and Comparative Example are shown in Figure 4 and Table 1. No antimicrobial was loaded in the cellooligosaccharide assembly of Comparative Example 1, in which the reducing end was not alkylated. In contrast, the antimicrobial was loaded in the cellooligosaccharide assemblies of Examples 1 to 4, in which the reducing end was alkylated. The loading rate increased with increasing carbon number up to 6 carbon atoms in the alkyl group, but decreased when the carbon number reached 8 carbon atoms. Among Examples 1 to 4, the cellooligosaccharide assembly having a cellulose I-type crystalline structure in which an alkyl group with 6 carbon atoms had been introduced showed the highest loading rate.

[0060]

[0061] [Antibacterial Drug Release Test by Cellulase] The cellooligosaccharide aggregate (CEL) of Example 1 was Ｉ -C ６ In detail, a cellulase degradation test was carried out on cellooligosaccharide aggregates (CEL) using PBS as a dispersion medium. Ｉ -C ６ A dispersion of rifampicin at a concentration of 0.2% (w / v) was prepared. A DMSO solution of rifampicin was added to the dispersion so that the rifampicin concentration was 20 μM and the DMSO concentration was 0.2% (v / v), and the mixture was allowed to stand at 25°C for 24 hours. Cellulase was then added to a concentration of 0.5 U / mL, and each sample was centrifuged (15,000 rpm, 10 minutes or more, 4°C) at 0, 1, 2, and 4 hours after the addition of cellulase. The supernatant was then dispensed and subjected to UV-visible absorption spectroscopy. Cellulase derived from Trichoderma reesei was used as the cellulase.

[0062] The method for measuring the ultraviolet-visible absorption spectrum was as described above, and the ratio of the absorbance at a wavelength of 475 nm to the absorbance at 0 hours after the addition of cellulase was calculated as the relative absorbance.

[0063] The results are shown in Figure 5. Two hours after the addition of cellulase, the relative absorbance reached almost its maximum value, suggesting that the cellooligosaccharide aggregates were almost completely decomposed and all of the antibiotic was released within two hours.

[0064] [Antibacterial Test of Antibacterial Drug-Carrying Cellooligosaccharide Assembly] The cellooligosaccharide assembly (CEL Ｉ -C ６ After carrying an antibacterial agent, the cellooligosaccharide aggregate (CEL) was subjected to an antibacterial test against Escherichia coli by treating it with cellulase. Ｉ -C ６ A dispersion of rifampicin at a concentration of 0.2% (w / v) was prepared. A DMSO solution of rifampicin was added to the dispersion so that the rifampicin concentration was 10 μM and the DMSO concentration was 0.2% (v / v), and the dispersion was allowed to stand at 25°C for 24 hours. Then, 0.5 U / mL of cellulase and 2.5 × 10 Escherichia coli were added. ５ The culture medium was then incubated at 37°C for 18 hours. After incubation, colony counting was performed to quantify the number of bacteria. The results are shown in Figure 6 as "Example 5 (Antibacterial agent: Present, Cellulase: Present)."

[0065] For comparison, as Control 1, the cellooligosaccharide assembly, rifampicin, and cellulase were not added, and the other conditions were the same as in Example 5. The results are shown in Figure 6 as "Control 1 (antibacterial agent: no, cellulase: no)."

[0066] For comparison, a control 2 was prepared without adding cellulase, and the same test as in Example 5 was otherwise carried out. The results are shown in Figure 6 as "Control 2 (antibacterial agent: present, cellulase: absent)."

[0067] The Escherichia coli used was ATCC 51813 strain. Colony counting was performed using "Compact Dry CF" (Shimadzu Diagnostics Co., Ltd.). As for cellulase, cellulase derived from Trichoderma reesei was used as described above.

[0068] As shown in Figure 6, Control 2, to which no cellulase was added, showed no bactericidal activity against E. coli, similar to Control 1, in which only E. coli was incubated. In contrast, in Example 5, in which an antimicrobial agent was carried and cellulase was added, the antimicrobial agent was released in response to cellulase, so E. coli was below the detection limit (N.D.), demonstrating bactericidal activity.

[0069] The various numerical ranges described in this specification can be arbitrarily combined with their respective upper and lower limit values, and all such combinations are considered to be preferred numerical ranges described in this specification. Furthermore, a numerical range described as "X to Y" means from X to Y.

[0070] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included within the scope and spirit of the invention, as well as within the scope of the invention described in the claims and their equivalents.

Claims

1. A drug carrier comprising an aggregate of cellooligosaccharides having a substituent containing an alkyl group at the anomeric position of the reducing end.

2. The drug carrier according to claim 1, wherein the alkyl group is an alkyl group having 4 or more carbon atoms.

3. The cellooligosaccharide is represented by the following general formula (1):

2. The drug carrier according to claim 1, wherein n in the formula is an average degree of polymerization and represents a number of 5 to 20, and m represents an integer of 4 or more.

4. The drug carrier according to claim 1, wherein the cellooligosaccharide aggregate has a cellulose type I crystalline structure.

5. The drug carrier described in claim 1, wherein the cellooligosaccharide aggregate has a cellulose type II crystalline structure.

6. A controlled release drug formulation comprising the drug carrier according to any one of claims 1 to 5 and a hydrophobic drug.

7. The controlled release drug formulation of claim 6, wherein the hydrophobic drug is an antibacterial drug.

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