Preventive or therapeutic agent for inflammatory bowel disease

Abstract

An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease that utilizes iR-29a and / or miR-29b and exhibits excellent preventive or therapeutic effects against inflammatory bowel disease. A preventive or therapeutic agent for inflammatory bowel disease, which comprises composite particles in which miR-29a and / or miR-29b is supported on carbonate apatite particles, and is administered systemically.

Description

[Technical field]

[0001] The present invention relates to a preventive or therapeutic agent for inflammatory bowel disease. More specifically, the present invention relates to a preventive or therapeutic agent for inflammatory bowel disease using miR-29a and / or miR-29b. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the gastrointestinal tract, and is mainly classified into ulcerative colitis and Crohn's disease (Non-Patent Documents 1 to 3). In particular, the incidence of inflammatory bowel disease is increasing in Europe and the United States (Non-Patent Document 2), and the high recurrence rate of inflammatory bowel disease is a serious problem. Conventionally, acute inflammatory bowel disease has basically been treated with anti-inflammatory drugs, immunosuppressants, glucocorticoids, etc. (Non-Patent Document 4). In addition, as a new treatment for inflammatory bowel disease, a method of inhibiting the mucosal inflammatory pathway targeting proinflammatory cytokines such as TNF-α or their cell surface receptors has been proposed (Non-Patent Documents 5 and 6).

[0003] However, even when inflammatory bowel disease is treated with drugs, approximately 30% of patients still require surgical treatment due to severe inflammation, intestinal stenosis, or toxic megacolon.

[0004] Although the etiology and pathology of inflammatory bowel disease have not been fully elucidated, it is believed that a combination of multiple factors, including oxidative stress disorders, congenital and adaptive immune disorders, genetic factors, intestinal bacteria, and diet, is involved.

[0005] On the other hand, nucleic acid medicines have been attracting attention as a next-generation treatment for various diseases. Among nucleic acid medicines, microRNAs (miRNAs) are small non-coding RNAs that regulate the expression of multiple target genes mainly by binding to complementary sequences in the 3'-UTR region. Recent studies have revealed the existence of miRNAs involved in epithelial barrier function (miRs-7, 21, 150), autophagy (miRs-13A, 93, 106, 142-3p), NF-kB signaling pathway (miRs-126, 146a), and IL23 / Th17 inflammatory axis (miRs-20, 23a, 29b, 155) (Non-Patent Document 7). Therefore, these miRNAs or anti-miRNAs are expected to be therapeutic tools for inflammatory bowel disease. However, conventionally, the diseases for which effective treatment with nucleic acid medicines using miRNAs has been recognized are limited to eye diseases, hyperlipidemia, and muscle degeneration diseases.

[0006] In addition, for nucleic acid medicines that use miRNA, it is important to efficiently deliver miRNA to target cells in vivo. Conventionally, liposomes have been widely used to deliver miRNA into cells in vitro, but the technology to efficiently deliver miRNA to the affected area in vivo for the treatment of inflammatory bowel disease has not been established.

[0007] On the other hand, it has been reported that in mice in which miR-29a and miR-29b have been knocked out, colitis induced by dextran sulfate (DSS) is aggravated (Non-Patent Document 8), and miR-29a and miR-29b are expected to be therapeutic agents for inflammatory bowel disease. However, as mentioned above, a technology for efficiently delivering miRNA to the affected area of ​​inflammatory bowel disease in vivo has not been developed, and in order to put miR-29a and miR-29b into practical use as therapeutic agents for inflammatory bowel disease, it is essential to establish an in vivo delivery technology (Non-Patent Document 9). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Nat Rev Immunol 3 : 521-33, 2003 [Non-Patent Document 2] J Clin Invest 117 : 514-21, 2007 [Non-Patent Document 3] Gastroenterology 115: 182-205, 1998 [Non-Patent Document 4] Drugs 65: 2253-86, 2005 [Non-Patent Document 5] Curr Opin Gastroenterol 23: 395-9, 2007 [Non-Patent Document 6] Surgery today 45: 933-8, 2015 [Non-Patent Document 7] Gut 64(6): 1008, 2015 [Non-Patent Document 8] Immunity 39: 521-36, 2013. [Non-Patent Document 9] Int J Nanomedicine 11: 5287-5310, 2016. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a preventive or therapeutic agent for inflammatory bowel disease, which utilizes miR-29a and / or miR-29b and shows excellent preventive or therapeutic effects against inflammatory bowel disease. [Means for solving the problem]

[0010] It is known that carbonate apatite particles have the effect of delivering nucleic acids into cells (JP Patent Publication 2007-63194). Carbonate apatite particles are not known to accumulate in inflamed tissues, and therefore, according to the prior art, even if composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles are administered systemically, it is considered that miR-29a and / or miR-29b cannot be efficiently delivered to the affected area of ​​inflammatory bowel disease. Therefore, the present inventors attempted to locally administer composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles to the affected area of ​​inflammatory bowel disease, but almost no improvement of inflammatory bowel disease was observed (see Reference Test Example 5 described later).

[0011] Under such circumstances, the present inventors have conducted intensive research and found that when composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles are administered systemically, not only a part of the miRNA is taken up in the affected area of ​​inflammatory bowel disease, but also a part of the miRNA is taken up in dendritic cells localized on the mucosa of the affected area, and inflammatory bowel disease can be effectively improved by the action of the miRNA directly taken up in the affected area and the action of the dendritic cells into which the miRNA has been taken up. That is, the present inventors have found that by administering composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles systemically, the miRNA acts not only on the affected area of ​​inflammatory bowel disease but also on dendritic cells involved in the early immune response of inflammation, thereby significantly improving the therapeutic effect of inflammatory bowel disease. The present invention was completed based on such findings and through further research.

[0012] That is, the present invention provides the following aspects. Item 1. Composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, A preventive or therapeutic agent for inflammatory bowel disease that is administered systemically. Item 2. The preventive or therapeutic agent for inflammatory bowel disease according to Item 1, which is administered intravascularly or subcutaneously. Item 3. The preventive or therapeutic agent for inflammatory bowel disease according to Item 1 or 2, wherein the carbonate apatite particles have an average particle size of 400 to 3,000 nm. Item 4. Composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, Use for the production of a preventive or therapeutic agent for inflammatory bowel disease to be administered systemically. Item 5. The use according to Item 4, wherein the systemic administration is intravascular administration or subcutaneous administration. Item 6. The use according to Item 4 or 5, wherein the carbonate apatite particles have an average particle size of 400 to 3,000 nm. Item 7. A method for preventing or treating inflammatory bowel disease, comprising systemically administering composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles to a patient in whom prevention or treatment of inflammatory bowel disease is required. Item 8. The method according to Item 7, wherein the systemic administration is intravascular administration or subcutaneous administration. Item 9. The method according to Item 7 or 8, wherein the carbonate apatite particles have an average particle size of 400 to 3000 nm. Effect of the Invention

[0013] According to the preventive or therapeutic agent for inflammatory bowel disease of the present invention, by systemically administering composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, the miRNA is taken up not only in the affected area of ​​inflammatory bowel disease but also in dendritic cells, thereby achieving significantly superior therapeutic effects.

[0014] miR-29a and / or miR-29b directly incorporated into the affected area of ​​inflammatory bowel disease contributes to alleviating or curing the symptoms of the affected area. In addition, dendritic cells incorporating miR-29a and / or miR-29b contribute to the therapeutic effect of inflammatory bowel disease by reducing the expression levels of cytokines (IL-6 and TGF-β) involved in the differentiation of naive T cells into Th-17 cells and cytokines (IL12 p40 and IL23 p19) that activate Th-17 cells and lead them to pathogenic Th-17 cells. That is, in the preventive or therapeutic agent for inflammatory bowel disease of the present invention, composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles are administered systemically, so that the agent not only acts directly on the affected area but also acts on dendritic cells, thereby making it possible to achieve a significantly superior therapeutic effect. Dendritic cells are an important factor involved in the early immune response of inflammatory bowel disease, and by inhibiting them, the preventive or therapeutic agent for inflammatory bowel disease of the present invention can effectively suppress the spread of inflammation throughout the intestinal tract mediated by interferon signals. [Brief description of the drawings]

[0015] [Figure 1] 1 shows the results of Reference Test Example 1. (a) shows the results of measuring the amount of MIRTX uptake in the rectum, transverse colon, cecum, and ileum after intravenous administration of MIRTX (miR-29b derivative (artificial miRNA)) / sCA complex to a DSS-induced mouse enteritis model. (b) shows images of the intestine stained with nuclei and observed under a fluorescent microscope after intravenous administration of Alexa647-miRNA / sCA complex to a mouse enteritis model induced by DSS. [Diagram 2]1 shows the results of Test Example 1. (a) shows the results of measuring the expression level of miR-29b-3p in the large intestine mucosa extracted from a mouse to which DSS and miR-29b / sCA complexes were intravenously administered. (b) shows the results of HE staining and microscopic observation of the rectum extracted from a mouse to which DSS and various miRNA / sCA complexes were intravenously administered. (c) shows the results of measuring the body weight of the mouse over time. (d) shows the results of measuring the body weight of the mouse over time 7 days after the start of the test. [Diagram 3] 1 shows the results of Test Example 1. (a) shows the results of measuring the length of the large intestine 7 days after the start of the test for mice that had been intravenously administered DSS and various miRNA / sCA complexes. (b) shows the results of scoring the degree of inflammation in the colorectum 7 days after the start of the test for the mice. (c) shows the results of measuring the expression level of miR-29b in the large intestine for a normal group at the start of the test and for a DSS group 4 and 6 days after the start of the test. [Figure 4] 1 shows the results of Reference Test Example 2. (a) is an image showing the results of observing the rectum under a fluorescent microscope after intravenous administration of the Alexa647-miRNA / sCA complex to a DSS-induced mouse colitis model and immunostaining it with an anti-CD11c antibody and DAPI. (b) is an image showing the results of observing the immunostained rectum under a confocal microscope. [Diagram 5] In Test Example 2, sCA-miR-29a / sCA complex or sCA-miR-29b / sCA complex was administered intravenously to a DSS-induced mouse colitis model, the large intestine was then collected, and the expression levels of messenger RNA for various cytokines (IL12 p40, IL23 p19, IL-6, and TGF-β) in dendritic cells isolated from the mucosa were measured. [Figure 6] FIG. 13 shows the results of analyzing gene expression levels by IPA using large intestines excised from mice to which DSS and various miRNA / sCA complexes were intravenously administered in Test Example 3. [Figure 7]This is an intracellular signal pathway map from Toll-like receptor (TLR) to the production of IFN and inflammatory cytokines. In Figure 7, molecules marked with an * were found to be activated by intravenous administration of DSS. [Figure 8] IFN signal pathway. In Fig. 8, molecules marked with * are those predicted to be activated by IPA, and molecules marked with ○ are those confirmed to be upregulated by mRNA array. [Figure 9] 1 shows the results of Test Example 4. a shows the results of measuring the body weight of mice subcutaneously administered with DSS and various miRNA / sCA complexes over time. b shows the results of measuring the body weight of the mice at the end of the test. c shows the results of measuring the length of the large intestine of the mice. d shows the results of scoring the degree of inflammation in the rectum of the mice. e shows the results of HE staining of the excised rectum of the DSS+sCA-miR-29b group and observing it under a microscope. [Figure 10] 5 shows the results of Test Example 5. (a) is an image showing the results of observing the rectum under a fluorescent microscope after subcutaneous administration of Alexa647-miR-29b / sCA complex into a DSS-induced mouse enteritis model and immunostaining it with anti-CD11c antibody and DAPI. (b) is a image showing the results of measuring the amount of miR-29b in CD11c-positive cells and CD11c-negative cells isolated from the large intestine of the mouse. (c) is an image showing the results of observing the rectum of the mouse under a fluorescent microscope after immunostaining it with anti-Ly-6G antibody, anti-F4 / 80 antibody, anti-CD4 antibody, anti-CD45R antibody, and DAPI. [Figure 11] In Test Example 5, after subcutaneous administration of the Alexa647-miR-29b / sCA complex to a DSS-induced mouse colitis model, the rectum was immunostained with anti-Ly-6G antibody, anti-F4 / 80 antibody, anti-CD4 antibody, anti-CD45R antibody, and DAPI and observed under a fluorescent microscope. [Figure 12]1 shows the results of Test Example 6. (a) shows the results of microscopic observation of the rectum stained with HE staining after intravenous administration of miR-29b / sCA complex (>50 nm) or miR-29b / sCA complex (≦50 nm) to a DSS-induced mouse colitis model. (b) shows the results of histological evaluation of the degree of inflammation in the large intestine of the mice. [Figure 13] 13 shows images of the rectum immunostained with anti-CD11c antibody and DAPI and observed under a fluorescent microscope in a DSS-induced mouse colitis model in Reference Test Example 3 after subcutaneous administration of the Alexa647-miRNA / sCA complex. [Figure 14] This figure shows the results of measuring the amount of MIRTX uptake in CD11c-positive cells (dendritic cells) and CD11c-negative cells isolated from the mucosa after subcutaneous administration of MIRTX / sCA complex to a DSS-induced mouse colitis model in Reference Test Example 4. [Figure 15] 1 shows the results of Reference Test Example 5. a shows the results of measuring the body weight of mice to which DSS and various miRNA / sCA complexes were locally administered over time. b shows the results of measuring the body weight of the mice at the end of the test. c shows the results of measuring the length of the large intestine of the mice. d shows the results of scoring the degree of inflammation in the rectum of the mice. e shows the results of HE staining of the excised rectum of the DSS+sCA-miR-29b group and microscopic observation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The preventive or therapeutic agent of the present invention is a drug used for preventing or treating inflammatory bowel disease, which contains composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, and is characterized by being administered systemically. The preventive or therapeutic agent of the present invention will be described in detail below.

[0017] miRNA The preventive or therapeutic agent of the present invention contains miR-29a and / or miR-29b as miRNA. It has been reported that in mice in which miR-29a and miR-29b have been knocked out, colitis induced by dextran sulfate (DSS) is aggravated (Non-Patent Document 8). However, it has not been known that these miRNAs have the effect of reducing the expression levels of cytokines such as IL-6, TGF-β, IL12 p40, and IL23 p19 in dendritic cells.

[0018] The miR-29a used in the present invention may be a mature miRNA, a hairpin precursor miRNA (pri-miRNA), or a pre-miRNA in which a part of the pri-miRNA is cleaved. The pri-miRNA or pre-miRNA is processed in cells to become mature miR-29a. It may form a double-stranded precursor consisting of an RNA having a complementary base sequence. The double-stranded RNA is unwound in cells to release mature miR-29a. The base sequence of the hairpin precursor miR-29a may be set so that it is processed in vivo to generate mature miR-29a, and such a base sequence can be appropriately set by those skilled in the art.

[0019] The origin of the miR-29a used in the present invention may be appropriately set according to the type of animal to which it is applied. For example, if it is used to treat inflammatory bowel disease in humans, human-derived miR-29a may be used. Specifically, the human-derived miR-29a used in the present invention may include the mature miR-29a shown below. The base sequence of mouse-derived miR-29a is the same as human-derived miR-29a. Mature has-miR-29a(sense):5'-UAG CAC CAU CUG AAA UCG GUU A-3' (SEQ ID NO: 1) Mature has-miR-29a (antisense):5'-UAA CCG AUU UCA GAU GGU GCU A-3' (SEQ ID NO: 2)

[0020] In addition, the miR-29b used in the present invention may be a mature miRNA, a hairpin precursor miRNA (pri-miRNA), or a pre-miRNA in which a part of the pri-miRNA is cleaved. The pri-miRNA or pre-miRNA is processed in cells to become mature miR-29b. It may form a double-stranded precursor consisting of an RNA having a complementary base sequence. The double-stranded RNA is unwound in cells to release mature miR-29b. The base sequence of the hairpin precursor miR-29b may be set so that it is processed in vivo to generate mature miR-29b, and such a base sequence can be appropriately set by those skilled in the art.

[0021] The origin of the miR-29b used in the present invention may be appropriately set according to the type of animal to which it is applied. For example, if it is used to treat inflammatory bowel disease in humans, human-derived miR-29b may be used. Specifically, the human-derived miR-29b used in the present invention may include the mature miR-29b shown below. The base sequence of mouse-derived miR-29b is the same as human-derived miR-29b. Mature has-miR-29b (sense): 5'-UAG CAC CAU UUG AAA UCA GUG UU-3' (SEQ ID NO: 3) Mature has-miR-29b(antisense):5'-AAC ACU GAU UUC AAA UGG UGC UA-3' (SEQ ID NO: 4)

[0022] miR-29a and / or miR-29b may be modified as necessary in a manner generally applied to nucleic acids in order to confer resistance to enzymatic degradation, etc. Examples of such modifications include modification of the sugar chain portion, such as 2'-O methylation, modification of the base portion, and modification of the phosphate portion, such as amination, lower alkylamination, and acetylation.

[0023] In the preventive or therapeutic agent of the present invention, either miR-29a or miR-29b may be used as the miRNA, or they may be used in combination.

[0024] Carbonate Apatite Particles Carbonate apatite is a type of hydroxyapatite (Ca 10 It has a structure in which some of the hydroxyl groups of (PO4)6(OH)2 are replaced with CO3, and has the general formula Ca 10-m X m (PO4)6(CO3) 1-n Y n Here, X is an element that can partially replace Ca in carbonate apatite, and examples thereof include Sr, Mn, and rare earth elements. m is usually a positive number of 0 to 1, preferably 0 to 0.1, more preferably 0 to 0.01, and even more preferably 0 to 0.001. Y is a group or element that can partially replace CO3 in carbonate apatite, and examples thereof include OH, F, and Cl. n is usually a positive number of 0 to 0.1, preferably 0 to 0.01, more preferably 0 to 0.001, and even more preferably 0 to 0.0001.

[0025] The average particle size of the carbonate apatite particles used in the present invention is not particularly limited as long as it is large enough to be administered into the body and transferred into cells, but since uptake in dendritic cells is mainly performed by phagocytosis, carbonate apatite particles larger than the average particle size (50 nm or less) of conventional carbonate apatite particles used as nucleic acid carriers administered into the blood can be preferably used. Specifically, the average particle size of the carbonate apatite particles used in the present invention is usually more than 50 nm (e.g., more than 50 nm and less than 3000 nm), preferably 100 to 3000 nm, more preferably 100 to 2000 nm, more preferably 200 to 2000 nm or 400 to 3000 nm, and particularly preferably 400 to 2000 nm.

[0026] The average particle size of carbonate apatite is a value measured by dynamic light scattering particle measurement (DLS). If there are large particles (e.g., particles with a particle size of 5 μm or more) that are not suitable for measurement using DLS, they are removed from the measurement range. In this specification, particle size means the particle size of an independent particle that can be recognized as a separate particle when measured with a scanning probe microscope. Therefore, when multiple particles are aggregated, the aggregate is determined to be one particle.

[0027] The carbonate apatite particles can be obtained according to a known method. For example, the carbonate apatite particles can be obtained by preparing the carbonate apatite particles by allowing calcium ions, phosphate ions, and hydrogen carbonate ions to coexist in an aqueous solution. The concentration of each ion in the aqueous solution is not particularly limited as long as the carbonate apatite particles are formed, and can be appropriately set with reference to the following.

[0028] The calcium ion concentration in the aqueous solution is usually 0.1 to 1000 mM, preferably 0.5 to 100 mM, and more preferably 1 to 10 mM.

[0029] The phosphate ion concentration in the aqueous solution is usually 0.1 to 1000 mM, preferably 0.5 to 100 mM, and more preferably 1 to 10 mM.

[0030] The hydrogen carbonate ion concentration in the aqueous solution is usually 1.0 to 10,000 mM, preferably 5 to 1,000 mM, and more preferably 10 to 100 mM.

[0031] The sources of calcium ions, phosphate ions, and bicarbonate ions are not particularly limited as long as they can supply these ions to the aqueous solution, and examples thereof include water-soluble salts of these ions. Specifically, CaCl2 can be used as the calcium ion source, NaH2PO4·2H2O can be used as the phosphate ion source, and NaHCO3 can be used as the carbonate ion source.

[0032] The aqueous solution for preparing carbonate apatite particles may contain components other than the above-mentioned ion sources and other substances, as long as carbonate apatite particles are formed. For example, fluorine ions, chloride ions, Sr, Mn, polyethylene glycol (PEG), etc. may be added to the aqueous solution to partially replace or modify Ca or CO3 in carbonate apatite. However, it is preferable that the amount of fluorine ions, chloride ions, Sr, Mn, PEG, etc. added is within a range that does not significantly affect the pH solubility and particle size range of the composite particles formed. In addition, the aqueous solution for preparing carbonate apatite particles may use water as a base, but various culture media and buffers for cell culture may also be used.

[0033] In the preparation of carbonate apatite particles used in the present invention, the order of mixing each ion source and other substances into the aqueous solution is not particularly limited, and the aqueous solution may be prepared in any order as long as the desired carbonate apatite particles are obtained. For example, a first solution containing calcium ions and other substances is prepared, and a second solution containing phosphate ions and hydrogen carbonate ions is separately prepared, and the first solution and the second solution are mixed to prepare the aqueous solution.

[0034] The carbonate apatite particles can be obtained by adjusting the pH of an aqueous solution containing the above ions to the range of 6.0 to 9.0 and leaving it (incubating) for a certain period of time. The pH of the aqueous solution when forming the carbonate apatite particles is, for example, 7.0 to 8.5, preferably 7.1 to 8.5, more preferably 7.2 to 8.5, even more preferably 7.3 to 8.5, particularly preferably 7.4 to 8.5, and most preferably 7.5 to 8.0.

[0035] The temperature condition of the aqueous solution when forming carbonate apatite particles is not particularly limited as long as carbonate apatite particles are formed, but is usually 4° C. or higher, preferably 25 to 80° C., and more preferably 37 to 70° C. or higher.

[0036] The incubation time of the aqueous solution for forming carbonate apatite particles is not particularly limited as long as carbonate apatite particles are formed, but is usually 1 minute to 24 hours, preferably 5 minutes to 1 hour. The presence or absence of particle formation can be confirmed, for example, by observation under a microscope.

[0037] In addition, the method of controlling the average particle size of carbonate apatite particles to the above range is not particularly limited, and examples thereof include a method of subjecting carbonate apatite particles formed in the above aqueous solution to ultrasonic vibration treatment. Specific examples of ultrasonic vibration treatment include a treatment in which an ultrasonic vibrator such as an ultrasonic crusher is directly brought into contact with a sample to apply ultrasonic waves; a treatment in which a liquid (e.g., water) is placed in an ultrasonic cleaner equipped with an ultrasonic vibrator and a water tank (cleaning tank), a container (e.g., a plastic tube) containing carbonate apatite particles is floated therein, and ultrasonic waves are applied to the aqueous solution containing carbonate apatite particles through the liquid, etc. By such ultrasonic vibration treatment, the particle size of carbonate apatite particles can be easily and efficiently refined to the above range. Furthermore, the particle size of carbonate apatite particles can also be adjusted to the above range by separating carbonate apatite particles of a desired particle size using a filter with a predetermined pore size after ultrasonic treatment.

[0038] The conditions of the ultrasonic vibration treatment are not particularly limited as long as the particle size can be controlled within a predetermined range. For example, when the ultrasonic vibration treatment is performed using an ultrasonic cleaner equipped with an ultrasonic vibrator and a water tank (cleaning tank), the following conditions are exemplified. Water bath temperature: for example, 5 to 45°C, preferably 10 to 35°C, and more preferably 20 to 30°C. High frequency output: for example, 10 to 500W, preferably 20 to 400W, further preferably 30 to 300W, and further preferably 40 to 100W. Oscillation frequency: for example, 10 to 60 Hz, preferably 20 to 50 Hz, and more preferably 30 to 40 Hz. Treatment time: for example, 30 seconds to 30 minutes, preferably 1 to 20 minutes, and more preferably 3 to 10 minutes.

[0039] The type of container for containing carbonate apatite particles used in ultrasonic vibration treatment is not limited as long as it is possible to reduce the particles to a predetermined particle size range, and can be appropriately selected depending on the volume of the aqueous solution and the purpose of use. For example, a plastic tube with a volume of 1 to 1000 ml can be used.

[0040] In addition, it is preferable to carry out the ultrasonic vibration treatment in the presence of a dispersant (i.e., in a state where the dispersant is added to the aqueous solution containing carbonate apatite particles). This is because by carrying out the ultrasonic vibration treatment in an environment where the dispersant and carbonate apatite particles coexist, carbonate apatite nanoparticles having a finer particle size can be obtained and re-aggregation of the particles can also be suppressed. The type of dispersant is not particularly limited as long as it can disperse carbonate apatite particles, and any dispersant that is generally added to pharmaceuticals may be used, and one example is albumin. The dispersant may be used alone or in combination of two or more types. The concentration of the dispersant in the aqueous solution containing carbonate apatite particles is not particularly limited as long as the effect of micronization and / or re-aggregation suppression can be obtained, and may be, for example, about 0.1 to 500 mg / ml, preferably about 1 to 100 mg / ml, more preferably about 1 to 10 mg / ml; or about 0.001 to 10% by weight.

[0041] (Composite particles of miR-29a and / or miR-29b and carbonate apatite particles) In the preventive or therapeutic agent of the present invention, composite particles are used in which miR-29a and / or miR-29b are supported and composited with carbonate apatite particles. By thus composited with carbonate apatite particles, miR-29a and / or miR-29b can be delivered to the affected area of ​​inflammatory bowel disease, and can also be efficiently taken up by dendritic cells. In addition, after the composite particles are introduced into cells, miR-29a and / or miR-29b can be released from the carbonate apatite particles in the cells, allowing miR-29a and / or miR-29b to exert the desired activity.

[0042] In the present invention, the composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles and composited refer to a state in which miR-29a and / or miR-29b are adsorbed and supported on carbonate apatite particles by ionic bonds, hydrogen bonds, etc. The method for forming composite particles of miR-29a and / or miR-29b and carbonate apatite particles is not particularly limited, and examples thereof include a method of forming the composite particles by allowing miR-29a and / or miR-29b to coexist with carbonate apatite particles in an aqueous solution; a method of simultaneously forming carbonate apatite particles and composite the miRNA with carbonate apatite particles by allowing miR-29a and / or miR-29b to coexist with calcium ions, phosphate ions, and hydrogen carbonate ions in an aqueous solution in which carbonate apatite particles are prepared; and the like.

[0043] When forming composite particles of miR-29a and / or miR-29b and carbonate apatite particles, and forming carbonate apatite particles and composite of miR-29a and / or miR-29b and carbonate apatite particles at the same time, miR-29a and / or miR-29b may be added to an aqueous solution used for preparing carbonate apatite at a concentration of, for example, 0.1 to 1000 nM, preferably 0.5 to 500 nM, more preferably 1 to 200 nM. Usually, about 60% of miR-29a and / or miR-29b present in the aqueous solution is encapsulated in carbonate apatite particles and composited.

[0044] In the composite particles of miR-29a and / or miR-29b and carbonate apatite particles, the ratio of miR-29a and / or miR-29b to carbonate apatite particles is not particularly limited and may be appropriately set depending on the dosage of miR-29a and / or miR-29b, etc. For example, when 2 mg of miR-29a and / or miR-29b is to be composited with carbonate apatite particles, 5 mg of miR-29a and / or miR-29b may be added to 2.5 L of the aqueous solution for preparing the carbonate apatite particles described above, thereby simultaneously forming carbonate apatite particles and composites of miR-29a and / or miR-29b with carbonate apatite particles.

[0045] In the present invention, the composite particles of miR-29a and / or miR-29b and carbonate apatite particles are used in a state of being dispersed in a solvent suitable for administration to a living body. As described above, carbonate apatite particles are obtained by dissolving various ion source substances in a solvent such as water, a medium, or a buffer, but the carbonate apatite particle dispersion solution obtained in this manner is not necessarily suitable for systemic administration to a living body in terms of osmotic pressure, buffer capacity, sterility, etc. In order to replace the solvent in which the carbonate apatite particles are dispersed with a solvent suitable for administration to a living body (e.g., physiological saline, etc.), it is usually necessary to separate the carbonate apatite particles from the solvent by centrifugation, filtration, gravitational sedimentation, etc., recover them, and replace the solvent. By adding the carbonate apatite particles to a solvent suitable for administration to a living body and then performing the above-mentioned ultrasonic vibration treatment, the composite particles of miR-29a and / or miR-29b and carbonate apatite particles can be dispersed in the solvent suitable for administration to a living body with the appropriate particle size. After dispersing in this way, it is desirable to administer the composite particles to the living body promptly before they aggregate. For example, administration is suitable within 1 minute, preferably within 30 seconds, after ultrasonic vibration treatment. However, as described above, when the aggregation of carbonate apatite particles is suppressed by adding a dispersant, it is also possible to administer the composite particles several minutes to several tens of minutes, or one day to several days after ultrasonic vibration treatment.

[0046] Purpose Since the composite particles of carbonate apatite particles carrying miR-29a and / or miR-29b can effectively cure or improve the symptoms of inflammatory bowel disease, the preventive or therapeutic agent of the present invention can be used for the treatment of inflammatory bowel disease. In addition, since inflammatory bowel disease may gradually progress while repeating recurrence and remission, the preventive or therapeutic agent of the present invention can also be used for the prevention or prevention of recurrence of inflammatory bowel disease.

[0047] Inflammatory bowel diseases are roughly classified into ulcerative colitis and Crohn's disease, and the preventive or therapeutic agent of the present invention can be applied to either of these.

[0048] Method of administration The preventive or therapeutic agent of the present invention is administered by systemic administration. Although local administration cannot deliver miR-29a and / or miR-29b to dendritic cells, systemic administration allows a portion of the miRNA to be delivered directly to the affected area and efficiently taken up by dendritic cells.

[0049] Specific examples of systemic administration include intravascular (intraarterial or intravenous) administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc. Among these, from the viewpoint of further improving the therapeutic effect of inflammatory bowel disease, preferred are intravascular administration and subcutaneous administration, and more preferred are intravenous injection.

[0050] The dosage of the preventive or therapeutic agent of the present invention cannot be determined in general terms because it is appropriately determined depending on the severity of inflammatory bowel disease, the sex, age, symptoms, etc. of the patient. For example, the dosage may be 1 to 100 mg / m per day in terms of the amount of mature miRNA of miR-29a and / or miR-29b. 2 (Body surface area) EXAMPLES

[0051] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. All of the tests shown below were performed with the approval of the Animal Care and Use Committee of the Osaka University School of Medicine and the Animal Experiment Committee of Osaka University (approval number: 260033-006).

[0052] Reference Example 1: Preparation of a complex of miRNA and carbonate apatite In all cases except Test Example 6, a complex of miRNA and carbonate apatite prepared by the following method was used. First, an inorganic aqueous solution (NaHCO3; 44 mM, NaH2PO4; 0.9 mM, CaCl2; 1.8 mM, pH 7.5) was prepared. 100 μg of miRNA was added to 50 ml of this inorganic aqueous solution and incubated at 37° C. for 30 minutes. Then, the mixture was centrifuged at 12,000 rpm for 3 minutes to obtain a pellet containing 60 μg of miRNA (composite particles in which miRNA is encapsulated in carbonate apatite particles; miRNA / sCA complex). Physiological saline (containing 0.5% by weight of albumin) was added to the pellet to make a total volume of 200 μL to disperse the pellet, which was subjected to ultrasonic vibration treatment (38 kHz, 80 W) for 10 minutes and immediately used for the test. The obtained miRNA / sCA complex was confirmed to have an average particle size of 400 to 2000 nm by dynamic light scattering particle measurement (DLS) using a Zetasizer Nano ZS (Malvern Instruments).

[0053] In the following test examples, the following mature miRNA (double strand) was used. In the following test examples, the amount of miRNA used is the total amount of the sense strand and the antisense strand. Hereinafter, carbonate apatite particles containing miR-29a may be referred to as "miRNA-29a / sCA complex." Carbonate apatite particles containing other miRNAs may also be described in the same format.

[0054] [Table 1]

[0055] Reference test example 1 7-week-old BALB / c mice (CLEA Japan, Inc.) were given water containing 2% by weight of dextran sulfate (DSS) for 7 days to develop inflammatory bowel disease, creating a DSS-induced mouse bowel disease model. MIRTX / sCA complex or Alexa647-miRNA / sCA complex was administered to the DSS-induced mouse bowel disease model via the tail vein at a dose of 60 μg / mouse.

[0056] In the group administered MIRTX / sCA complex, mice were sacrificed 4 hours after administration, and the rectum, transverse colon, cecum, and ileum were mashed to collect RNA. The amount of MIRTX taken up in each intestinal tract was then measured by quantitative RT-PCR (n=3). The amount of MIRTX was quantified as a relative value of the amount of MIRTX to the amount of RNU6B, using RNU6B as an internal standard.

[0057] In addition, in the group that received the Alexa647-miRNA / sCA complex, the rectum (the part of the rectum where inflammation is most severe) was removed from the mice 4 hours after administration, frozen sections were prepared, nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI), and the sections were observed under a fluorescent microscope.

[0058] For comparison, the test was performed under the same conditions as above, except that normal mice not administered DSS were used.

[0059] The results of administering the MIRTX / sCA complex and measuring the amount of MIRTX taken up in each intestinal tract are shown in Figure 1a. As a result, the amount of MIRTX taken up in the intestinal tract of the DSS-induced mouse colitis model was significantly higher than that of normal mice not given DSS, but the amount of MIRTX taken up itself was not that high.

[0060] The results of nuclear staining and microscopic observation of the rectum collected after administration of Alexa647-miRNA are shown in Fig. 1b. The scale bar in Fig. 1b is 50 μm. As a result, the DSS-induced mouse colitis model showed a slight increase in red signals (Alexa647 fluorescence) indicating the uptake of Alexa647-miRNA in the rectum compared to normal mice not given DSS, but the signals were still relatively small. These results confirmed that the amount of miRNA uptake in the intestinal tract did not reach a high level even when the miRNA / sCA complex was administered via the tail vein.

[0061] Test Example 1 Seven-week-old BALB / c mice (CLEA Japan, Inc.) were divided into six groups as shown in Table 2. DSS and the miRNA / sCA complex or miRNA were administered to the mice in each group under the conditions shown in Table 2.

[0062] [Table 2]

[0063] The body weight of the mice was measured from the start to the end of the test (n=5). In addition, the mice were sacrificed for the normal group at the start of the test, and the DSS and DSS+ sCA-miR-29b groups on the 4th and 6th days after the start of the test, and the colonic mucosa was collected and crushed to collect RNA. Next, the expression level of miR-29b-3p was measured by quantitative RT-PCR (n=3). The expression level of miR-29b-3p was quantified as a relative value of the expression level of miR-29b to the amount of RNU6B, using RNU6B as an internal standard.

[0064] The colons were also collected from mice sacrificed on the 9th day. The colon lengths were measured from the collected colons, and the rectal area where inflammation was most severe was stained with Hematoxylin-Eosin (HE) and the state of inflammation was observed under a microscope (n=5). The degree of inflammation in the rectal area where inflammation was most severe was scored according to the following criteria for mucosal damage, submucosal tissue damage, and muscular layer damage, and the sum of these scores was calculated as the histological inflammation score (n=5). <Mucous membrane damage> 0:normal 1:3-10 intraepithelial lymphocytes (IEL) / high power field (HPF) and focal damage 2:10 IEL / HPF and rare crypto abscesses 3:10 IEL / HPF, multiple crypt abscesses and erosion / ulceration <Submucosal tissue damage> 0:normal or widely scattered leukocytes 1:focal aggregates of leukocytes 2:diffuse leukocyte infiltration with expansion of submucosa 3: diffuse leukocyte infiltration <Muscle layer damage> 0:normal or widely scattered leukocytes 1:widely scattered leukocyte aggregates between muscle layers 2: leukocyte infiltration with focal effacement of the muscularis 3:extensive leukocyte infiltration with transmural effacement of the muscularis

[0065] The results of measuring the expression level of miR-29b-3p in the colon mucosa for the DSS group and the DSS + sCA-miR-29b group are shown in Figure 2A. As a result, it was confirmed that the expression level of miR-29b was reduced in the DSS group compared to the Normal group, but the reduction in the expression level of miR-29b was significantly suppressed in the DSS + sCA-miR-29b group compared to the DSS group.

[0066] The results of HE staining of the rectum are shown in Figure 2b. As a result, in the DSS, DSS+sCA-NC-miR, and DSS+miR-29b groups, the rectal mucosal structure was destroyed and numerous inflammatory cells were found to have infiltrated into the mucosa. In contrast, in the DSS+sCA-miR-29a and DSS+sCA-miR-29b groups, the colonic mucosal structure was maintained almost normally and little infiltration of inflammatory cells was observed.

[0067] The results of measuring the body weight of the mice over time are shown in Fig. 2c, and the results of measuring the body weight of the mice 7 days after the start of the test are shown in Fig. 2d. The results of measuring the length of the large intestine are shown in Fig. 3a, and the results of the histological inflammation score are shown in Fig. 3b. As a result, the DSS+sCA-miR-29a group and the DSS+sCA-miR-29b group showed significantly more suppressed weight loss and shortening of the large intestine, and lower histological inflammation scores, compared to the DSS group, the DSS+sCA-NC-miR group, and the DSS+miR-29b group.

[0068] From the above results, it was revealed that when miR-29a / sCA complex and miR-29b / sCA complex are administered intravenously, they can effectively improve inflammatory bowel disease, despite the low direct uptake of miRNA into the intestinal tract (results of Reference Test Example 1 above).

[0069] In addition, the expression level of miR-29b in the large intestine was measured for the normal group at the start of the test, and the DSS group 4 and 6 days after the start of the test. The results are shown in Fig. 3c. As a result, it was revealed that the expression level of miR-29b in the large intestine was reduced in mice that had developed inflammatory bowel disease.

[0070] Reference test example 2 7-week-old BALB / c mice (CLEA Japan, Inc.) were allowed to drink water containing 2% by weight of DSS for 7 days to develop inflammatory enteritis, and a DSS-induced mouse enteritis model was created. The Alexa647-miRNA / sCA complex was administered to the DSS-induced mouse enteritis model via the tail vein at a dose of 60 μg / mouse. Four hours after administration of the Alexa647-miRNA / sCA complex, the large intestine was removed from the mouse, and frozen sections were prepared from the rectum where inflammation was most pronounced, and immunostained with anti-CD11c antibody and DAPI (CD11c is green), and observed under a fluorescent microscope and a confocal microscope. For comparison, a test was performed under the same conditions as above, except that normal mice not administered DSS were used.

[0071] The results of observing the immunostained frozen sections with a fluorescence microscope are shown in Fig. 4a, and the results of observing the sections with a confocal microscope using a DSS-induced mouse colitis model are shown in Fig. 4b. The scale bar in Fig. 4a is 50 μm, and the scale bar in Fig. 4b is 5 μm. In Fig. 4a, the upper image shows the results of observing the red signal emitted by Alexa647-miRNA and the blue signal emitted by DAPI, the middle image shows the results of observing the green signal emitted by anti-CD11c antibody and the blue signal emitted by DAPI, and the lower image is a merged image of the upper and middle images. The coloring by the anti-CD11c antibody indicates the presence of dendritic cells.

[0072] As can be seen from Figure 4a, it was confirmed that the distribution of the red signal emitted by Alexa647-miRNA and the green signal emitted by the anti-CD11c antibody matched. In addition, from Figure 4b, it was found that the green signal emitted by the anti-CD11c antibody was present on the cell surface, and the red signal emitted by Alexa647-miRNA was filled in the cytoplasm. From these results, it was revealed that when the siRNA / sCA complex was administered intravenously, the siRNA / sCA complex was taken up by dendritic cells, and the dendritic cells were active in the intestinal tract.

[0073] Test Example 2 7-week-old BALB / c mice (CLEA Japan, Inc.) were allowed to drink water containing 2% by weight of DSS for 7 days to develop inflammatory bowel disease, and a DSS-induced mouse bowel disease model was created. The sCA-miR-29a / sCA complex or sCA-miR-29b / sCA complex was administered to the DSS-induced mouse bowel disease model via the tail vein on the first, second, and third days so that the dose of miRNA was 60 μg / mouse per administration, and the large intestine was removed from the mouse on the fourth day. Lamina propria cells were extracted from the mucosa of the large intestine, and CD11c-positive cells were separated using an anti-CD11c antibody and an autoMACS Pro Separator. RNA was extracted from the obtained CD11c-positive cells. The expression levels of various cytokines (IL12 p40, IL23 p19, IL-6, and TGF-β) were then measured by quantitative RT-PCR (n=3). The expression level of each cytokine was quantified as a relative value to the amount of β-actin, using β-actin as an internal standard.

[0074] For comparison, the expression levels of each cytokine were measured in the same manner as described above for normal mice (Normal) that were not administered DSS or miRNA / sCA complex, and for a DSS-induced mouse colitis model (DSS) that was administered only DSS but not the miRNA / sCA complex.

[0075] The results are shown in Figure 5. As a result, significant reductions in the expression levels of IL12 p40, IL23 p19, IL-6, and TGF-β were observed by intravenous administration of sCA-miR-29a / sCA complex or sCA-miR-29b / sCA complex to a DSS-induced mouse colitis model. IL-6 and TGF-β are cytokines known to be involved in the differentiation of naive T cells into Th-17 cells, and IL12 p40 and IL23 p19 are known to be cytokines that activate Th-17 cells and lead them to pathogenic Th-17 cells. In other words, it is inferred that intravenous administration of sCA-miR-29a / sCA complex or sCA-miR-29b / sCA complex causes miR-29a or miR-29b to accumulate in dendritic cells, thereby downregulating IL-6 and TGF-β, thereby inhibiting the differentiation of naive T cells into Th-17 cells, and downregulating IL12 p40 and IL23 p19, thereby inhibiting the activation of Th-17 cells into pathogenic Th-17 cells.

[0076] Test Example 3 Seven-week-old BALB / c mice (CLEA Japan, Inc.) were divided into four groups as shown in Table 3. DSS and miRNA / sCA complexes were administered to the mice in each group under the conditions shown in Table 3.

[0077] [Table 3]

[0078] Colons were harvested from sacrificed mice. RNA was extracted from the harvested colons, and mRNA libraries were prepared using the TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer's instructions. Sequencing was performed using the Illumina HiSeq 2500 platform (75-base single-end mode). Base calling was performed using Illumina Casava 1.8.2 software. Sequences were mapped to the mouse reference genome sequence (mm10) using TopHat v2.0.13, a combination of Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19. One million reads were mapped, and normalized gene expression levels were calculated using Cuffnorm version 2.2.1, where the length of the transcript (fragment) was taken as 1 kilobase.

[0079] Pathway analysis was then performed using QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.qiagen.com / ingenuity) (cutoff value was set at P < 0.05 for each gene).

[0080] The results of gene expression analysis by IPA are shown in Figure 6a. Figure 6 highlights molecules that were activated in the sCA-NC-miR group, in other words, molecules that were activated by administration of DSS, in comparison with the normal group and the sCA-NC-miR group. In Figure 6, the darker the color, the higher the degree of activation (expression level). Furthermore, the underlined molecules in Figure 6 are those that have been identified as many IFN pathway-related molecules. As a result, the sCA-miR-29a and sCA-miR-29b groups showed a tendency to lower the Z-scores (degree of activation) of molecules that were activated in the sCA-NC-miR group.

[0081] FIG. 7 shows an intracellular signal pathway map relating to the production of IFN and inflammatory cytokines from Toll-like receptor (TLR). In FIG. 7, the molecules marked with an * were found to be activated by administration of DSS. FIG. 8 shows a known IFN signal pathway. In FIG. 8, the molecules marked with an * are genes predicted to be activated by IPA, and the molecules marked with an ○ are molecules confirmed to be upregulated by mRNA array.

[0082] Test Example 4 Seven-week-old BALB / c mice (CLEA Japan, Inc.) were divided into four groups (n=3 per group) as shown in Table 4. DSS and miRNA / sCA complexes were administered to the mice in each group under the conditions shown in Table 4.

[0083] [Table 4]

[0084] The body weight of the mice was measured from the start to the end of the test. The large intestine was collected from the mice, and the length of the large intestine was measured. The rectal area where the inflammation was most severe was stained with Hematoxylin-Eosin (HE) and the state of inflammation was observed under a microscope. Furthermore, the degree of inflammation in the rectal area where the inflammation was most severe was scored in the same manner as in Test Example 1, and a histological inflammation score was obtained.

[0085] The results of measuring the body weight of the mice over time are shown in Fig. 9a, and the results of measuring the body weight of the mice at the end of the study (9 days after the start of administration of the miRNA / sCA complex) are shown in Fig. 9b. The results of measuring the length of the large intestine are shown in Fig. 9c, and the results of the histological inflammation score are shown in Fig. 9d. As a result, the DSS+sCA-miR-29b group showed significantly more suppressed weight loss and shortening of the large intestine, and lower histological inflammation scores, compared to the DSS and DSS+sCA-NC-miR groups.

[0086] The results of HE staining of the rectum in the DSS+sCA-miR-29b group are shown in Fig. 9E. As can be seen from Fig. 9E, in the DSS+sCA-miR-29b group, the mucosal structure of the colon was maintained almost normally, and infiltration of inflammatory cells was hardly observed.

[0087] These results confirmed that the miR-29b / sCA complex can effectively improve inflammatory bowel disease not only by intravenous administration but also by subcutaneous administration. In other words, these results strongly suggest that the miR-29a / sCA complex and the miR-29b / sCA complex can effectively treat inflammatory bowel disease by systemic administration.

[0088] Test Example 5 7-week-old BALB / c mice (CLEA Japan, Inc.) were given water containing 2% by weight of DSS for 7 days to develop inflammatory bowel disease, creating a DSS-induced mouse bowel disease model. Alexa647-conjugated miR-29b / sCA complex was subcutaneously administered to the DSS-induced mouse bowel disease model at a dose of 60 μg / mouse.

[0089] Four hours after administration of the complex, the large intestine was removed from the mice and frozen sections were prepared from the rectum, where inflammation was most severe. These sections were then immunostained with anti-CD11c antibody, anti-Ly-6G antibody, anti-F4 / 80 antibody, anti-CD4 antibody, anti-CD45R antibody, and DAPI, and observed under a fluorescent microscope.

[0090] Anti-CD11c antibody (green) and DAPI (blue) were used to visualize nuclei and CD11c. +The results of observing frozen sections immunostained for dendritic cells under a fluorescent microscope are shown in Figure 10a. As can be seen from Figure 10a, the distribution of the red signal emitted by Alexa647-miRNA and the green signal emitted by the anti-CD11c antibody were confirmed to be consistent. In other words, this result confirmed that when Alexa647-conjugated miR-29b / sCA complex was administered subcutaneously, miRNA was efficiently taken up by dendritic cells and localized in the inflamed intestinal tract.

[0091] In addition, lamina propria cells were extracted from the colon removed above, and separated into CD11c-positive cells and CD11c-negative cells using anti-CD11c antibody and autoMACS Pro Separator. RNA was collected from the obtained CD11c-positive cells and CD11c-negative cells. Next, the amount of miR-29b was measured by quantitative RT-PCR. The amount of miR-29b was quantified as a relative value, with the expression level of miR-29b in the absence of administration of the complex taken as 1. The results are shown in Figure 10b. As a result, it was confirmed that the amount of miR-29b was increased in CD11c-positive cells in the group administered with Alexa647-miRNA (DSS+sCA-miR-29b) compared to the group administered with only DSS (DSS alone). The increase in the amount of miR-29b is attributed to the uptake of exogenous miR-29b (i.e., Alexa 647-conjugated miR-29b) in CD11c-positive cells. These results demonstrated that the localization patterns of subcutaneously administered Alexa 647-conjugated miR-29b and CD11c-positive cells were consistent, and confirmed that subcutaneously administered Alexa 647-conjugated miR-29b was taken up by dendritic cells and localized in the inflamed intestinal tract.

[0092] In addition, anti-Ly-6G antibody (green) and DAPI (blue) were used to detect nuclei and Ly-6G. + Neutrophils were immunostained, and the nuclei and F4 / 80 were identified using anti-F4 / 80 antibody (green) and DAPI (blue). +Immunostaining of macrophages revealed that nuclei and CD4 were stained with anti-CD4 antibody (green) and DAPI (blue). + Immunostaining of helper T cells revealed that the nuclei and CD45R were stained with anti-CD45R antibody (green) and DAPI (blue). + The results of immunostaining of B cells are shown in Figure 11. As a result, the red color of Alexa 647-conjugated miR-29b did not overlap with the green color of each of the immune cells, confirming that subcutaneously administered Alexa 647-conjugated miR-29b was not taken up by immune cells other than dendritic cells.

[0093] Test Example 6 (1) Preparation of miR-29b / sCA complex First, an inorganic aqueous solution (NaHCO3; 44 mM, NaH2PO4; 0.9 mM, CaCl2; 1.8 mM, pH 7.5) was prepared. 100 μg of miR-29b was added to 50 ml of this inorganic aqueous solution and incubated at 37°C for 30 minutes. After that, the mixture was centrifuged at 12,000 rpm for 3 minutes to obtain a pellet containing 60 μg of miRNA (composite particles in which miRNA is encapsulated in carbonate apatite particles; miRNA / sCA complex). The pellet was dispersed in 200 μL of saline (containing 0.5% albumin by weight), subjected to ultrasonic vibration treatment (38 kHz, 80 W), and then separated using a membrane filter into particles greater than 50 nm and approximately 3000 nm or less (hereinafter, miR-29b / sCA complexes (>50 nm)) and particles between 10 nm and 50 nm or less (hereinafter, miR-29b / sCA complexes (≦50 nm)), and immediately used in the test.

[0094] (2) Test method Eight-week-old BALB / cAJcl mice (female) were divided into three groups (two mice per group) as shown in Table 5. DSS and miR-29b / sCA complex were administered to the mice in each group under the conditions shown in Table 5.

[0095] [Table 5]

[0096] The colon was collected from the sacrificed mice and stained with Hematoxylin-Eosin (HE), and the state of inflammation was observed under a microscope to evaluate the degree of inflammation histologically. The degree of inflammation was evaluated on a scale of 0 to 3 points for three items, "damage to the mucosal lamina propria," "damage to the submucosa," and "damage to the muscular layer," according to the following criteria, for a total of 9 points. The degree of inflammation was evaluated by dividing the entire length of the collected colon into 10 equal parts and averaging the evaluation results of 10 visual fields for each part. <Criteria for assessing damage to the lamina propria> 0 points : Normal finding. 1 point : Three to ten intraepithelial lymphocytes (IEL) are present per high power field (HPF), and localized mucosal damage is observed. 2 points : More than 10 IELs per HPF and a few crypt abscesses. 3 points More than 10 IELs per HPF, scattered crypt abscesses, and a few erosions and ulcers were observed. <Criteria for determining submucosal injury> 0 points Normal, or slight scattered signs of neutrophil infiltration. 1 point :Localized neutrophil infiltration is observed. 2 points : Diffuse neutrophil infiltration was observed, with expansion into the submucosal layer. 3 points :Neutrophil infiltration is observed throughout the submucosa. <Criteria for determining muscle layer injury> 0 points : Normal, or scattered neutrophils in some parts of the muscularis layer. 1 point The muscle layer is preserved, but diffuse neutrophil infiltration is observed between the muscle layers. 2 points :Due to neutrophil infiltration, there is disruption in the alignment of the muscle layer. 3 points: Due to extensive neutrophil infiltration, there is transmural disruption of the muscular layer arrangement.

[0097] (3) Test results The results of HE staining are shown in Fig. 12(a). As can be seen from Fig. 12(a), the degree of destruction of the colonic mucosal structure was lower in the DSS+sCA(>50nm)-miR-29b group and the DSS+sCA(≦50nm)-miR-29b group than in the DSS group, and in particular, the degree of destruction of the colonic mucosal structure was significantly lower in the DSS+sCA(>50nm)-miR-29b group than in the DSS+sCA(≦50nm)-miR-29b group.

[0098] The results of histological evaluation of the degree of inflammation are shown in Fig. 12b. The histological evaluation results also showed a significant difference in the DSS+sCA(>50nm)-miR-29b group compared to the DSS group, confirming that the inflammation suppression effect was significantly higher.

[0099] These results demonstrate that the therapeutic effect of inflammatory bowel disease can be further improved by using carbonate apatite particles with a particle size of more than 50 nm to carry miR-29b.

[0100] Reference test example 3 7-week-old BALB / c mice (CLEA Japan, Inc.) were allowed to drink water containing 2% by weight of DSS for 7 days to develop inflammatory enteritis, creating a DSS-induced mouse enteritis model. Alexa647-miRNA / sCA complex was subcutaneously administered to the DSS-induced mouse enteritis model at a dose of 60 μg / mouse. Four hours after administration of the Alexa647-miRNA / sCA complex, the large intestine was removed from the mouse, and frozen sections were prepared from the rectum, where inflammation is most pronounced, and immunostained with anti-CD11c antibody and DAPI (CD11c is green), followed by observation under a fluorescent microscope.

[0101] The results of observing the immunostained frozen sections under a fluorescent microscope are shown in Figure 13. As can be seen from Figure 13, it was confirmed that the distribution of the red signal emitted by Alexa647-miRNA and the green signal emitted by the anti-CD11c antibody matched. In other words, it was confirmed that even when the miRNA / sCA complex was administered subcutaneously, the miRNA was efficiently taken up by dendritic cells and localized in the inflamed intestinal tract, just as in the case of intravenous administration.

[0102] Reference test example 4 7-week-old BALB / c mice (CLEA Japan, Inc.) were given water containing 2% by weight of dextran sulfate (DSS) for 7 days to develop inflammatory bowel disease, creating a DSS-induced mouse bowel disease model. The MIRTX / sCA complex was administered subcutaneously to the DSS-induced mouse bowel disease model at a dose of 60 μg / mouse.

[0103] Four hours after administration of the MIRTX / sCA complex, the mice were sacrificed and the large intestine was removed. Lamina propria cells were extracted from the mucosa of the colon and separated into CD11c-positive cells and CD11c-negative cells using an anti-CD11c antibody and an autoMACS Pro Separator. RNA was collected from the resulting CD11c-positive cells and CD11c-negative cells. The amount of MIRTX taken up in each intestinal tract was then measured by quantitative RT-PCR. The amount of MIRTX was quantified as a relative value of the amount of MIRTX to the amount of RNU6B, using RNU6B as an internal standard.

[0104] For comparison, MIRTX was quantified under the same conditions as above, but without administration of the MIRTX / sCA complex.

[0105] The results are shown in Figure 14. As a result, when the MIRTX / sCA complex was administered subcutaneously, the amount of MIRTX was confirmed to be high in CD11c positive cells, but low in CD11c negative cells. In other words, this result also confirmed that when the miRNA / sCA complex was administered subcutaneously, it was efficiently taken up by dendritic cells and localized in the inflamed intestinal tract.

[0106] Reference Test Example 5 Seven-week-old BALB / c mice (CLEA Japan, Inc.) were divided into four groups (n=5 per group) as shown in Table 6. DSS and miRNA / sCA complexes were administered to the mice in each group under the conditions shown in Table 6.

[0107] [Table 6]

[0108] The body weight of the mice was measured from the start to the end of the test. The colons were collected from the mice, and the length of the colons was measured. Hematoxylin-Eosin (HE) staining was performed, and the state of inflammation was observed under a microscope. Furthermore, the degree of inflammation in the rectum, where inflammation was most severe, was scored in the same manner as in Test Example 1, and a histological inflammation score was obtained.

[0109] The results of measuring the body weight of the mice over time are shown in FIG. 15A, and the results of measuring the body weight of the mice at the end of the test (9 days after the start of administration of the miRNA / sCA complex) are shown in FIG. 15B. The results of measuring the length of the large intestine are shown in FIG. 15C, and the results of the histological inflammation score are shown in FIG. 15D. As a result, the DSS+sCA-miR-29b group, like the DSS and DSS+sCA-NC-miR groups, showed almost no inhibition of mouse body weight loss, inhibition of large intestinal shortening, or reduction in the histological inflammation score.

[0110] Moreover, the results of HE staining of the rectum in the DSS+sCA-miR-29b group are shown in Fig. 15e. This result also confirmed that inflammation in the colon was hardly improved in the DSS+sCA-miR-29b group.

[0111] In other words, from the results, it was confirmed that the miR-29b / sCA complex had almost no ameliorative effect when administered locally to inflammatory bowel disease.

Claims

1. Comprising composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, A preventive or therapeutic agent for inflammatory bowel disease, which is administered intravascularly or subcutaneously.

2. 2. The preventive or therapeutic agent for inflammatory bowel disease according to claim 1, wherein the carbonate apatite particles have an average particle size of 200 to 2000 nm.

3. Composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles, Use for the production of a preventive or therapeutic agent for inflammatory bowel disease, which is administered intravascularly or subcutaneously.

4. The use according to claim 3, wherein the carbonate apatite particles have an average particle size of 200 to 2000 nm.

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