JP2015089874A - Therapeutic agent for colon cancer using mir-340 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a colon cancer treatment technique which can treat colon cancer using microRNA, and can suppress cancer metastasis and the like to provide good prognosis.SOLUTION: Administration of a composite particle in which microRNA is composited with apatite carbonate can suppress in vivo proliferation of a colon cancer cell, and can exhibit an antitumor effect effectively.

Description

  The present invention relates to a therapeutic agent for colorectal cancer using miR-340. More specifically, the present invention relates to a therapeutic agent for colorectal cancer that can treat colorectal cancer and that can suppress cancer metastasis and the like to improve the prognosis. Furthermore, the present invention relates to a method for examining cancer metastasis to the liver in patients with intestinal cancer and a method for predicting prognosis of patients with colorectal cancer using miR-340 as a marker.

  In recent years, the mechanism of metastasis via bone marrow has attracted attention in the mechanism of cancer metastasis, and there are many reports that the presence or absence of cancer cells in the bone marrow is associated with prognosis or recurrence in various cancer types. The presumed mechanism of metastasis via the bone marrow is the theory that cancer cells in the primary tumor home to the bone marrow and then become dormant to acquire drug resistance. The theory of acquiring ability and transferring to other organs such as the liver is considered. However, the detailed mechanism of cancer metastasis, changes in the properties of cancer cells in the bone marrow, changes in the properties of host bone marrow cells themselves in a tumor-bearing state, etc. are still in the research stage and few reports have been recognized. On the other hand, colorectal cancer was once seen in many countries in Europe and the United States, but with the shift to Western lifestyles in Japan, it has become the third most common cancer disease among cancer patients in Japan. Yes. In addition, because colorectal cancer tends to cause cancer metastasis to the liver, lungs, etc., and metastasis that has become uncontrollable is a cause of death, colorectal cancer treatment has a prognosis such as suppression of metastasis. It is important to establish treatment strategies in

  On the other hand, microRNA is a short RNA of about 22 bases that exists in the body, plays an important role in the control of gene expression, and is a therapeutic agent for various diseases caused by abnormal gene expression. It is expected to be used as a prognostic marker. In particular, cancer treatment methods that supplement microRNAs whose expression is reduced in cancer-bearing states will administer microRNAs that were originally present in the body in non-cancer-bearing states, and it has been reported in animal experiments that there are few side effects. (For example, see Non-Patent Document 1), and application to humans is strongly expected.

  So far, various studies have been conducted on microRNAs showing relevance to colorectal cancer. For example, it has been reported that miR-340 can suppress the growth of colon cancer cells in vitro (see Non-patent Document 2), can be used as a diagnostic marker for colon cancer (see Patent Document 1), and the like. Thus, it has been reported that a plurality of microRNAs including miR-340 are related to colorectal cancer. However, the gene expression of bone marrow free cancer cells in colorectal cancer patients and the relationship between the prognosis and gene expression of colorectal cancer patients have not been analyzed, and microRNAs that play an important role in the prognosis of colorectal cancer metastasis, etc. Has not been identified. Therefore, regarding microRNA candidates that are related to colorectal cancer, what type of microRNA can be selected to establish colorectal cancer treatment that can treat colorectal cancer and improve the prognosis such as suppression of metastasis It has not been revealed.

  In addition, the drug delivery system (DDS) to the living body is the most successful in the practical application of cancer treatment using microRNA. Since microRNA is a short RNA molecule of about 22 bases, it is easily degraded in vivo by the action of RNase, etc. and does not have specific recognition ability for tumor cells, so microRNA is simply administered in vivo. Alone, the microRNA cannot be accumulated in tumor cells in vivo, and the action of the microRNA cannot be exhibited effectively. Therefore, the establishment of DDS technology that accumulates administered microRNA in tumor cells is indispensable for cancer treatment using microRNA. The present inventors have reported that complex particles obtained by complexing a polynucleotide to be introduced into tumor cells with carbonate apatite are effective as DDS for specifically accumulating and introducing the polynucleotide into tumor cells. (Refer nonpatent literature 3). However, a technique for applying the DDS technique to the treatment of colorectal cancer has not been studied yet.

Cancer Research 14, 70 (1): 5923-5930, 2010 Oncol. Rep., 28 (4): 1346-1352, 2012 Plos One 5, 376 (1): 87-94, 2013

US Patent Application Publication No. 2008/096084

  An object of the present invention is to establish a colorectal cancer treatment technique that can treat colorectal cancer using microRNA and that can suppress cancer metastasis and the like to improve the prognosis. Another object of the present invention is to establish a method for examining cancer metastasis to the liver in patients with intestinal cancer and a method for predicting prognosis of patients with colorectal cancer.

  In order to solve the above problems, the present inventor first made a comprehensive comparison of microRNA expression of free cancer cells in the bone marrow in patients with colorectal cancer liver metastases and patients with colorectal cancer non-liver metastases. It was found that expression was decreased in bone marrow free cancer cells of patients with cancer liver metastasis. That is, it was found that miR-340 expression reduction is likely to be involved in colon cancer metastasis. In addition, the present inventor confirmed the expression of miR-340 in the excised tissue from patients with colorectal cancer. The miR-340 low expression group had a poorer prognosis than the miR-340 high expression group. I found. That is, the present inventors have found that a colorectal cancer patient having a colorectal cancer cell in which miR-340 expression is reduced has a poor prognosis after surgical treatment of colorectal cancer. Based on these findings, it was found that miR-340 supplementation therapy is effective in suppressing metastasis of colorectal cancer and improving the prognosis after surgery for colorectal cancer.

  Furthermore, the present inventor has found that administration of composite particles in which microRNA is combined with carbonate apatite can suppress the growth of colon cancer cells in vivo and can effectively exert an antitumor effect. The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A therapeutic agent for colorectal cancer comprising, as an active ingredient, composite particles in which miR-340 is combined with carbonate apatite particles.
Item 2. Item 2. The therapeutic agent for colorectal cancer according to Item 1, wherein the carbonate apatite particles have an average particle size of 50 nm or less.
Item 3. Item 3. The therapeutic agent for colon cancer according to item 1 or 2, which is administered to a colon cancer patient in which free cancer cells in bone marrow are observed and miR-340 is lowly expressed in the bone marrow free cancer cells.
Item 4. Item 3. The therapeutic agent for colorectal cancer according to Item 1 or 2, which is administered to a colorectal cancer patient having colorectal cancer cells in which the expression level of miR-340 is decreased, after resection of the colorectal cancer.
Item 5. An agent for preventing cancer metastasis in colorectal cancer patients, comprising, as an active ingredient, composite particles in which miR-340 is combined with carbonate apatite particles.
Item 6. An agent for improving prognosis in a patient after resection of colorectal cancer, comprising as an active ingredient composite particles in which miR-340 is combined with carbonate apatite particles.
Item 7. A method for examining cancer metastasis to the liver in colorectal cancer patients, comprising detecting the expression level of miR-340 in free cancer cells in bone marrow of colorectal cancer patients.
Item 8. A method for predicting the prognosis of a colorectal cancer patient, comprising detecting the expression level of miR-340 in colorectal cancer cells of the colorectal cancer patient.

  According to the therapeutic agent for colorectal cancer of the present invention, by using miR-340, metastasis of colorectal cancer can be suppressed while suppressing the proliferation of colorectal cancer cells, so that colorectal cancer with a good prognosis is treated. It becomes possible. Furthermore, the therapeutic agent for colorectal cancer of the present invention uses miR-340 complexed with carbonate apatite to allow miR-340 administered in vivo to accumulate in colorectal cancer cells, thereby causing the intracellular Therefore, it is equipped with DDS technology required for practical use of colorectal cancer treatment, and is extremely useful in clinical practice.

  Further, according to the method for examining cancer metastasis of the present invention, by using the expression level of miR-340 in bone marrow free cancer cells as an index, cancer metastasis to the liver has occurred in the colon cancer patient or to the liver. Therefore, it is possible to presume colorectal cancer patients who have a high risk of cancer metastasis to the liver, and to perform appropriate treatment.

  Further, according to the method for predicting prognosis of colorectal cancer patients of the present invention, whether or not prognosis is likely to deteriorate after surgical operation of colorectal cancer by using the expression level of miR-340 in colorectal cancer cells as an index. Since it can be estimated, it becomes possible to perform appropriate treatment at an early stage for postoperative colorectal cancer patients who have a high risk of prognosis deterioration.

In Test Example 1, it is a diagram showing a cell sorting flow in which CD14 ⁻ CD45 ⁻ EpCAM ⁺ cells (BM4) were obtained from whole bone marrow cells (BM1). In Experiment 1, it is a figure which shows the result of having measured the amount of mRNA of E-cadherin, N-cadherin, Vimentin, CEA, and MYC in CD14 ^- CD45 ^- EpCAM ⁺ cell (BM4). In FIG. 2, the expression level of each mRNA level in CD14 ⁻ CD45 ⁻ EpCAM ⁺ cells (BM4) is shown as a relative expression ratio with respect to the expression level of each mRNA level in BM2 (monocyte (hairball) cell). . In Experimental example 1, it is a figure which shows the result of having performed immunostaining of EpCAM, DAPI, and cytokeratin about CD14 ^- CD45 ^- EpCAM ⁺ cell (BM4). In Test Example 2, in vitro colon cancer cell lines HCT116 and SW480 were treated with pre-miR-340 (indicated as “pre-miR-340” in the figure), Pre-miR negative It is a figure which shows the result of having measured cell proliferation on the conditions (denoted as "Pre-miR negative control" in a figure) processed by control, and the untreated conditions (denoted as "Parent" in a figure). In Experimental Example 3, it is a figure which shows the result of having compared the expression level of miR-340 in a colon cancer cell and a normal mucosa cell. In FIG. 5, the expression level of miR-340 is shown as a relative expression ratio with respect to the expression level of RNU6B. In Experimental example 4, it is a figure which shows the result of having divided into a miR-340 high expression group and a miR-340 low expression group, and putting together a 5-year disease-free survival rate and a 5-year total survival rate. In Test Example 5, conditions treated with a preparation containing composite particles of pre-miR-340 and carbonate apatite particles (indicated as “pre-miR-340” in the figure), Pre-miR negative control and carbonate apatite particles included Results of evaluating the therapeutic effect of colorectal cancer in vivo under conditions treated with the drug product (indicated as “Pre-miR negative control” in the figure) and untreated conditions (indicated as “Parent” in the figure) It is. A shows the change in tumor volume over time, and the arrows in A show the time points when each preparation was administered. B shows the photograph which image | photographed the back part of the nude mouse 36 days after administration of HCT116 cell.

1. Therapeutic Agent for Colorectal Cancer The therapeutic agent for colorectal cancer of the present invention is characterized by using composite particles obtained by complexing miR-340 with carbonate apatite particles as an active ingredient. Hereinafter, the therapeutic agent for colorectal cancer of the present invention will be described in detail.

miR-340
The miR-340 used in the present invention may be a mature miRNA (mature-miRNA), a hairpin type precursor miRNA (pri-miRNA), or a pre-miRNA in which a part of the pri-miRNA is cleaved. miRNA may be sufficient. The pri-miRNA and pre-miRNA are processed in the colon cancer cells to become mature miRNA. Moreover, miR-340 used in the present invention may form a double-stranded precursor consisting of RNA having a complementary base sequence. The double-stranded precursor breaks the double strand in colon cancer cells and releases mature miRNA.

What is necessary is just to set suitably about the origin of miR-340 used by this invention according to the kind of animal used as application object. For example, when used for the treatment of human colorectal cancer, human-derived miR-340 may be used. Specific examples of human-derived miR-340 used in the present invention include mature has-miR-340 shown below.
Mature has-miR-340 (sense); 5'-UUAUAAAGCAAUGAGACUGAUU-3 (SEQ ID NO: 1)
Mature has-miR-340 (antisense); 5'- AAUCAGUCUCAUUGCUUUAUAA-3 '(SEQ ID NO: 2)

Carbonate apatite particles Carbonate apatite has a structure in which hydroxyl group of hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) is partially substituted with CO ₃ , and has a general formula Ca _10-m X _m (PO ₄ ) ₆ (CO ₃ ) _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 a positive number of usually 0 or more and 1 or less, preferably 0 or more and 0.1 or less, more preferably 0 or more and 0.01 or less, and still more preferably 0 or more and 0.001 or less. Y is a group or element that can partially substitute CO ₃ in carbonate apatite, and examples thereof include OH, F, and Cl. n is usually a positive number from 0 to 0.1, preferably from 0 to 0.01, more preferably from 0 to 0.001, and even more preferably from 0 to 0.0001.

  The average particle size of the carbonate apatite particles used in the present invention is not particularly limited as long as it is a size that can be administered into a living body and transferred into colon cancer cells. From the viewpoint of efficient accumulation and migration into colorectal cancer cells, it is usually 50 nm or less, preferably 1 to 40 nm, more preferably 1 to 20 nm, and more preferably 5 to 10 nm.

  In addition, the average particle diameter of the said carbonate apatite is a value measured by observing using a scanning probe microscope. When measuring the particle size with a scanning probe microscope, confirm the measurement site with a CCD camera, and if there are huge particles (for example, a particle size of 5 μm or more) that are clearly unsuitable for measurement using a scanning probe microscope, They are removed from the measurement range. In the present specification, the 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 a plurality of particles are aggregated, the aggregate is determined as one particle.

  The carbonate apatite particles can be obtained according to a known method. For example, it can be obtained by preparing it in the presence of calcium ions, phosphate ions and bicarbonate ions 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.

  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.

  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.

  The bicarbonate ion concentration in the aqueous solution is usually 1.0 to 10,000 mM, preferably 5 to 1000 mM, and more preferably 10 to 100 mM.

The supply source of calcium ions, phosphate ions and hydrogen carbonate ions is not particularly limited as long as these ions can be supplied in an aqueous solution, and examples thereof include water-soluble salts of these ions. Specifically, CaCl ₂ can be used as the calcium ion source, NaH ₂ PO ₄ .2H ₂ O can be used as the phosphate ion source, and NaHCO ₃ can be used as the carbonate ion source.

The aqueous solution for preparing the carbonate apatite particles may contain components other than the above-described ion supply sources and other substances as long as the carbonate apatite particles are formed. For example, Ca or CO ₃ in the carbonate apatite may be partially substituted by adding fluorine ions, chlorine ions, Sr, Mn, or the like to the composition in the aqueous solution. However, the addition amount of fluorine ions, chlorine ions, Sr, and Mn is preferably in a range that does not significantly affect the pH solubility and particle size range of the composite particles to be formed. In addition, the aqueous solution for preparing the carbonate apatite particles may use water as a base, but various culture media and buffers for cell culture may be used.

  In the preparation of the carbonate apatite particles used in the present invention, the mixing order of each ion source and other substances in the aqueous solution is not particularly limited, and the aqueous solution may be mixed in any order as long as the target carbonate apatite particles are obtained. May be prepared. 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 Can be mixed to prepare an aqueous solution.

  The carbonate apatite particles can be obtained by adjusting the pH of the aqueous solution containing each of the above ions to a range of 6.0 to 9.0 and leaving (incubating) for a certain period of time. The pH of the aqueous solution in 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, still more preferably 7.3 to 8.5, particularly preferably 7.4 to 8.5, Most preferably, 7.5-8.0 is mentioned.

  The temperature condition of the aqueous solution in forming the carbonate apatite particles is not particularly limited as long as the carbonate apatite particles are formed, but is usually 10 ° C or higher, preferably 25 to 80 ° C, more preferably 37 to 70 ° C or higher. Is mentioned.

  The incubation time of the aqueous solution for forming the carbonate apatite particles is not particularly limited as long as the carbonate apatite particles are formed, but is usually 1 minute to 24 hours, preferably 10 minutes to 1 hour. The presence or absence of particle formation can be confirmed by observing under a microscope, for example.

  Further, the method for controlling the average particle size of the carbonate apatite particles to 50 nm or less is not particularly limited, and examples thereof include a method of ultrasonically treating the carbonate apatite particles formed in the aqueous solution. Here, the ultrasonic vibration treatment is not a treatment in which an ultrasonic vibrator such as an ultrasonic crusher or a homogenizer used for so-called microbial cell crushing is directly brought into contact with a sample, but generally a precision instrument or a test. This is a process using an ultrasonic cleaner in which an ultrasonic vibrator and a cleaning tank used for cleaning tubes and the like are integrated. Put a liquid (for example, water) into the cleaning tank (water tank) of the ultrasonic cleaner, float a container (for example, a plastic tube) containing carbonate apatite particles on it, and pass the liquid through the procedure to clean precision equipment. It means a treatment in which ultrasonic waves are applied to an aqueous solution containing carbonate apatite particles. Thereby, the particle size of the carbonate apatite particles can be reduced to 50 nm or less simply and efficiently.

  An apparatus that can be used for ultrasonic vibration treatment is capable of applying ultrasonic vibration to a container containing carbonate apatite particles indirectly through a solvent such as water, like the ultrasonic cleaner. If there is no particular limitation. From the viewpoint of versatility and ease of handling, it is preferable to use an ultrasonic cleaner equipped with an ultrasonic vibrator and a thermostatic bath.

  The conditions for the ultrasonic vibration treatment are not particularly limited as long as the particle diameter can be controlled within a predetermined range. For example, the temperature of the water tank can be appropriately selected from temperatures of 5 to 45 ° C, preferably 10 to 35 ° C, more preferably 20 to 30 ° C. The high frequency output of the ultrasonic vibration treatment can be appropriately set within a range of 10 to 500 W, for example, preferably 20 to 400 W, more preferably 30 to 300 W, and more preferably 40 to 100 W. The oscillation frequency is usually 10 to 60 Hz, preferably 20 to 50 Hz, and more preferably 30 to 40 Hz. The ultrasonic vibration treatment time is, for example, 30 seconds to 30 minutes, preferably 1 to 20 minutes, and more preferably 3 to 10 minutes.

  The type of container containing carbonate apatite particles used when performing ultrasonic vibration treatment is not limited as long as the particles can be refined to a predetermined particle diameter range, depending on the volume of the aqueous solution and the purpose of use. Can be selected as appropriate. For example, a plastic tube having a capacity of 1 to 1000 ml can be used.

  The ultrasonic vibration treatment is preferably performed in the presence of albumin (that is, in a state where albumin is added to an aqueous solution containing carbonate apatite particles). This is because ultrasonic vibration treatment is performed in an environment where albumin and carbonate apatite particles coexist, so that superapatite ultrafine nanoparticles with a finer particle diameter can be obtained and re-aggregation of particles can be suppressed. It is because it becomes. The concentration of albumin in the aqueous solution containing carbonate apatite particles is not particularly limited as long as the effect of miniaturization and / or reaggregation suppression is obtained, for example, 0.1 to 500 mg / ml, preferably 1 to 100 mg / ml, More preferably, about 1 to 10 mg / ml can be added.

Composite particles of miR-340 and carbonate apatite particles The therapeutic agent for colorectal cancer of the present invention uses composite particles in which the miR-340 and carbonate apatite particles are combined. By combining miR-340 with carbonate apatite particles in this way, miR-340 is efficiently accumulated in colon cancer cells in vivo by the action of carbonate apatite, and miR-340 is introduced into colon cancer cells. It becomes possible to make it. Moreover, since miR-340 can be released from the carbonate apatite particles in the cell after being introduced into the cell, it is possible to cause the miR-340 to exhibit a desired activity.

  In the present invention, the composite particle of miR-340 and carbonate apatite particles refers to a state in which miR-340 is adsorbed and supported on carbonate apatite particles by ionic bonds, hydrogen bonds, or the like. The method for forming the composite particles of miR-340 and carbonate apatite particles is not particularly limited. For example, a method of forming miR-340 and carbonate apatite particles by coexisting in an aqueous solution; in an aqueous solution for preparing carbonate apatite particles. Thus, there may be mentioned a method in which miR-340 coexists with calcium ions, phosphate ions and hydrogen carbonate ions to simultaneously form carbonate apatite particles and combine miR-340 and carbonate apatite particles.

  When miR-340 and carbonate apatite particles are combined, the formation of carbonate apatite particles and the combination of miR-340 and carbonate apatite particles simultaneously, miR-340 is added to the aqueous solution used for the preparation of carbonate apatite. For example, 0.1 to 1000 nM, preferably 0.5 to 500 nM, and more preferably 1 to 200 nM.

  In the composite particles of miR-340 and carbonate apatite particles, the ratio of miR-340 and carbonate apatite particles is not particularly limited, and may be set as appropriate according to the dose of miR-340. For example, when 2 mg of miR-340 is combined with carbonate apatite particles, 5 mg of miR-340 is added to 1 L of an aqueous solution for preparing the carbonate apatite particles described above to form carbonate apatite particles and miR-340. And carbonate apatite particles may be combined at the same time.

  In addition, the composite particles of miR-340 and carbonate apatite particles may be loaded with other drugs such as anticancer agents as long as the effects of the present invention are not impaired. Examples of such anticancer agents include, but are not limited to, alkylating agents such as cyclophosphamide hydrate, ifosfamide, thiotepa, busulphalan, melphalan, nimustine hydrochloride, ranimustine, dacarpadin, temozolomide; methotrexate, pemetrexed sodium Antimetabolites such as hydrate, fluorouracil, doxyfluridine, capecitabine, tagafur, cytarabine, gemcitabine hydrochloride, fludarabine phosphate, nelarabine, cladribine, levofolinate calcium; doxorubicin hydrochloride, daunorubicin hydrochloride, prrubicin hydrochloride, epirubicin hydrochloride isal Salt, aclarubicin hydrochloride, amrubicin hydrochloride, mitoxantrone hydrochloride, mitomycin C, actinomycin D, bleomaciin hydrochloride Antibiotics such as salt, pupelomachine hydrochloride, dinostatin timamarer, calicheamicin, microtubule inhibitors such as vincristine sulfate, vinblastine sulfate, vindesine sulfate, paclitaxel; anastrozole, exemestane, letrozole, fadrozole Aromatase inhibitors such as hydrochloride hydrate; platinum preparations such as cisplatin, carboplatin, nedaplatin, oxaliplatin; adrenal corticosteroids such as irinotecan hydrochloride hydrate, nogitecan hydrochloride, etoposide, sobuzoxane, etc., prednisolone, dexamethasone , Lenalidomide which is thalidomide and its derivatives, bortezomib which is a protease inhibitor, and the like. These anticancer agents may be used individually by 1 type, and may be used in combination of 2 or more type.

  In addition, the therapeutic agent for colorectal cancer of the present invention is used in a state where the composite particles of miR-340 and carbonate apatite particles are dispersed in a solvent suitable for administration to a living body. As described above, carbonate apatite particles can be obtained by dissolving various ion supply substances in a solvent such as water, a medium, or a buffer. From the viewpoint of osmotic pressure, buffer capacity, sterility, etc., it is not necessarily suitable for administration to a living body (intravascular administration). Therefore, in order to replace the solvent in which the carbonate apatite particles are dispersed with a solvent suitable for administration to a living body (for example, physiological saline), the carbonate apatite particles are usually separated from the solvent by centrifugation and collected. An operation to replace the solvent is necessary. However, when such an operation is performed, the carbonate apatite particles are aggregated and the particles become enormous, so that the state changes to a state unsuitable for administration to a living body. Therefore, by adding the agglomerated carbonate apatite particles to a solvent suitable for administration to the living body and then performing the ultrasonic vibration treatment described above, the composite particles of miR-340 and carbonate apatite particles can be used for administration to the living body. It can be dispersed in a suitable solvent with an appropriate particle size (preferably an average particle size of 50 nm or less).

Applicable disease, dose, usage, etc. The therapeutic agent for colorectal cancer of the present invention is applied to colon cancer patients for the purpose of treating colorectal cancer. In addition, the therapeutic agent for colorectal cancer of the present invention may be used as a preventive agent for cancer metastasis (especially, a preventive agent for metastasis to the liver) in colorectal cancer patients. Furthermore, the therapeutic agent for colorectal cancer of the present invention may be administered for the purpose of preventing recurrence or metastasis after removing colorectal cancer from a colorectal cancer patient, or an agent for improving prognosis in a patient after resection of colorectal cancer or It can also be used as a cancer metastasis preventive agent (particularly a metastasis preventive agent to the liver).

  In addition, as shown in Test Example 1 to be described later, a colon cancer patient in which free bone marrow cancer cells are observed by the present inventor and miR-340 is lowly expressed in the bone marrow free cancer cells is It has been elucidated that cancer metastasis is likely to occur. Therefore, the therapeutic agent for colorectal cancer of the present invention is administered to a colon cancer patient in which free cancer cells in bone marrow are observed and miR-340 is lowly expressed in the bone marrow free cancer cells. -340 replacement therapy can effectively treat colorectal cancer patients and suppress the transfer to the liver.

  Whether or not miR-340 is lowly expressed in bone marrow free cancer cells is determined by miR-340 in bone marrow free cancer cells obtained from patients with non-metastatic colorectal cancer (hereinafter referred to as “free cancer in liver non-metastatic bone marrow”). The expression level of miR-340 is sometimes measured in advance, and the amount of miR-340 in bone marrow free cancer cells obtained from colorectal cancer patients and the amount of free cancer cells miR in liver non-metastatic bone marrow -340 by comparing quantities. When the amount of miR-340 in bone marrow free cancer cells obtained from a colorectal cancer patient is lower than that of liver non-metastatic bone marrow free cancer cells miR-340, in particular, about 1/2 or less, about 1/4 or less, Alternatively, when the ratio is about 1 / 7.5 or less, it is estimated that the colorectal cancer patient has metastasized to the liver or is likely to occur, and administration of the therapeutic agent for colorectal cancer of the present invention is performed. MiR-340 replacement therapy is effective.

  The measurement of the amount of miR-34 in bone marrow free cancer cells can be performed according to a conventionally known method. For example, free cancer cells in bone marrow are separated from the bone marrow of colon cancer patients according to a known technique, and RNA samples are obtained from the free cancer cells in bone marrow by the guanidine-cesium chloride ultracentrifugation method, acidic guanidine-phenol-chloroform (AGPC) method, And measure the amount of miR-34. The method for measuring the amount of miR-34 is not particularly limited, and examples thereof include a microarray method, an RT-PCR method, a real-time RT-PCR method, and a northern blot method.

  In addition, as shown in Test Example 4 to be described later, the present inventor tends to deteriorate the prognosis of colorectal cancer patients having colorectal cancer cells in which the expression level of miR-340 is reduced after the colon cancer is excised. It has been elucidated. Therefore, the therapeutic agent for colorectal cancer of the present invention is supplemented with miR-340 by administering it after colorectal cancer resection to a colorectal cancer patient having colorectal cancer cells in which the expression level of iR-340 is decreased. The therapy can also effectively suppress the prognosis.

  Whether or not miR-340 is lowly expressed in colorectal cancer cells is determined by the average expression level of miR-340 in colorectal cancer cells derived from colorectal cancer patients with good prognosis (hereinafter referred to as “miR-good prognosis group miR”). -340 dose average value ”) in advance, and miR-340 amount in colon cancer cells of colon cancer tissue removed from colorectal cancer patients and average value of colon cancer cell miR-340 amount This is done by comparing When the amount of miR-340 in colon cancer cells obtained from a colorectal cancer patient is lower than the average value of miR-340 amount in the good prognosis group, in particular, about 1 / 1.2 or less, about 1/2 or less, or 1 When the ratio is about / 3 or less, it is presumed that the prognosis after surgery is likely to deteriorate in the colon cancer patient, and miR-340 supplementation therapy by administration of the therapeutic agent for colorectal cancer of the present invention is effective.

  Measurement of the amount of miR-34 in colorectal cancer cells can be performed according to a conventionally known method. For example, colon cancer cells are separated from the excised colon cancer tissue according to a known method, and RNA samples are prepared from the colon cancer cells in the same manner as in the case of free cancer cells in the bone marrow, and the miR-34 amount is measured. Just do it.

  The method for administering the therapeutic agent for colorectal cancer of the present invention is not particularly limited as long as the therapeutic agent for colorectal cancer of the present invention can be delivered to colorectal cancer in vivo. For example, it is intravascular (intraarterial or intravenous). Injection, continuous infusion, subcutaneous administration, local administration, intramuscular administration and the like can be mentioned. Of these, intraarterial and intravenous administration is preferable.

The dose of the colorectal cancer therapeutic agent of the present invention is appropriately set according to the patient's symptom level, the patient's gender, age, etc., and thus cannot be uniformly defined. For example, the weight of miR-340 About 15 to 70 mg / m ² (body surface area) per day in terms of conversion.

  Further, as described above, when the carbonate apatite particles used in the present invention have an average particle size of 50 nm or less, the carbonate apatite particles are efficiently accumulated in the colon cancer cells in the living body and transferred to the colon cancer cells efficiently. Therefore, the therapeutic agent for colorectal cancer of the present invention can be administered after the composite particles of miR-340 and carbonate apatite particles are dispersed in the form of fine particles by ultrasonic vibration treatment, and then the particles aggregate. It is desirable to do this promptly. For example, administration within 1 minute, preferably within 30 seconds after the ultrasonic vibration treatment is suitable. However, as described above, when the aggregation of carbonate apatite particles is suppressed by adding albumin, it can be administered after several minutes to several tens of minutes after the ultrasonic vibration treatment.

2. Method for Examining Cancer Metastasis to Liver in Colorectal Cancer Patients As shown in Test Example 1 described later, colon cancer in which free cancer cells in bone marrow are observed and miR-340 is lowly expressed in the bone marrow free cancer cells Patients have been shown to be prone to cancer metastasis to the liver. Therefore, the present invention further provides a method for examining cancer metastasis to the liver in colorectal cancer patients, wherein the expression level of miR-340 in free bone marrow cancer cells of colorectal cancer patients is detected. If the expression level of miR-340 is decreased in the test method, it is predicted that cancer metastasis to the liver has occurred or that the risk of cancer metastasis to the liver is high.

  Whether the expression level of miR-340 in bone marrow free cancer cells is reduced or not is determined by miR-340 in bone marrow free cancer cells obtained from patients with non-metastatic colorectal cancer (hereinafter referred to as “liver non-metastatic bone marrow free”). The expression level of "cancerous miR-340" may be measured in advance, and the amount of miR-340 in bone marrow free cancer cells obtained from patients with colorectal cancer and the amount of free cancer cells in non-metastatic bone marrow This is done by comparing the amount of miR-340. When the amount of miR-340 in bone marrow free cancer cells obtained from a colorectal cancer patient is lower than that of liver non-metastatic bone marrow free cancer cells miR-340, in particular, about 1/2 or less, about 1/4 or less, Alternatively, when the ratio is about 1 / 7.5 or less, the colorectal cancer patient is predicted to have cancer metastasis to the liver or have a high risk of cancer metastasis to the liver.

  In the test method, the method for measuring the amount of miR-340 in bone marrow free cancer cells is as described in the column of “Therapeutic agent for colorectal cancer”.

3. Prediction method for prognosis of colorectal cancer patients As shown in Test Example 4 to be described later, colorectal cancer patients having colorectal cancer cells in which the expression level of miR-340 is decreased tend to deteriorate prognosis after resection of colorectal cancer. It has been revealed that there is. Therefore, the present invention further provides a prognosis prediction method for colorectal cancer patients, characterized by detecting the expression level of miR-340 in colorectal cancer cells of colorectal cancer patients. If the expression level of miR-340 is decreased in the prediction method, the risk of recurrence and metastasis after surgery is high, and the prognosis is predicted to be easily deteriorated.

  Whether or not the expression level of miR-340 in colorectal cancer cells is reduced is determined in advance by measuring the mean value of miR-340 in the good prognosis group, in colorectal cancer cells of colorectal cancer tissue removed from colorectal cancer patients This is performed by comparing the amount of miR-340 and the average value of colorectal cancer cell miR-340. When the amount of miR-340 in colorectal cancer cells obtained from a colorectal cancer patient is lower than the mean value of miR-340 amount in the good prognosis group, particularly about 1 / 1.2 or less, about 1/2 or less, or 1 / When it is about 3 or less, it is presumed that the colorectal cancer patient has a high risk of postoperative recurrence and metastasis, and prognosis is likely to deteriorate.

  In the prediction method, the method for measuring the amount of miR-340 in colorectal cancer cells and normal colorectal mucosal cells is as described in the column of “Therapeutic agent for colorectal cancer”.

  Hereinafter, although this invention is demonstrated in detail based on a test example, an Example, etc., this invention is not limited by these. In the following studies, all patients who received samples were given informed consent according to guidelines approved by each institution. This test was conducted under the supervision of the Osaka University Hospital Ethics Committee.

Test Example 1: Comprehensive examination of microRNA expression in free bone marrow cancer cells derived from colon cancer patients Recovery of free cancer cells in bone marrow derived from colorectal cancer patients Free cancer cells in bone marrow were isolated from 7 patients with colorectal liver metastases and 12 patients with colorectal cancer non-liver metastases by the following method. Anesthesia is administered before surgery for colorectal cancer patients, and 20 mL of bone marrow fluid (total volume of 40 mL) is collected from the left and right iliac crests, and mononuclear cells (total bone marrow cells, BM1) by standard Ficoll-Hypaque gradient method Was recovered. Next, MACS ^™ pro using anti-CD14 immunobeads (Milteny Biotec) (Milteny Biotec, Bergisch Gladbach, Germany) performed magnetic bead cell sorting to remove CD14 ⁺ cells (BM2) from BM1 and collect CD14 ⁻ cells. Next, the magnetic bead cell sorting using anti-CD45 immunobead ^{(Milteny Biotec), CD14 - CD45} + cells were removed (BM3), CD14 ^{- -} CD14 from cells CD45 ^- cells were harvested. After incubating the obtained CD14 ^- CD45 ^- cells with FcR blocking reagent (Milteny Biotec), CD14 ^- CD45 ^- EpCAM ⁺ cells (BM4) are recovered by magnetic bead cell sorting using anti-EpCAM immunobeads (Milteny Biotec) did. FIG. 1 shows a cell sorting flow for obtaining CD14 ⁻ CD45 ⁻ EpCAM ⁺ cells (BM4) from whole bone marrow cells (BM1).

Subsequently, the mRNA levels of E-cadherin, N-cadherin, Vimentin, CEA and MYC were measured for CD14 ⁻ CD45 ⁻ EpCAM ⁺ cells (BM4). The results are shown in FIG. As is clear from FIG. 2, in BM4, the amount of mRNA of E-cadherin, which is an epithelial marker, is 9.25 times that of BM2 (monocytic (hairball-shaped) cells), and the amount of mRNA of Vimentin, which is a mesenchymal marker. 0.48 times, the amount of mRNA of N-cadherin was comparable. In addition, in BM4, the amount of mRNA of CEA, which is a tumor marker, and the amount of mRNA of MYC, which is an oncogene, are 19.5 times and 2.26 times that of BM2 (monocyte (hairball) cells), respectively. It was. The result of the gene expression of BM4 is consistent with the gene expression of cancer cells, and it was confirmed that the cancer cells were recovered as BM4. Furthermore, BM4 was immunostained using antibodies against EpCAM, DAPI, and cytokeratin. The results are shown in FIG. From the result of this immunostaining, it was confirmed that cancer cells were recovered as BM4.

2. Analysis of microRNA expression characteristics of bone marrow free cancer cells Total RNA was isolated from the bone marrow free cancer cells obtained above using miRNeasy kit (Applied Biosystems, Carlsbad, CA) according to the attached manual. Total RNA concentration and purity were analyzed with a spectrophotometer and RNA integrity was confirmed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, Calif.). Total RNA (100 ng) was labeled directly with cyanine 3-CTP without fractionation or amplification using an Agilent protocol that can accurately measure microRNA from 0.2 amol to 2 fmol in a linear dynamic range. Each 100 ng of total RNA was competitively hybridized to a microRNA array (Agilent Microarray Kit; G4470C) with 886 microRNAs (version 12.0 of the Sanger miRNA database; http://www.mirbase.org/) . The signal intensity of each hybridization was measured using Extraction Software Version A.7.5.1 (Agilent Technologies). P value was calculated using paired t-test.

  As a result, 10 types of microRNAs shown in Table 1 were detected as microRNAs having a difference in expression level of 1.5 times or more between patients with colorectal cancer liver metastases and patients with colorectal cancer non-liver metastases. As can be seen from Table 1, the expression level of miR-340 in the free bone marrow cancer cells derived from patients with colorectal cancer liver metastases was significantly lower than that in patients with colorectal cancer non-liver metastases. From this result, it became clear that colon cancer patients whose miR-340 expression level is low in bone marrow free cancer cells are likely or likely to undergo cancer metastasis to the liver. It is also clear that miR-340 replacement therapy is effective for patients with colorectal cancer whose miR-340 expression level is low in bone marrow free cancer cells to suppress cancer metastasis to the liver. became.

Test Example 2: Inhibition effect of miR-340 on colon cancer cell line in vitro The following tests were performed using HCT116 strain and SW480 strain (both purchased from ATCC) as human colon cancer cells.

Human colon cancer cells were treated with Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS (fetal bovine serum) and 1% antibiotic solution (Sigma, St. Louis, MO) at 37 ° C, 5 ° C. Cultured in a% CO ₂ atmosphere. Then, using Lipofectamine iMax (Invitrogen, Darmstadt, Germany) according to the manufacturer's protocol, pre-miR-340 (MC12670, Applied Biosystems; said SEQ ID NO: 1 and the precursor of has-miR-340 in a 12-well dish) The precursor of mature hsa-miR-340 consisting of 2) 30 nmol / L was transfected into human colon cancer cells. In addition, Pre-miR negative control (Applied Biosystems; sense 5'-UAAAUGUACUGCGCGUGGAGAGGAA-3 '(sequence number 3), antisense 5'-UUCCUCUCCACGCGCAGUACAUUUA-3' (sequence number 4)) was used as a control.

Transfected colon cancer cells were seeded in a 2-well dish at 3 to 4 × 10 ⁴ cells / well and cultured for 72 hours. After the culture, the number of cells was measured using NucleoCounter kit (Chemometec, Gydevang 43, DK-3450 Alleroed, Denmark).

  The obtained results are shown in FIG. As is clear from FIG. 4, cell growth was significantly inhibited in both HCT116 and SW480 strains when treated with pre-miR-340 as compared to untreated (P <0.001). That is, from this result, it was confirmed that miR-340 has an action of suppressing the growth of colon cancer cells.

Test Example 3: Comparison of miR-340 expression in colorectal cancer cells and normal mucosal cells miR-340 expression levels of colorectal cancer cells and normal mucosal cells (derived from normal colorectal mucosa and submucosa of colorectal cancer patients) were compared with those in Test Example 1 above. It measured by the same method.

  The obtained results are shown in FIG. From these results, it was revealed that miR-340 expression in colon cancer cells was significantly reduced compared to normal colon mucosa cells (P = 0.010). That is, this result also confirmed that miR-340 replacement therapy is effective for the treatment of colorectal cancer.

Test Example 4: Correlation analysis of miR-340 expression in colorectal cancer cells with clinicopathological findings and prognosis
Samples of 136 primary colon cancer cells were collected from patients with colorectal cancer who had undergone surgical treatment at Osaka University Hospital and two related hospitals between 1999 and 2010. Each sample was stored at −80 ° C. with RNAlater ™ until RNA extraction was performed. Of the 136 colon cancer patients, stage 0 was 4 cases, stage I was 15 cases, stage II was 38 cases, stage IIIA / B was 52 cases, and stage IIIC was 27 cases.

  The miR-340 expression level in each colorectal cancer cell was measured by the same method as in Test Example 1, and the average value of the miR-340 expression level was determined. Next, miR-340 expression level is lower than the average value (miR-340 low expression group, n = 73) and miR-340 expression level is higher than the average value (miR-340 high expression group, n = 63) Divided into. Table 2 shows the results of a summary of the clinicopathological findings divided between the miR-340 low expression group and the miR-340 high expression group. The results were divided into the miR-340 low expression group and the miR-340 high expression group for 5 years. FIG. 6 shows the results of summarizing the disease-free survival rate (5-years Disease Free Survival) and the 5-year overall survival rate (5-years Overall Survival).

  As is clear from Table 2, in the tumor size, the miR-340 low expression group was larger than the miR-340 high expression group (P <0.001), but other factors showed low miR-340 expression. There was no difference between the group and miR-340 high expression group. As is clear from FIG. 6, both the 5-year disease-free survival rate and the 5-year total survival rate were lower in the miR-340 low expression group than in the miR-340 high expression group. That is, it was revealed that colon cancer patients having colon cancer cells in which miR-340 is lowly expressed have a high risk of recurrence and metastasis after surgery and tend to have a poor prognosis. In addition, it was also clarified from this result that prognosis worsening can be suppressed by colorectal cancer patients having colorectal cancer cells in which miR-340 is lowly expressed by miR-340 supplementation therapy.

  In addition, univariate analysis and multivariate analysis were performed for factors related to 5-year disease-free survival. Statistical analysis was performed using the JMP9 program (SAS Institute, Cary, NC). Tumor recurrence was estimated from colorectal cancer using the Kaplan-Meier method and the Cox hazard model, and the statistical significance was determined using the log-rank test and the Cox hazard ratio. Associations between discrete variables were analyzed using the chi-square test. Average values were compared using Student's t-test. For continuous variables used for in vitro analysis, data were expressed as mean ± SE and analyzed with the Wilcoxon's rank test. A statistically significant difference was considered when the P value was <0.05.

  The results are shown in Table 3. From univariate analysis, lymph node metastasis (P = 0.003), serosal surface infiltration (P = 0.001), and miR-340 low expression (P = 0.020) are significantly associated with 5-year disease-free survival I understood that. In addition, from the results of multivariate analysis, miR-340 low expression was observed in the serosa layer surface infiltration (RR, 3.053; 95% CI, 1.311-7.041; P = 0.011) and lymph node metastasis (RR, 2.918; 95% CI, 1.081-10.16; P = 0.033), which was also found to be an independent predictor of 5-year disease-free survival.

Test Example 5: Treatment effect of colon cancer with miR-340 and carbonate apatite particles in vivo HCT116 per 100 μL of medium / Matrigel solution containing Matrigel (BD Biosciences, San Jose, Calif.) And medium at a volume ratio of 1: 1 The cells were mixed to 1 × 10 ⁶ cells, and 100 μL of the cells were injected subcutaneously into the lower part of the back on the left and right sides of female nude mice (NIHON CLEA, Tokyo, Japan). When the nude mouse is bred and the tumor volume on the back reaches 75 to 80 mm ³ , a preparation containing the composite particles of Pre-miR-340 and carbonate apatite particles obtained as described below is administered per administration. The tail vein was injected so that the particles were 30 μg. The administration of the formulation was repeated once a day for 3 days. Further, as a control, a test was similarly performed using a preparation containing composite particles prepared using Pre-miR negative control (SEQ ID NO: 3 and SEQ ID NO: 4) instead of Pre-miR-340.

(Preparation method of preparation containing composite particles of Pre-miR-340 and carbonate apatite particles)
In 100 ml of distilled water, 0.37 g of NaHCO ₃ , 90 μl of NaH ₂ PO ₄ .2H ₂ O (1M), and 180 μl of CaCl ₂ (1M) are added and dissolved in this order, and the pH is adjusted to 7.5 with 1N HCl. Adjusted. This was filtered through a 0.2 μm diameter filter. 2 μg of Pre-miR-340, 4 μl of CaCl ₂ (1M) was mixed per 1 ml of the obtained buffer, and incubated in a 37 ° C. water bath for 30 minutes. Thereafter, the solution is centrifuged at 15000 rpm × 5 minutes, and the resulting pellet is dispersed in physiological saline to obtain a dispersion of composite particles in which miR340 is encapsulated in carbonate apatite particles. This is subjected to ultrasonic vibration treatment for 10 minutes. By applying, a preparation containing a complex composed of carbonate apatite nanoparticles including miR340 was obtained. In the ultrasonic vibration treatment, the dispersion liquid contained in a plastic container is floated on water set at 20 ° C. using a water bath having an ultrasonic vibration function, and the high frequency output is 55 W and the oscillation frequency is 38 kHz. Went for a minute. The formulation thus obtained was immediately used for the test. In addition, it has been confirmed in the measurement using a scanning probe microscope that the thus obtained preparation has an average particle size of 50 nm or less of the complex composed of carbonate apatite nanoparticles including iR340.

  From the time of administration of the medium / Matrigel solution, the tumor size (major axis x minor axis x minor axis x 1/2) of the back of the mouse was measured over time. The obtained results are shown in FIG. As is apparent from FIG. 7, when the composite particles of miR-340 and carbonate apatite particles were administered via the tail vein, tumor growth was significantly suppressed compared to the control (P <0.05). From this result, when miR-340 and carbonate apatite composite particles are administered intravenously, miR-340 is efficiently delivered to colon cancer cells and transferred to colon cancer cells. It was revealed that the anti-tumor action by -340 can be exerted effectively.

Claims (8)

  A therapeutic agent for colorectal cancer comprising, as an active ingredient, composite particles in which miR-340 is combined with carbonate apatite particles.   The therapeutic agent for colorectal cancer according to claim 1, wherein the average particle diameter of the carbonate apatite particles is 50 nm or less.   The therapeutic agent for colorectal cancer according to claim 1 or 2, which is administered to a colon cancer patient in which free cancer cells in bone marrow are observed and miR-340 is lowly expressed in the bone marrow free cancer cells.   The therapeutic agent for colorectal cancer according to claim 1 or 2, which is administered to a colorectal cancer patient having colorectal cancer cells in which the expression level of miR-340 is decreased, after resection of the colorectal cancer.   An agent for preventing cancer metastasis in colorectal cancer patients, comprising, as an active ingredient, composite particles in which miR-340 is combined with carbonate apatite particles.   An agent for improving prognosis in a patient after resection of colorectal cancer, comprising as an active ingredient composite particles in which miR-340 is combined with carbonate apatite particles.   A method for examining cancer metastasis to the liver in colorectal cancer patients, comprising detecting the expression level of miR-340 in free cancer cells in bone marrow of colorectal cancer patients.   A method for predicting the prognosis of a colorectal cancer patient, comprising detecting the expression level of miR-340 in colorectal cancer cells of the colorectal cancer patient.