Peritoneal dissemination inhibitors and methods for treating peritoneal dissemination

Abstract

Provided is a peritoneal seeding inhibitor that efficiently inhibits peritoneal seeding. The peritoneal seeding inhibitor comprises an adeno-associated virus vector loaded with miR-29b. The serotype of this adeno-associated virus vector includes at least one type selected from AAV1, AAV2, AAV4, and AAV-DJ. More specifically, the serotype of the adeno-associated virus vector is AAV1, AAV2, AAV5, or AAV-DJ in humans (Homo sapiens), and is AAV1, AAV2, AAV5, AAV9, or AAV-DJ in mice (Mus musculus). Through this peritoneal seeding inhibitor, miR-29b is expressed in cells constituting the peritoneal cavity, and peritoneal seeding is treated or prevented.

Description

[Technical Field]

[0001] The present invention relates in particular to a peritoneal dissemination inhibitor, a method for inhibiting peritoneal dissemination, a method for preventing peritoneal dissemination, and a method for treating peritoneal dissemination. [Background technology]

[0002] Peritoneal dissemination is a common form of metastasis and recurrence in cancers (malignant tumors) that develop in the abdominal cavity, such as gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, and ovarian cancer. Peritoneal dissemination is a serious condition that can lead to a poor prognosis, as it creates numerous small nodules in the abdominal cavity, causing ascites and gastrointestinal obstruction. However, until now, there has been no effective treatment for peritoneal dissemination, and no special treatment method targeting peritoneal dissemination has existed. Furthermore, even when curative surgery can be performed macroscopically, peritoneal dissemination often appears several months after surgery. For this reason, systemic chemotherapy with intravenous administration of anticancer drugs and molecular targeted drugs has been the standard treatment for cancers with peritoneal dissemination. However, these drugs have little transfer from the blood to the abdominal cavity, and sufficient therapeutic effects are often not obtained. Therefore, there has been a need for more effective treatment methods.

[0003] Referring to Patent Document 1, there is a description of a conventional attenuated herpes simplex virus effective in treating cancer and a cancer treatment composition containing the same, in which an attenuated herpes simplex virus incorporating an exogenous suicide gene is described. This virus attacks the cancer itself, which involves peritoneal dissemination, but it could not prevent peritoneal dissemination itself.

[0004] Thus, since there has been no reliable method to control peritoneal dissemination until now, establishing a method to efficiently prevent the recurrence of peritoneal dissemination has been considered the most important challenge in improving the prognosis of cancer patients who develop peritoneal dissemination, such as in the digestive system.

[0005] Referring to Non-Patent Document 1, it is described that the function of miR-29b, a microRNA that is reduced in the peritoneal cavity of gastric cancer patients with peritoneal dissemination, was investigated. According to Non-Patent Literature 1, the miR-29b mimic, which is the RNA of miR-29b itself, was transfected into human peritoneal mesothelial cells (HPMCs) by lipofection, and its effects were evaluated in vitro. When miR-29b was applied to human peritoneal mesothelial cells (HPMCs) stimulated with Transforming Growth Factor-β (TGF-β), proliferation was suppressed, and the ability of cancer cells to peritoneal mesothelial cells and their adhesion ability to peritoneal mesothelial cells was significantly reduced. As a result, miR-29b suppressed mesothelial-mesenchymal transition (MMT) induced by TGF-β. This was expected to suppress peritoneal dissemination. Therefore, when miR-29b mimic was combined with atelocollagen and administered to a mouse peritoneal dissemination model, it suppressed peritoneal dissemination on the omentum, but failed to suppress peritoneal dissemination on the mesentery. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2002-218975 [Non-patent literature]

[0007] [Non-Patent Document 1] Kimura Y, Ohzawa H, Miyato H et al., “MiR-29b may suppresses peritoneal metastases through inhibition of the mesothelial-mesenchymal transition (MMT) of human peritoneal mesothelial cells,” Scientific Reports, 2022, 12(1):205 [Overview of the project] [Problems that the invention aims to solve]

[0008] However, the technology described in Non-Patent Document 1 (hereinafter referred to as "prior art") was not able to suppress peritoneal dissemination sufficiently in vivo using miR-29b to the extent that it could be used as a therapeutic agent. Therefore, there was a need for a peritoneal dissemination inhibitor that could efficiently introduce miR-29b into peritoneal mesothelial cells and suppress peritoneal dissemination.

[0009] This invention has been made in view of these circumstances and aims to resolve the above-mentioned problems. [Means for solving the problem]

[0010] The peritoneal dissemination inhibitor of the present invention comprises an adeno-associated virus vector carrying miR-29b, wherein the adeno-associated virus vector is selected for its serotype which is directional to peritoneal mesothelial cells covering the surface of the host's peritoneal cavity, and suppresses tumor formation by inhibiting mesothelial-mesenchymal transition of the peritoneal mesothelial cells and inhibiting peritoneal fibrosis, rather than targeting cancer cells, and the adeno-associated virus vector is administered once, followed by multiple administrations of paclitaxel, and the serotype of the adeno-associated virus vector is AAV2 or AAV-DJ in humans (Homo sapiens) and AAV1, AAV2, AAV4, or AAV-DJ in mice (Mus musculus). 。 The peritoneal seeding treatment method of the present invention is a peritoneal seeding treatment method for animals other than humans. By using an adeno-associated virus vector carrying miR-29b, miR-29b is expressed in cells constituting the abdominal cavity. The adeno-associated virus vector is selected with a serotype having directivity to peritoneal mesothelial cells covering the abdominal surface of the host. It suppresses the mesenchymal transformation of the peritoneal mesothelial cells rather than cancer cells, suppresses peritoneal fibrosis, and thus suppresses tumor formation. After the adeno-associated virus vector is administered once, paclitaxel is administered multiple times. The serotype of the adeno-associated virus vector is AAV2 or AAV-DJ in humans (Homo sapiens), and AAV1, AAV2, AAV4, or AAV-DJ in mice (Mus musculus).

Advantages of the Invention

[0011] According to the present invention, by including an adeno-associated virus vector carrying miR-29b, it is possible to provide a peritoneal seeding inhibitor that can more effectively introduce miR-29b into peritoneal mesothelial cells and the like and suppress peritoneal seeding.

Brief Description of the Drawings

[0012] [Figure 1A] It is a conceptual diagram showing the structure of the vector according to Example 1 of the present invention. [Figure 1B] It is a conceptual diagram showing the structure of the vector according to Example 1 of the present invention. [Figure 2] It is a photograph showing cultured human peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 3A] It is a conceptual diagram showing the results of a serotype selection experiment on human peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 3B] It is a photograph showing the results of a serotype selection experiment on human peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 3C] It is a graph showing the results of a serotype selection experiment on human peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 4A]It is a conceptual diagram showing the results of a serotype selection experiment on mouse peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 4B] It is a photograph showing the results of a serotype selection experiment on mouse peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 4C] It is a graph showing the results of a serotype selection experiment on mouse peritoneal mesothelial cells according to Example 1 of the present invention. [Figure 5] It is a photograph showing the results of an experiment on suppressing mesothelial-to-mesenchymal transition of peritoneal mesothelial cells by TGF-β according to Example 1 of the present invention. [Figure 6A] It is a conceptual diagram showing the results of an AAV introduction experiment into mouse peritoneum according to Example 1 of the present invention. [Figure 6B] It is a photograph showing the results of an AAV introduction experiment into mouse peritoneum according to Example 1 of the present invention. <00001​​​​​​​​​​​​​​​​​​​​​​​​​This graph shows the changes in the mouse peritoneum caused by AAV-DJ-miR-29b according to Example 1 of the present invention. [Figure 12A] This is a conceptual diagram of an experiment to suppress peritoneal dissemination in pancreatic cancer according to Example 2 of the present invention. [Figure 12B] This is a photograph showing the results of an experiment to suppress peritoneal dissemination in pancreatic cancer according to Example 2 of the present invention. [Figure 12C] This graph shows the results of an experiment to suppress peritoneal dissemination in pancreatic cancer according to Example 2 of the present invention. [Figure 12D] This is a photograph showing the changes in the mouse peritoneum during an experiment to suppress peritoneal dissemination in pancreatic cancer according to Example 2 of the present invention. [Figure 12E] This graph shows the changes in the mouse peritoneum during an experiment to suppress peritoneal dissemination in pancreatic cancer according to Example 2 of the present invention. [Figure 13A] This is a conceptual diagram of an experiment to suppress peritoneal dissemination by using an anticancer agent in combination with an anticancer agent according to Example 2 of the present invention. [Figure 13B] This photograph shows the results of an experiment to suppress peritoneal dissemination by using an anticancer agent in combination with an anticancer agent according to Example 2 of the present invention. [Figure 13C] This graph shows the results of an experiment to suppress peritoneal dissemination by using an anticancer agent in combination with an anticancer agent according to Example 2 of the present invention. [Modes for carrying out the invention]

[0013] <Embodiment> Cancer, especially gastric cancer, can metastasize in three ways: hematogenous, lymphatic, and disseminated. Gastric cancer that originates from the mucosal surface of the stomach lumen is said to invade deeper and, when exposed to the serosal side (=outside), scatter cancer cells into the abdominal cavity, forming peritoneal dissemination on the peritoneum. Peritoneal dissemination is a condition that often leads to the development of malignant ascites and intestinal obstruction, and because adequate treatment outcomes are sometimes not achieved, it has been a major clinical problem. Furthermore, in advanced cancer, adjuvant chemotherapy is administered after curative surgery, but peritoneal dissemination recurrence can still occur. However, another problem is that there is no treatment to prevent peritoneal dissemination in high-risk groups for peritoneal dissemination recurrence.

[0014] In response to this, the inventors comprehensively analyzed exosome miRNAs in ascites from gastric cancer patients with peritoneal dissemination and in peritoneal lavage fluid from patients with early-stage gastric cancer without peritoneal dissemination, and discovered that miR-29 levels were significantly reduced in gastric cancer patients with peritoneal dissemination. The inventors found that in patients with gastric cancer that had reached the serosa (T4) and had peritoneal dissemination, the expression of miR-29b was significantly reduced. They then confirmed that miR-29b is a "tumor-suppressor miRNA" that acts to suppress cancer. Therefore, the inventors conceived the idea that making miR-29b act on various cells in the peritoneal cavity could be a new treatment for peritoneal dissemination, and they diligently proceeded with experiments.

[0015] The inventors then investigated the expression of miR-29b in various cells using digital PCR and found that it was highly expressed in human peritoneal mesothelial cells (HPMCs). Since treatment of HPMCs with TGF-β reduced miR-29b expression, it was hypothesized that the decrease in miR-29b in the peritoneal cavity might be due to decreased expression in peritoneal mesothelial cells.

[0016] Therefore, the inventors conceived a therapeutic strategy of supplementing the peritoneal cavity with miR-29b. In conducting experiments with miR-29b, they focused on mesothelial-mesenchymal transition (MMT), a process in which peritoneal mesothelium transforms into mesenchymal cells, promoting cancer progression, and investigated its mechanism of action. miR-29b, which was previously expressed with reduced levels, was highly expressed in peritoneal mesothelial cells on the surface of the peritoneum, and its expression decreased upon stimulation by cancer cells. Furthermore, its expression was low in cancer cells and immune cells.

[0017] Therefore, as described in Non-Patent Document 1, miR-29b was enhanced by lipofection of peritoneal mesothelium stimulated with TGF-β with the mimic RNA itself. As a result, HPMC proliferation was suppressed, and the migratory ability and adhesion ability of cancer cells to peritoneal mesothelial cells were significantly reduced. Furthermore, miR-29b suppressed TGF-β-induced mesothelial-mesenchymal transition (MMT).

[0018] Therefore, the inventors conducted further experiments and performed a local administration experiment of miR-29b using an allogeneic tumor transplantation model. In this experiment, miR-29b was mixed with atelocollagen and injected into the peritoneal cavity of mice every three days, and the number of peritoneal dissemination nodules on the omentum and mesentery was counted and evaluated. Here, although peritoneal dissemination in mice is often evaluated by the number of peritoneal dissemination nodules on the mesentery, in this experiment, the number of peritoneal dissemination nodules on the mesentery did not change. On the other hand, the number of peritoneal dissemination nodules on the omentum of mice was significantly reduced. Unfortunately, however, miR-29b mimic did not produce sufficient efficacy to sustain mesothelial-mesenchymal transition (MMT) and to be used as a therapeutic agent for peritoneal dissemination.

[0019] Based on this, the inventors considered it necessary to efficiently deliver miR-29b into the peritoneal cavity and proceeded with further experiments. In the process, they conceived the idea of ​​delivering miR-29b to peritoneal mesothelial cells, which make up the peritoneal cavity, using an adeno-associated virus vector. The inventors then actually fabricated an adeno-associated virus vector carrying miR-29b and diligently repeated experiments. Specifically, they selected a serotype of adeno-associated virus that is directional to peritoneal mesothelial cells covering the surface of the peritoneal cavity, inserted the precursor sequence of miR-29b, which is degraded in the peritoneal cavity of patients with peritoneal dissemination, and fabricated an adeno-associated virus vector carrying miR-29b, "AAV-miR-29b," as described in Examples 1 and 2 below. This AAV-miR-29b was administered intraperitoneally to a mouse peritoneal dissemination model using the mouse gastric cancer cell line YTN16P. As a result, the number of peritoneal dissemination nodules on the mesentery was significantly reduced compared to the control group, showing an inhibitory effect on the progression of peritoneal dissemination. Furthermore, simultaneous administration of the cancer cell line and AAV-miR-29b also significantly reduced the number of peritoneal dissemination nodules, indicating a preventive effect.

[0020] As a result, we have demonstrated that it is possible to actually suppress the establishment and progression of peritoneal dissemination, thereby completing the present invention. While most conventional gene therapies for cancer have been developed to target cancer cells, the present invention can control peritoneal dissemination by modifying quantitative changes in miR-29b in the host's peritoneal environment. Specifically, by expressing miR-29b in cells constituting the host's peritoneal cavity using an adeno-associated virus vector, peritoneal dissemination can be treated and prevented. Thus, the present invention can provide useful therapeutic agents and methods targeting cancer peritoneal dissemination, a difficult-to-treat condition. The embodiments of the present invention will be described in more detail below.

[0021] [Peritoneal dissemination inhibitor] Specifically, the peritoneal dissemination inhibitor according to this embodiment is characterized by containing an adeno-associated virus vector carrying miR-29b.

[0022] Here, microRNA (also called "miRNA" or "miR") is a type of functional ncRNA (non-coding RNA) encoded on the genome. It is a single-stranded RNA molecule of about 20-25 nucleotides (nt) in length, produced through a multi-step process. MicroRNA is involved in post-transcriptional gene expression regulation in eukaryotes. In this embodiment, the precursor sequence of miR-29b, which acts as a cancer suppressor and whose expression is reduced in the peritoneal cavity of patients with peritoneal dissemination, is introduced into the vector as the microRNA. An example of the sequence of miR-29b (transcribed into microRNA) common to humans and mice used in this embodiment is shown as Sequence ID 1 in the sequence listing.

[0023] However, the miR-29b according to this embodiment does not necessarily have to be a DNA precursor sequence of miR-29b as shown in Sequence ID No. 1. It may be a sequence similar to the miRNA gene on the genome, a sequence similar to pre-miRNA or mature-miRNA, or a sequence that is activated by other intracellular processing to ultimately synthesize miR-29b. Furthermore, the miR-29b sequence may be modified by modifying it or by enhancing its inhibitory effect on mesothelial-mesenchymal transition (MMT). In addition, the miR-29b sequence may be a sequence from an animal species other than humans or mice, or a sequence modified to suit the animal species, and the copy number and enhancer sequence loaded into the AAV may also be arbitrary. Moreover, the miR-29b according to this embodiment may contain other genes, such as other tumor suppressor miRNAs, on its sequence. Such other miRNAs with tumor suppressive effects may be, for example, miR-34.

[0024] On the other hand, adeno-associated viruses (AAVs) are small, non-enveloped viruses (approximately 20 nm) belonging to the Dependvirus genus of the Parvoviridae family that infect humans and primates. They rely on helper viruses. In this embodiment, miR-29b is loaded onto the AAV. The AAV has the characteristics of low toxicity, low immunogenicity, and the ability to sustain expression for a period of time. Furthermore, since the AAV is a method that has actually been used in several clinical applications and is highly safe, loading it with miR-29b will further enhance its safety when actually applied in clinical settings. In this embodiment, as mentioned above, the AAV loaded with miR-29b is also referred to as "AAV-miR-29b".

[0025] Here, compared to the administration of the miR-29b mimic in Non-Patent Document 1 described above, which forms a complex with atelocollagen, the peritoneal dissemination inhibitor using the delivery method utilizing AAV-miR-29b according to this embodiment shows an extremely high peritoneal dissemination inhibitory effect in in vivo experiments using a mouse peritoneal dissemination model, as shown in Examples 1 and 2 described later, to an extent that could not be predicted by those skilled in the art, and exhibits a remarkable effect compared to the prior art.

[0026] Furthermore, the peritoneal dissemination inhibitor according to this embodiment is characterized in that the serotype of AAV includes one of the group consisting of AAV1, AAV2, AAV4, AAV5, AAV9, and AAV-DJ. In particular, as the serotype of AAV according to this embodiment, in humans (Homo sapiens), AAV2 is particularly preferred, as shown in the results of selecting adeno-associated virus serotypes that are directional to peritoneal mesothelial cells covering the surface of the peritoneal cavity in Examples 1 and 2 described later. That is, AAV2 has the highest directional properties towards peritoneal mesothelial cells. In addition, those skilled in the art have found that AAV2 also has damaging properties against cancer cells themselves. Therefore, a synergistic effect can be expected, and the use of this AAV2 type vector is particularly preferred. On the other hand, for mice (Mus musculus), AAV1, AAV2, AAV4, AAV5, AAV9, or AAV-DJ are preferred. Of these, AAV-DJ is particularly preferred for mice. Hereafter, when distinguishing between AAV-miR-29b proteins mounted on AAVs of each serotype, they will be referred to as AAV2-miR-29b, AAV-DJ-miR-29b, etc.

[0027] To explain in more detail, the peritoneal dissemination inhibitor according to this embodiment is characterized by causing miR-29b to be expressed in cells constituting the peritoneal cavity, thereby treating or preventing peritoneal dissemination. In other words, by delivering miR-29b to cells constituting the peritoneal cavity using AAV and causing its expression, it is possible to suppress or prevent the establishment and progression of peritoneal dissemination.

[0028] The cells constituting the peritoneal cavity targeted by the peritoneal dissemination inhibitor according to this embodiment may be, for example, various cells that constitute the membranous tissue covering the surface of body cavities such as the peritoneal cavity, thoracic cavity, and pericardial sac. In this embodiment, peritoneal mesothelial cells that constitute the mesothelium of the peritoneum are described as the main target cells, but other cells may also be used. That is, the cells constituting the peritoneal cavity according to this embodiment may be various cells that constitute membranous tissue that becomes fibrotic when other miR-29b levels decrease, making it easier for cancer cells to metastasize (hereinafter, these cells will be referred to as "peritoneal mesothelial cells, etc.").

[0029] Furthermore, the peritoneal dissemination inhibitor according to this embodiment is characterized by suppressing mesothelial-mesenchymal transition (MMT) of cells constituting the peritoneal cavity and thereby suppressing peritoneal fibrosis. That is, the peritoneal dissemination inhibitor of this embodiment suppresses the occurrence of mesothelial-mesenchymal transition (MMT) in peritoneal mesothelial cells, etc., due to stimulation by cancer cells or inflammatory substances, etc. This significantly suppresses the migratory ability of peritoneal mesothelial cells, etc., and suppresses peritoneal fibrosis of the entire peritoneum. Therefore, the peritoneal dissemination inhibitor according to this embodiment can reduce the adhesion ability of cancer cells to peritoneal mesothelial cells, etc., and treat or prevent peritoneal dissemination.

[0030] Furthermore, the cancers targeted by the peritoneal dissemination inhibitor according to the embodiments of the present invention include cancers that occur in the abdominal cavity, such as gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, and ovarian cancer. Furthermore, the peritoneal dissemination inhibitor according to the embodiment of the present invention can also be applied to other cancers that may cause peritoneal dissemination. For example, the therapeutic agent of this embodiment can also be applied to peritoneal dissemination caused by various cancers such as head and neck cancer, esophageal cancer, liver cancer, gallbladder cancer, lung cancer, kidney cancer, testicular cancer, breast cancer, prostate cancer, bladder cancer, skin cancer, thyroid cancer, malignant lymphoma, and other sarcomas. In other words, the peritoneal dissemination inhibitor according to this embodiment can also be used to suppress the occurrence of peritoneal dissemination due to invasion and metastasis of these cancers.

[0031] For the treatment or prevention of this condition, the peritoneal dissemination inhibitor according to this embodiment is preferably administered simultaneously with or after surgery for the tumor. As shown in Example 1 described later, the peritoneal dissemination inhibitor according to this embodiment can suppress the number of peritoneal dissemination nodules whether administered on the third day after cancer cell dissemination or simultaneously with cancer cell dissemination. In addition, as shown in Example 2 described later, even when peritoneal dissemination has already occurred, the number of peritoneal dissemination nodules can be suppressed by administering the inhibitor simultaneously with an anticancer drug into the peritoneal cavity.

[0032] Therefore, as a very early prevention measure for peritoneal dissemination, it can be administered in situations where "peritoneal dissemination was not observed at the time of surgery, but a very small number of cancer cells remain that are difficult to detect by normal examinations," such as T4 gastric cancer with CY0 (negative peritoneal lavage cytology). In other words, by administering the peritoneal dissemination inhibitor according to this embodiment in such a state, it is expected that the establishment and progression of peritoneal dissemination can be suppressed. Alternatively, the peritoneal dissemination inhibitor according to this embodiment can be administered during surgery. Specifically, the inventors have found that peritoneal dissemination is common in T4 gastric cancer patients with low miR-29b levels. Therefore, administering the peritoneal dissemination inhibitor according to this embodiment during surgery can prevent the occurrence and recurrence of postoperative peritoneal dissemination.

[0033] In this case, the peritoneal dissemination inhibitor according to this embodiment may be administered directly into the abdominal cavity during or after the above-mentioned surgery. With this configuration, as shown in Examples 1 and 2 described later, the peritoneal dissemination inhibitor of this embodiment can help suppress or prevent the establishment and progression of peritoneal dissemination due to mesothelial-mesenchymal transition. That is, as in Examples 1 and 2, where AAV was injected intraperitoneally, the peritoneal dissemination inhibitor of this embodiment can be directly administered into the peritoneal cavity using means common to those skilled in the art, such as injection, intravenous drip, or surgical spray.

[0034] Thus, the peritoneal dissemination inhibitor according to this embodiment can be used as a pharmaceutical agent, such as a therapeutic agent (therapeutic composition) or a preventive agent (preventive composition) for peritoneal dissemination. In other words, the peritoneal dissemination inhibitor according to this embodiment can be used as an agent to suppress peritoneal dissemination and to prevent recurrence of surgically treatable cancer due to peritoneal dissemination. In fact, as shown in Examples 1 and 2 described later, it is possible to inhibit mesothelial-mesenchymal transition by using miR-29b, thereby suppressing the establishment and progression of peritoneal dissemination. This makes it possible to use miR-29b as a therapeutic agent for peritoneal dissemination.

[0035] In addition, as described above, in this embodiment, by expressing miR-29b in cells constituting the peritoneal cavity using an AAV equipped with miR-29b, it is possible to use it as a method for suppressing and preventing peritoneal dissemination. By configuring the peritoneal dissemination inhibitor of this embodiment and using it as a therapeutic agent or treatment method, it becomes possible to suppress or prevent the establishment and progression of peritoneal dissemination due to mesothelial-mesenchymal transition, and also suppress the proliferation of peritoneal disseminations with a poor prognosis. In other words, the therapeutic agent of this embodiment can be provided as a means of treating peritoneal dissemination caused by cancer metastasis occurring in the abdominal cavity by preventing peritoneal fibrosis.

[0036] Furthermore, the peritoneal dissemination inhibitor according to this embodiment can be combined with drugs related to standard clinical cancer treatment methods. In other words, the peritoneal dissemination inhibitor according to this embodiment may be used in combination with an anticancer agent. Specifically, it can be used in combination with anticancer agents used for chemotherapy by those skilled in the art. That is, by using the peritoneal dissemination inhibitor according to this embodiment in combination with anticancer agents used to treat peritoneal dissemination, it becomes possible to suppress or prevent the establishment and progression of peritoneal dissemination more effectively than with treatment using anticancer agents alone.

[0037] Furthermore, the peritoneal dissemination inhibitor according to this embodiment may include not only miR-29b itself, but also miR-29 synthesis promoters, degradation inhibitors, etc. That is, the peritoneal dissemination inhibitor according to this embodiment targets the biosynthesis system of miR-29b as the therapeutic target for peritoneal dissemination, including cancer occurring in the abdominal cavity, and includes synthesis promoters, which are substances that promote the synthesis of miR-29b, and degradation inhibitors, which are substances that inhibit the degradation of miR-29b.

[0038] Specifically, these miR-29b synthesis promoters and degradation inhibitors may also be miR-29b gene expression regulators. More specifically, various gene regulators targeting the synthesis and degradation pathways of miR-29b can be used as gene expression regulators. This gene modifier may be, for example, a peptide containing an antibody against nucleic acids (nucleotides) such as DNA or RNA that regulate transcription and modification in the biosynthetic pathway from the miR-29b gene, or proteins such as various enzymes (hereinafter referred to as "target proteins"), PNA, nucleic acids or proteins involved in genome editing, etc. Of these, the nucleic acid may be antisense DNA or RNA, siRNA, shRNA, ribozyme, etc. Furthermore, the gene expression regulator of this embodiment can be identified from the amino acid sequence, mRNA sequence, genomic DNA sequence, etc., of the target protein and manufactured by a method easily accessible to those skilled in the art.

[0039] Thus, various compositions affecting the biosynthetic pathway related to the synthesis and degradation of miR-29b, including miR-29b synthesis promoters and degradation inhibitors, can also be provided as therapeutic agents for peritoneal dissemination. In other words, these can be provided as peritoneal dissemination inhibitors that suppress the establishment and progression of peritoneal dissemination. In addition, as a peritoneal dissemination inhibitor in this embodiment, it is also possible to use synthesis promoters or degradation inhibitors that target the same pathway as gene expression regulation by miR-29b.

[0040] Furthermore, the peritoneal dissemination inhibitor of this embodiment can utilize various medical compositions such as low-molecular-weight compounds targeting enzymes related to mesothelial-mesenchymal transition (MMT), antibody drugs, nucleic acids, and PNAs. For example, low-molecular-weight compounds may be synthesized and used in combination as peritoneal dissemination inhibitors. Furthermore, the peritoneal dissemination inhibitor of this embodiment may also include medical compositions that promote cancer apoptosis, stimulate the cancer immune system, or enable gene modification techniques such as genome editing.

[0041] Furthermore, if the peritoneal dissemination inhibitor according to the embodiment of the present invention is a low-molecular-weight compound, it may be provided as various salts. These salts may be produced by reacting the low-molecular-weight compound with an acid or base that can be used in the manufacture of pharmaceuticals.

[0042] Furthermore, in the case of peritoneal dissemination inhibitors according to this embodiment, for inoperable peritoneal dissemination, i.e., highly advanced cases, strengthening the peritoneal mesothelium with miR-29b alone may not be sufficient to suppress progression. Therefore, an AAV vector expressing anti-miRNA or miRNA inhibitor may be administered simultaneously.

[0043] Furthermore, as a peritoneal dissemination inhibitor according to this embodiment, it is also possible to use other modifiers, such as those that suppress mesothelial-mesenchymal transition in the cells constituting the peritoneal cavity. Furthermore, the therapeutic agent of this embodiment may be used in combination with various tumor adhesion inhibitors, tumor angiogenesis inhibitors, etc., to suppress the adhesion of cancer cells and the formation of new angiogenesis within peritoneal dissemination, thereby enhancing its effectiveness.

[0044] Furthermore, it is possible to use various medical vectors other than AAV in addition to AAV. Examples of such viral vectors include adenovirus vectors, lentiviral vectors, and retrovirus vectors. By using a vector that can hold longer DNA than AAV, it becomes possible to achieve genome editing specific to the target tumor, such as the type of cancer from which peritoneal dissemination originated.

[0045] Furthermore, the peritoneal dissemination inhibitor according to the embodiment of the present invention also includes prodrugs. Here, a prodrug refers to a derivative that, after administration, is decomposed and converted under physiological conditions, such as specific pH conditions or the action of enzymes. In addition, although the prodrug may be inactive when administered to the patient, it is converted into an active therapeutic agent in the body.

[0046] Furthermore, the peritoneal dissemination inhibitor according to the embodiment of the present invention may contain, for example, physiological saline, atelocollagen, or other collagen as any formulation-acceptable carrier. In addition, it can be administered together with glucose, D-sorbitol, D-mannose, D-mannitol, ethanol, polyalcohols, such as propylene glycol, polyethylene glycol, or nonionic surfactants as a carrier.

[0047] Furthermore, the peritoneal dissemination inhibitor according to the embodiment of the present invention may contain a suitable pharmaceutically acceptable carrier in order to prepare a formulation-acceptable carrier. This carrier may include biocompatible materials such as silicone, collagen, and gelatin. Furthermore, the formulation may contain, for example, diluents, fragrances, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers, and plasticizers. The therapeutic agent of this embodiment may also contain appropriate excipients. Furthermore, the peritoneal dissemination inhibitor according to the embodiment of the present invention may be formulated using a pharmaceutically acceptable carrier known in the art in a dosage form suitable for administration.

[0048] [Peritoneal dissemination treatment method] Furthermore, the therapeutic method according to an embodiment of the present invention is a therapeutic method for peritoneal dissemination, characterized by administering the above-mentioned peritoneal dissemination inhibitor. Specifically, the peritoneal dissemination treatment method according to this embodiment is a method for treating peritoneal dissemination in animals, characterized by expressing miR-29b in cells constituting the peritoneal cavity using an adeno-associated virus vector carrying miR-29b. In other words, it can be used as a treatment method for peritoneal dissemination in animals that express miR-29 in cells constituting the peritoneum and are capable of developing peritoneal dissemination. Furthermore, the therapeutic agents of the embodiments of the present invention can be used to treat living organisms or parts of living organisms. These living organisms are not particularly limited, but include, for example, humans, domesticated animals, wild animals, and other animals.

[0049] In this case, the therapeutic agent according to the embodiment of the present invention can be used primarily for treating humans and various other vertebrates. These non-human animals are not particularly limited and include various mammals encompassing placental mammals of the infraclass Eutheria. Specifically, they may include, for example, rodents such as mice, rats, ferrets, hamsters, guinea pigs, or rabbits, dogs, cats, sheep, pigs, cattle, horses, or non-human transgenic primates. In other words, the peritoneal dissemination treatment agent according to the embodiment of the present invention can be used not only for the treatment of humans but also as a method for suppressing or preventing peritoneal dissemination in various animals. Furthermore, in countries where human treatment is industrially available (and therefore patentable), the therapeutic method according to this embodiment of the present invention may also be applicable to humans.

[0050] The administration route of the pharmaceutical composition according to the embodiment of the present invention is not particularly limited, but it can be administered parenterally or orally. Parenteral administration methods include, for example, intraperitoneal injection with the aforementioned carrier, dispersal onto the peritoneum during surgery, as well as intravenous, intra-arterial, subcutaneous, intradermal, and intramuscular administration.

[0051] Furthermore, in order to use the therapeutic agent according to the embodiment of the present invention for the above-mentioned treatment, the administration interval and dosage can be appropriately selected and changed according to various conditions such as the state of the tumor and the condition of the subject. In this case, when treating the condition, a dosage that slows tumor growth can be used. Furthermore, when used in combination with other therapeutic agents, it can be effectively utilized by suppressing nausea and other serious side effects.

[0052] The therapeutic agent according to the embodiment of the present invention allows for the appropriate selection and modification of the dosage and number of administrations for cancer patients who may develop peritoneal dissemination, depending on the purpose of administration and various conditions such as the patient's age and weight, symptoms, and the severity of the disease. The number and duration of these administrations will be determined by monitoring the patient's condition, and if necessary, further or repeated administrations may be required.

[0053] The therapeutic agent according to the embodiment of the present invention is characterized by being used in combination with an anticancer agent. That is, the therapeutic agent according to the embodiment of the present invention can be used in combination with other therapeutic compositions such as conventional chemotherapeutic agents and anticancer agents. This anticancer agent may include, for example, paclitaxel, as shown in Example 2 described later, which is used by those skilled in the art for the treatment of peritoneal dissemination, or other types may be used, or a combination (cocktail) of multiple therapeutic compositions may be used. This is expected to provide therapeutic and preventive effects for peritoneal dissemination. Furthermore, the therapeutic agent according to this embodiment can be used even when treatment has already begun. Furthermore, it can be used in combination with other cancer treatments and methods such as radiation therapy, particle beam therapy, and surgery. This could make it an effective treatment for peritoneal dissemination, which has a poor prognosis.

[0054] In addition to the therapeutic agent of this embodiment, therapeutic compositions that alleviate pain or improve quality of life may be added to the treatment. The composition of the present invention may be administered simultaneously with other compositions, or at intervals, but the order of administration is not particularly important. Furthermore, in the embodiments of the present invention, the period during which the disease is improved or alleviated is not particularly limited; it may be a temporary improvement or alleviation, or it may be an improvement or alleviation for a certain period of time.

[0055] [Effects of the invention] Traditionally, chemotherapy has been the only treatment for peritoneal carcinomatosis, and no effective drugs or treatment methods targeting peritoneal carcinomatosis have been developed. In contrast, when AAV-miR-29b was administered intraperitoneally to a mouse peritoneal dissemination model using the mouse gastric cancer cell line YTN16P, as shown in Example 1 below, the number of peritoneal dissemination nodules on the mesentery was significantly reduced compared to the control group, demonstrating an inhibitory effect on the progression of peritoneal dissemination. Furthermore, as shown in Example 2 below, even in other cancers such as highly malignant pancreatic cancer, the number of peritoneal dissemination nodules on the mesentery was significantly reduced compared to the control group, demonstrating an inhibitory effect on the progression of peritoneal dissemination. In other words, even in pancreatic cancer, where the prognosis for peritoneal dissemination is as poor as or worse than that of gastric cancer, the progression of peritoneal dissemination can be suppressed. In summary, the peritoneal dissemination inhibitor according to this embodiment can efficiently suppress tumor formation by acting on peritoneal mesothelial cells rather than cancer cells.

[0056] Furthermore, the number of peritoneal dissemination nodules was significantly lower when cancer cell lines and AAV-miR-29b were administered simultaneously, suggesting a preventive effect. These results suggest that a single intraperitoneal administration of AAV-miR29b at the end of surgery for T4CYO gastric cancer patients with deep wall invasion (T4) and negative lavage cytology (CYO) may prevent future peritoneal dissemination recurrence. In other words, it becomes possible to perform treatment aimed at preventing peritoneal dissemination, a difficult-to-treat condition.

[0057] Specifically, the therapeutic and preventive effects include preventing metastatic lesions of cancer that have disseminated to the peritoneum from growing to cover the peritoneum or as nodules, thereby preventing obstruction of the digestive tract and other organs and tissues. Furthermore, it is expected to improve cachexia and prevent the occurrence of new peritoneal dissemination. In addition, it is expected that the cancer itself may go into remission by eliminating, removing, or surgically removing each metastatic lesion with chemotherapy, anticancer drugs, or surgery. In fact, as shown in Example 2 below, it is possible to further suppress the progression of peritoneal dissemination by using it in combination with chemotherapy (anticancer drugs).

[0058] [Other embodiments] Furthermore, as a viral vector according to another embodiment of the present invention, a method for suppressing peritoneal dissemination according to another embodiment of the present invention may be characterized by measuring the expression level of miR-29b in the collected intraperitoneal fluid and predicting the prognosis. By configuring the system in this way and measuring the expression level of miR-29b in peritoneal cell samples taken during surgery or examination, it becomes possible to determine the likelihood of peritoneal dissemination (prediction of prognosis) and the effectiveness of treatment. In other words, the measurement of miR-29b and other factors in the peritoneum can be used as predictive markers for prognosis and treatment effectiveness.

[0059] Specifically, by measuring the expression levels of miR-29b and other molecules in peritoneal cells obtained during cancer surgery using various methods such as RT-PCR, digital PCR, and Northern blotting, it is possible to determine the likelihood of peritoneal dissemination and identify the prognosis. This provides a diagnostic method that allows for the prediction of the likelihood of metastasis due to peritoneal dissemination of tumors and the prognosis, and enables the selection of treatment methods based on the measurement of miR-29b expression levels. This expression level may be an absolute expression level, a relative expression level, or a concentration based on a specific range of expression levels. By configuring the system in this way and combining it with prognosis prediction methods, it becomes possible to investigate the likelihood of peritoneal dissemination and then treat it effectively. Furthermore, after drug administration, the expression level of miR-29b and other factors in the peritoneum can be used as an indicator to determine the effectiveness of the treatment.

[0060] In this embodiment, "one or more" means either one or a combination of several of the components. That is, any one of them may be used, or any combination of two to all of them may be included. [Examples]

[0061] Next, the present invention will be further described with reference to the drawings, but the following specific examples are not intended to limit the present invention.

[0062] [Experimental Method] (Construction of plasmid DNA) pAAV-CMV-[mir-29b-1] was created by cleaving pAAV-CMV-MCS (manufactured by Takara Bio Inc.) with restriction enzymes BamHI and XbaI, and introducing a sequence containing the precursor sequence of miR-29b (hereinafter referred to as the "mir-29b-1 sequence," which is shown as Sequence ID No. 1) obtained by cleaving pLV-[hsa-mir-29b-1] (manufactured by Biosettia), which expresses miR-29b, with BamHI and XbaI. Figure 1A shows the structure of pAAV2-CMV-MCS, and Figure 1B shows the structure of the fabricated pAAV-CMV-[mir-29b-1] vector.

[0063] (AAV vector recovery) HEK293 (Human Embryonic Kidney Cells 293) cells were cultured and transfected with pAAV-CMV-[mir-29b-1] and AAV plasmids and helper plasmids (manufactured by Takara Bio Inc.) encoding AAV serotypes 1-10 and AAV-DJ using the calcium phosphate method. After 72 hours, HEK293 cells were detached and collected, the cells were destroyed to release the AAV vector, and treated with endonucleases. The supernatant was collected after centrifugation. The recovered AAV vector solution was mixed with cesium chloride, and density gradient ultracentrifugation was performed to collect the fraction containing the AAV vector. After dialysis with vector buffer, PCR quantification was performed to determine the vector concentration in the sample.

[0064] (Isolation of peritoneal mesothelial cells) Human peritoneal mesothelial cells were isolated and cultured after obtaining approval from the Jichi Medical University Ethics Review Committee, and after explaining the process to the patient and obtaining their informed consent. The cells were collected from 2-4 cm of patients undergoing non-cancer surgery (obesity surgery). 3Omental tissue was obtained and immediately immersed in TrypLE Express (Thermo Fisher Scientific) diluted to half its concentration with phosphate-buffered saline (PBS), and cultured at 37°C for 2 hours. The supernatant was filtered through a 100 μm nylon mesh filter and centrifuged at 1500 rpm at 4°C for 5 minutes. The harvested cells were resuspended in DMEM medium (Cosmo Bio Inc.) containing 20% ​​FBS (Fetal Bovine Serum, Sigma), 100 U / ml penicillin, and 100 mg / ml streptomycin, and cultured in an incubator at 37°C and 5% CO2. Figure 2 shows cultured human peritoneal mesothelial cells.

[0065] For mice, the omentum was collected after sacrificial death of C57BL / 6N mice (manufactured by Crea Japan Co., Ltd.), and peritoneal mesothelial cells were isolated and cultured using the same method as for humans.

[0066] 〔result〕 (Serotype selection experiments for peritoneal mesothelial cells and gastric cancer cell lines) First, human or mouse peritoneal mesothelial cells were infected with a control vector expressing GFP, and the directivity of AAV serotypes to peritoneal mesothelial cells was investigated. Therefore, control AAV vectors were prepared by incorporating the GFP gene as a reporter into AAV serotypes AAV1-10 and AAV-DJ. These will be referred to as AAV1-10 and AAV-DJ vectors, respectively. In a 24-well plate, human and mouse peritoneal mesothelial cells and mouse gastric cancer cell line YTN16P (provided by Dr. S. Nomura of the University of Tokyo) were placed in 1 × 10⁶ wells. 4 Each seed was sown individually. After culturing overnight, the next day each AAV vector was added to 1 x 10⁶ wells. 5 Each vector genome (vg) was added, and human peritoneal mesothelial cells were observed with a fluorescence microscope after 3 days, and mouse peritoneal mesothelial cells after 7 days. The percentage of GFP-expressing cells was then measured by flow cytometry.

[0067] Figures 3A to 3C show the results of serotype selection experiments in which human peritoneal mesothelial cells were infected with a control vector expressing GFP. Figure 3A is a conceptual diagram of this experiment. Figure 3B is a photograph showing GFP expression for each vector. Figure 3C is a graph showing the median Fluorescence Intensity (MFI) of GFP expression as measured by flow cytometry. As a result, the AAV2 vector was found to be the most directive. In other words, in humans, serotype 2 showed the highest transduction efficiency. In addition, AAV1, AAV5, and AAV-DJ were also directive and usable in humans.

[0068] Figures 4A to 4C show the results of a similar serotype selection experiment performed on mouse peritoneal mesothelial cells. Figure 4A is a conceptual diagram of the experiment, Figure 4B is a photograph of GFP expression for each vector, and Figure 4C is a graph of the MFI of GFP expression. As a result, in mice, the AAV-DJ vector showed the highest directivity to the peritoneal mesothelium. In other words, the DJ serotype was the most efficient in introducing the virus into mice. In addition, AAV1, AAV2, AAV4, AAV5, and AAV9 also showed directivity and were usable in mice.

[0069] Furthermore, the inventors conducted similar experiments with the mouse gastric cancer cell line YTN16P and confirmed that GFP expression was weak in all serotypes. In other words, it is thought that AAV is not easily introduced into mouse gastric cancer cell lines, regardless of serotype.

[0070] (Experiment on suppressing mesothelial-mesenchymal transition in peritoneal mesothelial cells using TGF-β) Next, we investigated whether AAV2-miR-29b suppresses the transformation of peritoneal mesothelial cells into mesothelial-mesenchymal cells by transforming growth factor-β (TGF-β).

[0071] In this experiment, human peritoneal mesothelial cells were placed in a 24-well plate at a rate of 1 × 10⁶4 Each seed was sown individually, and TGF-β (manufactured by Protein Tech Japan Co., Ltd.) was added at a concentration of 10 ng / ml. The following day, the AAV2-miR-29b vector was added to each well at a rate of 1 × 10⁶. 5 Each vector genome (vg) was added, and after 3 days, immunofluorescence staining was performed to observe the expression of epithelial and mesenchymal cell markers using a fluorescence microscope.

[0072] Figure 5 shows the results. "No Treat" is the control with no additives, "TGF-β" is the control with only TGF-β added, "TGF-β1+AAV2-miR-29b" is the result with the AAV2-miR-29b vector from this example added, and "TGF-β1+AAV2-NC" is the result with the pAAV2-negative control vector. The pAAV2-negative control vector is an AAV vector prepared from a plasmid DNA vector that does not contain the miR-29b precursor sequence. In each column, "Bright Field" shows a micrograph, and "E-cadherin," "Calretinin," "Vimentin," "Fibronectin," and "DAPI" show epithelial and mesenchymal cell markers, respectively. Fibronectin was added for confirmation, as Non-Patent Literature 1 showed that one of the mechanisms by which miR-29b reduces cancer cell adhesion involves a mechanism mediated by fibronectin and integrins.

[0073] As a result, it was found that miR-29b suppresses the upregulation of the mesenchymal marker Vimentin caused by TGF-β. In other words, even when TGF-β was added to peritoneal mesothelial cells, the AAV2-miR-29b vector was able to suppress mesothelial-mesenchymal transition.

[0074] (AAV introduction experiment into mouse peritoneum) As mentioned above, in vitro experiments using human peritoneal mesothelial cells showed results that suppressed mesothelial-mesenchymal transition, so we decided to conduct in vivo experiments using mice. First, we introduced the AAV-DJ-miR-29b vector, which showed the highest transduction efficiency in mice, and confirmed that it actually expressed miR-29b in the peritoneum.

[0075] In this experiment, 5 × 10¹⁶ C57BL / 6N mice were placed in the peritoneal cavity of 8-week-old mice. 10 Mutant mice were injected with vg AAV-DJ-miR-29b and the control vector AAV-DJ-GFP, and sacrificially killed on day 28. In the group administered AAV-DJ-GFP, GFP expression in peritoneal mesothelial cells was observed using a fluorescence microscope. In the group administered AAV-DJ-miR-29b, miR-29b expression in peritoneal mesothelial cells was examined using a digital PCR method (Thermo Fisher Scientific kit) similar to that described in Non-Patent Document 1, in cells obtained from the mouse omentum (the source of peritoneal mesothelial cell culture).

[0076] Figure 6A shows a conceptual diagram of this experiment. Figure 6B shows the GFP expression level on the peritoneum of AAV-DJ-GFP. Even after 28 days, the no-treat group showed no GFP expression, while the group that received the control vector on the peritoneum (AAV-DJ-GFP group) showed GFP expression. In other words, it was confirmed that the DJ serotype of AAV is indeed introduced into the peritoneum of mice. Figure 6C shows the miR-29b expression level (copy number / μL) in mouse peritoneal mesothelial cells (MPMCs) obtained from the peritoneum. Compared to the control group (no treatment) and the negative control group (AAV-DJ-NC) administered only with the AAV-DJ vector, the group administered with AAV-DJ-miR-29b (AAV-DJ-miR-29b) showed significantly higher miR-29b expression (p<0.01) when tested using 1-way ANOVA.

[0077] (Experiment on suppressing peritoneal dissemination using AAV-DJ-miR-29b) Next, we examined whether AAV-DJ-miR-29b actually suppresses peritoneal seeding in vivo by changing the administration time between administration on the third day and simultaneous administration. As shown in Fig. 7, first, the outline of this peritoneal seeding suppression experiment (administered on the third day after cancer cell seeding) will be described. Here, we envisioned prevention at a very early stage of peritoneal seeding formation. Cancer cells were intraperitoneally administered, and three days later, AAV-DJ-miR-29b was diluted with phosphate-buffered saline (PBS) and intraperitoneally injected. The number of peritoneal seeding nodules on the greater omentum and mesentery was counted and evaluated. Specifically, 1×10 5 cells of the mouse gastric cancer cell line YTN16P2 were administered intraperitoneally to 8-week-old C57BL / 6N mice. Three days later, 5×10 5 vg of AAV-DJ-miR-29b and AAV-DJ-GFP were intraperitoneally injected, and the mice were sacrificed three weeks later. For these, the number of peritoneal seeding nodules on the mesentery and greater omentum was measured for each of (1) the control group without AAV vector administration (No treat), (2) the group administered with AAV-DJ-miR-29b (AAV-DJ-miR-29b), and (3) the negative control group administered with only the AAV-DJ vector (AAV-DJ-NC), with n = 5 for each group.

[0078] Fig. 8A shows photographs of the mesentery of each group in the peritoneal seeding suppression experiment (administered on the third day after cancer cell seeding). Fig. 8B shows a comparison of the number of nodules in each group. In the group administered with AAV-DJ-miR-29b, the number of peritoneal seeding nodules on the mesentery was significantly (p < 0.05) less. That is, when AAV-miR-29b was administered intraperitoneally in the mouse peritoneal seeding model, the peritoneal seeding nodules on the mesentery were significantly reduced compared to the control group, and an inhibitory effect on the progression of peritoneal seeding was observed.

[0079] As shown in Fig. 9, the outline of the peritoneal seeding suppression experiment (administered simultaneously with cancer cell seeding) will be described. Here, we envisioned preventive administration during surgery. AAV-DJ-miR-29b was administered simultaneously with intraperitoneal administration of cancer cells, and the number of peritoneal seeding nodules was counted and evaluated. Specifically, similar to the experimental example in Fig. 8 above, 1×10 5Each mouse was administered 5 × 10⁶ mice containing the YTN16P2 gastric cancer cell line. 5 Vg AAV-DJ-miR-29b and AAV-DJ-GFP were injected intraperitoneally, and after 3 weeks, the subjects were sacrificially killed, and the number of peritoneal dissemination nodules on the mesentery and omentum was measured.

[0080] Figure 10A shows photographs of the mesentery in each group of the peritoneal dissemination suppression experiment (administered simultaneously with cancer cell dissemination). Figure 10B shows a comparison of the number of nodules in each group, and the group administered AAV-DJ-miR-29b had significantly fewer peritoneal dissemination nodules on the mesentery (p<0.05). In other words, the number of peritoneal dissemination nodules was significantly lower even with the simultaneous administration of cancer cell lines and AAV-miR-29b, suggesting a preventive effect.

[0081] (Changes in mouse peritoneum caused by AAV-DJ-miR-29b) Next, we confirmed the inhibitory effect of AAV-DJ-miR-29b on peritoneal fibrosis in mice. Specifically, frozen sections of mouse peritoneal tissue collected during the peritoneal dissemination suppression experiment described above were observed using a fluorescence microscope. In addition, Masson's trichrome staining, which detects collagen connective tissue fibers in the tissue sections, was performed using the Trichrome Stain Kit (ScyTek Laboratories), and the thickness of the peritoneum was measured.

[0082] Figure 11A is a photograph showing the changes that occur in the peritoneum of mice in this experiment. Figure 11B shows the results of comparing peritoneal thickness in each group. No peritoneal thickening was observed in the group administered AAV-DJ-miR-29b. In other words, peritoneal fibrosis was significantly suppressed (p<0.05) in the group administered AAV-miR-29b. [Examples]

[0083] (Effects on pancreatic cancer) In Example 1 described above, the effect was demonstrated on a mouse gastric peritoneal tumor model, so we confirmed the effect on different types of cancer. In this example, we used a pancreatic cancer peritoneal dissemination model using mouse pancreatic cancer cells PAN02 to confirm the effect of suppressing peritoneal dissemination in pancreatic cancer.

[0084] Figure 12A shows a conceptual diagram of this experiment. In this experiment, mouse pancreatic cancer cells PAN02 were introduced into the peritoneal cavity of C57BL / 6N mice in 5 × 10⁶ units. 5 Administer as one dose, and simultaneously administer AAV-DJ-miR-29b 5 × 10⁶ times. 10 VG (vector genome) was administered. After 14 days, the cells were euthanized, and the status of peritoneal dissemination was evaluated by counting the number of peritoneal dissemination nodules on the mesentery. Since the status of peritoneal dissemination differed between the pancreatic cancer cell line and the gastric cancer cell line used in the experiment, the evaluation was performed at 14 days in the pancreatic cancer model. The parietal peritoneum was fixed with 4% formalin, embedded in paraffin, and the thickness of the abdominal wall was measured by Masson's trichrome staining on slides sectioned to a thickness of 4 μm.

[0085] Figure 12B shows photographs of the mesentery of each group of patients with pancreatic cancer. "No treatment" indicates the group that received only cancer cells. AAV indicates the group that did not receive any treatment. "AAV-DJ-NC" indicates the negative control group that received only the AAV-DJ vector. "AAV-DJ-miR-29b" indicates the group that received AAV-DJ-miR-29b as described in this example. Figure 12C shows the results of measuring the number of mesenteric dissemination nodules when pancreatic cancer cells and AAV-miR-29b were administered simultaneously and the formation of peritoneal dissemination was evaluated on day 14. In the AAV-miR29b-treated group, the number of peritoneal disseminations on the mesentery was significantly suppressed. "***" in the figure indicates p<0.01.

[0086] Figure 12D is a photograph of frozen sections of peritoneal tissue observed under a fluorescence microscope, showing peritoneal changes, similar to the gastric cancer model in Example 1. Figure 12E shows the results of comparing peritoneal thickness in each group, measured by Masson's trichrome staining. When examining wall thickening associated with peritoneal fibrosis, the AAV-miR-29b-administered group showed a significant decrease in peritoneal thickness. The "**" in the figure indicates p<0.05.

[0087] Thus, the AAV-miR-29b according to this embodiment is thought to be effective in preventing postoperative peritoneal dissemination recurrence even in pancreatic cancer, which has particularly high tissue penetration and is prone to generating peritoneal dissemination. For this reason, similar effects can be expected from AAV-miR-29b in other cancer types that cause peritoneal dissemination.

[0088] (Therapeutic effects when used in combination with anticancer drugs) As mentioned above, the treatment demonstrated a preventive effect against peritoneal dissemination in gastric and pancreatic cancer models. Therefore, we investigated the therapeutic effect in the advanced stage of peritoneal dissemination, where peritoneal dissemination is already present at the time of diagnosis. Specifically, the efficacy of AAV-miR29b in this study was investigated by combining it with intraperitoneal chemotherapy, which is being conducted as a clinical trial at a limited number of facilities. More specifically, it was administered in combination with paclitaxel, an anticancer drug used in intraperitoneal chemotherapy for peritoneal dissemination of gastric and pancreatic cancer.

[0089] Figure 13A shows a conceptual diagram of this experiment. In this experiment, mouse gastric cancer cells YTN16P were introduced into the peritoneal cavity of C57BL / 6N mice in 5 × 10⁶ units. 5 Individual doses were administered. Then, on the 7th day, 5 x 10 10 Vg AAV-DJ-miR-29b was administered, and simultaneously, 200 μg of paclitaxel (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 167-28166) was administered at the usual dose (1st dose). On day 14, 200 μg of paclitaxel was administered (2nd dose). Then, 21 days later, the animals were sacrificially killed, and the formation of peritoneal dissemination was evaluated by counting the number of peritoneal dissemination nodules on the mesentery. In other words, AAV-miR-29b was administered only once, while paclitaxel was administered multiple times.

[0090] Figure 13B shows photographs of the mesentery of each group. "No treatment" indicates that only cancer cells were administered, and no AAV was administered. "AAV-DJ-NC" indicates the negative control, in which only the AAV-DJ vector was administered. "AAV-DJ-miR-29b" indicates that the AAV-DJ-miR-29b used in this example was administered. "Mock" indicates that the solvent for paclitaxel was administered. Each group is shown as a combination of administration with paclitaxel "PTX (200 μg)". Figure 13C shows the results of measuring the number of mesenteric dissemination nodules after 21 days. The group treated with AAV-miR-29b showed significantly fewer peritoneal dissemination nodules. The asterisk (*) in the figure indicates p<0.05.

[0091] These results suggest that AAV-miR-29b in this embodiment can be expected to have an effect not only in preventing peritoneal dissemination, but also in treating already formed peritoneal dissemination, and can be used in combination with anticancer drugs to suppress or prevent the establishment and progression of peritoneal dissemination.

[0092] It goes without saying that the configuration and operation of the above embodiment are examples and can be modified as appropriate without departing from the spirit of the present invention. [Industrial applicability]

[0093] The present invention provides a peritoneal dissemination inhibitor that can be used as a peritoneal dissemination inhibitor or preventive agent, or therapeutic agent, for patients with peritoneal dissemination who have a poor prognosis and are expected to be cured with a single dose, and can be used industrially.

Claims

1. Contains an adeno-associated virus vector equipped with miR-29b, The adeno-associated virus vector described above is selected from a serotype that is directional to peritoneal mesothelial cells covering the surface of the host's peritoneal cavity. By suppressing mesothelial-mesenchymal transition in the peritoneal mesothelial cells, rather than cancer cells, and inhibiting peritoneal fibrosis, tumor formation is suppressed. The adeno-associated virus vector is administered once, followed by multiple doses of paclitaxel. The serotype of the adeno-associated virus vector is: In humans (Homo sapiens), it is AAV2 or AAV-DJ. For the mouse (Mus musculus), the options are AAV1, AAV2, AAV4, or AAV-DJ. A peritoneal dissemination inhibitor characterized by the following features.

2. The adeno-associated virus vector is selected from a serotype that is directional to peritoneal mesothelial cells obtained by isolation and culture from peritoneal tissue. The peritoneal dissemination inhibitor according to feature 1.

3. Expressing miR-29b in cells constituting the peritoneal cavity to treat or prevent peritoneal dissemination. The peritoneal dissemination inhibitor according to feature 1.

4. The adeno-associated virus vector is It is administered simultaneously with or after surgery for the tumor. The peritoneal dissemination inhibitor according to feature 1.

5. The adeno-associated virus vector is It is administered directly into the peritoneal cavity. The peritoneal dissemination inhibitor according to feature 1.

6. A method for treating peritoneal dissemination in animals other than humans, Using an adeno-associated virus vector equipped with miR-29b, By expressing miR-29b in the cells that make up the peritoneal cavity, The adeno-associated virus vector described above is selected from a serotype that is directional to peritoneal mesothelial cells covering the surface of the host's peritoneal cavity. By suppressing mesothelial-mesenchymal transition in the peritoneal mesothelial cells, rather than cancer cells, and inhibiting peritoneal fibrosis, tumor formation is suppressed. The adeno-associated virus vector is administered once, followed by multiple doses of paclitaxel. The serotype of the adeno-associated virus vector is: In humans (Homo sapiens), it is AAV2 or AAV-DJ. For the mouse (Mus musculus), the options are AAV1, AAV2, AAV4, or AAV-DJ. A method for treating peritoneal dissemination characterized by the following features.

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