Esophageal stricture inhibitor

Abstract

To provide a safe and novel stricture inhibitor for benign esophageal stricture.SOLUTION: The present invention relates to an esophageal stricture inhibitor containing siRNA that inhibits CHST15 gene expression as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel esophageal stricture inhibitor containing CHST15 siRNA as an active ingredient.

Background Art

[0002] Minimally invasive endoscopic treatment for esophageal cancer and Barrett's esophagus that enables preservation of esophageal function has become established in recent years as a radical treatment for superficial esophageal cancer and Barrett's esophagus (Literatures 1-5). In particular, the progress of the technique of endoscopic submucosal dissection (ESD) has been remarkable, and it has been established as a new treatment method that can be a radical treatment even for extensive lesions. However, since scar stricture occurs at a high rate after ESD, patients may not only have dysphagia due to stricture, but may even require surgery due to stricture, and although the cancer has been eradicated, the subsequent QOL is significantly impaired. Therefore, suppression (prevention and treatment) of stricture after ESD has newly become a clinical problem along with the progress of endoscopic treatment techniques (Literatures 1-6). It has become clear that the development of stricture depends on the range of esophageal mucosa resection, and evidence has accumulated that the critical point is whether the resection range is 3 / 4 circumference, that is, 75% or more, or less (Literatures 2, 4). That is, when ESD is performed in a range of 3 / 4 circumference or more, postoperative esophageal stricture is inevitable, and it is becoming clear that different clinical treatments are required for 3 / 4 circumference or more and less.

[0003] As strategies for suppressing stricture after esophageal ESD, protection of the esophageal mucosa against gastric acid reflux, anti-inflammation, anti-fibrosis, promotion of mucosal regeneration, and mechanical dilation are considered. Attempts have been made with proton pump inhibitors (esophageal mucosal protection), steroid preparations (anti-inflammation), cell sheets (promotion of regeneration), endoscopic balloon dilation (mechanical dilation), esophageal stents (mechanical dilation), etc., but disease control is still difficult (References 5, 6). Although balloon dilation is associated with pain and the risk of esophageal rupture and thus has to be performed at present, it causes restenosis and recurrent restenosis at a high rate, so repeated dilation is often necessary, it is intractable over several months, and QOL is significantly inhibited. Therefore, local injection of steroids has been carried out in recent years (References 6, 7). This involves injecting a steroid solution endoscopically into the area of the ESD ulcer with a local injection needle immediately after ESD, and it is said to be safer compared to balloon dilation. However, steroids not only have a risk of infection including abscess formation, but it has also been reported that local administration can induce delayed esophageal perforation (References 2-6, 8). Since this is caused by damaging the muscular layer, careful administration avoiding the muscular layer after ESD is required. However, it is difficult to administer anatomically avoiding the muscular layer during injection into the large artificial ulcer area associated with extensive ESD, and there is always a risk of perforation. Furthermore, definitely, although there are reports that it has a certain effect on stricture after ESD in a relatively small range of less than 3 / 4 circumference, it has been clarified that it has no effect at all on stricture after ESD in a wide range of more than 3 / 4 circumference (References 2-6). Therefore, for stricture after ESD in a range of more than 3 / 4 circumference, there is no drug therapy including steroids, and at present, mechanical dilation such as balloon dilation and stents has to be carried out. On the other hand, not only in esophageal cancer, but especially in Barrett's esophagus, which has been increasing in recent years, there are many cases presenting lesions in a wide range of more than 3 / 4 circumference or circumferentially, so endoscopic treatment and ESD treatment in a wide range of more than 3 / 4 circumference are required. In addition, esophageal stricture after ESD is completely different from cancerous stricture. The fact that it is a benign stricture after radical resection of cancer is extremely important in clinical practice. For a benign stricture, stents that palliatively prevent progressive esophageal stricture caused by cancer that cannot be completely resected and grows, let alone iatrogenic risks such as perforation and mediastinal abscess induced by muscle layer destruction, must be avoided as much as possible. That is, in clinical practice, a treatment with extremely high hurdles that ensures high safety while having a stricture inhibitory effect is required. Therefore, how to safely suppress esophageal stricture that is inevitable after extensive ESD as described above has become an urgent clinical issue, including the development of new drugs.

[0004] CHST15 (Carbohydratesulfotransferase 15), a glycosulfotransferase, is a type II transmembrane Golgi protein that transfers a sulfate group to the 6-position of the GalNAc(4SO4) residue of chondroitin sulfate A (CS-A) to synthesize highly sulfated chondroitin sulfate E (CS-E) (References 9, 10). Although it is hardly expressed in normal human tissues, its expression is known to be enhanced in inflammation, fibrosis, and cancer. CS-E has been reported to promote collagen fiber (fibril) formation (Reference 11), and it is thought to be involved in the maintenance and enhancement of local fibrotic lesions. Furthermore, it has been reported that highly sulfated CS binds to molecules CD44 involved in fibroblast adhesion, chemokines MCP-1 and SDF-1 involved in fibroblast migration, and PDGF and TGF-β involved in fibroblast proliferation (References 12, 13), suggesting that it is also involved in fibroblast settlement and activation through the concentration of these molecules at the lesion site.

[0005] In the esophagus, in a porcine semi-circular (50%) ESD model, it has been reported that mRNA of CHST15 increases in the stenotic esophageal tissue after ESD, and fibrosis is suppressed when the expression of mRNA is inhibited by CHST15 siRNA (Reference 14). Therefore, it is suggested that, similar to steroids, it may have an effect of suppressing stenosis after ESD of less than 3 / 4 circumference. However, as clearly stated as a limitation in Non-Patent Document 14, it is unknown whether stenosis after ESD of 3 / 4 circumference or more, and even more so, total circumference can be suppressed. Similar to the ineffectiveness of steroids for stenosis of 3 / 4 circumference or more, it is extremely difficult to show a suppressing effect on stenosis of 3 / 4 circumference or more and even more so, total circumference, so prediction is difficult. Also, in Non-Patent Document 14, ESD was performed at two sites in the same individual, CHST15 siRNA was administered to one site, and negative control siRNA was administered to the other site. Since the influence of the stricture lesion presented by the negative control siRNA administration part on the clinical symptoms was mixed, it was completely unknown how CHST15 siRNA would affect the clinical symptoms (such as weight loss associated with dysphagia).

Prior Art Documents

Non-Patent Documents

[0006] 1) Siersema PD. Treatment option for esophageal strictures. Nature Gastroenterol Hepatol 5: 142-152, 2008. 2) Japanese Esophageal Society, Clinical Practice Guidelines for Esophageal Cancer, 2017 Edition. 3) Sami SS, Haboubi N, Ang Y et al. UK guidelines on esophageal dilatation in clinical practice. Gut 67: 1000-1023, 2018. 4) Draganov PV, Wang AY, Othman MO et al. AGA institute clinical practice update: Endoscopic submucosal dissection in the united states. Clin Gastroenterol Hepatol 17: 16-25, 2019. 5) Barret M< Beye S, Leblanc S et al. Systematic review: the prevention of oesophageal stricture after endoscopic resection. Aliment Pharmacol Ther 42: 20-39, 2015. 6) Jain D, Singhal S. Esophageal stricture prevention after endoscopic submucosal dissection. Clin Endosc 49: 241-256, 2016. 7) Hashimoto S, Kobayashi M, Takeuchi M, et al. The efficacy of endoscopic triamcinolone injection for the prevention of esophageal stricture after endoscopic submucosal dissection. Gastrointest Endosc 74: 1389-1393, 2011. 8) Yamashita S, Kato M, Fujimoto A, et al. Inadequate steroid injection after esophageal ESD might cause mural necrosis. Endosc int Open 7: E115-E121. 2019. 9) Ohtake S, Kondo S, Morisaki T, et al. Expression of sulfotransferase involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells. Biochemical Biophysica Acta 1780: 687-95, 2008. 10)Habuchi O, Moroi R, Ohtake S, et al. Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage. Anal Biochem 310: 129-36, 2002. 11)Kvist AJ, Hohnson AE, Morgelin M et al. Chondroitin sulfate perlecan enhances collagen fibril formation. JBC 281: 33127-33139, 2006. 12)Yamada S and Sugahara K. Potential therapeutic Application of chondroitin sulfate / dermatan sulfate. Current Drug Discovery Technologies 5: 289-301, 2008. 13)Mizumoto S and Sugahara K. Glycosaminoglycans are functional ligands for advanced glycation end-products in tumors. FEBS Journal 280: 2462-2470, 2013. 14) Sato H, Sagara S, Nakajima N, et al. Prevention of esophageal stricture after endoscopic submucosal dissection using RNA-based silencing of carbohydrate sulfotransferse 15 in a pig model. Endoscopy 49: 1-9, 2017.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a safe and novel stricture inhibitor for benign esophageal stricture.

Means for Solving the Problems

[0008] The present inventors examined the effect of local administration of CHST15 siRNA using a circumferential (100%) ESD model and found that it has a stricture inhibitory effect that could not be predicted from conventional treatments. Further, it was revealed that the effect is not based only on the anti-fibrotic effect of CHST15 siRNA, but is due to completely different effects such as the appearance of mature epithelium and almost perfect protection of the muscular layer. It has been found that the drug exhibits a stricture inhibitory effect against circumferential ESD, which was extremely difficult, and has no risk of perforation or mediastinal abscess associated with muscle layer destruction, and exhibits an extremely excellent effect in terms of actual clinical safety, and is expected to be applied to the treatment of benign esophageal stricture associated with extensive endoscopic treatment. The inventors of the present invention have found that in esophageal strictures after porcine circumferential (100%) ESD at a level where known drug therapies containing steroids cannot exert effective effects, administration of CHST15 siRNA alone can achieve a remarkable stricture inhibitory effect. Furthermore, the inventors have found that the effect is accompanied by a muscular layer protective effect, and have also discovered that it can avoid the risk of muscular layer destruction associated with known steroid preparations and mechanical dilation. In benign esophageal strictures after circumferential ESD where there has been no symptomatic treatment method other than mechanical dilation, CHST15 siRNA has been demonstrated to have both high safety and a remarkable stricture inhibitory effect simultaneously.

[0009] More specifically, the present invention provides the following [1] to [8]. [1] A pharmaceutical composition for treating or preventing benign esophageal stricture, comprising as an active ingredient an siRNA that suppresses the expression of the CHST15 gene. [2] The pharmaceutical composition according to [1], wherein the esophageal stricture is a stricture selected from the group consisting of strictures due to achalasia, peptic stricture, Schatzki ring, stricture due to endoscopic treatment, stricture due to eosinophilic esophagitis, postoperative stricture, stricture associated with radiotherapy, corrosive stricture, and refractory stricture. [3] The pharmaceutical composition according to [1] or [2], wherein the esophageal stricture occurs in 3 / 4 or more circumferences of the esophagus. [4] The pharmaceutical composition according to any one of [1] to [3], characterized by not being accompanied by injury to the muscular layer of the esophagus. [5] The pharmaceutical composition according to any one of [1] to [4], not combined with steroids. [6] The pharmaceutical composition according to any one of [1] to [5], not combined with endoscopic balloon dilation or with the number of times of combined use of endoscopic balloon dilation reduced. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the siRNA is contained at 100 to 10,000 nM. [8] The pharmaceutical composition according to any one of [1] to [7], administered as a single dose or at one-week intervals.

[0010] The present invention further relates to the following. A method for treating esophageal stricture, comprising the step of administering siRNA that suppresses the expression of the CHST15 gene. B-1. siRNA that suppresses the expression of the CHST15 gene for use in the treatment of esophageal stricture. C-1. Use of siRNA that suppresses the expression of the CHST15 gene in the manufacture of an esophageal stricture inhibitor. D-1. A method for manufacturing an esophageal stricture inhibitor, comprising the step of using siRNA that suppresses the expression of the CHST15 gene.

Advantages of the Invention

[0011] Since more than three-fourths circumferential, especially full circumferential ESD of the esophagus shows extremely strong stricture, a drug that suppresses it has not been known until now. Surprisingly, it has been revealed for the first time by the present invention that a stricture inhibitory effect can be obtained by locally administering CHST15 siRNA. In addition, CHST15 siRNA has hitherto been known only to have an anti-fibrotic effect limited to the submucosal layer, and it has also been revealed for the first time by the present invention that it has a use for protecting the muscular layer (muscularis propria), which is different from fibrosis. Thus, a novel, effective and safe treatment method has been provided for esophageal stricture that was impossible to treat with conventional drug therapies.

Brief Description of the Drawings

[0012]

Figure 1

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Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail. The present inventor has found that by suppressing the expression of the CHST15 (Carbohydrate sulfotransferase 15) gene, an inhibitory effect on esophageal stricture after circumferential ESD of the esophagus is exerted. More specifically, the present inventor has found that by suppressing the expression of the CHST15 gene by the RNAi (RNA interferance; RNA interference) effect, esophageal stricture is suppressed. Furthermore, the present inventor has found that even for esophageal stricture after circumferential ESD at a level where no significant therapeutic effect is recognized with existing steroid preparations used, the stricture is significantly suppressed by siRNA that suppresses the expression of the CHST15 gene, and the effect is safely exerted by the muscle layer protecting action.

[0014] The CHST15 gene of the present invention is not particularly limited, but is usually derived from an animal, more preferably from a mammal, and most preferably from a human. In addition, CHST15 of the present invention is also called GalNAc4S-6ST (N-acetylgalactosamine 4-sulfate 6-O sulfotransferase) as an alias.

[0015] The sequence of CHST15 (GalNAc4S-6ST) of the present invention can be obtained, for example, based on the accession number NM_015892. As an example, the nucleotide sequence of the CHST15 gene of the present invention is described in SEQ ID NO: 3, and the amino acid sequence encoded by the gene is described in SEQ ID NO: 4. Even a protein consisting of an amino acid sequence other than the above, for example, having a high identity (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequence described in SEQ ID NO: 4 and having the function of the above protein is included in the CHST15 protein of the present invention. The above protein is, for example, a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted in the amino acid sequence described in SEQ ID NO: 4, and usually the number of amino acids that change is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.

[0016] The genes in the present invention include, for example, endogenous genes in other organisms corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3 (such as homologs of the above genes in humans). In addition, the endogenous DNAs of other organisms corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3 generally have high identity (homology) with the DNA set forth in SEQ ID NO: 3, respectively. High identity means preferably 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95% or more, even 96%, 97%, 98% or 99% or more) homology. This homology can be determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). Further, when the DNA is isolated from a living body, it is considered to hybridize with the DNA set forth in SEQ ID NO: 3 under stringent conditions. Here, examples of "stringent conditions" include "2×SSC, 0.1% SDS, 50°C", "2×SSC, 0.1% SDS, 42°C", "1×SSC, 0.1% SDS, 37°C", and more stringent conditions such as "2×SSC, 0.1% SDS, 65°C", "0.5×SSC, 0.1% SDS, 42°C" and "0.2×SSC, 0.1% SDS, 65°C".

[0017] In the present specification, "siRNA that suppresses the expression of the CHST15 gene" can also be expressed as "CHST15 siRNA", and preferably, it is an siRNA having a structure in which the RNAs set forth in SEQ ID NO: 1 (5'-ggagcagagc aagaugaaua caaucag-3') and SEQ ID NO: 2 (5'-gauuguauuc aucuugcucu gcuccau-3') hybridize.

[0018] In the siRNA of the present invention, not all nucleotides necessarily have to be ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides as long as they have the function of suppressing the expression of the CHST15 gene as the molecule itself. This "corresponding" means that although the structure of the sugar moiety is different, they are the same base species (adenine, guanine, cytosine, thymine (uracil)). For example, the deoxyribonucleotide corresponding to a ribonucleotide having adenine refers to a deoxyribonucleotide having adenine. Also, the "plurality" is not particularly limited, but preferably refers to a small number of about 2 to 5.

[0019] The siRNA of the present invention can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. Also, for the synthesis of the desired RNA, it is possible to use a general contract synthesis service.

[0020] Since the CHST15 siRNA of the present invention has an inhibitory effect on benign esophageal stricture as a single agent, the present invention provides an esophageal stricture inhibitor (pharmaceutical composition for treating or preventing benign esophageal stricture) containing CHST15 siRNA as an active ingredient. Alternatively, the present invention provides a method for treating benign esophageal stricture including the step of administering CHST15 siRNA, CHST15 siRNA for use in the treatment of benign esophageal stricture, the use of CHST15 siRNA in the manufacture of an esophageal stricture inhibitor, and a method for manufacturing an esophageal stricture inhibitor including the step of using (formulating and / or mixing with a pharmaceutically or physiologically acceptable carrier) CHST15 siRNA.

[0021] The esophageal stricture to be treated or prevented in the present invention is not particularly limited as long as it is a benign esophageal stricture in which the anti-CHST15 siRNA of the present invention exerts a therapeutic effect, but is a stricture selected from the group consisting of strictures due to achalasia, peptic stricture, Schatzki ring, stricture due to endoscopic treatment, stricture due to eosinophilic esophagitis, postoperative stricture, stricture associated with radiotherapy, corrosive stricture, and refractory stricture, and preferably is an esophageal stricture after extensive esophageal ESD of 3 / 4 circumference or more. In addition, the "treatment" in the present invention is not necessarily limited to the case having a complete therapeutic effect, and may be the case having a partial effect.

[0022] The esophageal stricture inhibitor or pharmaceutical composition of the present invention can be mixed with a pharmaceutically or physiologically acceptable carrier, excipient, diluent, etc., and administered orally or parenterally. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, or suspensions can be used. As parenteral preparations, dosage forms such as injections, drip infusions, topical agents, inhalants (nebulizers), or suppositories can be selected. Injections can include subcutaneous injections, intramuscular injections, intraperitoneal injections, intracranial administration injections, or intranasal administration injections, etc. Topical agents can include intranasal administration agents, or ointments, etc. Formulation techniques for the above dosage forms to include the anti-cancer agent or pharmaceutical composition of the present invention as the main component are well-known.

[0023] For example, tablets for oral administration can be manufactured by adding an excipient, disintegrant, binder, lubricant, etc. to the esophageal stricture inhibitor or pharmaceutical composition of the present invention, mixing them, and compression molding. Lactose, starch, or mannitol, etc. are generally used as excipients. Calcium carbonate, calcium carboxymethylcellulose, etc. are generally used as disintegrants. Gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone are used as binders. Talc, magnesium stearate, etc. are well-known as lubricants.

[0024] The tablets containing the esophageal stricture inhibitor or pharmaceutical composition of the present invention can be coated with a known coating for masking or enteric preparation. Ethyl cellulose, polyoxyethylene glycol, etc. can be used as the coating agent.

[0025] Also, the injection can be obtained by dissolving or dispersing the esophageal stricture inhibitor or pharmaceutical composition of the present invention, which is the main component, together with a suitable dispersant in a dispersion medium. Depending on the selection of the dispersion medium, it can be made into either an aqueous solvent or an oily solvent dosage form. For an aqueous solvent, distilled water, physiological saline, or Ringer's solution, etc. can be used as the dispersion medium. For an oily solvent, various vegetable oils, propylene glycol, etc. are used as the dispersion medium. At this time, a preservative such as paraben can be added as necessary. Also, known isotonic agents such as sodium chloride and glucose can be added to the injection. Furthermore, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

[0026] Also, the esophageal stricture inhibitor or pharmaceutical composition of the present invention can be made into an external preparation by making it into a solid, liquid, or semi-solid composition. For the solid or liquid composition, it can be made into an external preparation by using the same composition as described above. The semi-solid composition can be prepared by adding a thickening agent to a suitable solvent as necessary. Water, ethyl alcohol, polyethylene glycol, etc. can be used as the solvent. Generally, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone, etc. are used as the thickening agent. A preservative such as benzalkonium chloride can be added to this composition. Also, it can be made into a suppository by combining an oily base material such as cocoa butter or an aqueous gel base material such as a cellulose derivative as a carrier.

[0027] When the esophageal stricture inhibitor or pharmaceutical composition of the present invention is used as a gene therapy agent, in addition to the method of directly administering the esophageal stricture inhibitor or pharmaceutical composition of the present invention by injection, there is a method of administering a vector incorporated with a nucleic acid. Examples of the above vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, etc., and by using these virus vectors, administration can be carried out efficiently.

[0028] In addition, it is also possible to introduce the esophageal stricture inhibitor or pharmaceutical composition of the present invention into lipid vesicles such as liposomes and administer the vesicles. Vesicles retaining siRNA are introduced into predetermined cells by the lipofection method. Then, the obtained cells are systemically administered, for example, intravenously or intraarterially.

[0029] The present invention also provides a method for treating or preventing esophageal stricture in a subject, which includes the step of administering the esophageal stricture inhibitor or pharmaceutical composition of the present invention to an individual (for example, a patient) or the esophageal tissue thereof. The individual targeted by the treatment or prevention method of the present invention is not particularly limited as long as it is an organism capable of developing esophageal stricture, but is preferably a human. Administration to the subject can be carried out by methods known to those skilled in the art, such as oral administration, intradermal administration, or subcutaneous administration, or intravenous injection. Systemic administration or direct local administration into the esophageal tissue is possible. In addition, a commercially available gene transfection kit can also be used to introduce the siRNA of the present invention into the target tissue or organ.

[0030] The esophageal stricture inhibitor or pharmaceutical composition of the present invention is administered in a necessary amount (effective amount) to mammalian subjects including humans within a range of a safe dosage. The dosage of the esophageal stricture inhibitor or pharmaceutical composition of the present invention can be finally appropriately determined by the judgment of those skilled in the art (physicians or veterinarians) in consideration of the type of dosage form, administration method, age and weight of the subject, symptoms of the subject, and the like. For example, although it varies depending on age, sex, symptoms, administration route, number of administrations, and dosage form, for example, the dosage of the CHST15 siRNA of the present invention in local administration is about 100 to 10,000 nM per day per administration, and is administered as a single administration or at intervals of one week to one month (for example, at intervals of one week, two weeks, or one month).

[0031] All prior art documents cited in this specification are incorporated herein by reference.

Examples

[0032] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples. The "CHST15 siRNA" used in this example is an siRNA having a structure in which the RNAs described in SEQ ID NOs: 1 and 2 are hybridized.

[0033] <Example 1> For the preparation of a full-circumferential (100%) ESD model of the esophagus, experimental minipigs were used. In mice and rats, which are commonly used as experimental animals, there is no stratified squamous epithelium like that of humans anatomically, and physiological reflux does not occur, so it is difficult to reflect the elements involved in the important pathological conditions of human esophageal diseases. On the other hand, the esophagus of a minipig weighing about 40 kg is about 30 cm in total length, which is comparable to the esophagus length of about 40 cm in adult humans. It has a stratified squamous epithelium like that of humans, physiological reflux also occurs, and there is also the advantage that an ESD experiment can be performed using a human upper gastrointestinal endoscope set. Mini pigs were divided into three groups: a physiological saline administration group, a steroid preparation (Kenacort) administration group, and a CHST15 siRNA (manufactured by Hokkaido System Science Co., Ltd., lot number 40481638) administration group (n = 1 / group). Under general anesthesia and respiratory management, the upper gastrointestinal endoscope was orally inserted into the mini pigs of each group, and a full-circumferential (100%) ESD with a total length of 5 cm was performed using a dual knife between 15 cm and 25 cm from the incisor row. For the full-circumferential artificial ulcer immediately after ESD, a total of 2 mL of the administration solution (physiological saline, steroid preparation, CHST15 siRNA, 250 nM solution) was injected into a total of 20 sites evenly covering the entire area from the ulcer bottom to the surrounding area using an endoscopic injection needle. If esophageal stricture develops within 2 weeks without treatment after ESD and food cannot pass through, the mini pigs will die within 4 weeks thereafter. Therefore, the observation period after single administration was set to 2 weeks, and all groups were sacrificed 2 weeks later (Day 14). The degree of esophageal stricture was examined by gross findings and pathological histological analysis (Masson staining). Results: Normal saline administration group : <Gross findings> Shortening of the esophageal length and complete obstruction were observed. The state was one in which ingestion was impossible. <Pathological findings> Although the appearance of some immature regenerated epithelium was observed, the artificial ulcer caused by ESD remained, and extensive fibrosis deep to the ulcer bottom and necrosis of the muscular layer were observed. Steroid preparation (Kenacort) administration group : <Gross findings> The degree of shortening of the esophageal length was mild compared to the physiological saline administration group, but complete obstruction was observed. The state was one in which ingestion was impossible. <Pathological findings> Although the artificial ulcer caused by ESD remained, compared with the physiological saline administration group, repair findings by relatively mature regenerated epithelium were observed. However, extensive fibrosis was observed throughout the entire layer deeper, and the degree was stronger than that of the physiological saline administration group. In addition, extensive destruction of the muscular layer with defects was observed, and fibrosis extended to the serosa layer. CHST15 siRNA administration group : <Gross findings> There was almost no shortening of the esophageal length. Although there was some narrowing of a part of the lumen, there was no stenosis or obstruction, and the patient was able to ingest food. <Pathological findings> The artificial ulcer caused by ESD was almost covered with regenerated epithelium, which was a more mature regenerated epithelium with regularly constructed stratified squamous epithelium even when compared with the steroid administration group. There was no pathological fibrosis deeper than this, and the fibrosis was at almost a physiological level supporting the epithelium. Furthermore, no damage to the muscular layer was observed at all. Discussion: In the steroid preparation administration group, there was no effect of suppressing esophageal stenosis after circumferential ESD, which was considered to be the same result as in the clinical trial. Although a tendency for the reduction of the ulcer bottom with regenerated epithelium was observed when compared with the physiological saline administration group, extensive damage to the muscular layer was presented. This was considered to be due to the protein catabolic effect of steroids, and it was suggested that this could be the cause of delayed perforation observed clinically. On the other hand, the stenosis suppression effect observed in the CHST15 siRNA administration group was an effect that could be obtained while keeping the muscular layer in almost a normal state. This indicated that stenosis was suppressed while keeping the peristaltic movement of the esophagus, that is, the swallowing function, almost normal, and strongly suggested that it could be treated extremely safely also in actual clinical practice. In the case of esophageal stenosis after ESD in actual clinical practice, it is the current situation that balloon dilation is repeatedly performed, and since damage to the muscular layer must be avoided as much as possible, it is considered that multiple administrations of steroids should be avoided. Also, in esophageal cancer, there is recurrence, and in order to be able to safely perform ESD treatment on the recurrent esophageal cancer, protection of the muscular layer is an absolute condition. It was considered that the clinical usefulness of CHST15 siRNA was extremely high compared with steroid preparations also for performing repeated endoscopic treatments.

Industrial applicability

[0034] In a test using an esophageal stricture model after total circumferential (100%) ESD of pigs, which is completely ineffective with existing therapies, the CHST15 siRNA of the present invention showed a remarkable stricture inhibitory effect. In addition, since it has a muscular layer protective effect, it was considered that it could lead to a clinical scene that has never been envisioned before, enabling maintenance of the function of the muscular layer (swallowing function) or repeated endoscopic treatment, along with high safety (no risk of perforation like existing treatments). From these results, it was shown that the CHST15 siRNA of the present invention is useful as a novel esophageal stricture inhibitor.

Claims

Claim 1 A pharmaceutical composition for treating or preventing esophageal stricture, comprising as an active ingredient an siRNA that suppresses the expression of the CHST15 gene, wherein the siRNA is administered locally at 100 to 10,000 nM once a day, by single administration or at intervals of one week to one month, and the esophageal stricture occurs in more than three-fourths of the esophagus. The pharmaceutical composition. Claim 2 The pharmaceutical composition according to claim 1, wherein the esophageal stricture is a stricture selected from the group consisting of achalasia-related stricture, peptic stricture, Schatzki ring, stricture due to endoscopic treatment, eosinophilic esophagitis-related stricture, postoperative stricture, radiation therapy-associated stricture, corrosive stricture, and refractory stricture. Claim 3 The pharmaceutical composition according to claim 1 or 2, characterized by not being accompanied by injury to the muscular layer of the esophagus. Claim 4 The pharmaceutical composition according to any one of claims 1 to 3, not combined with steroids. Claim 5 The pharmaceutical composition according to any one of claims 1 to 4, not combined with endoscopic balloon dilation or with a reduced number of times of combined use of endoscopic balloon dilation. Claim 6 The pharmaceutical composition according to any one of claims 1 to 5, administered by single administration or at intervals of one week.

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