WO2020225871A1 - 食道狭窄抑制剤 - Google Patents
食道狭窄抑制剤 Download PDFInfo
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- WO2020225871A1 WO2020225871A1 PCT/JP2019/018365 JP2019018365W WO2020225871A1 WO 2020225871 A1 WO2020225871 A1 WO 2020225871A1 JP 2019018365 W JP2019018365 W JP 2019018365W WO 2020225871 A1 WO2020225871 A1 WO 2020225871A1
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- A—HUMAN NECESSITIES
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- the present invention relates to a novel esophageal stricture inhibitor containing CHST15 siRNA as an active ingredient.
- Endoscopic treatment for esophageal cancer which is minimally invasive to esophageal cancer and Barrett's esophagus and enables maintenance of esophageal function preservation, has become established in recent years as a curative treatment for superficial esophageal cancer and Barrett's esophagus (Reference 1-5).
- ESD endoscopic submucosal dissection
- Strategy for suppressing stenosis after esophageal ESD includes protection of esophageal mucosa against gastric acid reflux, anti-inflammatory, antifibrotic, promotion of mucosal regeneration, and mechanical dilation.
- Proton pump inhibitor protection of esophageal mucosa
- steroids Attempts have been made for preparations (anti-inflammatory), cell sheets (promoting regeneration), endoscopic balloon dilatation (mechanical dilation), and esophageal stents (mechanical dilation), but pathological control is still difficult (" Documents 5 and 6).
- balloon dilatation involves pain and risk of esophageal rupture, it has to be performed at present, but it often requires repeated dilatation because it causes restenosis and re-stenosis at a high rate. It is refractory for months and QOL is significantly impaired. Therefore, in recent years, local injection of steroids has been performed (References 6 and 7). This is to inject a steroid solution endoscopically into the ulcer part after ESD with a local injection needle immediately after ESD, and it is said to be safer than balloon dilatation. However, it has been reported that steroids not only carry the risk of infection including abscess formation, but also induce delayed esophageal perforation by topical administration (References 2-6, 8).
- CHST15 Carbohydratesulfotransferase 15
- CS-A chondroitin sulfate A
- CS- highly sulfated chondroitin sulfate E
- It is a type II transmembrane Golgi protein that synthesizes E) (References 9 and 10). Although it is rarely expressed in normal human tissues, it is known that its expression is enhanced in inflammation / fibrosis and cancer.
- CS-E promotes collagen fiber (fibril) formation (Reference 11), and CS-E is considered to be involved in the maintenance and enhancement of locally fibrotic lesions.
- highly sulfated CS includes the molecule CD44 involved in fibroblast adhesion, chemokines MCP-1, SDF-1 involved in fibroblast migration, and PDGF and TGF- ⁇ involved in fibroblast proliferation. It has been reported that they bind (References 12 and 13), suggesting that they are also involved in the colonization and activation of fibroblasts through the enrichment of these molecules in the lesion area.
- Non-Patent Document 14 two ESDs are prepared in the same individual, CHST15 siRNA is administered to one place, and negative control siRNA is administered to the other place, and the narrowing exhibited by the negative control siRNA administration portion. Since the effects of lesions on clinical symptoms were mixed, it was completely unclear what kind of effect CHST15 siRNA had on clinical symptoms (weight loss associated with dysphagia, etc.).
- An object of the present invention is to provide a safe novel stenosis inhibitor for benign esophageal stricture.
- CHST15 siRNA has both high safety and a remarkable stenosis-suppressing effect in benign esophageal stricture after circumferential ESD, for which there has been no symptomatic method other than mechanical dilatation.
- [1] A pharmaceutical composition for treating or preventing benign esophageal stricture, which comprises siRNA that suppresses the expression of the CHST15 gene as an active ingredient.
- Esophageal stenosis includes acarasia stenosis, digestive stenosis, shacky ring, endoscopic treatment stenosis, eosinophil esophagitis stenosis, postoperative stenosis, radiation therapy stenosis, corrosive stenosis
- the pharmaceutical composition of [1] which is a stenosis selected from the group consisting of intractable stenosis.
- [3] The pharmaceutical composition according to [1] or [2], wherein esophageal stricture occurs in 3/4 or more laps of the esophagus.
- [4] The pharmaceutical composition according to any one of [1] to [3], which is not accompanied by muscular layer injury of the esophagus.
- [5] The pharmaceutical composition according to any one of [1] to [4], which is not used in combination with steroids.
- [6] The pharmaceutical composition according to any one of [1] to [5], wherein the endoscopic balloon dilatation is not used in combination or the number of times of endoscopic balloon dilation is reduced.
- [7] The pharmaceutical composition according to any one of [1] to [6], wherein siRNA is contained at 100 to 10,000 nM.
- [8] The pharmaceutical composition according to any one of [1] to [7], which is administered as a single dose or at weekly intervals.
- the present invention further relates to the following.
- A-1 A method for treating esophageal stricture, which comprises a step of administering siRNA that suppresses the expression of the CHST15 gene.
- B-1 A 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 production of an esophageal stricture inhibitor.
- D-1 A method for producing an esophageal stricture inhibitor, which comprises a step of using siRNA that suppresses the expression of the CHST15 gene.
- CHST15 siRNA Since the esophagus exhibits extremely strong stenosis over 3/4 circumference, especially after circumferential ESD, no drug has been known to suppress it, but surprisingly, CHST15 siRNA is locally administered. It was clarified for the first time by the present invention that the stenosis suppressing effect can be obtained. In addition, CHST15 siRNA has been known to have an antifibrotic effect limited to the submucosal layer, and this time, it is the first time according to the present invention that CHST15 siRNA has a muscle layer (unique muscle layer) protection application different from fibrosis. It became clear. As described above, a new effective and safe treatment method for esophageal stricture, which cannot be treated by conventional drug therapy, has been provided.
- the present inventor has found that by suppressing the expression of the CHST15 (Carbohydrate sulfotransferase 15) gene, an inhibitory effect on esophageal stenosis after esophageal circumflex ESD is exhibited. More specifically, the present inventor has found that esophageal stricture is suppressed by suppressing the expression of the CHST15 gene by the RNAi (RNA interferance) effect. Furthermore, the present inventor remarkably narrows the esophageal stricture after circumscribed ESD at a level where no significant therapeutic effect is observed with the existing steroid preparations, due to siRNA that suppresses the expression of the CHST15 gene. It was found that the effect is safely exerted by the muscle layer protective action.
- CHST15 Carbohydrate sulfotransferase 15
- CHST15 gene of the present invention is not particularly limited, but is usually derived from animals, more preferably from mammals, and most preferably from humans.
- CHST15 of this invention is also called GalNAc4S-6ST (N-acetylgalactosamine 4-sulfate 6-0 sulfotransferase) as another name.
- the sequence of CHST15 (GalNAc4S-6ST) of the present invention can be obtained, for example, based on accession number NM_015892.
- the nucleotide sequence of the CHST15 gene of the present invention is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 4.
- Even a protein consisting of an amino acid sequence other than the above has high identity (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95) with the sequence set forth in, for example, SEQ ID NO: 4. % Or more) and the function of the above protein is included in the CHST15 protein of the present invention.
- the above-mentioned 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 shown in SEQ ID NO: 4, and the number of amino acids that usually changes is 30 amino acids. Within, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.
- the gene in the present invention includes, for example, an endogenous gene in another organism (such as a homologue of the human gene) corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3.
- the endogenous DNA of other organisms corresponding to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 generally has high identity (homology) with the DNA of SEQ ID NO: 3, respectively. ..
- High identity is preferably 70% or higher, more preferably 80% or higher, more preferably 90% or higher (eg, 95% or higher, further 96%, 97%, 98% or 99% or higher) homology.
- Means. This homology is based on the MBLAST algorithm (Altschul et al.
- siRNA that suppresses the expression of CHST15 gene can also be expressed as "CHST15 siRNA", preferably SEQ ID NO: 1 (5'-ggagcagagc aagaugaaua caaucag-3') and SEQ ID NO: 2 It is a siRNA having a hybridized structure of the RNA described in (5'-gauuguauuc aucuugcucu gcuccau-3').
- RNA ribonucleotides
- one or more ribonucleotides constituting siRNA may be the corresponding deoxyribonucleotides as long as the molecule itself has a function of suppressing the expression of the CHST15 gene.
- This "corresponding" refers to the same base species (adenine, guanine, cytosine, thymine (uracil)), although the structure of the sugar moiety is different.
- the deoxyribonucleotide corresponding to the ribonucleotide having adenine refers to the deoxyribonucleotide having adenine.
- the above-mentioned "plurality” is not particularly limited, but preferably refers to a small number of about 2 to 5.
- siRNA of the present invention can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer.
- general synthetic contract services can be used for the synthesis of desired RNA.
- the present invention contains a CHST15 siRNA as an active ingredient and is a pharmaceutical composition for treating or preventing benign esophageal stricture. Things) to provide.
- the present invention relates to a method for treating benign esophageal stricture including a step of administering CHST15 siRNA, CHST15 siRNA for use in the treatment of benign esophageal stricture, use of CHST15 siRNA in the production of an esophageal stricture inhibitor, and CHST15 siRNA.
- a method for producing an esophageal stricture inhibitor which comprises a step of using (formulating and / or mixing with a pharmaceutically or physiologically acceptable carrier).
- the esophageal stenosis to be treated or prevented in the present invention is not particularly limited as long as it is a benign esophageal stenosis in which the anti-CHST15 siRNA of the present invention exerts a therapeutic effect, but stenosis due to acarasia, digestive stenosis, shacky ring, etc. It is a stenosis selected from the group consisting of stenosis due to endoscopic treatment, stenosis due to eosinophilic esophageal inflammation, stenosis after surgery, stenosis associated with radiotherapy, corrosive stenosis, and intractable stenosis, preferably 3/4 lap. This is esophageal stricture after extensive esophageal ESD.
- the "treatment" in the present invention is not necessarily limited to the case of having a complete therapeutic effect, but may be a case of having a partial effect.
- 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.
- the oral preparation may be a dosage form such as a granule, a powder, a tablet, a capsule, a solvent, an emulsion, or a suspension.
- a dosage form such as an injection, a drip, an external preparation, an inhalant (nebulizer) or a suppository can be selected.
- the injection include a subcutaneous injection, an intramuscular injection, an intraperitoneal injection, an intracranial injection, an intranasal injection, and the like.
- Examples of the external preparation may be a nasal administration agent, an ointment, or the like.
- the formulation technology of the above dosage form is known so as to contain the anticancer agent or pharmaceutical composition of the present invention which is the main component.
- tablets for oral administration are produced by adding excipients, disintegrants, binders, lubricants, etc. to the esophageal stricture inhibitor or pharmaceutical composition of the present invention, mixing them, and compressing and shaping them.
- excipients lactose, starch, mannitol and the like are generally used.
- disintegrant calcium carbonate, carboxymethyl cellulose calcium and the like are generally used.
- binder gum arabic, carboxymethyl cellulose, or polyvinylpyrrolidone is used.
- talc, magnesium stearate and the like are known.
- the tablet containing the esophageal stricture inhibitor or the pharmaceutical composition of the present invention can be coated with a known coating in order to make it a masking or enteric-coated preparation.
- a coating agent ethyl cellulose, polyoxyethylene glycol or the like can be used.
- the injection can be obtained by dissolving the esophageal stricture inhibitor or the pharmaceutical composition of the present invention, which is the main component, together with an appropriate dispersant, dissolving in a dispersion medium, or dispersing.
- an aqueous solvent or an oily solvent can be used.
- distilled water, physiological saline, Ringer's solution or the like is used as the dispersion medium.
- various vegetable oils, propylene glycol, etc. are used as the dispersion medium.
- a preservative such as paraben can be added as needed.
- a known tonicity agent such as sodium chloride or glucose can be added to the injection.
- painkillers such as benzalkonium chloride and procaine hydrochloride can be added.
- the esophageal stricture inhibitor or pharmaceutical composition of the present invention can be used as an external preparation by making it a solid, liquid or semi-solid composition.
- the solid or liquid composition can be used as an external preparation by using the same composition as described above.
- the semi-solid composition can be prepared by adding a thickener to a suitable solvent as needed.
- a suitable solvent water, ethyl alcohol, polyethylene glycol or the like can be used.
- the thickener bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, polyvinylpyrrolidone and the like are generally used.
- a preservative such as benzalkonium chloride can be added to this composition.
- it can also be used as a suppository by combining an oil-based base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.
- a vector containing a nucleic acid is administered.
- the vector include an adenovirus vector, an adeno-associated virus vector, a herpesvirus vector, a vaccinia virus vector, a retrovirus vector, a lentiviral vector, and the like, and these viral vectors can be used for efficient administration.
- phospholipid endoplasmic reticulum such as liposomes
- the endoplasmic reticulum carrying siRNA is introduced into a predetermined cell by the lipofection method. Then, the obtained cells are systemically administered to, for example, intravenously or intraarterial.
- the present invention also provides a method for treating or preventing esophageal stricture in a subject, which comprises the step of administering the esophageal stricture inhibitor or pharmaceutical composition of the present invention to an individual (for example, a patient) or its esophageal tissue.
- the individual to be treated or prevented by the 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 a subject can be performed by a method known to those skilled in the art, such as oral administration, intradermal administration, subcutaneous administration, or intravenous injection. It can be administered systemically or directly into the esophageal tissue.
- a commercially available gene transfer kit can also be used to introduce the siRNA of the present invention into a target tissue or organ.
- the esophageal stenosis inhibitor or pharmaceutical composition of the present invention is administered in a required amount (effective amount) to a mammalian subject including humans within a safe dose range.
- the dose of the esophageal stricture inhibitor or the pharmaceutical composition of the present invention is finally determined by a person skilled in the art (doctor or veterinarian) in consideration of the type of dosage form, administration method, age and weight of the subject, symptoms of the subject, and the like. ) Can be appropriately determined.
- the dose of CHST15 siRNA of the present invention in local administration is about 100 to 10,000 nM per day, although it varies depending on age, gender, symptom, route of administration, frequency of administration, and dosage form.
- Single dose or 1 week to 1 month interval eg 1 week interval, 2 week interval or 1 month interval).
- CHST15 siRNA used in this example is a siRNA having a structure in which the RNAs described in SEQ ID NOs: 1 and 2 are hybridized.
- Example 1 For the production of the esophageal circumference (100%) ESD model, experimental miniature pigs were used. In mice and rats, which are widely used as experimental animals, there is no stratified squamous epithelium like humans anatomically, and physiological reflux does not occur, so it is difficult to reflect the factors involved in the important pathophysiology of human esophageal disease. .. On the other hand, the esophagus of a miniature pig weighing about 40 kg has a total length of about 30 cm, which is comparable to the esophageal length of a human adult of about 40 cm, has stratified squamous epithelium like humans, causes physiological reflux, and is also human.
- Saline administration group ⁇ Macroscopic findings> Shortening of esophageal length and complete obstruction were observed. It was inedible. ⁇ Pathological findings> Although some immature regenerated epithelium appeared, artificial ulcer due to ESD remained, and extensive fibrosis deep below the ulcer floor and muscular crush were observed. Steroid preparation (Kenacort) administration group : ⁇ Macroscopic findings> Although the degree of shortening of the esophageal length was mild compared to the saline-administered group, complete obstruction was observed. It was inedible.
- the ulcer base with regenerated epithelium tended to shrink, but the muscular layer was extensively damaged. This is thought to be due to the protein catabolism of steroids, suggesting that it causes clinically observed delayed perforation.
- the stenosis suppressing effect observed in the CHST15 siRNA administration group was an effect that can be obtained while maintaining the muscle layer in an almost normal state. It showed that stenosis was suppressed while maintaining the peristaltic movement of the esophagus, that is, swallowing function, and strongly suggested that it could be treated extremely safely in clinical practice.
- the CHST15 siRNA of the present invention showed a remarkable stenosis-suppressing effect in a test using a esophageal stenosis model after porcine esophageal circumference (100%) ESD, which was completely ineffective with existing therapies.
- it since it has a muscular layer protective effect, it enables high safety (there is no risk of perforation unlike existing treatments), maintenance of muscular layer function (swallowing function), or repeated endoscopic treatment. It was thought that it would be possible to derive the appearance of a clinical site that could not be drawn at all in the past. From these results, it was shown that the CHST15 siRNA of the present invention is useful as a novel esophageal stricture inhibitor.
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Abstract
Description
そこで、近年ステロイドの局注が行われつつある(文献6、7)。これはESD施行直後に局注針でESD後潰瘍の部分にステロイド溶液を内視鏡下に注入するものであり、バルーン拡張と比較すると安全性が高いとされている。しかしながら、ステロイドは膿瘍形成を含む感染リスクがあるだけでなく、局所投与により遅延性の食道穿孔を誘発することが報告されている(文献2-6、8)。筋層を傷害することがその原因であるため、ESD後に筋層を避けて慎重に投与することが要求されるが、広範ESDに伴う巨大な人工潰瘍部分への注射時に、解剖学的に筋層を避けて投与することは困難であり、常時穿孔リスクが伴う。さらに決定的には、3/4周以下の比較的小さい範囲のESD後の狭窄には一定の効果を有するとの報告もあるが、3/4周以上の広範囲のESD後の狭窄には全く効果がないことが明らかになっている(文献2-6)。したがって、3/4周以上のESD後の狭窄に関しては、ステロイドを含めて薬物療法が皆無であり、バルーン拡張やステントなどの機械的拡張を余儀なくされざるを得ないのが現状である。一方で、食道癌のみならず、とりわけ近年増加の一途を辿るバレット食道においては3/4周以上の広範囲ないしは全周性に病変を呈するケースが多いため、3/4周以上の広範囲の内視鏡治療・ESD治療が要求される。
また、ESD後の食道狭窄は、癌性狭窄とは全く異なり、癌を根治させた後の良性狭窄であるという事実は、実臨床的には極めて重要である。切除しきれず増大する癌による進行性の食道狭窄を姑息に防止するステントや、ましてや筋層破壊による穿孔や縦隔膿瘍を誘発する医原性リスクは極力回避しなければならないのが良性狭窄である。すなわち、狭窄抑制効果と同時に、高い安全性を確保するという極めてハードルの高い治療が実臨床では要求される。そこで、上述のような広範囲ESD後に必発する食道狭窄を如何に安全に抑制するかが、新規薬剤の開発も含めて喫緊の臨床課題となっている。
2) 日本食道学会 食道癌診療ガイドライン 2017年版.
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.
本発明者らは、ステロイドを含む公知の薬物療法では有効な効果を発揮できないレベルであるブタ食道全周性(100%)ESD後の食道狭窄において、CHST15 siRNAを単独で投与することにより、著しい狭窄抑制効果が得られることを見出した。さらに本発明者は、その効果は筋層保護作用を伴っていることを見出し、公知のステロイド製剤や機械的拡張に伴う筋層破壊のリスクを回避し得ることも発見した。従来は機械的拡張以外に対症方法がなかった全周性ESD後の良性食道狭窄において、CHST15 siRNAは高い安全性と著しい狭窄抑制効果を同時に有することを実証した。
〔1〕CHST15遺伝子の発現を抑制するsiRNAを有効成分として含む、良性食道狭窄を治療又は予防するための医薬組成物。
〔2〕食道狭窄が、アカラシアによる狭窄、消化性狭窄、シャッキー・リング、内視鏡治療による狭窄、好酸球性食道炎による狭窄、外科手術後狭窄、放射線療法に伴う狭窄、腐食性狭窄、及び難治性狭窄からなる群より選ばれる狭窄である、〔1〕の医薬組成物。
〔3〕食道狭窄が、食道の3/4周以上で生じている、〔1〕又は〔2〕の医薬組成物。
〔4〕食道の筋層傷害を伴なわないことを特徴とする、〔1〕~〔3〕のいずれか一項記載の医薬組成物。
〔5〕ステロイドを併用しない、〔1〕~〔4〕のいずれか一項記載の医薬組成物。
〔6〕内視鏡的バルーン拡張術を併用しない又は内視鏡的バルーン拡張術の併用回数を減らした、〔1〕~〔5〕のいずれか一項記載の医薬組成物。
〔7〕siRNAが、100~10,000nMで含まれる、〔1〕~〔6〕のいずれか一項記載の医薬組成物。
〔8〕単回投与又は1週間間隔で投与される、〔1〕~〔7〕のいずれか一項記載の医薬組成物。
[A-1]CHST15遺伝子の発現を抑制するsiRNAを投与する工程を含む、食道狭窄を治療する方法。
[B-1]食道狭窄の治療において用いるための、CHST15遺伝子の発現を抑制するsiRNA。
[C-1]食道狭窄抑制剤の製造における、CHST15遺伝子の発現を抑制するsiRNAの使用。
[D-1]CHST15遺伝子の発現を抑制するsiRNAを使用する工程を含む、食道狭窄抑制剤の製造方法。
本発明者は、CHST15(Carbohydrate sulfotransferase 15)遺伝子の発現を抑制することにより、食道全周性ESD後の食道狭窄に対する抑制効果が発揮されることを見出した。より詳しくは、本発明者は、CHST15遺伝子の発現をRNAi(RNA interferance;RNA干渉)効果によって抑制することにより、食道狭窄が抑制されることを見出した。さらに、本発明者は、既存で使用されているステロイド製剤では有意な治療効果が認められないレベルの全周性ESD後の食道狭窄に対しても、CHST15遺伝子の発現を抑制するsiRNAにより著しく狭窄が抑制され、かつ、筋層保護作用により安全に効果が発揮されることを見出した。
なお、本実施例において使用した「CHST15 siRNA」は、配列番号:1および2に記載されたRNAがハイブリダイズした構造のsiRNAである。
食道全周性(100%)ESDモデルの作製については、実験用ミニブタを使用した。実験動物として汎用されるマウス・ラットにおいては、解剖学的にヒトのような重層扁平上皮がなく、生理的に逆流が起こらないため、ヒト食道疾患の重要な病態に関与する要素を反映しにくい。一方、体重40kgほどのミニブタの食道は全長30cmほどであり、ヒト成人の食道長約40cmにくらべても遜色なく、ヒト同様に重層扁平上皮を有し、生理的逆流も起こる上に、ヒトの上部消化管内視鏡セットを用いたESD実験を行える利点もある。
ミニブタは、生理食塩水投与群、ステロイド製剤(ケナコルト)投与群、CHST15 siRNA(北海道システムサイエンス社製、ロット番号40481638)投与群、の3群を使用した(n=1/群)。各群のミニブタを全身麻酔呼吸器管理下に、上部消化管内視鏡を経口挿入し、門歯列より15cmから25cmの間に、デュアルナイフを用いて全長5cmの全周性(100%)ESDを施行した。ESD実施直後の全周性人工潰瘍に対して、潰瘍底から周囲の全体に均等に覆うように合計20か所に、内視鏡用注射針を用いて投与溶液(生理食塩水、ステロイド製剤、CHST15 siRNA, 250nM溶液)を合計2mL注入した。
ESD後無処置であると2週間以内に食道狭窄が発現し、食物も通過しなくなるため、それ以降4週以内にミニブタが死亡することから、単回投与後の観察期間を2週間に設定し、2週間後(Day 14)に全群屠殺し、肉眼所見、並びに病理組織学的解析(マッソン染色)により、食道狭窄の程度を検討した。
結果:
生理食塩水投与群:
<肉眼所見>
食道長の短縮と完全閉塞を認めた。摂食不可の状態であった。
<病理所見>
一部未熟な再生上皮の出現を認めるものの、ESDによる人口潰瘍が残存し、潰瘍底以深の広範な線維化と筋層の挫滅を認めた。
ステロイド製剤(ケナコルト)投与群:
<肉眼所見>
食道長の短縮の程度は生理食塩水投与群と比較して軽度ではあったが、完全閉塞を認めた。摂食不可の状態であった。
<病理所見>
ESDによる人口潰瘍が残存するものの、生理食塩水投与群と比較すると、比較的成熟した再生上皮による修復所見を認めた。しかし、以深は全層性に広範な線維化を認め、その程度は生理食塩水投与群よりも強度であった。また、欠損を伴う広範囲の筋層の破壊を認め、漿膜層まで線維化が及んでいた。
CHST15 siRNA投与群:
<肉眼所見>
食道長の短縮はほぼ認めず、一部内腔の狭小化は認めるものの、狭窄・閉塞はなく、摂食可能の状態であった。
<病理所見>
ESDによる人口潰瘍はほぼ再生上皮に被覆され、ステロイド投与群と比較しても重層扁平上皮が規則正しく構築された、より成熟した再生上皮であった。以深は病的線維化はなく、上皮を支持するほぼ生理的なレベルの線維であった。さらに、筋層の障害を全く認めなかった。
考察:
ステロイド製剤投与群においては全周性ESD後の食道狭窄抑制効果はなく、これは臨床治験と同じ結果と考えられた。生理食塩水投与群と比較して、再生上皮を伴う潰瘍底の縮小傾向は認めたものの、筋層の広範な傷害を呈していた。これはステロイドの持つタンパク異化作用によるものと考えられ、臨床で認められる遅発性穿孔の原因となることが示唆された。
一方、CHST15 siRNA投与群において認められた狭窄抑制効果は、筋層をほぼ正常状態に保持したまま獲得できる効果であった。それは食道の蠕動運動すなわち嚥下機能をほぼ正常に保ったまま狭窄が抑制されていることを示すとともに、実臨床的にも極めて安全に治療できることが強く示唆された。
実臨床におけるESD後食道狭窄においては、バルーン拡張を繰り返し実施しているのが現状であり、筋層へのダメージは極力回避しなければならないため、ステロイドの複数回投与は回避すべきと考えられる。また、食道癌においては再発があり、その再発食道癌に対してもESD治療を安全に施せるようにしておくためには、筋層の保護は絶対的条件となる。繰り返す内視鏡治療を実施するためにも、ステロイド製剤に比較してCHST15 siRNAの臨床的有用性が極めて高いと考えられた。
これらの結果より、本発明のCHST15 siRNAは新規食道狭窄抑制剤として有用であることが示された。
Claims (8)
- CHST15遺伝子の発現を抑制するsiRNAを有効成分として含む、食道狭窄を治療又は予防するための医薬組成物。
- 食道狭窄が、アカラシアによる狭窄、消化性狭窄、シャッキー・リング、内視鏡治療による狭窄、好酸球性食道炎による狭窄、外科手術後狭窄、放射線療法に伴う狭窄、腐食性狭窄、及び難治性狭窄からなる群より選ばれる狭窄である、請求項1記載の医薬組成物。
- 食道狭窄が、食道の3/4周以上で生じている、請求項1又は2記載の医薬組成物。
- 食道の筋層傷害を伴なわないことを特徴とする、請求項1~3のいずれか一項記載の医薬組成物。
- ステロイドを併用しない、請求項1~4のいずれか一項記載の医薬組成物。
- 内視鏡的バルーン拡張術を併用しない又は内視鏡的バルーン拡張術の併用回数を減らした、請求項1~5のいずれか一項記載の医薬組成物。
- siRNAが、100~10,000nMで含まれる、請求項1~6のいずれか一項記載の医薬組成物。
- 単回投与又は1週間間隔で投与される、請求項1~7のいずれか一項記載の医薬組成物。
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