JP2024157057A - Compositions for treating Barrett's esophagus - Google Patents

Abstract

[Problem] The present invention aims to provide a therapeutic composition for Barrett's esophagus, more specifically, a therapeutic composition that restores Barrett's esophagus to a normal esophagus, and a method for treating Barrett's esophagus using the therapeutic composition.
The present invention relates to a composition for treating Barrett's esophagus, the composition comprising a MEK inhibitor. The present invention also relates to a composition for preventing esophageal adenocarcinoma, the composition comprising a MEK inhibitor.
[Selection diagram] None

Description

The present invention relates to a composition for treating Barrett's esophagus. More specifically, the present invention relates to a composition for treating Barrett's esophagus that promotes the regeneration of squamous epithelium, thereby restoring columnar epithelium, which is a tissue morphology characteristic of Barrett's esophagus, to squamous epithelium, which is the original tissue morphology of the esophagus.

Barrett's esophagus is a condition in which the mucosal tissue of the lower to upper esophagus changes from squamous epithelium to columnar epithelium, and is caused by gastroesophageal reflux disease (GERD). It is known that the development of Barrett's esophagus increases the risk of developing esophageal adenocarcinoma. If esophageal adenocarcinoma that develops from Barrett's esophagus progresses, the prognosis is poor. Treatments for Barrett's esophagus include resection and ablation of mucosal tissue that has changed to columnar epithelium. However, circumferential mucosal resection can cause esophageal stenosis. In ablation therapy, only the surface layer is ablated, and carcinoma can develop from the Barrett's esophageal mucosal tissue remaining in the depths. In such cases, the tumor area may be covered by the regenerated surface layer, which may delay detection. It is considered very important to restore Barrett's esophagus to its original state in order to prevent the development of esophageal adenocarcinoma, which has a poor prognosis. However, to date, no other non-invasive and effective treatments have been developed other than the surgical treatments mentioned above.

Meanwhile, the molecular mechanisms that function in the process of Barrett's esophagus progressing to esophageal adenocarcinoma have also been elucidated. Dulak et al. identified 26 mutated genes thought to be related to esophageal adenocarcinoma by mutation spectrum analysis (Non-Patent Document 1). These included, for example, TP53 , CDKN2A , SMAD4 , PIK3CA , SPG20 , TLR4 , ELMO1 and DOCK2 . Chao et al. reported that biallelic inactivation of TP53 is involved in the progression of Barrett's esophagus to esophageal adenocarcinoma (Non-Patent Document 2). In addition, Sommerer et al. found that approximately 32% of esophageal adenocarcinomas developed from Barrett's esophagus had BRAF and KRAS2 mutations, and concluded that abnormalities in the Raf/MEK/ERK (MAPK) kinase pathway are involved in the early stages of esophageal adenocarcinoma developed from Barrett's esophagus (Non-Patent Document 3).

As described above, genetic mutations that may be involved in the progression of Barrett's esophagus to esophageal adenocarcinoma are gradually being elucidated, but the molecular mechanism that causes a normal esophagus to change to Barrett's esophagus is currently unknown. As a result, there has been little progress in the development of effective non-invasive treatments other than surgical methods for the treatment of Barrett's esophagus.

Dulak et al., Nat Genet., 45:478-486 2013. Chao et al., Clin Cancer Res., 14:6988-6995 2008. Sommerer et al., Oncogene 23:554-558 2004.

In view of the above circumstances, the present invention aims to provide a composition for treating Barrett's esophagus, more specifically, a composition for treating Barrett's esophagus that restores the esophagus to a normal state, and a method for treating Barrett's esophagus using the composition.

The inventors found that when a MEK inhibitor was administered to a rat model of gastroesophageal reflux disease, the length of Barrett's esophagus was shortened and squamous epithelium was regenerated within the erosions of Barrett's esophagus.
MEK (MAPK/ERK kinase) is a kinase that phosphorylates and activates ERK (extracellular signal-regulated kinase) in the MAPK signaling pathway. Numerous active mutations and gene amplifications of Ras, Raf, MEK, and other components of the MAPK signaling pathway have been reported in many cancers. The inventors have now found for the first time that factors constituting the MAPK signaling pathway are also involved in the process in which a normal esophagus changes to Barrett's esophagus, and have completed the present invention.

That is, the present invention relates to the following (1) to (5).
(1) A composition for treating Barrett's esophagus, comprising a MEK inhibitor.
(2) The composition according to (1) above, wherein the MEK inhibitor is one or more selected from the group consisting of trametinib, binimetinib, selumetinib, cobimetinib, pimasertib, refametinib, ulixertinib, abemaciclib, U0126, PD184352 (CI-1040), PD98059 and BIX 02189.
(3) A composition for preventing esophageal adenocarcinoma, comprising a MEK inhibitor.
(4) The composition according to (3) above, characterized in that the MEK inhibitor is one or more selected from the group consisting of trametinib, binimetinib, selumetinib, cobimetinib, pimasertib, refametinib, ulixertinib, abemaciclib, U0126, PD184352 (CI-1040), PD98059 and BIX 02189.
(5) The composition according to (3) or (4) above, which is administered to a patient suffering from Barrett's esophagus.
In this specification, the symbol "to" indicates a numerical range including both the values on either side of it.

The present invention provides a composition for treating Barrett's esophagus and a non-invasive method for treating Barrett's esophagus using the composition.

Effect of MEK inhibitor on the length of Barrett's esophagus. The results of measuring the length of Barrett's esophagus in "placebo-treated rats" (group administered placebo after reflux surgery) and "MEK inhibitor-treated rats" (group administered MEK inhibitor after reflux surgery) are shown. Therapeutic effect of MEK inhibitor on Barrett's esophagus (1). Esophageal mucosal tissue from a MEK inhibitor-treated rat was stained with hematoxylin and eosin and observed under a microscope. Regeneration of squamous epithelium is observed in the area enclosed by the dashed line. Therapeutic effect of MEK inhibitor on Barrett's esophagus (2). Esophageal tissue from a placebo-treated rat (A) and esophageal tissue from a MEK inhibitor-treated rat (B) were stained with hematoxylin and eosin and observed under a microscope. In A and B, columnar epithelium was observed in the area surrounded by the dashed line, and squamous epithelium was observed in the area surrounded by the solid line. Confirmation of the effect of MEK inhibitor. The results of immunostaining esophageal tissue from a "placebo-treated rat" (A) and esophageal tissue from a "MEK inhibitor-treated rat" (B) with an anti-pERK antibody are shown.

Barrett's esophagus is an esophagus in which at least a portion of the mucosal tissue from the lower to upper esophagus changes from squamous epithelium to columnar epithelium, and it often develops in association with gastroesophageal reflux disease. The present inventors have shown that when a MEK inhibitor was administered to a rat model of gastroesophageal reflux disease, the length of Barrett's esophagus was shortened and that squamous epithelium was regenerated in the erosions of Barrett's esophagus, restoring Barrett's esophagus to a normal esophagus. In other words, the present inventors have shown for the first time that a MEK inhibitor is effective as a therapeutic agent for Barrett's esophagus.

MEK (MAPK/ERK kinase) is a dual-specificity serine/threonine kinase that phosphorylates serine and threonine residues of ERK (extracellular signal-regulated kinase) 1 and ERK2 in the RAS/RAF/MEK/ERK signaling pathway, activating ERK1/2. In normal cells, Ras activated by extracellular growth factor stimulation binds to Raf to promote its activation, Raf phosphorylates and activates MEK, and MEK then phosphorylates and activates ERK. Activated ERK translocates from the cytoplasm to the nucleus, where it activates transcription factors such as ELK, CREB, c-Myc, c-Fos, and Sp-1 to induce gene expression. The pathway in which information for gene expression is transmitted by the phosphorylation cascade from RAS to ERK is called the RAS/RAF/MEK/ERK signaling pathway. There are two subtypes of MEK: MEK1 and MEK2. "MEK" as described herein includes both MEK1 and MEK2.

The first embodiment is a composition for treating Barrett's esophagus, which contains a MEK inhibitor (hereinafter also referred to as the "therapeutic composition according to this embodiment"). "Treatment" of Barrett's esophagus means returning Barrett's esophagus to its original esophagus, specifically returning the mucosal tissue of Barrett's esophagus, which is mainly composed of columnar epithelium, to the original esophageal mucosal tissue composed of squamous epithelium, or regenerating mucosal tissue composed of squamous epithelium in the esophagus.

MEK inhibitors in this embodiment are not particularly limited, but include, for example, trametinib (CAS: 871700-17-3), binimetinib (CAS: 606143-89-9), selumetinib (CAS: 606143-52-6), cobimetinib (CAS: 934660-93-2), pimasertib (CAS: 1236699-92-5), and refametinib. (CAS: 923032-37-5), ulixertinib (CAS: 869886-67-9), abemaciclib (CAS: 1231929-97-7), U0126 (CAS: 1173097-76-1), PD184352 (CI-1040) (CAS: 212631-79-3), PD98059 (CAS: 167869-21-8) and BIX 02189 (CAS: 1094614-85-3). In addition to these compounds, other examples include antibodies and peptide aptamers that suppress or inhibit MEK activity, as well as substances that suppress or inhibit the expression of the MEK gene, such as siRNA and miRNA.

As described above, MEK inhibitors have the effect of restoring Barrett's esophagus to a normal esophagus, and can prevent the progression of Barrett's esophagus to esophageal adenocarcinoma.
The second embodiment is a composition for preventing esophageal adenocarcinoma, which comprises a MEK inhibitor (hereinafter also referred to as the "preventive composition according to this embodiment"). The preventive composition according to this embodiment is particularly effective in preventing the onset of esophageal adenocarcinoma in individuals with Barrett's esophagus.
The therapeutic composition and preventive composition according to this embodiment (hereinafter also referred to as "the composition, etc. according to this embodiment") may contain, in addition to the MEK inhibitor as an active ingredient, one or more formulation additives, pharma- ceutically acceptable carriers, etc., and may also contain other drugs, etc. that are effective for improving the pathology of Barrett's esophagus.

The dosage forms of the composition according to this embodiment include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents when used. Tablets and granules may be coated by known methods. Injections are prepared by dissolving the active ingredient in water, but may also be dissolved in saline or glucose solution as necessary, and buffers and preservatives may be added.

Preparations for oral or parenteral administration are provided in any formulation form. For example, the preparation may be prepared as a therapeutic agent or composition for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or liquids, or as a therapeutic agent or composition for parenteral administration in the form of an injection, drip infusion, transdermal absorption agent, transmucosal absorption agent, nasal drops, inhalants, suppositories, or the like for intravenous, intramuscular, or subcutaneous administration. Injections and drip infusions may be prepared in a powder form, such as a lyophilized form, and dissolved in an appropriate aqueous medium, such as physiological saline, when used.

The type of formulation additive used in the production of the composition according to this embodiment, the ratio of the formulation additive to the active ingredient, or the production method of the therapeutic composition and the preventive composition can be appropriately selected by a person skilled in the art depending on the form. The formulation additive can be an inorganic or organic substance, or a solid or liquid substance, and can generally be blended at between 1% and 90% by weight of the active ingredient. Specific examples of additives for formulations include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

To produce solid preparations for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to produce powders, or, if necessary, binders such as sucrose, hydroxypropylcellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and calcium carboxymethylcellulose are added and then wet or dry granulated to produce granules. To produce tablets, these powders and granules can be compressed as is or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets can be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylic acid-methyl methacrylate polymer to produce enteric-coated preparations, or coated with ethylcellulose, carnauba wax, hardened oil, and the like to produce sustained-release preparations. To produce capsules, the powders or granules can be filled into hard capsules, or the active ingredient can be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like and then coated with a gelatin membrane to produce soft capsules.

To produce an injection, the active ingredient is dissolved in distilled water for injection, if necessary, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, and an isotonic agent such as sodium chloride or glucose, and then sterile filtered and filled into an ampule, or mannitol, dextrin, cyclodextrin, gelatin, etc. are added and the mixture is vacuum freeze-dried to produce an injection that is dissolved when used. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. can be added to the active ingredient and emulsified in water to produce an emulsion for injection.

To manufacture a rectal preparation, the active ingredient is dissolved in a suppository base such as cacao butter, tri-, di-, or monoglycerides of fatty acids, or polyethylene glycol, and poured into a mold and cooled, or the active ingredient is dissolved in polyethylene glycol, soybean oil, or the like, and then coated with a gelatin film.

The dosage and frequency of administration of the composition etc. of this embodiment are not particularly limited, and can be appropriately selected at the discretion of a physician depending on conditions such as the purpose of treatment and prevention, the type of disease, and the weight and age of the patient.
In general, the daily oral dose for adults is about 0.01-1000 mg (weight of active ingredient), and can be administered once a day or in divided doses, or every few days. When used as an injection, it is desirable to administer 0.001-100 mg (weight of active ingredient) to adults continuously or intermittently.

The compositions of the present invention may be prepared as sustained release formulations, such as implants and microencapsulated delivery systems, using pharma- ceutically acceptable carriers that can prevent immediate removal from the body. Such carriers include biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Suspensions of liposomes can also be used as pharma-ceutically acceptable carriers. Liposomes can be prepared as lipid compositions, including but not limited to phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanol (PEG-PE), filtered through a filter of appropriate pore size to a size suitable for use, and purified by reverse phase evaporation.

The composition according to the present embodiment may be provided in the form of a kit together with instructions on the administration method, etc. The drug contained in the kit is supplied in a container made of a material that effectively maintains the activity of the drug for a long period of time, does not adsorb to the inside of the container, and does not alter the components. For example, a sealed glass ampoule may contain a buffer or the like sealed in the presence of a neutral, non-reactive gas such as nitrogen gas.
The kit may also include an instruction manual. The instruction manual may be provided to the user in the form of a printed sheet of paper or in the form of an electromagnetically readable medium such as a CD-ROM or DVD-ROM.

A third embodiment is a method for treating Barrett's esophagus, comprising administering to a patient the therapeutic composition according to this embodiment (first embodiment).
Here, "treatment" refers to restoring Barrett's esophagus to its original state, specifically, returning the mucosal tissue of Barrett's esophagus, which is mainly composed of columnar epithelium, to the original esophageal mucosal tissue composed of squamous epithelium, or regenerating mucosal tissue composed of squamous epithelium.

The fourth embodiment is a method for preventing esophageal adenocarcinoma, comprising administering to a patient the preventive composition according to this embodiment (the second embodiment).
Here, "prevention" means preventing the onset of esophageal adenocarcinoma in a mammal at risk of developing the disease, and in particular, means preventing the onset of esophageal adenocarcinoma in a mammal that contains tissue made of columnar epithelium characteristic of Barrett's esophagus in at least a portion of its esophageal mucosal tissue.

The "mammal" that is the subject of the method for treating Barrett's esophagus and the method for preventing esophageal adenocarcinoma according to the present embodiment means any animal classified as a mammal, and is not particularly limited, and includes, for example, humans, pet animals such as dogs, cats, rabbits, and ferrets, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is humans.

Where this specification is translated into English and contains the singular words "a,""an," and "the," these are intended to include the plural as well as the singular, unless the context clearly indicates otherwise.
The present invention will be further described below with reference to examples. However, these examples are merely illustrative of embodiments of the present invention and are not intended to limit the scope of the present invention.

1. Gastroesophageal reflux disease rat model A rat gastroesophageal reflux disease model was created by surgical operation according to previous reports (Miyashita et al., Dig Dis Sci 56:1309-1314; Miyashita et al., Surgery 164:49-55 2018).
In this example, 6-week-old male Sprague-Dawley rats (Japan CLEA) were used. After acclimatization for 2 weeks, the rats were kept at 20-22°C, 30-50% humidity, and a 12-hour light-dark cycle. After overnight fasting, the rats were anesthetized with diethyl ether, and then the abdomen was opened. The esophagogastric junction was ligated, and the esophagus was cut on the esophageal side. The jejunum 5 cm anal side from the Treitz ligament was elevated to the esophageal stump using a loop. The esophageal stump and the elevated jejunum were anastomosed end-to-side, and treatment was performed to allow gastric juice and intestinal juice to flow back into the esophagus. It has been reported that Barrett's esophagus was observed in almost 100% of rats that underwent the above surgical procedure (Miyashita et al., Dig Dis Sci 56:1309-1314). Seventeen weeks after surgery, a sustained-release pellet containing trametinib or a placebo pellet (without trametinib) was subcutaneously inserted. Twenty weeks after surgery, the rats were euthanized and histological observations of the esophagus were performed. The sustained-release pellet of trametinib contains 3.4 mg of trametinib and releases trametinib over a period of 28 days. Trametinib was purchased from Selleck, and the production of the sustained-release pellets was outsourced to Innovative Research of America.
Hereinafter, rats that did not undergo any surgical procedures or other treatment are referred to as "control rats" (n = 3), rats that had a placebo pellet inserted subcutaneously are referred to as "placebo-treated rats" (n = 12), and rats that had a trametinib-containing pellet inserted subcutaneously are referred to as "MEK inhibitor-treated rats" (n = 12).

2. Histological Observation of the Esophagus Measurement of the length of the Barrett's esophagus segment exhibiting histological characteristics of columnar epithelium revealed that the length of the Barrett's esophagus in MEK inhibitor-treated rats was significantly shortened compared with that in placebo-treated rats (Fig. 1).
In addition, in the esophageal tissue of MEK inhibitor-treated rats, regeneration of squamous epithelium was observed in the erosion (area enclosed by dashed line in Fig. 2). On the other hand, no regeneration of squamous epithelium was observed in the esophageal tissue of placebo-treated rats. Comparing the esophageal tissue images of placebo-treated rats and MEK inhibitor-treated rats, columnar epithelium was observed in the esophageal tissue of placebo-treated rats (area enclosed by dashed line in Fig. 3A), whereas regenerated squamous epithelium was observed in addition to columnar epithelium in the esophageal tissue of MEK inhibitor-treated rats (area enclosed by dashed line in Fig. 3B, columnar epithelium; area enclosed by solid line, squamous epithelium).
These results confirmed that MEK inhibitors shorten the length of Barrett's esophagus and promote the regeneration of squamous epithelium in esophageal mucosal tissue.

3. Confirmation of the effect of MEK inhibitors Next, we confirmed whether the MEK inhibitor trametinib inhibited MEK kinase activity in the administered esophageal tissue. MEK is a kinase that phosphorylates and activates ERK in the MAPK signaling pathway. MEK inhibitors such as trametinib inhibit MEK kinase activity. Therefore, the function of MEK inhibitors in esophageal tissue can be confirmed by the presence or absence of ERK phosphorylation as an indicator.
Esophageal tissues excised from MEK inhibitor-treated and placebo-treated rats were fixed with 4% paraformaldehyde, and paraffin blocks were prepared and paraffin sections were prepared. The prepared sections were attached to slides and then air-dried. After deparaffinization with Histoclear (registered trademark) (Cosmo Bio Co., Ltd.), the sections were hydrated with 100% ethanol, blocked with 3% hydrogen peroxide, and then antigen activation was performed using microwaves. The sections were washed with PBS and then blocked by leaving them in 3% goat serum at room temperature for 1 hour. After washing the slides with PBS, anti-pERK antibody (Abcam) solution was added and incubated overnight at 4°C. After washing with PBS, biotin-labeled secondary antibody (Vector) was added to the slides by the ABC method and incubated at room temperature for 60 minutes. After washing the slides with PBS, HRP-labeled avidin-biotin complex was added to the slides and incubated at room temperature for 30 minutes. After washing the slides with PBS, the staining was carried out using DAB (3,3'-diaminobenzidine tetrahydrochloride) as a substrate for HRP. The immunostained slides were observed under a confocal microscope.

Phosphorylated ERK stained with anti-pERK antibody was distributed in the esophageal tissue from placebo-treated rats (Fig. 4A), whereas the staining intensity was reduced in the esophageal tissue from MEK inhibitor-treated rats (Fig. 4B). These results confirmed that MEK kinase activity was inhibited by the MEK inhibitor in the esophageal tissue from MEK inhibitor-treated rats.

These results indicate that MEK inhibitors have the effect of shortening the length of Barrett's esophagus, which is made up of columnar epithelium, and promoting the regeneration of squamous epithelium.

The therapeutic composition of the present invention is believed to restore the mucosal tissue of Barrett's esophagus to normal esophageal mucosal tissue and further exerts the effect of preventing the progression to esophageal adenocarcinoma. Therefore, the present invention is expected to be used in the medical field (especially in the field of cancer treatment).

Claims (5)

A composition for treating Barrett's esophagus, the composition comprising a MEK inhibitor. The composition according to claim 1, characterized in that the MEK inhibitor is one or more selected from the group consisting of trametinib, binimetinib, selumetinib, cobimetinib, pimasertib, refametinib, ulixertinib, abemaciclib, U0126, PD184352 (CI-1040), PD98059 and BIX 02189. A composition for preventing esophageal adenocarcinoma, the composition comprising a MEK inhibitor. The composition according to claim 3, characterized in that the MEK inhibitor is one or more selected from the group consisting of trametinib, binimetinib, selumetinib, cobimetinib, pimasertib, refametinib, ulixertinib, abemaciclib, U0126, PD184352 (CI-1040), PD98059 and BIX 02189. The composition according to claim 3 or 4, which is administered to a patient suffering from Barrett's esophagus.