Pharmaceutical composition for Hailey-Hailey disease and its uses

Abstract

This invention provides a novel pharmaceutical composition for Hailey-Hailey disease. [Solution] The pharmaceutical composition for Hailey-Hailey disease of the present invention contains 5-methyl-2-(1-piperazinyl)benzenesulfonic acid. The compound is used as an active ingredient in the form of its anhydride, salt, hydrate or solvate, or hydrate or solvate of its salt. The hydrate is, for example, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate. The pharmaceutical composition of the present invention can treat or prevent Hailey-Hailey disease.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for Hailey-Hailey disease and a method for treating or preventing Hailey-Hailey disease.

Background Art

[0002] Hailey-Hailey disease is a kind of skin disease, in which blister-like eruptions (vesicles) frequently appear in intertriginous areas such as the neck, axilla, and groin due to acantholysis in the epidermis (Non-Patent Document 1). The vesicles are accompanied by itching, and when the condition worsens, the vesicles rupture and the mucosa becomes exposed, forming an erosive state. In these states, crusts, pustules, pigmentation, or secondary infections are added, forming a situation similar to impetigo and generating a bad odor. And because of the possibility of chronicity and recurrence, it is accompanied by a decline in the quality of life (hereinafter also referred to as QOL) of patients, and social handicaps are also feared.

[0003] Currently, there is no fundamental treatment method recommended in the guidelines for Hailey-Hailey disease, and disease management and symptom management by avoiding exacerbating factors are the mainstays. For example, patients may receive lifestyle guidance to reduce factors that exacerbate symptoms, such as friction, sweating, and sunlight exposure. At the mild stage, treatment with steroid ointments or vitamin D preparations may be administered, and when the condition worsens, treatment with immunosuppressants or retinoids may be administered. Also, laser treatment and surgical peeling may be performed.

[0004] Regarding treatment methods such as administering retinoids, steroids, vitamin D preparations, etc., there are also reports of side effects, and there is no treatment that is safe and provides a consistent effect. Therefore, for Hailey-Hailey disease, there remains a high unmet medical need for a safe and effective treatment.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] Therefore, the present invention aims to provide a novel pharmaceutical composition for Hailey-Hailey disease, and a method for treating or preventing it. [Means for solving the problem]

[0007] The pharmaceutical composition for Hailey-Hailey disease of the present invention contains 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (5-methyl-2-(piperazin-1-yl)benzenesulfonic acid).

[0008] The present invention provides a method for treating or preventing Hailey-Hailey disease, comprising the step of administering 5-methyl-2-(1-piperazinyl)benzenesulfonic acid to a subject.

[0009] The present invention is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid for use in the treatment or prevention of Hailey-Hailey disease.

[0010] The present invention relates to 5-methyl-2-(1-piperazinyl)benzenesulfonic acid in the manufacture of pharmaceutical compositions for Hailey-Hailey disease. [Effects of the Invention]

[0011] According to the pharmaceutical composition of the present invention, by including 5-methyl-2-(1-piperazinyl)benzenesulfonic acid, it is possible to treat or prevent Hailey-Hailey disease. [Brief explanation of the drawing]

[0012] [Figure 1A]Figure 1A is a graph showing the relationship between the administration of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate and the expression of differentiation marker molecules in an in vitro HHD 3D cultured epidermal model in Example 1. The vertical axis represents the relative gene expression level relative to the gene expression level in keratinocytes on Day 0. [Figure 1B] Figure 1B is a graph showing the relationship between the administration of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate and the expression of differentiation marker molecules in an in vitro HHD 3D cultured epidermal model in Example 1. The vertical axis represents the suppression rate of the increase in gene expression of differentiation markers due to ATP2C1 siRNA treatment. [Figure 2] Figure 2 is a graph showing the relationship between the administration of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate and cell adhesion in an in vitro epidermal cell adhesion disorder model in Example 2. [Modes for carrying out the invention]

[0013] The present invention can be exemplified by the following embodiments. [1] A pharmaceutical composition for Hailey-Hailey disease comprising 5-methyl-2-(1-piperazinyl)benzenesulfonic acid. [2] The pharmaceutical composition according to [1], wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydride, its salt, its hydrate or solvate, or its hydrate or solvate. [3] The pharmaceutical composition according to [1], wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydrous, its salt, its hydrate, or its salt hydrate. [4] The pharmaceutical composition according to [2] or [3], wherein the hydrate is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate. [5] A pharmaceutical composition according to any one of [1] to [4] that suppresses acantholysis in Hailey-Hailey disease. [6] The pharmaceutical composition according to any one of [1] to [5], which improves the IGA score in Hailey-Hailey disease. [7] The pharmaceutical composition according to any one of [1] to [6], which suppresses or improves at least one selected from the group consisting of lesioned skin area, itching, pain, and odor in Hailey-Hailey disease. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the dosage of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is 1 to 1000 mg / day, and the dosage is the amount converted as the anhydride of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid.

[0014] [9] A method for treating or preventing Hailey-Hailey disease, which includes the step of administering 5-methyl-2-(1-piperazinyl)benzenesulfonic acid to a subject.

[10] The method according to [9], wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydride, its salt, its hydrate or solvate, or the hydrate or solvate of its salt.

[11] The method according to [9], wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydride, its salt, its hydrate, or the hydrate of its salt.

[12] The pharmaceutical composition according to

[10] or

[11] , wherein the hydrate is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate.

[13] The method according to any one of [9] to

[12] , wherein the administration method is oral administration.

[14] The method according to any one of [9] to

[13] , wherein the dosage per day is 1 to 1000 mg / day, and the dosage is the amount converted as the anhydride of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid.

[15] The method according to any one of [9] to

[14] , wherein the number of administrations per day is 1 to  3 times.

[16] The method according to any one of [9] to

[15] , wherein the administration interval is daily administration.

[0015]

[17] 5-Methyl-2-(1-piperazinyl)benzenesulfonic acid for use in the treatment or prevention of Hailey-Hailey disease.

[18] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[17] , which is an anhydride thereof, a salt thereof, a hydrate or solvate thereof, or a hydrate or solvate of a salt thereof.

[19] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[17] , which is an anhydride thereof, a salt thereof, a hydrate thereof, or a hydrate of a salt thereof.

[20] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[18] or

[19] , wherein the hydrate is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate.

[21] 5-Methyl-2-(1-piperazinyl)benzenesulfonic acid in the manufacture of a pharmaceutical composition for Hailey-Hailey disease.

[22] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[21] , which is an anhydride thereof, a salt thereof, a hydrate or solvate thereof, or a hydrate or solvate of a salt thereof.

[23] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[21] , which is an anhydride thereof, a salt thereof, a hydrate thereof, or a hydrate of a salt thereof.

[24] The 5-methyl-2-(1-piperazinyl)benzenesulfonic acid according to

[22] or

[23] , wherein the hydrate is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate.

[0016]

[25] A pharmaceutical composition for acantholysis, comprising 5-methyl-2-(1-piperazinyl)benzenesulfonic acid.

[26] The pharmaceutical composition according to

[25] , wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydride, its salt, its hydrate or solvate, or its hydrate or solvate.

[27] The pharmaceutical composition according to

[25] , wherein the 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is its anhydrous, its salt, its hydrate, or its salt hydrate.

[28] The pharmaceutical composition according to

[26] or

[27] , wherein the hydrate is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate.

[0017] In this specification, "treatment of a disease" includes, for example, the meaning of curing a disease, achieving remission of a disease, alleviating a disease, or suppressing the progression of a disease, and also includes, for example, the meaning of treating symptoms caused by the disease (e.g., curing, achieving remission, alleviating, or suppressing symptoms). "Prevention of a disease" includes, for example, the meaning of preventing contracting a disease, preventing the onset of a disease, or preventing the recurrence of a disease, and also includes, for example, the meaning of preventing symptoms caused by the disease (e.g., preventing the onset of symptoms or preventing the recurrence of symptoms). In this specification, "treatment or prevention of a disease" can also be expressed as, for example, the suppression of a disease, and "treatment or prevention of symptoms caused by a disease" can also be expressed as, for example, the suppression of symptoms caused by a disease.

[0018] The epidermis of the skin is typically composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale.

[0019] In this specification, treatment refers to, for example, a procedure performed on a subject diagnosed by a physician as having a disease or symptoms of a disease. In this specification, prevention refers to, for example, a procedure performed on a subject who does not have a disease or symptoms of a disease, and is intended to prevent the onset of the disease or symptoms of a disease. The subject may also be referred to as a test subject or subject, and may be a human (patient) or a non-human animal (sick animal). In this specification, the term "patient" may also include, for example, a non-human animal that is sick, and can be read as "sick animal". Furthermore, if the subject does not have the disease or symptoms of the disease in question, it may also be referred to as a healthy person (healthy human or healthy non-human animal) with respect to the disease or symptoms of the subject.

[0020] The present invention will be described below with specific examples, but the present invention is not limited to these examples. Unless otherwise specified, each embodiment described below can be used interchangeably with one another. Hereinafter, Hailey-Hailey disease will also be referred to as HHD.

[0021] [Medicinal composition for HHD] The pharmaceutical composition for HHD of the present invention is characterized by containing 5-methyl-2-(1-piperazinyl)benzenesulfonic acid. The pharmaceutical composition for HHD of the present invention is characterized by containing the above compound, but other components and conditions are not particularly limited.

[0022] As described above, the pharmaceutical composition for HHD of the present invention can treat or prevent HHD. The pharmaceutical composition for HHD of the present invention may be used, for example, for therapeutic purposes, for preventive purposes, or for both therapeutic and preventive purposes. Hereinafter, the term "treatment / prevention" can be interpreted as meaning treatment, prevention, or treatment and prevention. The pharmaceutical composition for HHD of the present invention allows for safe treatment / prevention with fewer side effects, for example.

[0023] In this specification, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid is referred to as MPBS. The form of MPBS is not limited and may include MPBS anhydride (also called MPBS anhydride), MPBS salt (also called MPBS salt), MPBS hydrate or hydrate of the MPBS salt (each also referred to as MPBS hydrate), and MPBS solvate or solvate of the MPBS salt (each also referred to as MPBS solvate). These are collectively referred to as the MPBS compounds of the present invention. The MPBS compounds contained in the pharmaceutical composition of the present invention may be, for example, MPBS anhydride, MPBS salt, MPBS hydrate, or MPBS solvate, and may contain only one of these, or two or more of these. The pharmaceutical composition of the present invention preferably contains the MPBS compounds as an active ingredient.

[0024] The type of MPBS salt is not particularly limited and includes, for example, alkali metal salts, alkaline earth metal salts, amphoteric element salts, amine salts, inorganic acid salts, or organic acid salts. Examples of alkali metal salts include sodium salts or potassium salts, examples of alkaline earth metal salts include magnesium salts or calcium salts, and examples of amphoteric element salts include aluminum salts. Examples of amine salts include lower alkylamine salts such as triethylamine salt; hydroxy lower alkylamine salts such as 2-hydroxyethylamine salt, bis-(2-hydroxyethyl)amine salt, tris(hydroxymethyl)aminomethane salt, or N-methyl-D-glucamine salt; cycloalkylamine salts such as dicyclohexylamine salt; benzylamine salts such as N,N-dibenzylethylenediamine salt; or dibenzylamine salts. Examples of the inorganic salt include hydrochloride, hydrobromide, sulfate, or phosphate, and examples of the organic salt include fumarate, succinate, oxalate, or lactate.

[0025] The type of MPBS hydrate is not particularly limited, and is, for example, a monohydrate. The type of MPBS solvate is not particularly limited. The solvent that can form the MPBS solvate is not particularly limited, and is, for example, a non-aqueous solvent, and specific examples include alcohols such as methanol, ethanol, or isopropyl alcohol, acetone, ethyl acetate, or methylene chloride.

[0026] In the HHD pharmaceutical composition of the present invention, the MPBS is preferably the anhydrous MPBS or the hydrated MPBS, more preferably the hydrated MPBS, and more specifically, the monohydrate MPBS.

[0027] The aforementioned MPBS compounds are known compounds. These MPBS compounds can be synthesized, for example, by the methods described in Japanese Patent Publication No. 3-7263, Japanese Patent Publication No. 9-221479, European Patent Application Publication No. 390654, European Patent Application Publication No. 779283, U.S. Patent Publication No. 5053409, and U.S. Patent Publication No. 5990113, and are compounds readily available to those skilled in the art.

[0028] The aforementioned MPBS compounds may be substituted with compounds represented by the following general formula (I) or their salts, or their hydrates or solvates, as described in International Publication No. WO03 / 011296. [ka]

[0029] In the formula, R1 is a hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a C1-C4 halogenated alkyl group, a halogen atom, or a C6-C 12 R2 represents an aryl group; R2 is a hydrogen atom, a C1-C6 alkyl group, or a C7-C 12 The aralkyl group is an aralkyl group, and the aralkyl group may have one or more substituents selected from the group consisting of a cyano group, a nitro group, a C1-C6 alkoxy group, a halogen atom, a C1-C6 alkyl group, and an amino group, where n represents an integer from 1 to 4.

[0030] Examples of C1-C6 alkyl groups defined by R1 in the general formula (I) include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, and isohexyl groups. Examples of C3-C7 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups. Examples of C1-C4 halogenated alkyl groups include trifluoromethyl, trifluoroethyl, and pentafluoroethyl groups. Examples of halogen atoms include fluorine, chlorine, and bromine atoms. C6-C 12 Examples of aryl groups include the phenyl group and the naphthyl group.

[0031] Preferred examples of R1 include, for example, a hydrogen atom, a C1-C6 alkyl group, a C5-C6 cycloalkyl group, a trifluoromethyl group, a halogen atom, or a phenyl group. More preferred examples include, for example, a C1-C3 alkyl group, a cyclohexyl group, a trifluoromethyl group, a chlorine atom, a bromine atom, or a phenyl group. Even more preferred examples include a methyl group or a propyl group, with a methyl group being particularly preferred.

[0032] Examples of C1-C6 alkyl groups defined in R2 include the alkyl groups defined in R1 above. C7-C 12Examples of aralkyl groups include benzyl, phenethyl, and naphthylmethyl groups. These aralkyl groups may have one or more substituents selected from the group consisting of C1-C6 alkoxy groups such as cyano, nitro, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, tert-pentyloxy, and hexyloxy groups; halogen atoms as defined by R1 above; alkyl groups as defined by R1 above; and amino groups.

[0033] Preferred examples of R2 include, for example, a hydrogen atom, a C1-C3 alkyl group, and a C7-C 12 Examples include aralkyl groups, and the aralkyl group may have one or more substituents selected from C1-C3 alkyl groups, C1-C3 alkoxy groups, and halogen atoms. More preferred examples include hydrogen atoms, C7-C 12 An aralkyl group is an example, and the aralkyl group may have one or more substituents selected from C1-C3 alkoxy groups, and is particularly preferably a hydrogen atom. Furthermore, in the above general formula (I), n is preferably 2.

[0034] In the HHD pharmaceutical composition of the present invention, the MPBS may, for example, be in an ionized form. If the HHD pharmaceutical composition of the present invention is, for example, a liquid as described later, and contains an aqueous solvent, a non-aqueous solvent, or a mixture thereof, the MPBS may be ionized regardless of whether it is an anhydrous, salt, hydrate, or solvate. The molecular form of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) is, for example, represented by the following formula (II), and can become an ionized molecular species in a proton solvent such as water. Specifically, the molecular species may take the form of a monovalent cation of the following formula (IIIa), a twin ion (twitter) having a positive and negative charge of the following formula (IIIb), or a monovalent anion of the following formula (IIIc). The monovalent cation molecular species can, for example, form a salt with an acid (anion), and the monovalent anion can, for example, form a salt with a base (cation).

[0035] [ka] [ka]

[0036] The pharmaceutical composition for HHD of the present invention can be used, for example, for the treatment or prevention of HHD, and the treatment and prevention are, for example, as described above. The pharmaceutical composition for HHD of the present invention can be rephrased as, for example, a pharmaceutical composition used for the symptoms of HHD, and more specifically, as a pharmaceutical composition used for the treatment or prevention of the symptoms of HHD. Furthermore, the treatment or prevention of HHD can also be said to be, for example, the suppression of HHD.

[0037] The subject of this invention is HHD, a disease characterized by acantholysis as a pathological symptom. Acantholysis is a condition in which keratinocytes and other cells lose intercellular adhesion, for example, in the spinous layer of the skin, and can be observed as clefts within the epidermis or as blister formation. Furthermore, acantholytic cells (Tsanck cells), which are floating, round keratinocytes with concentrated nuclei, can be observed within the blisters. In the skin lesions of HHD patients, for example, abnormal differentiation of keratinocytes is known to cause acantholysis. Acantholysis can be observed, for example, by taking a sample of the diseased skin from a patient and following a method known to those skilled in the art.

[0038] The pharmaceutical composition for HHD of the present invention can, for example, suppress the pathological symptoms of HHD. The pathological symptoms to be suppressed are, for example, acantholysis, and may also include abnormal keratinization in addition to acantholysis. That is, the pharmaceutical composition for HHD of the present invention may suppress acantholysis, or it may suppress both acantholysis and abnormal keratinization. According to the pharmaceutical composition for HHD of the present invention, for example, acantholysis can be suppressed, and both acantholysis and abnormal keratinization can be suppressed.

[0039] Abnormal keratinization refers to the individual keratinization of keratinocytes in the stratum spinosum of the skin, resulting in abnormal keratinocytes. In the case of HHD, this can be observed as granulosi or acantholytic cells with concentrated nuclei. Abnormal keratinization can be observed, for example, by taking a sample of the patient's diseased skin and following a method known to those skilled in the art.

[0040] According to the pharmaceutical composition for HHD of the present invention, for example, it is possible to improve a patient's IGA (Investigator's Global Assessment) score. Here, the IGA score is not particularly limited. The IGA score is generally classified into multiple stages, with higher scores indicating more severe symptoms.

[0041] The pharmaceutical composition for HHD of the present invention can, for example, suppress or improve the physical clinical symptoms caused by HHD, specifically, for example, at least one selected from the group consisting of lesional skin area, itching, pain, and odor in HHD. According to the pharmaceutical composition for HHD of the present invention, for example, by suppressing or improving the aforementioned pathological symptoms, the physical clinical symptoms exemplified herein can be suppressed or improved.

[0042] The lesional skin area is, for example, the area where erythema with clusters of small vesicles is observed. This erythema is similar to impetigo, where vesicles rupture and become erosions, accompanied by crusting, pustules, hyperpigmentation, or secondary infection. The HHD pharmaceutical composition of the present invention can suppress or improve the lesional skin area compared to before administration to, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.

[0043] Pruritus can be evaluated, for example, by using a numerical rating scale for pruritus, where the pruritus felt by the subject is assessed. In a numerical rating scale, for example, 0 indicates no pruritus and 10 indicates the worst possible pruritus the subject can imagine, and the subject uses this scale to evaluate pruritus. The pharmaceutical composition for HHD of the present invention can reduce the post-administration value of the numerical rating scale for pruritus by, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more, compared to the pre-administration value.

[0044] Pain can be evaluated, for example, by using a numerical rating scale for pain, where the pain felt by the subject is assessed. In a numerical rating scale, for example, 0 indicates no skin pain, and 10 indicates the worst skin pain the subject can imagine, and the subject uses this scale to evaluate skin pain. The HHD pharmaceutical composition of the present invention can reduce the post-administration value of the numerical rating scale for pain by, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more compared to the pre-administration value.

[0045] Odor can be evaluated, for example, by using a numerical rating scale for odor, based on the odor perceived by the subject. In a numerical rating scale, for example, 0 indicates no odor, and 10 indicates the worst odor the subject can imagine, and the subject uses this scale to evaluate the odor. The pharmaceutical composition for HHD of the present invention can reduce the post-administration value of the numerical rating scale for odor by, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more, compared to the pre-administration value.

[0046] The pharmaceutical composition for HHD of the present invention improves, for example, at least one selected from the group consisting of PGIS (Patient Global Impression of Severity), PGIC (Patient Global Impression of Change), CGIS (Clinician Global Impression of Severity), CGIC (Clinician Global Impression of Change), DLQI (Dermatology Life Quality Index), and Skindex-29 in the HHD.

[0047] (1) The PGIS is a single questionnaire that evaluates the subject's overall impression of severity using a score, and the pharmaceutical composition for HHD of the present invention can improve the PGIS and improve the score. (2) PGIC is a single questionnaire that evaluates the degree of overall improvement in a subject's health status using a score, and the HHD pharmaceutical composition of the present invention can improve PGIC and improve the score. (3) The CGIS is a single questionnaire in which a physician evaluates the overall impression of a subject's severity using a score, and the HHD pharmaceutical composition of the present invention can improve the CGIS and improve the score. (3) CGIC is a single questionnaire used by physicians to score the degree of overall improvement in a subject's health condition, and the HHD pharmaceutical composition of the present invention can improve CGIC and improve the score. (4) The DLQI is a questionnaire in which subjects evaluate each question regarding their quality of life with a score. The pharmaceutical composition for HHD of the present invention can improve the DLQI and improve the score. (5) The Skindex-29 is a questionnaire in which subjects evaluate each question regarding their quality of life using a score. The pharmaceutical composition for HHD of the present invention can improve the Skindex-29 and improve the score.

[0048] The method of administering the HHD pharmaceutical composition of the present invention is not particularly limited and may be administered orally or parenterally. Parenteral administration may be, for example, transdermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, and intestinal.

[0049] The dosage form of the HHD pharmaceutical composition of the present invention is not particularly limited and can be appropriately determined, for example, depending on the method of administration. The dosage form may be, for example, a liquid, gel, cream, or solid. Examples of oral dosage forms include granules, fine granules, powders, tablets, capsules (e.g., hard capsules, soft capsules), syrups, emulsions, suspensions, liquids, and jellies. Examples of parenteral dosage forms include injections, suppositories, transdermal preparations, etc.

[0050] The pharmaceutical composition for HHD of the present invention may contain the aforementioned MPBSs, but its other components are not particularly limited. For example, the pharmaceutical composition for HHD of the present invention may contain only the aforementioned MPBSs as an active ingredient, or it may contain, in addition to the aforementioned MPBSs, other active ingredients for HHD.

[0051] The HHD pharmaceutical composition of the present invention may, for example, contain only the active ingredient, or may contain additives in addition to the active ingredient. The additive is preferably a pharmaceutically acceptable substance. The type of additive is not particularly limited and can be appropriately selected, for example, depending on the dosage form. Examples of the additive include carriers, excipients, stabilizers, lubricants, sweeteners, preservatives, suspending agents, dispersants, thickeners, pH adjusters, antifoaming agents, and fragrances. Examples of the carrier include liquids, solids, gels, and creams.

[0052] The target recipients of the HHD pharmaceutical composition of the present invention are not particularly limited, and include, for example, humans and non-human animals, with humans being preferred. Non-human animals include, for example, non-human mammals such as mice, rats, rabbits, and horses.

[0053] The administration conditions for the HHD pharmaceutical composition of the present invention are not particularly limited and can be appropriately determined, for example, according to the method of administration and patient information. The patient information may include, for example, age, sex, weight, presence or absence of HHD and its symptoms, the severity of HHD and its symptoms, and medical history.

[0054] When administering the HHD pharmaceutical composition of the present invention orally, the following conditions are exemplifiable. The following examples can be appropriately adjusted according to patient information, etc., for infants, toddlers, children, adults, or the elderly. The dosage of the MPBS can be expressed, for example, as the dosage obtained by converting the MPBS into anhydrous MPBS. The dosages exemplified below are the dosages of the MPBS converted into anhydrous MPBS, and as a specific example, the dosage of MPBS monohydrate converted into anhydrous MPBS.

[0055] • Daily dose of the aforementioned MPBS (calculated as the amount of anhydrous MPBS) Lower limit: For example, 1 mg, 10 mg, 20 mg, 25 mg, 30 mg, 50 mg, 60 mg, 100 mg, 150 mg, 200 mg, or 300 mg Upper limit: For example, 300mg, 400mg, 500mg, 600mg, 800mg, 1000mg, or 1200mg Range: For example, 1-1000mg, 10-1000mg, 20-1000mg, 25-1000mg, 30-1000mg, 50-1000mg, 60-1000mg, 100-1000mg, 1-600mg, 10-600mg, 25-600mg, 30-600mg, 50-600mg, 100-600mg, 10-500mg, 20-500mg, 25-500mg , 30~500mg, 50~500mg, 60~500mg, 100~500mg, 200~500mg, 10~400mg, 20~400mg, 25~400mg, 30~400m g, 50~400mg, 60~400mg, 100~400mg, 200~400mg, 10~300mg, 20~300mg, 25~300mg, 30~300mg, 50~300m g, 60~300mg, 100~300mg, 200~300mg, 10~200mg, 20~200mg, 25~200mg, 30~200mg, 50~200mg, 60~200 mg, 100~200mg, 10~100mg, 20~100mg, 25~100mg, 30~100mg, 50~100mg, 60~100mg, 300~400mg, 300~5 00mg, 300-600mg, 300-800mg, 300-1000mg, 300-1200mg, 400-600mg, 400-800mg, 400-1000mg, 400-1200mg, 500-600mg, 500-800mg, 500-1000mg, 500-1200mg, 600-800mg, 600-1000mg, or 600-1200mg Examples of specific dosages: For example, 1 mg, 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, or 1200 mg • Number of doses per day: For example, 1 to 3 times, preferably 1 or 2 times, more preferably 2 times. • Frequency of administration (interval): For example, daily

[0056] When administering the HHD pharmaceutical composition of the present invention orally, the timing of administration can be freely set, for example, before meals, during meals, immediately after meals, after meals, between meals, upon waking, or before going to bed, and administration between meals is preferable. Inter-meal administration of the HHD pharmaceutical composition of the present invention means, for example, that the time interval from food intake to administration is at least 1 hour after food intake, preferably at least 2 hours after, and / or, for example, that the time interval from administration to the intake of the next meal is at least 1 hour, 2 hours, 3 hours, 4 hours, or 4.5 hours after, preferably 1 hour after, when the next meal is consumed. That is, the HHD pharmaceutical composition of the present invention is administered, for example, between 1 or 2 hours after food intake and 1 hour, 2 hours, 3 hours, 4 hours, or 4.5 hours before the intake of the next meal.

[0057] As described above, the pharmaceutical composition for HHD of the present invention can be used to treat or prevent the symptoms of HHD, which is a disease that causes acantholysis. For this reason, the present invention can also be described as, for example, a pharmaceutical composition for acantholysis that treats or prevents acantholysis. Furthermore, the present invention can also be described as, for example, a pharmaceutical composition for acantholysis and abnormal keratinization that treats or prevents both acantholysis and abnormal keratinization simultaneously.

[0058] [Methods of treating or preventing HHD] The present invention provides a method for treating or preventing high hemoglobin (HHD), comprising the step of administering 5-methyl-2-(1-piperazinyl)benzenesulfonic acid to a subject. As stated above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) may be any of the MPBS derivatives, i.e., MPBS anhydride, MPBS salt, MPBS hydrate, and / or MPBS solvate. Unless otherwise specified, the present invention provides a method for treating or preventing high hemoglobin (HHD), hereinafter referred to as the "HHD treatment / prevention method of the present invention." The treatment / prevention method of the present invention is characterized by the administration of the MPBS derivatives, and other conditions and steps are not particularly limited.

[0059] In the treatment and prevention method of the present invention, the subject is a patient, and the patient may be, for example, a patient who has developed HHD or a patient who has not developed HHD.

[0060] In the therapeutic and preventive methods of the present invention, the administration of MPBS is, for example, the administration of the pharmaceutical composition for HHD of the present invention. In the present invention, the MPBS, its composition, and the method of administration can be described by reference to the description of the pharmaceutical composition for HHD of the present invention.

[0061] [Use of MPBS-type products] The present invention relates to 5-methyl-2-(1-piperazinyl)benzenesulfonic acid for use in the treatment or prevention of high hemorrhagic disease (HHD). As stated above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) may be any of the MPBS derivatives, i.e., MPBS anhydride, MPBS salt, MPBS hydrate, and / or MPBS solvate. In the present invention, the MPBS derivatives, their compositions, and methods of use thereof can be described by reference to the description in the HHD pharmaceutical composition of the present invention.

[0062] Furthermore, the present invention relates to the use of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid in the manufacture of a pharmaceutical composition for HHD. As described above, the form of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) is not limited and may be any of the MPBS compounds, i.e., the MPBS anhydride, the MPBS salt, the MPBS hydrate, and / or the MPBS solvate. In the present invention, the MPBS compounds, their compositions, and methods of use thereof can be described by reference to the description of the pharmaceutical composition for HHD of the present invention.

[0063] [Medicinal compositions for agonisthraxation and their uses] The pharmaceutical composition for acantholysis of the present invention contains 5-methyl-2-(1-piperazinyl)benzenesulfonic acid. As described above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) is not limited in form and may be any of the MPBS compounds, i.e., MPBS anhydride, MPBS salt, MPBS hydrate, and / or MPBS solvate. The pharmaceutical composition for acantholysis of the present invention may contain, for example, only one of the MPBS compounds or two or more of them. The MPBS compounds can be used for the treatment or prevention of acantholysis. The pharmaceutical composition for acantholysis of the present invention is characterized by containing the MPBS compounds, but no other conditions are limited. It is preferable that the pharmaceutical composition for acantholysis of the present invention contains the MPBS compounds as an active ingredient. When the pharmaceutical composition for acantholysis of the present invention is used for the treatment or prevention of acantholysis, the type of disease that causes acantholysis as a pathological symptom is not particularly limited.

[0064] As described above, the acantholytic pharmaceutical composition of the present invention can treat or prevent acantholysis. The acantholytic pharmaceutical composition of the present invention may be used, for example, for therapeutic purposes, for preventive purposes, or for both therapeutic and preventive purposes. Hereinafter, the term "treatment / prevention" can be interpreted as meaning treatment, prevention, or treatment and prevention. The acantholytic pharmaceutical composition of the present invention allows for safe treatment / prevention with fewer side effects, for example. An example of a disease in which acantholysis occurs is Hailey-Hailey disease.

[0065] In the pharmaceutical composition for acantholysis of the present invention, the MPBSs, their composition, and method of use are not particularly limited, and for example, the description in the pharmaceutical composition for HHD of the present invention described above can be referenced. When referencing, "HHD" and "symptoms of HHD" can be read as "acantholysis."

[0066] The present invention provides a method for treating or preventing acantholysis, comprising the step of administering 5-methyl-2-(1-piperazinyl)benzenesulfonic acid to a subject. As described above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) is not limited in form and may be any of the MPBS derivatives, i.e., the MPBS anhydride, the MPBS salt, the MPBS hydrate, and / or the MPBS solvate. Unless otherwise specified, the present invention provides a method for treating or preventing acantholysis, hereinafter referred to as the "method for treating / preventing acantholysis of the present invention." The method for treating / preventing acantholysis of the present invention is characterized by the administration of the MPBS derivatives, and other conditions and steps are not particularly limited. The subjects targeted by the present invention are, for example, patients who have developed or are likely to develop acantholysis as a pathological symptom, and the type of disease that causes acantholysis as a pathological symptom is not particularly limited.

[0067] In the present invention, the MPBSs, their compositions, and methods of administration thereof are not particularly limited, and the description of the pharmaceutical composition for HHD of the present invention can be incorporated by reference. When incorporating such descriptions, "HHD" and "symptoms of HHD" can be replaced with "acantholysis."

[0068] The present invention relates to 5-methyl-2-(1-piperazinyl)benzenesulfonic acid for use in the treatment or prevention of acantholysis. As stated above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) may be any of the MPBS derivatives, i.e., MPBS anhydride, MPBS salt, MPBS hydrate, and / or MPBS solvate, without limiting its form. In the present invention, the MPBS derivatives, their compositions, and methods of use can be described by reference to the description in the above-mentioned pharmaceutical composition for HHD. When referenced, "HHD" and "symptoms of HHD" can be read as "acantholysis."

[0069] Furthermore, the present invention relates to the use of 5-methyl-2-(1-piperazinyl)benzenesulfonic acid in the manufacture of a pharmaceutical composition for acantholysis. As described above, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid (MPBS) is not limited in form and may be any of the MPBS compounds, i.e., the MPBS anhydride, the MPBS salt, the MPBS hydrate, and / or the MPBS solvate. In the present invention, the MPBS compounds, their compositions, and methods of use can be described by reference to the description in the pharmaceutical composition for HHD of the present invention. When referencing, "HHD" and "symptoms of HHD" can be read as "cantholysis." [Examples]

[0070] In the following examples, 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate (hereinafter sometimes referred to as Compound A in the examples) was used as the MPBS.

[0071] [Example 1] Suppression of differentiation abnormalities in an in vitro HHD 3D cultured epidermal model The inhibitory effect of compound A on differentiation abnormalities in a three-dimensional cultured epidermal model of HHD was evaluated. In skin lesions of HHD patients, keratinocyte differentiation abnormalities are known to cause acantholysis. In this model, the effect of compound A on changes in the gene expression of differentiation markers was investigated.

[0072] <Test cells and study design> Since HHD is known to be associated with mutations in the ATP2C1 gene, an ATP2C1 siRNA-induced in vitro HHD three-dimensional cultured epidermal model was constructed using the following method. Cells and culture medium were prepared using the LabCyte EPI-KIT (Japan Tissue Engineering Co., Ltd., 401810), a human epidermal model preparation kit. Human primary keratinocytes in the aforementioned human epidermal model preparation kit were transfected with siRNA. For transfection, Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778), a cationic lipid preparation, was used as the transfection reagent, and Opti-MEM® Reduced Serum Medium (Thermo Fisher Scientific, 31985-062) was used as the culture medium. As an example, ATP2C1 siRNA (Thermo Fisher Scientific, J-006119-05-0005) was used, and as a control, Non-targeting control siRNA (Thermo Fisher Scientific, D-001810-01-05) was used. The final concentration of the siRNA was 100 nmol / L. In this specification, "final concentration" for each test substance solution and reagent refers to the concentration in the culture medium at the final stage after all test substance solutions and reagents have been added.

[0073] Transfection cells into which the siRNA was introduced were cultured in three dimensions using a gas-liquid interface culture insert and a 24-well plate (Day 0), and in vitro epidermal tissue was prepared by culturing for 8 days (Day 0 to Day 8). The culture environment was maintained at 37°C and 5% CO2. From the start of the three-dimensional culture (Day 0), an aqueous solution of compound A (solvent: phosphate-buffered saline) was added to the culture medium of the in vitro epidermal tissue so that the final concentration of compound A was 10 μmol / L. The culture medium was changed daily, and the aqueous solution of compound A was added at the same time as the change. Eight days after the start of the three-dimensional culture (Day 8), the in vitro epidermal tissue was sampled and used for evaluation. Three groups were established: a control siRNA-treated group, an ATP2C1 siRNA-treated group, and an ATP2C1 siRNA + compound A-treated group, which was treated with ATP2C1 siRNA and compound A, as in the example group. The number of samples was 9 epidermal tissues per group.

[0074] <Evaluation Method> RNA was extracted from the sampled in vitro epidermal tissue using the RNeasy® Mini QIAcube Kit (QIAGEN, 74116). The concentration of the RNA was measured using a micro-spectrophotometer (DropSense 96® (Trinean) or LUNATIC (Unchained Labs)), and then reverse-transcribed to cDNA. SuperScript® IV VILO® Master Mix (Thermo Fisher Scientific, 11756050) or High-capacity RNA to cDNA® kit (Thermo Fisher Scientific, 4387406) was used for the reverse transcription reaction. Using the cDNA as a template, RT-qPCR analysis was performed using the QuantStudio® 6 Flex Real-Time PCR System (Applied Biosystems). For the RT-qPCR reaction, TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, 4444557) or TaqMan® Fast Universal PCR Master Mix (Thermo Fisher Scientific, 4352042) was used as the master mix, and the TaqMan® probe (Thermo Fisher Scientific) listed in Table 1 was used. As differentiation markers, the basal layer markers KRT5 and KRT14, and the granular layer marker IVL were used.

[0075] [Table 1]

[0076] In the aforementioned RT-qPCR analysis, RPLP0 or HPRT1 was used as the internal standard gene. Using the ΔΔCt method, relative gene expression levels, their mean values, and standard errors were calculated based on the average gene expression levels in keratinocytes on Day 0. The suppression rates were calculated as follows. Suppression rate (%)=100×(AB) / (AC) A: Mean relative gene expression levels in the ATP2C1 siRNA treatment group B: Relative gene expression levels in the ATP2C1 siRNA + compound A treatment group C: Mean relative gene expression level of the control siRNA-treated group

[0077] <Statistical analysis> Statistical analysis was performed using SAS, with a significance level of 5% (two-sided). The success of inducing an in vitro HHD 3D cultured epidermal model was determined by comparing the control siRNA treatment group with the ATP2C1 siRNA treatment group. The pharmacological effect of compound A was determined by comparing the ATP2C1 siRNA treatment group with the example group (ATP2C1 siRNA + compound A treatment group). Student's t-test was used for all statistical analyses.

[0078] Figures 1A and 1B are graphs showing the results of gene expression analysis, specifically the relationship between the administration of MPBS monohydrate and the expression of differentiation marker molecules in an in vitro HHD 3D cultured epidermal model. The vertical axis of Figure 1A represents the relative gene expression level relative to the gene expression level in keratinocytes on Day 0, and the vertical axis of Figure 1B represents the suppression rate of the increase in gene expression of differentiation markers due to ATP2C1 siRNA treatment.

[0079] In the ATP2C1 siRNA treatment group, statistically significant increases in the expression of basal layer markers KRT5 and KRT14, as well as granular layer marker IVL, which has been reported to have abnormal expression in HHD patients, were observed compared to the control siRNA treatment group. In contrast, in the example group, the increase in the expression of these differentiation markers was suppressed by the addition of compound A, and the abnormal expression of these marker genes was corrected.

[0080] [Example 2] Improvement of intercellular adhesion disorder in an in vitro epidermal intercellular adhesion disorder model The effect of compound A on improving intercellular adhesion in an epidermal intercellular adhesion disorder model was evaluated. It is known that acantholysis occurs when intercellular adhesion is impaired. The impairment of intercellular adhesion was evaluated by measuring the change in electrical resistance (Cell Index) using a real-time cell analyzer.

[0081] <Test cells and study design> An in vitro model of intercellular adhesion disorder in epidermal cells was created using 2,2'-methylenebis(6-t-butyl-4-methylphenol) (hereinafter referred to as bisphenol) by the following method. Bisphenol has been reported to selectively inhibit the ATPase activity of SPCA1, the molecule responsible for HHD. Human primary keratinocytes derived from Caucasian females (KURABO, KK-4109) were used as the cells. HuMedia-KG2 medium (KURABO, KK-2150S) was used to culture the aforementioned human primary keratinocytes. A real-time cell analyzer, xCELLigence Real-time Cell Analyzer (Agilent), was used and placed in an incubator under conditions of 37°C and 5% CO2.

[0082] First, HuMedia-KG2 medium was added at a rate of 50 μl / well to an E-Plate VIEW 96 PET (Agilent) plate. The plate was then placed in the real-time cell analyzer, and the background value of electrical resistance was measured. Next, the plate was removed, and human primary keratinocytes were seeded onto the plate at a rate of 100 μl / well to a total of 25,000 cells / well. The plate was then placed back into the real-time cell analyzer. Simultaneously with the setup, measurement of electrical resistance was started and recorded at 15-minute intervals (Day 0).

[0083] On Day 1, the plate was removed. After removing 50 μl / well of the culture supernatant from the wells of the plate, 100 μl / well of calcium solution was added to achieve a final calcium concentration of 1.2 mmol / L. The plate was then re-inserted into the real-time cell analyzer, and the measurement of electrical resistance was resumed. Next, on Day 2, the plate was removed. After removing 100 μl / well of the culture supernatant from the wells of the plate, 50 μl / well of compound A was added. The plate was then re-inserted into the real-time cell analyzer, and the measurement of electrical resistance was resumed. Three hours after the addition of compound A, the plate was removed, and 50 μl / well of bisphenol solution was added to the plate. The plate was then re-inserted into the real-time cell analyzer, and the measurement of electrical resistance was resumed.

[0084] The group composition consisted of three groups: a group without bisphenol addition, a group with bisphenol addition (without compound A addition), and the example group with bisphenol addition / compound A addition. At the time of bisphenol addition, the final concentration of compound A in the well was 0 μmol / L (without compound A addition), 0.01 μmol / L, 0.1 μmol / L, or 1 μmol / L, and the final concentration of bisphenol was 0 μmol / L (without bisphenol addition) or 4 μmol / L.

[0085] Then, the normalized electrical resistance value (normalized Cell Index; nCi) 60 hours after the addition of bisphenol was used as an indicator of the suppression of intercellular adhesion disorders in epidermal cells. The nCi was normalized so that the electrical resistance value (Cell Index) immediately before the addition of bisphenol was 1.

[0086] <Statistical analysis> Statistical analysis was performed using SAS (Statistical Analysis System). Student's t-test was used to compare the bisphenol-free group with the bisphenol-added group. The pharmacological effect of compound A was determined by comparing the bisphenol-added group with the bisphenol-added / compound A-added group, using Williams' multiple comparison test.

[0087] Compound A was evaluated in an in vitro epidermal cell adhesion disorder model induced by bisphenol. The aforementioned test was performed four times, and the mean value and standard error of nCi at 60 hours after bisphenol addition were calculated and statistically analyzed. The results are shown in Figure 2.

[0088] Figure 2 is a graph showing the relationship between the administration of MPBS monohydrate and cell adhesion in an in vitro epidermal cell adhesion disorder model, with the vertical axis representing normalized electrical resistance. As shown in Figure 2, the bisphenol-added group, in which bisphenol was added to cultured human primary keratinocytes under a final calcium concentration of 1.2 mmol / L, showed a statistically significant decrease in nCi compared to the group without bisphenol addition. ## (p<0.01). Furthermore, compared to the bisphenol-added group, it was found that the bisphenol-added / compound A-added group could recover the nCi levels that had decreased due to bisphenol addition by further pretreatment with compound A. In particular, pretreatment with compound A at a concentration of 0.1-1 μmol / L resulted in a statistically significant recovery of the nCi levels that had decreased due to bisphenol addition. ** (p<0.005). These results suggest that compound A has an ameliorative effect on bisphenol-induced intercellular adhesion disorders of epidermal cells.

[0089] [Example 3] Transcriptome analysis in an in vitro model of intercellular adhesion disorder in epidermal cells For compound A, the transcriptome, i.e., the changes in gene transcript expression, was comprehensively evaluated in the epidermal cell adhesion disorder model. Gene expression changes were evaluated using the NextSeq500 / 550 sequencer by measuring changes in RNA count values ​​or changes in RNA expression levels normalized from RNA count values.

[0090] <Experimental Design and Data Analysis Methods> Except as described below, the cells were cultured under the same conditions as in Example 2 to create the epidermal cell adhesion disorder model.

[0091] The experimental groups (1) to (3) of this embodiment are shown below. (1) Group 1 (control group) On Day 1, calcium was added to the wells, and the cells were incubated for 75 hours in the presence of a final calcium concentration of 1.2 mmol / L. This experimental group did not undergo bisphenol treatment and simulated keratinocytes in a normal state in the presence of calcium. (2) Group 2 (Bisphenol-added group) On Day 1, calcium was added to the wells, and the cells were incubated for 24 hours in the presence of a final calcium concentration of 1.2 mmol / L. DMSO solution was then added. After incubation for 3 hours following the addition of DMSO, bisphenol (final concentration of 4 μmol / L) was added, and the cells were incubated for another 48 hours. This experimental group was exposed to bisphenol, simulating the conditions under which HHD would occur. (3) Group 3 (compound A addition group) On Day 1, calcium was added to the wells, and the cells were incubated for 24 hours under conditions of a final calcium concentration of 1.2 mmol / L. Then, compound A (final concentration 1 μmol / L) was added. After incubation for 3 hours following the addition of compound A, bisphenol (final concentration 4 μmol / L) was added, and the cells were incubated for 48 hours. This experimental group simulated the conditions under which compound A was administered to subjects who had developed HHD.

[0092] After the culture was completed, cells were collected from each of the experimental groups (1) to (3) described above, and total RNA was extracted using an RNeasy® Mini QIAcube column (Qiagen) according to the product protocol. All extracted RNA samples were treated with DNase. Subsequently, using NanoDrop (Thermo Fisher Scientific) and Bioanalizer RNA6000 nanochip (Agilent), it was confirmed that all of the extracted RNA samples were of high quality, with an RIN value of 9.4 or higher and a total RNA amount of 1.3 μg or higher.

[0093] Using the extracted total RNA (10 ng) as a template, cDNA libraries were prepared according to the respective product protocols using NEBNext® Ultra II Directional RNA Library prep for Illumina (NEB), NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB), and NEBNext® Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, NEB).

[0094] Next, the cDNA library was sequenced using the NextSeq500 / 550 system (Illumina) according to the product protocol. Quality control was performed using FASTQC (version 0.12.1) and Trimmomatic-0.39, and the results were converted to FASTQ files.

[0095] Next, using StrandNGS software (version 4.0, Strand Life Science) and Star (version 2.7.10b), we referenced the human genome assembly and transcript annotation (release 101) obtained from the Ensembl database and identified the types of genes from the 150 nucleotide sequence information contained in the FASTQ file. Then, we calculated the expression levels of those transcripts. Transcript expression levels were calculated using read count values ​​and normalized TPM values.

[0096] Differential expression analysis was performed using DESeq2 (a two-group comparison package) in the statistical analysis software R (version 4.3.2) to calculate the log2 ratio of RNA expression levels and significance levels. Significance testing was performed based on p-values ​​calculated by the Wald test with multiple test correction based on the Benjamini-Hochberg method (BH method). In this analysis, genes with an absolute value of the log2 ratio of 0.585 or higher (expression variation ratio of 1.5 times or more) or 0.263 or higher (expression variation ratio of 1.2 times or more), and with a p-value of less than 0.1 from the Wald test between any two groups, were identified as "genes with altered expression levels" (differential expression genes; DEGs). DEGs include both up-expression and down-expression genes.

[0097] Enrichment analysis of the gene ontology involved comparing ontology terms defined as Biological Processes in the Gene Ontology database with the genetic information associated with those ontology terms and DEGs. Ontology terms with a p-value of less than 0.05 using Fisher's exact test with multiple testing correction based on the BH method were considered significant, and annotation was performed for DEGs. This identified ontology terms related to biological functions associated with DEGs.

[0098] The expression levels of transcripts in each experimental group were compared. As a result, when comparing Group 1 and Group 2, 4827 genes were identified as DEGs (log2 ratio criterion of 0.585 or higher; 2025 genes increased and 2802 genes decreased in Group 2, the disease group). Furthermore, when comparing Group 2 and Group 3, 1515 genes were identified as DEGs (log2 ratio criterion of 0.263 or higher; 538 genes increased and 977 genes decreased in Group 3, the drug-added group).

[0099] The results of the enrichment analysis are shown in Tables 2 and 3. Table 2 shows the ontological terms of biological functions that increased with bisphenol and decreased with compound A, and Table 3 shows the ontological terms of biological functions that decreased with bisphenol and increased with compound A. Of the obtained ontological terms of biological functions associated with DEG, six ontological terms were identified that were associated with DEG that increased in the bisphenol-added group (Group 2; disease group), while conversely, six ontological terms were associated with DEG that decreased in the compound A-added group (Group 3) (Table 2). In addition, ten ontological terms were identified that were associated with DEG that decreased in the bisphenol-added group (Group 2; disease group), while conversely, ten ontological terms were associated with DEG that increased in the compound A-added group (Group 3) (Table 3). These results suggest that bisphenol addition activates endoplasmic reticulum (ER) stress and alters Golgi apparatus function, and that compound A has an inhibitory effect on these changes. Furthermore, the addition of bisphenol suppressed epidermal differentiation and weakened intercellular adhesion, suggesting that compound A normalized these changes. Therefore, the effects of compound A on improving bisphenol-induced epidermal intercellular adhesion disorders were suggested.

[0100] [Table 2]

[0101] [Table 3]

[0102] Although the present invention has been described above with reference to embodiments and examples, the present invention is not limited to the above embodiments. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention.

[0103] This application claims priority based on Japanese Patent Application No. 2024-104617, filed on 28 June 2024, and incorporates all of its disclosures herein. [Industrial applicability]

[0104] According to the pharmaceutical composition of the present invention, by including 5-methyl-2-(1-piperazinyl)benzenesulfonic acid, it is possible to treat or prevent Hailey-Hailey disease.

Claims

[Claim 1] The inventions described herein.

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