WO2025095039A1 - Pharmaceutical composition for skin diseases, and method for searching novel use of medicine - Google Patents

Abstract

A comparative transcriptome analysis according to the present invention is a very useful method as a method for searching a novel use of an existing drug. As employing the comparative transcriptome analysis to use a protective action of keratinocytes as an indicator, it is found that κ-opioid agonists and Chinese herbal medicines can become therapeutic agents for skin disorders that are side effects of anticancer drugs.

Description

Pharmaceutical composition for skin diseases and method for exploring new uses of pharmaceuticals

The present invention relates to a pharmaceutical composition for treating skin diseases and a method for exploring new uses of pharmaceuticals.

The epidermis is the outermost layer of the skin, and acts as a barrier to prevent the intrusion of foreign substances and the evaporation of moisture from the body, protecting the inside. The epidermis is a stratified squamous epithelium with a thickness of approximately 0.1 to 0.2 mm, composed of the keratinocyte layer, granular cell layer, spinous cell layer, and basal cell layer from the outer layer. The majority of these are keratinocytes (keratinized cells), which differentiate as they move from the lower layer to the upper layer (outer layer), keratinizing and differentiating into the keratinocyte layer. The main function of keratinocytes is to act as a barrier against skin damage caused by heat, ultraviolet rays, dehydration, pathogenic bacteria, fungi, parasites, viruses, etc. Therefore, inhibition of keratinocyte proliferation or dysfunction can lead to various skin disorders.

Skin disorders occur for various reasons, but may occur as a side effect of cancer treatments such as anticancer drug therapy and radiation therapy. In particular, during chemotherapy, skin disorders called hand-foot syndrome (HFS) or its synonym hand-foot skin reaction (HFSR) occur as a side effect, which significantly impairs the quality of life of patients (hereafter, all terms will be referred to as "HFS"). HFS has long been a problem in Japan, and the Ministry of Health, Labor and Welfare has issued the "Manual for Treating Severe Side Effects: Hand-Foot Syndrome" (Non-Patent Document 1) to raise awareness, but currently there is no satisfactory treatment or therapy.

Skin disorders due to side effects are frequently observed with multikinase inhibitors (sorafenib, sunitinib, axitinib, pazopanib, regorafenib, etc.), anti-epidermal growth factor receptor (EGFR) antibody drugs, and EGFR tyrosine kinase inhibitors (Non-Patent Document 2), but are also known to occur with platinum-based DNA synthesis inhibitors such as cisplatin, carboplatin, and oxaliplatin, pyrimidine-based DNA synthesis inhibitors such as fluorouracil (5-FU), gemcitabine, capecitabine, TS-1 (S-1), doxorubicin, and docetaxel, and immune checkpoint inhibitors (nivolumab, pembrolizumab, atezolizumab, durvalumab, etc.) (Non-Patent Document 3).

Symptoms of HFS include redness, edema, hyperkeratosis (hyperkeratosis), sensory abnormalities, and pain on the palms and soles of the feet, and in severe cases, blisters and bleeding may occur, and sometimes the patient may have difficulty grasping the hands or walking, resulting in significant limitations in activities of daily living. HFS is a finding of impaired skin barrier function, and histopathologically, necrosis of epidermal keratinocytes is observed in a band-like pattern from the spinous layer to the granular layer of the epidermis, and hyperkeratosis (hyperkeratosis) and parakeratinization are observed in the stratum corneum, and is characterized by severe impairment of the skin barrier function and abnormal keratinization in keratinocytes (Non-Patent Document 4). As the site of impairment is in the stratum corneum, a keratinocyte protective agent with skin barrier function could be used as a preventive drug for HFS and a therapeutic drug to alleviate the pathology.

Despite progress in cancer chemotherapy, the details of the mechanism behind HFS have remained unclear for over 20 years, and no method of prevention has been established. Treatments for HFS have attempted to reduce physical irritation to the skin, keratinizing with emollients containing urea or salicylic acid, and using pyridoxamine (vitamin B6) or COX2 inhibitors, but clinical trials have not shown clear efficacy (Non-Patent Document 5).

Medicines for treating HFS include a pharmaceutical composition containing allopurinol and pyridoxamine, which are used to treat gout, etc. (Patent Document 1), histone deacetylase inhibitors such as phenylbutyrate (Patent Document 2), and a treatment containing an azurophilic antifungal drug as an active ingredient (Patent Document 3), but none of these have been put to practical use yet.

Skin disorders caused by new anticancer drugs have also been reported. Immune checkpoint inhibitors have recently come into use in cancer treatment, and attention has been drawn to the occurrence of skin disorders such as vitiligo and reduced skin pigmentation that are thought to be caused by immune reactions. However, no effective drug therapy for vitiligo has yet been found (Non-Patent Document 6).

One of the reasons for the lack of effective drugs for skin disorders is the lack of an appropriate drug evaluation system. As for drug evaluation systems, there is a problem that it is difficult to create appropriate animal models for skin disorder research, because the metabolism and resistance of epidermal cells differs in some areas between humans, who have little body hair, and animals, who are covered in hair and have strong skin tissue. There is an example of creating a mouse HFS model using capecitabine, but the non-toxic dose for mice is about 10 times the human dose, which shows low drug reactivity, and the drug candidate discovered there required about 60 times the human clinical dose (Non-Patent Document 7). Thus, in cases where the sensitivity of anticancer drugs such as HFS or drug metabolites are involved, or where an immune response is involved, evaluation is difficult using animal models with different sensitivities, and it is desirable to first evaluate using the same type of human cells.

JP 2012-180370 A Special Publication No. 2012-530076 JP 2021-11453 A

Ministry of Health, Labor and Welfare, "Manual for dealing with serious side effects: Hand-foot syndrome," September 2019 Heidary et al, J Am Acad Dermatol, 2008, 58: 545-570 Ellis et al, J Am Acad Dermatol, 2020, 83:1130-1143 Lacouture etal, Ann Oncol, 2008, 19: 1955-1961 Kwakman et al, Oncology Reviews, 2020, 14:57-63 Geisler et al., Am Acad Dermatol. 2020; 83(5): 1255-1268. doi:10.1016/j.jaad.2020.03.132. Hiromoto et al., Sci Rep. 2021 Apr 26;11:8964. doi:10.1038/s41598-021-88460-9 Yamamizu et al, Sci Rep, 2013, 3 : 3213, doi: 10.1038/srep0321310.1038/s41598-021-88118-6. Shokirova et al, Sci Rep. 2021, 11(1):8647. doi:10.1038/s41598-021-88118-6. Zhang et al., Front Immunol. 2022, 13:991594. doi:10.3389/fimmu.2022.991594. Yang et al., Exp Dermatol. 2023, 32(4):511-520. doi:10.1111/exd.14743. Ueno et al., Biomed Res Int., 2015, 2015:960840. doi:10.1155/2015/960840 ) Zhou et al., Nature Communication 2019, 10:1523. doi:10.1038/s41467-019-09234-6 Chen et al., BMC Bioinformatics 2013, 14:128. doi:10.1186/1471-2105-14-128 Tanaka et al. Clin Cosmet Investig Dermatol.2023:16:2829-2839. doi:10.2147/CCID.S428170. Lee wt al. Dev Cell. 2014 14;29(1):47-58. doi:10.1016/j.devcel.2014.03.005. Wang et al. Nat Commun. 2018 8;9(1):4684. doi:10.1038/s41467-018-07037-9. Weglowska et al., Int J Mol Sci 2022, 23, 238, https://doi.org/10.3390/ijms23010238 Urakawa et al., J Cancer. 2019, 10(20):4846-4851. doi:10.7150/jca.31059.

As mentioned above, it is difficult to use animal models to evaluate the development of therapeutic drugs for skin disorders. Therefore, in the development of preventive and therapeutic drugs involving keratinocytes, it is important to develop an evaluation system for normal human keratinocytes without relying on animal models. The pharmaceutical composition shown in the following examples relates to a protective agent for keratinocytes, which have the barrier function of the skin, and aims to provide a compound that protects keratinocyte damage in HFS caused by anticancer drugs in particular. In addition, to achieve this, it aims to provide a method for exploring new uses for existing pharmaceuticals without relying on animal models.

This specification discloses the following pharmaceutical compositions and discovery methods.
(1) A topical agent for treating skin disorders, comprising a κ-opioid agonist and/or a herbal medicine component as an active ingredient, said active ingredient having a keratinocyte protecting effect.
Kappa opioid agonists and herbal medicines, which are oral preparations of complex herbal medicines, have never been used as topical agents for skin disorders. By exploring new uses for existing drugs, we found that these drugs are effective in protecting keratinocytes and can treat and prevent skin disorders.

(2) The topical preparation according to (1), wherein the κ opioid agonist is at least one selected from the group consisting of nalfurafine (TRK-820), difelikefalin (CR845), YNT-1612, CR665 (FE200665), HSK21542, GR89696, U69593, Salvinorin A, EOM salvinorin B, and pharma- ceutically acceptable salts thereof.
Since κ opioid agonists have a common site of action, it is presumed that all κ opioid agonists have a keratinocyte-protecting effect.

(3) The topical preparation according to (1), wherein the protective effect of keratinocytes is at least one of promotion of proliferation during cell damage, inhibition of expression of inflammatory cytokine genes, and inhibition of expression of pigmentation-inducing genes.
The comparative transcriptome analysis shown below revealed that the topically administered agent has at least one of the following effects: promoting proliferation during cell damage, suppressing the expression of inflammatory cytokine genes, and suppressing the expression of pigmentation-inducing genes, thereby treating or preventing skin disorders.

(4) The topical preparation according to (1), wherein the herbal ingredient is at least one of licorice, bougainvillea root, and keiskei root.
Analysis of herbal prescriptions has shown that licorice, Boufu, and Keigai have keratinocyte-protecting effects.

(5) The disease to which the present invention is applied is Hand-Foot Syndrome (HFS), perivascular dermatitis, lichenoid dermatitis, erythematous dermatitis, bullous pemphigoid (BP), bullous rash, Stevens-Johnson syndrome-like reaction, psoriasis, hemolytic dermatitis, acute generalized exanthematous pustulosis (AGEP), folliculitis, acne-like reaction, epiphora, pigmentation, radiation dermatitis, which is a disorder of the skin and tissue mucosal epithelium due to radiation therapy, mucositis, stomatitis, angular cheilitis, pigmentation, or skin disorder of atopic dermatitis due to scratching,
The topical preparation according to any one of (1) to (4), which is intended for protecting keratinocytes.
There are various types of skin disorders, and the present invention is applicable to those skin disorders in which the symptoms are alleviated by protecting keratinocytes.

(6) A topical administration agent for treating vitiligo, vitiligo vulgaris, and hypopigmentation of the skin, which is a herbal medicine containing either one or both of the herbal ingredients of Daiso and Zingiber officinale.
Skin disorders such as vitiligo and skin pigment loss caused by the melanin production system have also been found, and these can also be evaluated using keratinocytes. Melanocytes, which produce pigment, originate from neural crest cells and exist in the basement membrane, while neural crest-derived cells can also differentiate into keratinocytes. In addition, melanosomes, organelles that synthesize and store melanin pigment, are transferred from melanocytes to keratinocytes. Therefore, since skin pigment loss such as vitiligo and vitiligo vulgaris is deeply related to keratinocytes, it is possible to select effective drugs using an evaluation system using keratinocytes.

(7) A method for exploring new uses of pharmaceutical compositions by extracting opposing intracellular processes from the results of transcriptome analysis of a plurality of pharmaceutical compositions, comprising the steps of: obtaining a gene expression profile (Gene Ontology-Biological Process, GO-BP) reflecting a predetermined side effect from the results of transcriptome analysis of the target pharmaceutical composition; obtaining a gene expression profile (GO-BP) of a candidate compound for treating/preventing the side effect; comparatively analyzing the GO-BP reflecting the side effect of the target pharmaceutical composition with the GO-BP of the candidate compound, selecting a candidate compound that exhibits a GO-BP that counteracts the side effect of the target pharmaceutical composition, and verifying the effect of the candidate compound against the side effect in an in vitro cell evaluation system.
According to this method, transcriptome analysis of multiple existing pharmaceutical compositions can be performed, and a pharmaceutical compound capable of treating the side effects of the target pharmaceutical composition can be selected by comparative transcriptome analysis. This is a very efficient method for exploring new uses of GO-BP as a phenotype of a pharmaceutical composition.

(8) A method for exploring a new use of a pharmaceutical composition, the new use of which is keratinocyte protection, the target pharmaceutical composition being an anticancer drug, and the side effect being HFS caused by the anticancer drug, the method comprising the steps of: obtaining a gene expression profile (GO-BP) reflecting damage to keratinocytes by the anticancer drug; obtaining a gene expression profile (GO-BP) of a candidate compound for protecting keratinocytes and treating/preventing the same; comparatively analyzing the GO-BP reflecting the side effects of the anticancer drug and the GO-BP of the candidate compound, selecting a candidate compound that exhibits keratinocyte protective effect from the GO-BP, and verifying the keratinocyte protective effect of the candidate compound from the cell viability in an in vitro human normal keratinocyte proliferation evaluation system.
By using this screening method, the present inventors have discovered that κ opioid agonists and herbal medicines have a keratinocyte-protecting effect.

FIG. 1 is a schematic diagram showing a method for discovering keratinocyte-protecting drugs by comparative transcriptome analysis. A diagram showing the protective effect of single agents against sorafenib-induced cell proliferation inhibition. Sor: sorafenib, NFN: nalfurafine. Cytoprotective effect of kappa opioid agonists against the inhibition of keratinocyte proliferation by regorafenib and capecitabine. Regorafenib (A) or capecitabine (B) strongly inhibits keratinocyte proliferation, while the kappa opioid agonists nalfurafine (NFN) and difelikephalin (DKN) have the effect of reducing proliferation inhibition. A diagram showing the protective effect of herbal medicines against cell proliferation inhibition by regorafenib. Reg: regorafenib, JHT: Jumihaidokuto, SFS: Shofusan, USI: Unseiin. A diagram showing the expression induction effect of keratinocyte-related genes by drugs. Regorafenib (Reg) strongly suppresses expression, but the kappa opioid agonists nalfurafine (NFN) and difelikephalin (DKN) also enhance expression, and have the effect of maintaining keratinocyte keratinization. FIG. 1 shows the gene expression induction effect of drugs in exacerbating factors related to keratinocyte skin disorders. Regorafenib (Reg) strongly enhances expression in some areas, but κ opioid agonists nalfurafine (NFN) and difelikephalin (DKN) tend to suppress expression, suggesting an inhibitory effect on keratinocyte skin disorders. A diagram showing the expression induction effect of keratinocyte pigmentation-related genes by Kampo medicines. KKB (Keishi-ka-jutsu-fu-to), GYT (Goshu-yu-to), and KDB (commonly known as the Kandabashi prescription = a combination of Keishi-ka-shakuyaku-to and Shimotsu-to) have the effect of promoting the melanin production process.

The inventors of the present invention have noticed that the side effects of drugs are controlled by gene expression in cells, just like pharmacological actions, and have come up with the idea of using transcriptome analysis to screen drugs that reduce side effects. Once a combination of a compound and a target cell is determined, the transcriptome analysis will cover all pathways and biological processes of the main and secondary actions. Specifically, the method involves performing transcriptome analysis of a drug with side effects to find intracellular processes that reflect the side effects, extracting the opposing intracellular processes from the transcriptome analysis of a candidate compound that treats the side effects, and exploring new uses from the comparative actions of the compound (hereinafter, this method is referred to as the comparative transcriptome method or comparative transcriptome analysis). In other words, the method involves performing transcriptome analysis of a drug with side effects and transcriptome analysis of a candidate compound that treats the side effects, comparing them, and exploring compounds that can complement the gene expression that causes side effects. If the causative gene that causes the side effects is clear, it is sufficient to search for compounds that induce gene expression that complements it. Even if the causative gene is not clear, the results of gene expression analysis obtained from transcriptome analysis can be further analyzed by GO-BP analysis or pathway analysis to search for compounds that induce gene expression that can counteract side effects. Since the intracellular processes and pathways to be compared can also be referenced from information extracted from databases, it is also possible to compare database information with compounds analyzed independently.

Intracellular processes and the genes involved in them can be identified by querying public databases of gene expression biological processes (GO-BP), such as GENEONTOLYOGY. In drug discovery research, determining target genes, one of the most important tasks, can be efficiently achieved by comparative transcriptomics.

Transcriptome analysis, which analyzes comprehensive gene expression, is widely used to investigate cell responses and phenotypes, but the traditional method of use is to explain the mechanisms of drug stimulation and cell interactions, using information from "selected stimulus → gene expression results." In contrast, comparative transcriptome analysis is an analytical method that combines information from the opposite direction, "gene expression results → selection of stimulus," in order to select drugs that can counter side effects based on the results of gene expression analysis of drugs; by performing this on normal cells, drugs that treat side effects can be found. If disease-derived cells are used, compounds for treating the disease can also be searched for.

Figure 1 shows a method for searching for keratinocyte-protecting drugs using comparative transcriptome analysis. In a similar manner, once the biological process of interest is determined, the pharmacological action of each drug can be estimated from the gene expression profile, and the drug with the desired effect can be selected, making it an effective screening method.

Here, we searched for protective drugs to prevent and reduce skin disorders, but it goes without saying that similar analyses can be used to search for preventive and therapeutic drugs for all kinds of side effects. In the following examples, in order to search for protective drugs to prevent and reduce skin disorders, human normal keratinocytes were used as target cells, and all pharmacological actions, including beneficial and harmful effects, that occur in normal skin were analyzed. Human normal keratinocytes were treated with evaluation compounds, and the transcriptome analysis results were subjected to gene ontology enrichment analysis (particularly GO-BP analysis) using bioinformatics techniques to detect the damage caused by anticancer drugs to keratinocytes. Meanwhile, compounds that can detect events that lead to the activation and survival of keratinocytes were selected as protective drug candidates. Furthermore, the validity of the comparative transcriptome analysis of the selected protective drug candidates was verified by the cell viability using an in vitro keratinocyte evaluation system.

The specific steps are as follows:
(1) Obtain a gene expression profile (GO-BP) that reflects damage to keratinocytes as an anticancer drug-induced HFS event. (2) Analyze the gene expression profile (GO-BP) of candidate compounds that can be applied to treatment and prevention. (3) Select candidate compounds that counteract HFS events by matching comparative GO-BPs. (4) Verify the protective effect of candidate compounds on keratinocytes (cell survival rate) in an in vitro cell proliferation evaluation system.

This reverse engineering approach, using normal human keratinocytes, makes it possible to evaluate all kinds of skin disorders that occur in normal skin, and to search for compounds that prevent or alleviate these disorders. Furthermore, if existing drugs are used as candidate compounds, this corresponds to drug repositioning, which finds new applicable diseases.

GO-BP analysis using comparative transcriptome analysis can also reveal information on various cellular phenotypes. For example, if we assume that pain occurs during skin disorders, we can investigate "GO-BPs in response to stimuli" and select medicines that are expected to have analgesic effects as a phenotype. In addition, in skin diseases, scarring and pigmentation after healing can have a significant impact on cosmetic appearance, sometimes reducing the patient's quality of life (QOL). Vitiligo and hypopigmentation as diseases and symptoms are also similarly problematic. Predictive assessment of the alleviation and prevention of such skin conditions can also be evaluated from gene expression profiles.

In this specification, the term "skin disorders as a side effect of anticancer drugs" includes various disorders that occur on the skin as a side effect of anticancer drugs, such as redness, erythema, edema, bleeding, pain, pruritus, inflammation, paresthesia, rash, skin desquamation, stratum corneum peeling, blisters, and vitiligo, but particularly refers to the decreased function, damage, and necrosis of keratinocytes. One typical example of such a disease is HFS, which is pathologically characterized by keratinocyte necrosis.

In this specification, the term "protection of keratinocytes" includes healing, alleviating, or reducing skin damage that has already occurred due to the administration of anticancer drugs, as well as preventively suppressing the occurrence of skin damage due to the administration of anticancer drugs, and suppressing inflammation and pigmentation during the repair of skin damage.

The primary skin disorder of interest here is a skin disorder called HFS, which occurs as a result of the administration of anticancer drugs. There are no particular limitations on the type of anticancer drug, and this includes various anticancer drugs that can cause skin disorders as a side effect, including molecular targeted drugs as well as anticancer drugs that are not classified as molecular targeted drugs.

The therapeutic or preventive effect on skin disorders caused by the administration of anticancer drugs can be evaluated, for example, by the action of restoring cell viability that has been reduced by anticancer drug treatment, using cultured skin cells such as epidermal keratinocytes. As described in the Examples below, it can be evaluated using monolayer cultured cells or a cultured three-dimensional human epidermal model, but considering the species differences in skin structure and keratinocyte responsiveness, it is important to evaluate using normal human keratinocytes. Animal cells are not recommended due to differences in metabolic enzymes and melanin synthesis systems, and human cell lines may have genetic mutations. In addition, since topical administration agents such as topical agents are envisioned, the intended purpose can be achieved by directly evaluating in vitro keratinocyte sheets.

　Target applicable diseases include, in the case of skin diseases, HFS, perivascular dermatitis, lichenoid dermatitis, erythematous dermatitis, bullous pemphigoid (BP), bullous rash, Stevens-Johnson syndrome-like reaction, psoriasis, hemolytic dermatitis, acute generalized exanthematous pustulosis (AGEP), folliculitis, acne-like reaction, epiphora, pigmentation, and skin disorders such as radiation dermatitis, which is a damage to the skin and tissue mucosal epithelium caused by radiation therapy, mucositis, stomatitis, angular cheilitis, pigmentation, and atopic dermatitis caused by scratching. Diseases in the case of reduced skin pigmentation include vitiligo, vitiligo vulgaris, and depigmentation.

The topical preparation of the present invention is applied to the site of skin disorders for therapeutic or preventive purposes, and may be in the form of an ointment, cream, lotion, external liquid, gel, cataplasm, patch, sheet, etc. It may be a single agent or a complex combination agent, and may be manufactured by adding excipients, binders, lubricants, disintegrants, surfactants, suspending agents, emulsifiers, stabilizers, etc. that are commonly used in the pharmaceutical field, as necessary.

The dosage of the topical agent of the present invention can be appropriately selected depending on the age and weight of the patient, the state of the skin disorder, and the site. For example, the daily amount of active ingredient for an adult weighing approximately 60 kg is about 1 μg to 100 mg in the case of topical application, and the daily administration may be once or in several divided doses. It may be administered daily or every few days. The administration period of the keratinocyte protective agent is until a certain therapeutic effect is obtained for the skin disorder (until the skin disorder disappears or is reduced to a level that does not interfere with daily life), or it may be administered prophylactically to the hands and feet, where HFS is likely to occur, before the onset of skin disorders after the start of anticancer drug administration.

The inventors used the above-mentioned methods to search for anticancer drug-based preventive and therapeutic agents for HFS, and surprisingly found that κ opioid agonists have a protective effect on keratinocytes, and at the same time have an inhibitory effect on events such as inflammation induction, mechanical pain sensation, and pigmentation that are presumed to be involved in skin disorders and nerve disorders. κ opioid agonists are known to have analgesic and antipruritic effects, and are therefore suitable compounds for reducing and preventing the skin disorders of HFS that occur as a side effect of cancer treatment, while suppressing itching and pain.

Kappa opioid agonists have also been reported to have effects that lead to the inhibition of cell proliferation. For example, nalfurafine, a representative kappa opioid agonist, has been reported to inhibit tumor angiogenesis (Non-Patent Document 8), and to suppress immune cell migration and angiogenesis in the cornea (Non-Patent Document 9). However, since wound healing occurs through the process of "inflammation - keratinocyte proliferation - epithelialization - angiogenesis - granulation tissue formation - scarring," it is necessary to focus on keratinocytes.

The inventors also found that nalfurafine suppresses the expression of IL37 (Non-Patent Document 10) and DCT (Non-Patent Document 11), which are genes that induce pigmentation in the skin. In addition to protecting keratinocytes, κ-opioid agonists such as nalfurafine also have pigmentation inhibition, antipruritic effects, and analgesic effects, so they can be used more preferably when protecting keratinocytes as a combination agent with other compounds.

Here, the kappa opioid agonist is, for example, selected from the group consisting of nalfurafine (TRK-820), difelikefalin (CR845), YNT-1612, CR665 (FE200665), HSK21542, GR89696, U69593, Salvinorin A, EOM salvinorin B, and pharma- ceutically acceptable salts thereof, but any kappa opioid agonist may be used.

GO-BP analysis can be applied not only to single-ingredient compounds (single drugs), but also to compounds that are compounds of multiple ingredients. For example, the most typical compound drug among existing pharmaceuticals is traditional Chinese medicine. Western medicines basically contain one active ingredient per drug, and the site of action (mechanism of action) is also clearly defined, but traditional Chinese medicines contain multiple active ingredients per drug, and their major feature is that they are effective for a variety of symptoms. Prescriptions are based on traditional Chinese medicine, and place emphasis on the patient's constitution and condition, as represented by "deficiency/excess" and "qi/blood/water," and are prescribed based on the results. Traditional Chinese medicines are prescribed as decoctions, administered orally and systemically, and are also prescribed for the purpose of reducing the side effects of cancer treatment.

In cancer treatment, attention must also be paid to the form of administration. For example, in the case of HFS, a side effect of anticancer drugs, it is assumed that the reason this side effect appears on the hands and feet is that the palms and soles have many blood vessels and a high rate of skin cell division, and that they have many eccrine glands, which make them susceptible to damage when the anticancer drug is excreted through sweat (Non-Patent Document 5). As most cancer treatment drugs are administered systemically, it is preferable to apply drugs to prevent and treat skin disorders on the hands and feet locally in order to avoid drug interactions with cancer treatment drugs. However, there have been no reports of the local administration of herbal prescriptions based on their protective effect on keratinocytes.

The inventors applied the GO-BP analysis method to the response of keratinocytes to herbal medicines, and as a result of evaluating several herbal medicines, they found that Juumifudokuto (herbal medicine ingredients: Bupleurum Root, Glycyrrhiza Root, Cherry Bark (Pueraria Root), Platycodon Root, Szechuan Kyusu, Ginger, Angelica Root, Bofeng, Jingjiang, and Poria Cocos) and Shofusan (herbal medicine ingredients: Rehmannia Root, Gypsum, Angelica Root, Syringa Root, Atractylodes Root, Bofeng, Atractylodes Root, Sesame, Anemone Rhizome, Glycyrrhiza Root, Sophora Root, Jingjiang, and Cicada Mitica) had a significant protective effect on keratinocytes against the skin damaging effects of regorafenib. Unsei-in (Unsei-in, herbal ingredients: Rehmannia Root, Peony Root, Szechuan Qiu, Angelica Root, Scutellaria Root, Phellodendron Bark, Coptis Rhizome, Gardenia Fruit), a prescription for skin diseases with a different herbal composition, had a slightly lower effect on protecting keratinocytes against regorafenib.

It has been found that herbal medicines containing the three herbs liquorice (kanzo), bofu, and keigai, which are common to Jumi-haidoku-to and Shofu-san, which have shown a strong keratinocyte-protecting effect, are preferable for treating and preventing HFS. In addition to Jumi-haidoku-to and Shofu-san, other herbal prescriptions that contain these three herbs include Keigairengyo-to and Bofu-tsusho-san, so these can also be used to protect keratinocytes.

On the other hand, it has been reported that immune checkpoint inhibitors, a type of anti-cancer drug, cause skin damage, resulting in vitiligo due to impaired melanin production. This is thought to be a different mechanism from HFS, which is accompanied by dermatitis, and treatment could involve the use of drugs that promote melanin synthesis, melanosome formation, and prostaglandin F2α activation to promote pigmentation.

The inventors investigated herbal medicines that have the ability to induce these related genes, and found that Keishikajutsubuto (herbal ingredients: cinnamon bark, peony root, licorice root, ginger, jujube, atractylodes umbellata, aconite), Goshuyuto (herbal ingredients: jujube, ginger, ginseng, goshuyu), and Keishikashakuyakuto + Shimotsuto (a combination of Keishikashakuyakuto and Shimotsutou, commonly known as the Kandabashi prescription, herbal ingredients: cinnamon bark, peony root, jujube, ginger, licorice root, rehmannia, angelica, and szechuan radish) have a melanin production promoting effect. The common herbal medicines are jujube (daiso) and ginger (shokyo), and medicines containing these are useful for treating vitiligo on the skin.

The following provides a detailed explanation with data, but the present invention is not limited to these.

[Culture of human keratinocytes and drug treatment]
Normal human epidermal keratinocytes (NHEK-Ad, Lonza) were seeded at 2 x ¹⁰⁵ cells/mL in serum-free cell culture medium (KGM-Gold™ Bullet Kit, Lonza) at 2 mL/well in a 6-well culture plate and cultured overnight in a _CO2 incubator (37°C, 5% _CO2 ). After confirming that the cells were confluent, the drug to be evaluated was added to the treatment medium Advanced DMEM + GlutaMAX™ Supplement (ThermoFisher) to a predetermined concentration and cultured overnight. Thereafter, RNA was extracted and subjected to gene expression analysis for transcriptome analysis. On the other hand, when cell damage was induced by an anticancer drug and the protective effect was analyzed based on the survival rate, keratinocytes were treated with the drug to be evaluated for 2 hours, then sorafenib or regorafenib was added, and the cells were cultured overnight, and the cell survival rate was examined.

[Drugs to be evaluated]
Each evaluation drug was evaluated at the following final concentration: nalfurafine hydrochloride 1 μM (MedChemExpress), pregabalin 1 μM (Tokyo Chemical Industry Co., Ltd.), bucladesine sodium 10 μM (Tokyo Chemical Industry Co., Ltd.), 5′-TMPS 100 μM (BioLog), sorafenib 10 μM (Santa Cruz), and regorafenib 20 μM (Tokyo Chemical Industry Co., Ltd.), all were dissolved in DMSO and adjusted to 0.1% DMSO when added to the cells. The herbal medicines, granules or tablets of Jumifudokuto, Shofusan, Unseiin, Keishikajutsufuto, Keishikashakuyakuto+Shimotsuto, Yokukansan (Tsumura Corporation), and Goshuyuto (Ominedo Pharmaceuticals), were dissolved in 30% ethanol, and after removing insoluble matter, the final evaluation concentration was adjusted to 100 μg/mL and added to the keratinocytes.

[Transcriptome analysis in keratinocytes]
Drugs were added to keratinocytes cultured to confluence in a 6-well culture plate and cultured overnight. The medium was removed from the plate and washed with ice-cold D-PBS(-), after which 600 μL/well of 2-mercaptoethanol-containing Buffer RLT Plus (included in RNeasy™ Plus Mini Kit) for RNA extraction was added and collected in a tube. Total RNA was extracted according to the extraction method in the kit protocol (RNeasy™ Plus Mini Kit, QIAGEN). Total RNA was eluted with 30-50 μL of nuclease-free water, and the concentration (ng/μL) and A260/A280 were measured using NanoDrop One. The degree of total RNA degradation was confirmed using Agilent RNA6000 Nano Kit according to the kit's protocol, and the RNA degradation was confirmed to be problem-free using Agilent 2100 Bioanalyzer System (RIN value 9.6 or higher). Human mRNA transcriptome analysis was performed as described in Non-Patent Document 12 using a microarray (3D-Gene mRNA Oligo chip, AROS (trademark) equipped with 24,460 probes, Toray Industries, Inc.).

[GO-BP analysis method]
In the gene ontology analysis of expressed genes from the transcriptome analysis results, the bioinformatics web tool Metascape (Non-Patent Document 13) was used as a biological process suitable for analyzing pharmacological actions. Metascape is a state-of-the-art high-precision analysis tool that has been used in more than 4,300 research papers as of March 2023 since its publication in 2019. In addition, a similar web tool, Enrichr (Non-Patent Document 14), also performed some GO analysis to confirm the commonality of the analysis results. Since Enrichr can also perform KEGG Pathway and Elsevier Pathway Analysis, the response of keratinocytes to the evaluation drug can be analyzed in more detail by performing pathway analysis alongside GO-BP. As a result, side effects and pharmacological actions occurring on the skin can be detected from the gene expression of normal human keratinocytes.

[Evaluation of keratinocyte protection in the presence of anticancer drugs]
We examined the protective effect of keratinocytes in the presence of multikinase inhibitors, including nalfurafine and herbal medicines. Keratinocytes were exposed to sorafenib or regorafenib, which are multikinase inhibitors that induce HFS at a very high frequency, to induce cytotoxicity, and the survival rate of keratinocytes was examined.

Human epidermal keratinocytes (NHEK-Ad) were seeded in a 96-well culture plate, and after they became confluent, the drugs to be evaluated were added at a given concentration to the treatment medium Advanced DMEM + GlutaMAX (trademark) Supplement (Lonza Co., Ltd.). After 2 hours of treatment, sorafenib was added at 10 μM or regorafenib at 20 μM, and the cells were cultured overnight. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL/well to the 96-well culture plate, and the cells were cultured for 1 to 4 hours. When the reaction became constant, the absorbance at 450 nm was measured using EnVision (PerkinElmer) or SpectraMax M3 (Molecular Devices) to calculate the cell survival rate. Statistical analysis of survival rate comparisons was performed using one-way ANOVA (Dunnett's multiple comparison test) using EZR software.

[Evaluation of keratinocyte protective effect of κ opioid agonists]
Multikinase inhibitors frequently cause skin disorders called HFS during cancer chemotherapy. Sorafenib was used to examine cytotoxicity using gene expression against keratinocytes as an index by transcriptome analysis. As a result, it was shown that the biological process of cytokine induction was activated by GO-BP from 1,747 genes whose gene expression rate was 2.0 or more and expression was increased, as shown in Table 1 (the relevant description part is shown in a thick outer frame. The same applies below). Pathway analysis showed that activation of cytokine pathways and complement pathways and inflammatory reactions were induced. On the other hand, as shown in Table 2, in 970 genes whose gene expression rate was suppressed to 0.5 or less, cell division was suppressed by GO-BP, and pathway analysis also showed that the cell cycle was suppressed. In other words, the cellular events shown by GO-BP and pathway analysis show results that directly reflect the symptoms of HFS, namely, the induction of dermatitis and the inhibition of keratinocyte proliferation.

　Assuming that the protection of keratinocytes would be performed with a single drug, the biological process of gene expression of an antipruritic drug (nalfurafine) and an analgesic drug (pregabalin) was investigated. This is because itching and pain are symptoms associated with skin disorders, and suppressing them is clinically required. GO-BP analysis was performed on the top 300 genes whose expression was enhanced by both drugs, and the results surprisingly revealed that nalfurafine has an effect of promoting epithelial cell proliferation (Table 3). This result suggests that comparative transcriptome analysis can suppress HFS, a side effect of sorafenib. In addition, when the gene group annotated by the GO-BP analysis was identified, it included PPARD, ZEB1, and IFT172, as shown in Table 4. These three genes have been found to support keratinocyte proliferation in the literature, and are thought to be the target molecules of nalfurafine's keratinocyte protective effect.

Next, the protective effect of nalfurafine against the cell proliferation inhibition by sorafenib was examined in an in vitro cell proliferation evaluation system. As comparative drugs, bucladesine sodium (BD, commercialized as Actosin ointment containing bucladesine sodium as the active ingredient), a treatment for pressure ulcers and skin ulcers, and thymidine nucleoside 5'-O-monophosphate (TMPS, Non-Patent Document 17), which has been reported to have a keratinocyte proliferation effect, were used. As a result, as shown in Figure 2, in the presence of 10 μM sorafenib, the survival rate of keratinocytes significantly decreased to 82.1% (p<0.01), but it recovered to 90.7% with pretreatment with nalfurafine and 91.7% with pretreatment with BD. Conversely, TMPS decreased to 70.3% under these conditions. Nalfurafine and BD were not statistically significantly different from the control group (no sorafenib treatment) and showed a protective effect on keratinocytes. The protective effect of kappa opioid agonists such as nalfurafine on keratinocytes has not been reported to date.

When used clinically as a therapeutic or preventive drug, events that lead to undesirable symptoms must be avoided, so we investigated the gene expression of aggravating factors related to keratinocyte skin disorders caused by nalfurafine, BD, and TMPS. It is known that such skin disorder events significantly impair the patient's QOL (Non-Patent Document 19). Here, we investigated the gene expression status of each drug during sorafenib treatment with respect to the effects on each factor in three categories: dermatitis induction factors (IL4, IL6, IL13, IL17A, IL23A, IL24, IL31, IL33), mechanical pain sensation factors (PIEZO1, PIEZO2, TRPA1, TRPV1), and melanin synthesis/pigmentation induction factors (IL37, MITF, TYR, TYRP1, DCT, MC1R, POMC). The gene expression rate (fold) relative to the untreated condition is summarized in Table 5. Gene induction by sorafenib (Sor) treatment was modified by each drug. The conditions under which the gene expression rate relative to the untreated condition was lower than that of sorafenib alone (suppressing exacerbation of each category) are shown in the bold outer frame. Nalfurafine (NFN) had a stronger inhibitory effect than BD and TIMP in all categories of dermatitis inducers, mechanical pain receptors, and melanin synthesis/pigmentation inducers, showing a favorable profile (when the gene expression rate number is smaller than Sor = bold outer frame = there are more, and when the number is larger than Sor = underlined part = there are fewer, it is preferable as a protective drug).

As for mechanical pain sensitivity factors, BD, a treatment for bedsores and skin ulcers, increases PIEZO2, which may be one of the factors contributing to the undesirable side effect of Actosin ointment, which is the induction of pain (as stated in the interview form for the drug). The antipruritic drug nalfurafine not only tends to inhibit PIEZO2, but also has an analgesic effect as a kappa agonist, so it is expected to have a clinical effect of suppressing itching and pain.

In addition, in the treatment of skin diseases, pigmentation induction is a major cosmetic concern for patients. BD is thought to have a tendency to exacerbate pigmentation induction, but nalfurafine has a favorable profile in that it shows a tendency to inhibit it. There have been no reports to date of such an inhibitory effect of κ opioid agonists on keratinocytes.

From the above, nalfurafine has a favorable effect on protecting keratinocytes in sorafenib-induced skin damage, and can therefore be used as a therapeutic and preventive drug for HFS.

Furthermore, because the protective effect on keratinocytes was thought to be a common effect of κ opioid agonists, the protective effects of nalfurafine and difelikephalin were examined against the cell proliferation inhibition of two anticancer drugs, regorafenib (a multikinase inhibitor) and capecitabine (a 5-FU DNA synthesis inhibitor). As shown in Figure 3, both nalfurafine and difelikephalin reduced the inhibition of keratinocyte proliferation and demonstrated a cell protective effect.

[Evaluation of keratinocyte protective effect of the combination agent]
The incidence of skin disorders as a side effect of multikinase inhibitors is 47-59% for sorafenib and 50-81% for regorafenib, with regorafenib showing a slightly higher incidence (all data taken from the drug interview form). We investigated what kind of gene expression reflects the skin disorder action of regorafenib as an event on keratinocytes. As a result, as shown in Table 6, 2149 types of genes with a gene expression rate of 2.0 or higher and increased expression were shown to induce strong inflammation with GO-BP, and pathway analysis showed that activation of cytokine pathways and complement pathways and inflammatory responses were induced. On the other hand, as shown in Table 7, in 659 types of genes whose expression was suppressed with a gene expression rate of 0.5 or less, epithelial development was strongly suppressed by GO-BP (the event GO:0008544 "epidermal formation and development" showed a Log ₁₀ (P) value of -28.27, indicating an extremely strong characteristic event), and pathway analysis also showed that cell cycle was suppressed and cell proliferation was suppressed. In other words, the cellular events shown by GO-BP and pathway analysis showed results that directly reflect the symptoms of HFS, namely the induction of dermatitis and strong inhibition of keratinocyte proliferation.

For such complex events, a prescription of a complex drug may be preferable to a single drug for treatment or prevention, so we evaluated a representative complex prescription, a herbal medicine. To date, there have been no reports of the application of orally administered herbal medicines in the form of decoctions or extracts for local administration (external application) for treatment or prevention.

Five herbal medicines with the potential for anti-inflammatory and analgesic effects were selected based on their intended use, and selection was made based on GO-BP events (keratinization, cell proliferation, cell-cell adhesion, wound healing, etc.) that lead to cell proliferation of keratinocytes and maintenance of barrier function. Table 8 shows the GO-BPs of 300 top genes whose expression is induced in keratinocytes when treated with five herbal medicines (Jumihaidokuto: Jyuumihaidokuto/JHT, Shofusan: Shoufusan/SFS, Keishikajutsubuto: Keishikajutsubuto/KKT, Keishigoshuyuto: Goshuyuto/GYT, Yokukansan: Yokukansan/YKS). Among these, multiple events related to cell proliferation and maintaining barrier function (bold outer frame) were detected in JHT and SFS, indicating that they have a strong effect.

The GO-BP that was most suppressed in keratinocytes by regorafenib treatment was epidermis development (GO:0008544), and 50 genes involved in this biological process were extracted (Table 9). Most of these were genes related to skin keratinization, which was consistent with the pathology of HFS. It is believed that de-suppression of these genes will lead to the treatment of HFS, and it was found that JHT or SFS treatment strongly increased the expression of 48 of the 50 genes, excluding IL17A and WHRN, and acted to restore the inhibition of epithelial development (14 genes with JHT and 7 with SFS showed a 2-fold or greater increase in expression = underlined part).

Next, keratinocytes were actually cultured in the presence of JHT or SFS for 2 hours, after which regorafenib was added and cell viability was examined in vitro. As a result, as shown in Figure 4, without the herbal medicine, cell viability dropped to 12.4% due to the cell proliferation inhibition of regorafenib, whereas in the presence of JHT and SFS, the cell viability was 23.4% and 23.0%, respectively, showing a recovery of approximately two-fold in both cases, demonstrating a protective effect on keratinocytes. The results of cell viability were in good agreement with the results of the GO-BP analysis, demonstrating the validity of the comparative transcriptome analysis.

Furthermore, to investigate the effect of herbal composition, Unseiin (USI), which is used for skin diseases and contains different herbal ingredients from JHT and SFS, was evaluated as a comparative example. USI had a cell survival rate of 15.3%, which was weaker than JHT and SFS. The difference in effect was the result of the difference in herbal composition, and the herbal ingredients that were common to JHT and SFS but not included in USI were liquorice (kanzo), bofu (bofu), and keigai (keigai) (Table 10). Not all herbal medicines for skin diseases are good, and it was thought that these three herbal medicines contributed greatly to the keratinocyte protective effect. Herbal medicines are generally administered orally as decoctions, and there have been no examples to date of evaluating the keratinocyte protective effect assuming topical use.

[Protective effect of κ opioid agonists on keratinocytes against inhibition of epithelial development]
Comparative transcriptome analysis is also useful for identifying target molecules (genes) of drugs. Once a notable GO-BP is identified, a group of genes involved in the process can be extracted from a public database (e.g., GENEONTOLYOGY, etc.), and the gene expression ratio can be used to determine which genes are affected by the drug. It was found that exposure to regorafenib in keratinocytes strongly suppressed the expression of 50 genes related to the GO-BP "epithelial development (GO:0008544)" (Table 9). Of these, five genes (KLK14, KRT25, LCE2A, LCE2C, NOTCH1) showed enhanced expression in JHT and SFS, which exerted a protective effect. In particular, KRT25, LCE2A, and LCE2C are known to be genes that are expressed specifically in the skin and that constitute the cornified envelope necessary for forming the barrier structure of the skin. These results suggest that regorafenib inhibits the expression of genes related to the keratinocyte keratinization envelope over a wide range, inhibiting keratinization and damaging the skin barrier structure, which leads to the development of HFS. In addition, JHT and SFS act to restore the keratinization envelope and exert a protective effect on keratinocytes.

When the effect of single-agent kappa agonists on regorafenib's inhibition of epidermal development was examined from the perspective of gene expression, as shown in Figure 5, regorafenib suppressed gene expression to 0.5-fold or less, but kappa opioid agonists (nalfurafine and difelikephalin) induced many genes to an expression rate of 1.0 or more, demonstrating their protective effect on keratinocytes.

Furthermore, when we looked at the expression of genes related to inflammation, pain, and pigmentation, which are undesirable symptoms of HFS, as shown in Figure 6, κ opioid agonists tended to suppress all of these, and it was found that they can also be expected to have an effect of alleviating HFS symptoms. In Figure 6, PIEZO02 is a receptor that detects mechanical pain, and while it is strongly expressed with regorafenib, which is undesirable, it is strongly suppressed by nalfurafine, which is particularly preferable.

[Melanin production promoting effect of herbal medicines for vitiligo]
Pigmentation associated with inflammation reduces QOL, but on the other hand, skin pigment loss, such as in vitiligo vulgaris, also reduces QOL, so a therapeutic drug is needed. Vitiligo caused by immune checkpoint inhibitors is also a problem in cancer treatment, but no drug has been found. When focusing on herbal medicines and examining them with comparative transcriptome analysis, it was found that melanin production is activated by three types of herbal medicines (KKB, GYT, and KDB) as shown in Figure 7. In addition, the herbal ingredients of these three types of herbal medicines are as shown in Table 11, and since they have in common Daiso and Shokyo, it was found that preparations containing either or both of these two herbal medicines can be used to treat vitiligo. In most cases, herbal medicines are generally administered orally as decoctions, and there have been no examples of evaluating the therapeutic effect of vitiligo assuming topical use, and this was discovered for the first time by the present invention.

As described above, comparative transcriptome analysis is effective for identifying drug target genes and discovering compounds for treating side effects, and is also an extremely useful technique in drug discovery research.

Claims (8)

A topical preparation for skin disorders, comprising:
The active ingredient is a kappa opioid agonist and/or a herbal medicine ingredient,
A topical administration agent, wherein the active ingredient has a keratinocyte protecting effect. The kappa opioid agonist is
The topical preparation according to claim 1, which is at least one selected from the group consisting of nalfurafine (TRK-820), difelikefalin (CR845), YNT-1612, CR665 (FE200665), HSK21542, GR89696, U69593, Salvinorin A, EOM salvinorin B, and pharma- ceutically acceptable salts thereof. The protective effect of the keratinocytes is
2. The topical preparation according to claim 1, which has at least one of the effects of promoting cell proliferation during cell damage, suppressing the expression of inflammatory cytokine genes, and suppressing the expression of pigmentation-inducing genes. The herbal medicine ingredient is
2. The topical preparation according to claim 1, which comprises at least one of licorice, burdock root and kettle root. The applicable diseases are:
Skin disorders such as hand-foot syndrome (HFS), perivascular dermatitis, lichenoid dermatitis, erythematous dermatitis, bullous pemphigoid (BP), bullous rash, Stevens-Johnson syndrome-like reaction, psoriasis, hemolytic dermatitis, acute generalized exanthematous pustulosis (AGEP), folliculitis, acne-like reaction, epiphora, pigmentation, radiation dermatitis, which is a disorder of the skin and tissue mucosal epithelium due to radiation therapy, mucositis, stomatitis, angular cheilitis, pigmentation, or atopic dermatitis due to scratching,
The topical preparation according to any one of claims 1 to 4, which is used for protecting keratinocytes. A topical preparation for the treatment of vitiligo, vitiligo vulgaris, and skin hypopigmentation, containing at least one herbal ingredient from either Daiso or Zingiber officinale. A method for exploring novel uses of pharmaceutical compositions by extracting corresponding intracellular processes from transcriptome analysis results of multiple pharmaceutical compositions, comprising:
Obtaining a gene expression profile (GO-BP) reflecting a predetermined side effect from the results of transcriptome analysis of the target pharmaceutical composition;
Obtaining a gene expression profile (GO-BP) of a candidate compound for treating or preventing side effects;
A GO-BP reflecting the side effects of the target pharmaceutical composition is compared with a GO-BP of a candidate compound;
Selecting a candidate compound that exhibits GO-BP that counteracts the side effects of the pharmaceutical composition of interest;
A method for exploring new uses of a pharmaceutical composition, comprising verifying the effect of a candidate compound on side effects in an in vitro cell evaluation system. The novel use of the pharmaceutical composition is protection of keratinocytes,
The pharmaceutical composition is an anticancer drug, and the side effect is HFS caused by the anticancer drug,
Obtaining a gene expression profile (GO-BP) reflecting damage caused by an anticancer drug to keratinocytes;
Obtaining a gene expression profile (GO-BP) of a candidate compound for protecting keratinocytes and treating or preventing the disease;
The GO-BP that reflects the side effects of anticancer drugs and the GO-BP of candidate compounds were compared and analyzed.
Select candidate compounds that exhibit keratinocyte protective effects from GO-BP,
A method for exploring new uses of a pharmaceutical composition, comprising verifying the protective effect of a candidate compound on keratinocytes based on cell viability in an in vitro human normal keratinocyte proliferation evaluation system.