Pharmaceutical composition for skin diseases, and method for searching novel use of medicine

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

[0001] The present invention relates to a pharmaceutical composition for treating skin diseases and a method for exploring new uses of pharmaceuticals.

[0002] The epidermis is the outermost layer of the skin and acts as a barrier to prevent the intrusion of foreign substances and evaporation of body moisture, protecting the interior. The epidermis is a stratified squamous epithelium approximately 0.1-0.2 mm thick, composed of the keratinocyte layer, granular cell layer, spinous cell layer, and basal cell layer from the outer layer. The majority of these cells are keratinocytes, which differentiate and migrate from the lower layer to the upper (outer) layer, keratinizing and differentiating into the keratinocyte layer. The main function of keratinocytes is to provide 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.

[0003] Skin disorders can occur for a variety of reasons, but can also occur as a side effect of cancer treatments such as anticancer drug therapy and radiation therapy. During chemotherapy in particular, skin disorders called hand-foot syndrome (HFS) or its synonym, hand-foot skin reaction (HFSR), can occur as a side effect, significantly impairing the patient's quality of life (hereinafter referred to as "HFS"). HFS has long been a problem in Japan, and the Ministry of Health, Labor and Welfare has issued a manual for dealing with serious side effects, "Hand-Foot Syndrome" (Non-Patent Document 1), to raise awareness, but currently there are no satisfactory treatments or therapies.

[0004] 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). It is also known that skin disorders due to side effects can 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).

[0005] Symptoms of HFS include redness, edema, hyperkeratosis (hyperkeratosis), paresthesia, pain, etc. on the palms and soles of the feet, and in severe cases, blisters and bleeding may also occur, sometimes causing difficulty grasping the hands and difficulty walking, significantly limiting activities of daily living. HFS is a finding of impaired skin barrier function, and histopathologically, band-like necrosis of epidermal keratinocytes is observed from the spinous layer to the granular layer of the epidermis, and hyperkeratosis (hyperkeratosis) and parakeratosis are observed in the stratum corneum, characterized by severe impairment of the skin barrier function and abnormal keratinization in keratinocytes (Non-Patent Document 4). Because the impaired site is in the stratum corneum, keratinocyte protective agents with skin barrier function can be used as preventive agents for HFS and therapeutic agents for alleviating the pathology.

[0006] Although cancer chemotherapy has progressed, the details of the mechanism of HFS development have remained unclear for over 20 years, and no preventative measures have been established. Treatments for HFS have included reducing physical irritation to the skin, exfoliating with emollients containing urea or salicylic acid, and using pyridoxamine (vitamin B6) or COX2 inhibitors, but clinical trials have not demonstrated clear efficacy (Non-Patent Document 5).

[0007] Disclosed therapeutic agents for HFS include a pharmaceutical composition containing allopurinol and pyridoxamine, which are used to treat gout and other conditions (Patent Document 1), a histone deacetylase inhibitor such as phenylbutyrate (Patent Document 2), and a therapeutic agent containing an azurophilic antifungal drug as an active ingredient (Patent Document 3), but these have not yet been put to practical use.

[0008] Skin disorders caused by new anticancer drugs have also been reported. Immune checkpoint inhibitors have recently begun to be used in cancer treatment, and attention has been drawn to the occurrence of skin disorders such as vitiligo and skin hypopigmentation, which are thought to be caused by immune reactions. However, no effective drug therapy for vitiligo has been found (Non-Patent Document 6).

[0009] One of the reasons for the lack of effective therapeutic agents for skin disorders is the lack of an appropriate drug evaluation system. The metabolic and resistance characteristics of epidermal cells differ between humans, who have little body hair, and animals, who are covered in hair and have strong skin tissue. This makes it difficult to create appropriate animal models for skin disorder research. While a mouse HFS model has been created using capecitabine, the nontoxic dose in mice was approximately 10 times the human dose, resulting in low drug responsiveness. Furthermore, the therapeutic candidate identified required approximately 60 times the human clinical dose (Non-Patent Document 7). Thus, in cases involving the sensitivity of anticancer drugs, such as HFS, or drug metabolites, or when immune responses are involved, evaluation using animal models with different sensitivities is difficult. Therefore, it is desirable to first evaluate using homologous human cells.

[0010] Japanese Patent Application Laid-Open No. 2012-180370 Japanese Patent Application Laid-Open No. 2012-530076 Japanese Patent Application Laid-Open No. 2021-11453

[0011] Ministry of Health, Labor and Welfare, “Hand-foot syndrome: a comprehensive guide to serious adverse reactions”, 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 et al, 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. al., Sci Rep. 2021 Apr 26;11:8964. doi:10.1038 / s41598-021-88460-9Yamamizu 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 / s41467-019-09234-6Chen et al., BMC Bioinformatics 2013, 14:128. doi:10.1186 / 1471-2105-14-128Tanaka et al. 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 / ijms23010238Urakawa et al., J Cancer. 2019, 10(20):4846-4851. doi:10.7150 / jca.31059.

[0012] As mentioned above, it is difficult to evaluate the development of therapeutic agents for skin disorders using animal models. Therefore, in the development of preventive and therapeutic agents involving keratinocytes, it is important to develop an evaluation system for normal human keratinocytes without relying on animal models. The pharmaceutical compositions shown in the following examples relate to protective agents for keratinocytes, which have the barrier function of the skin. In particular, the objective of the present invention is to provide compounds that protect keratinocyte damage in HFS caused by anticancer drugs. Another objective of the present invention is to provide a method for exploring new uses of existing pharmaceuticals without relying on animal models.

[0013] This specification discloses the following pharmaceutical compositions and discovery methods. (1) A topical agent for skin disorders, which contains a κ-opioid agonist and / or a herbal ingredient as an active ingredient, and the active ingredient has a keratinocyte-protecting effect. κ-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, it was discovered that these drugs are effective in protecting keratinocytes and can treat and prevent skin disorders.

[0014] (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 pharmaceutically 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.

[0015] (3) The topical agent according to (1), wherein the protective effect of keratinocytes is at least one of promoting proliferation during cell damage, suppressing expression of inflammatory cytokine genes, and suppressing expression of pigmentation-inducing genes. Comparative transcriptome analysis shown below revealed that the topical agent has at least one of the effects of promoting proliferation during cell damage, suppressing expression of inflammatory cytokine genes, and suppressing expression of pigmentation-inducing genes, thereby treating or preventing skin disorders.

[0016] (4) The topical preparation according to (1), wherein the herbal ingredient is at least one of licorice, Boufu, and Keigai. Analysis of herbal prescriptions has shown that licorice, Boufu, and Keigai have a keratinocyte-protecting effect.

[0017] (5) The topical administration agent according to any one of (1) to (4), for treating 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, acneiform reaction, epiphora, pigmentation, radiation dermatitis, which is a damage to the skin and tissue mucosal epithelium caused by radiation therapy, mucositis, stomatitis, angular cheilitis, pigmentation, or atopic dermatitis caused by scratching, wherein the topical administration agent is for protecting keratinocytes. There are various types of skin disorders, and the topical administration agent is applicable to those whose symptoms are alleviated by protecting keratinocytes.

[0018] (6) A herbal medicine for the treatment of vitiligo, vitiligo vulgaris, and skin hypopigmentation, comprising a topical formulation containing one or both of the herbal ingredients of Daiso and Zingiber officinale. Skin disorders such as vitiligo and skin hypopigmentation caused by the melanin production system have also been found, and these can also be evaluated using keratinocytes. Pigment-producing melanocytes originate from neural crest cells and reside in the basement membrane, while neural crest-derived cells can also differentiate into keratinocytes. Furthermore, melanosomes, organelles that synthesize and store melanin, are transferred from melanocytes to keratinocytes. Therefore, since skin hypopigmentation in conditions such as vitiligo and vitiligo vulgaris is also closely related to keratinocytes, effective drugs can be selected using an evaluation system using keratinocytes.

[0019] (7) A method for exploring new uses of pharmaceutical compositions by extracting counteractive intracellular processes from the results of transcriptome analysis of multiple pharmaceutical compositions, the method 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 or 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 effectiveness of the candidate compound against the side effect in an in vitro cell evaluation system. This method involves performing transcriptome analysis of multiple existing pharmaceutical compositions, and by comparative transcriptome analysis, it is possible to select a pharmaceutical compound that can treat the side effect of the target pharmaceutical composition. This is a very efficient method for exploring new uses of GO-BP as a phenotype of pharmaceutical compositions.

[0020] (8) A method for discovering a novel use of a pharmaceutical composition, wherein the novel use of the pharmaceutical composition is keratinocyte protection, the target pharmaceutical composition is an anticancer drug, and the side effect is 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 the gene expression profile (GO-BP) of a candidate compound for keratinocyte protection and treatment / prevention; comparatively analyzing the GO-BP reflecting the side effect of the anticancer drug with the GO-BP of the candidate compound, selecting a candidate compound that exhibits keratinocyte protective activity from the GO-BP, and verifying the keratinocyte protective effect of the candidate compound based on cell viability in an in vitro human normal keratinocyte proliferation evaluation system. The present inventors have discovered by this discovery method that kappa opioid agonists and herbal medicines have a keratinocyte protective effect.

[0021] Schematic diagram showing a method for discovering keratinocyte-protective drugs using comparative transcriptome analysis. Diagram showing the protective effect of single agents against cell proliferation inhibition by sorafenib. Sor: sorafenib, NFN: nalfurafine. Cytoprotective effect of kappa opioid agonists against keratinocyte proliferation inhibition by regorafenib and capecitabine. Regorafenib (A) or capecitabine (B) strongly inhibit keratinocyte proliferation, while the kappa opioid agonists nalfurafine (NFN) and difelikefalin (DKN) have the effect of alleviating proliferation inhibition. Diagram showing the protective effect of herbal medicines against cell proliferation inhibition by regorafenib. Reg: regorafenib, JHT: Jumi-haidoku-to, SFS: Shofu-san, USI: Unsei-in. Diagram showing the drug-induced expression of keratinocyte-related genes. Regorafenib (Reg) strongly suppresses expression, but the kappa opioid agonists nalfurafine (NFN) and difelikephalin (DKN) also enhance expression, maintaining keratinocyte keratinization. Figure showing the gene expression induction effect of drugs on exacerbating factors related to keratinocyte skin disorders. Regorafenib (Reg) strongly enhances expression in some areas, but the kappa opioid agonists nalfurafine (NFN) and difelikephalin (DKN) tend to suppress expression, suggesting an inhibitory effect on keratinocyte skin disorders. Figure showing the induction effect of keratinocyte pigmentation-related genes by herbal medicines. KKB (Keishi-ka-shutsu-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.

[0022] The inventors of the present invention have focused on the fact that drug side effects, like pharmacological actions, are controlled by cellular gene expression and have conceived the idea of ​​using transcriptome analysis to screen for drugs that alleviate side effects. Once a combination of a compound and target cell is determined, transcriptome analysis can cover all pathways and biological processes related to the primary and secondary effects. Specifically, transcriptome analysis of a drug with side effects is performed to identify intracellular processes that reflect the side effects, and transcriptome analysis of a candidate compound for treating the side effects is performed to extract opposing intracellular processes. Based on the comparative effects of the compound, new uses can be explored (hereinafter, this method is referred to as comparative transcriptome analysis or comparative transcriptome analysis). In other words, transcriptome analysis of a drug with side effects and that of a candidate compound for treating the side effects are performed, and the results are compared to identify compounds that can complement the gene expression that causes the side effects. If the causative gene for the side effects is identified, compounds that induce the complementing gene expression can be searched for. Even if the causative gene is unknown, 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.

[0023] 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.

[0024] Transcriptome analysis, which analyzes global gene expression, is widely used to investigate cellular responses and phenotypes. However, the traditional approach is to explain the mechanisms of drug stimulation and cellular interactions, using information from the "selected stimulus → gene expression results" chain. In contrast, comparative transcriptome analysis combines information from the reverse chain, "gene expression results → stimulus selection," to select drugs that can counteract side effects based on the results of drug gene expression analysis. By performing this on normal cells, drugs that treat side effects can be discovered. Disease-derived cells can also be used to search for compounds that can treat the disease.

[0025] Figure 1 shows a method for searching for keratinocyte-protecting drugs using comparative transcriptome analysis. Similarly, once the biological process of interest is determined, the pharmacological action of each drug can be estimated from the gene expression profile of each drug, and drugs with the desired effects can be selected using this method as an efficient screening method.

[0026] Here, we explored protective drugs for preventing and alleviating skin disorders. It goes without saying that similar analyses can be used to explore preventive and therapeutic drugs for all kinds of side effects. In the following examples, to explore protective drugs for preventing and alleviating skin disorders, human normal keratinocytes were used as target cells, and all pharmacological effects, including beneficial and harmful effects, occurring 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 capable of detecting events leading to keratinocyte activation and survival were selected as candidate protective drugs. Furthermore, the validity of comparative transcriptome analysis of the selected candidate protective drugs was verified by measuring cell viability using an in vitro keratinocyte evaluation system.

[0027] The specific steps are as follows: (1) Obtain a gene expression profile (GO-BP) that reflects damage to keratinocytes as an HFS event caused by anticancer drugs; (2) Analyze the gene expression profiles (GO-BP) of candidate compounds that can be applied to treatment and prevention; (3) Select candidate compounds that counteract HFS events by matching contrasting GO-BPs; (4) Verify the protective effect of candidate compounds on keratinocytes (cell survival rate) in an in vitro cell proliferation evaluation system.

[0028] This reverse engineering approach, using normal human keratinocytes, allows us to evaluate all types 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 identifies new indications for these disorders.

[0029] GO-BP analysis using comparative transcriptome analysis can also reveal information about diverse cellular phenotypes. For example, if pain occurs during a skin disorder, the "GO-BP in response to stimuli" can be examined to select a drug with a potential analgesic effect as a phenotype. Furthermore, in skin diseases, post-healing scarring and pigmentation can significantly impact cosmetic appearance and sometimes reduce a patient's quality of life (QOL). Vitiligo and hypopigmentation as diseases and symptoms are also similarly problematic. Gene expression profiles can also be used to predict and evaluate the alleviation and prevention of these skin conditions.

[0030] In this specification, the term "skin disorder as a side effect of anticancer drugs" encompasses various disorders that occur in the skin as a side effect of anticancer drugs, such as redness, erythema, edema, bleeding, pain, itching, inflammation, paresthesia, rash, skin scaling, stratum corneum peeling, blisters, and vitiligo, but particularly refers to decreased function, damage, and necrosis of keratinocytes, and a typical example of such a disease is HFS, which is pathologically characterized by keratinocyte necrosis.

[0031] As used herein, the term "protecting keratinocytes" includes healing, alleviating, or reducing skin damage that has already occurred due to the administration of an anticancer drug, as well as preventatively suppressing the occurrence of skin damage due to the administration of an anticancer drug, and suppressing inflammation and pigmentation during the repair of skin damage.

[0032] The primary target skin disorder here is a skin disorder called HFS, which occurs due to the administration of anticancer drugs. The type of anticancer drug is not particularly limited, and includes various anticancer drugs that may cause skin disorders as a side effect, including molecular targeted drugs as well as anticancer drugs that are not classified as molecular targeted drugs.

[0033] The therapeutic or preventive effect on skin disorders caused by anticancer drug administration can be evaluated, for example, by using cultured skin cells such as epidermal keratinocytes to measure the ability to restore cell viability reduced by anticancer drug treatment. As described in the Examples below, evaluation can be performed using monolayer cultured cells or a cultured three-dimensional human epidermal model. However, given 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 are not recommended due to the possibility of genetic mutations. Furthermore, since topical agents such as topical preparations are anticipated, the intended purpose can be achieved by directly evaluating them using in vitro keratinocyte sheets.

[0034] Targeted 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, acneiform 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 skin hypopigmentation include vitiligo, vitiligo vulgaris, and depigmentation.

[0035] The topical preparation of the present invention is applied to the site of skin disorders for therapeutic or preventive purposes, and its dosage form may be an ointment, cream, lotion, external liquid preparation, gel, cataplasm, patch, sheet, etc. It may be a single agent or a complex formulation, and may contain excipients, binders, lubricants, disintegrants, surfactants, suspending agents, emulsifiers, stabilizers, etc. that are commonly used in the pharmaceutical field, as needed.

[0036] The dosage of the topical agent of the present invention can be selected appropriately depending on the patient's age, body weight, condition of the skin disorder, and the site of the disorder. For example, the daily amount of active ingredient for an adult weighing approximately 60 kg is approximately 1 μg to 100 mg when administered topically by application, and the daily administration may be once or in divided doses. The agent may be administered daily or every few days. The keratinocyte protective agent may be administered until a certain therapeutic effect on the skin disorder is achieved (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 disorder after the start of anticancer drug administration.

[0037] The present 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 the effect of protecting keratinocytes, and at the same time have inhibitory effects on events such as inflammation induction, mechanical pain sensation, and pigmentation that are thought to be involved in skin disorders, nerve disorders, etc. κ-opioid agonists are known to have analgesic and antipruritic effects, and are therefore suitable compounds for reducing and preventing HFS skin disorders that occur as a side effect of cancer treatment, while suppressing itching and pain.

[0038] κ-opioid agonists have also been reported to have effects that lead to the inhibition of cell proliferation. For example, nalfurafine, a representative κ-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," attention must be paid to keratinocytes.

[0039] The inventors have also found that nalfurafine suppresses the expression of IL37 (Non-Patent Document 10) and DCT (Non-Patent Document 11), which are genes involved in skin pigmentation. κ-opioid agonists such as nalfurafine have an inhibitory effect on pigmentation and have antipruritic and analgesic effects in addition to a protective effect on keratinocytes, and therefore can be more preferably used when protecting keratinocytes as a combination agent with other compounds.

[0040] Here, the κ-opioid agonist is selected from the group consisting of, for example, nalfurafine (TRK-820), difelikefalin (CR845), YNT-1612, CR665 (FE200665), HSK21542, GR89696, U69593, salvinorin A, EOM salvinorin B, and pharmaceutically acceptable salts thereof, but any κ-opioid agonist may be used.

[0041] GO-BP analysis is not limited to single-ingredient compounds (single drugs), but can also be applied to compounds that are compounds of multiple ingredients. For example, the most typical compound drug among existing pharmaceuticals is traditional Chinese medicine. Western medicines generally contain one active ingredient per drug, and their site of action (mechanism of action) is also clearly defined. However, traditional Chinese medicines contain multiple active ingredients, making them uniquely effective for a wide range of symptoms. Prescriptions are based on traditional Chinese medicine, and focus on the patient's constitution and condition, as represented by "deficiency / excess" and "qi, blood, and water," and are prescribed based on these results. Traditional Chinese medicines are prescribed as decoctions, administered orally and systemically, and are also prescribed to reduce the side effects of cancer treatment.

[0042] In cancer treatment, careful attention must be paid to the dosage form. Taking HFS, a side effect of anticancer drugs, as an example, the reason for its appearance on the hands and feet is thought to be that the palms and soles have many blood vessels, a high rate of skin cell division, and a large number of eccrine glands, making them susceptible to interference with the excretion of anticancer drugs through sweat (Non-Patent Document 5). Because cancer treatment drugs are primarily administered systemically, topical application of drugs for the prevention and treatment of skin disorders on the hands and feet is preferred to avoid drug interactions with cancer treatment drugs. However, there have been no reports of topical administration of herbal prescriptions based on keratinocyte protective effects.

[0043] 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 Jumifudokuto (Jumihaidokuto, herbal ingredients: Bupleurum Root, Glycyrrhiza, Cherry Bark (Pueraria), Platycodon grandiflorum, Szechuan Qiu, Ginger, Angelica Root, Bofeng, Jingjiang, Poria Cocos) and Shofusan (Shofusan, herbal ingredients: Rehmannia Root, Gypsum, Angelica Root, Cangfu Root, Atractylodes Rhizome, Bofeng, Mucor, Sesame, Anemone Rhizome, Glycyrrhiza Root, Sophora Root, Jingjiang, Cicada Miscanthus) have a significant protective effect on keratinocytes against the skin damaging effects of regorafenib. Unseiin (Unseiin, herbal ingredients: Rehmannia Root, Peony Root, Szechuan Gum, 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.

[0044] It has been found that herbal medicines containing the three herbal medicines common to Jūmi-haidoku-to and Sōfu-san, which have shown a strong keratinocyte-protecting effect, namely, liquorice (kanzō), bofu (bofu), and keigai (keigai), are preferable for the treatment and prevention of HFS. In addition to Jūmi-haidoku-to and Sōfu-san, other herbal prescriptions containing these three herbal medicines include Keigairengyōto (keigairengyōto) and Bofu-tsushōsan (bofutsushosan), and these can also be used to protect keratinocytes.

[0045] On the other hand, immune checkpoint inhibitors, a type of anti-cancer drug, have been reported to cause skin damage, with vitiligo occurring 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.

[0046] The inventors investigated herbal medicines capable of inducing these related genes and found that Keishikajutsubuto (herbal ingredients: cinnamon bark, peony root, licorice root, ginger, jujube, atractylodes chinensis, and aconite root), Goshuyuto (herbal ingredients: jujube, ginger, ginseng, and 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 tree, and szechuan ginseng) have melanin production-promoting effects. The common herbal ingredients are jujube (daiso) and ginger (shokyo), and medications containing these are useful for treating vitiligo.

[0047] The present invention will be specifically explained below with reference to the following data, but is not limited to these.

[0048] [Culture of human keratinocytes and drug treatment] Normal human-derived epidermal keratinocytes (NHEK-Ad, Lonza) were cultured at 2 × 10 in serum-free cell culture medium (KGM-Gold™ Bullet Kit, Lonza). ５ The cells were seeded at 2 mL / well in a 6-well culture plate and incubated under CO ２ In an incubator (37°C, 5% CO ２ ) overnight. 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. Then, for transcriptome analysis, RNA was extracted and subjected to gene expression analysis. On the other hand, when analyzing the protective effect based on survival rate after cell damage caused by an anticancer drug, keratinocytes were treated with the drug to be evaluated for 2 hours, followed by the addition of sorafenib or regorafenib, and cultured overnight, and cell survival rate was examined.

[0049] [Drugs to be evaluated] Each 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.). Each drug was dissolved in DMSO to give a final concentration of 0.1% DMSO when added to the cells. The herbal medicines were Jumifudokuto, Shofusan, Unseiin, Keishikajutsubukuto, Keishikashakuyakuto+Shimotsuto, Yokukansan (Tsumura Corporation), and Goshuyuto (Ominedo Pharmaceutical) in granules or tablets, which were dissolved in 30% ethanol, and after removing insoluble matter, the solution was adjusted to a final evaluation concentration of 100 μg / mL and added to the keratinocytes.

[0050] [Transcriptome analysis in keratinocytes] Keratinocytes were cultured to confluence in a 6-well culture plate, and the drug was added and cultured overnight. The medium was removed from the plate and washed with ice-cold D-PBS(-). 600 μL / well of 2-mercaptoethanol-containing Buffer RLT Plus (included in the RNeasy™ Plus Mini Kit) for RNA extraction was added, and the RNA was 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 a NanoDrop One. The degree of total RNA degradation was confirmed using an Agilent RNA6000 Nano Kit prepared according to the kit's protocol, and the Agilent 2100 Bioanalyzer System confirmed that there were no problems with RNA degradation (RIN value of 9.6 or higher). Human mRNA transcriptome analysis was performed using a microarray (3D-Gene mRNA Oligo chip, AROS™ equipped with 24,460 probes, Toray Industries, Inc.) as described in Non-Patent Document 12.

[0051] [GO-BP Analysis Method] In the gene ontology analysis of expressed genes from transcriptome analysis results, the bioinformatics web tool Metascape (Non-Patent Document 13) was used to identify biological processes suitable for analyzing pharmacological effects. Metascape is a state-of-the-art, high-precision analysis tool that has been used in over 4,300 research papers as of March 2023 since its publication in 2019. Partial GO analysis was also performed using a similar web tool, Enrichr (Non-Patent Document 14), to confirm the consistency of the analysis results. Since Enrichr also supports KEGG Pathway and Elsevier Pathway Analysis, pathway analysis can be performed alongside GO-BP to analyze the response of keratinocytes to evaluation drugs in more detail. This allows for the detection of side effects and pharmacological effects on the skin from gene expression in normal human keratinocytes.

[0052] [Evaluation of keratinocyte protective effect in the presence of anticancer drugs] The protective effect of keratinocytes in the presence of multikinase inhibitors was examined for the drugs under evaluation, nalfurafine and herbal medicines. Keratinocytes were exposed to sorafenib or regorafenib, multikinase inhibitors that induce HFS at a very high frequency, to cause cytotoxicity, and the survival rate of keratinocytes was examined.

[0053] Human epidermal keratinocytes (NHEK-Ad) were seeded onto 96-well culture plates. After reaching confluence, the target drug was added at a predetermined concentration to the treatment medium, Advanced DMEM + GlutaMAX™ Supplement (Lonza Inc.). After 2 hours of treatment, sorafenib (10 μM) or regorafenib (20 μM) was added and cultured overnight. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well to the 96-well culture plate. After 1 to 4 hours of culture, once the reaction reached a steady state, the absorbance at 450 nm was measured using EnVision (PerkinElmer) or SpectraMax M3 (Molecular Devices) to calculate cell viability. Statistical analysis of the survival rate comparison was performed using one-way ANOVA (Dunnett's multiple comparison test) using EZR software.

[0054] [Evaluation of the Protective Effect of κ-Opioid Agonists on Keratinocytes] Multikinase inhibitors frequently cause a skin disorder called HFS during cancer chemotherapy. Using sorafenib, transcriptome analysis was performed to examine cytotoxicity in keratinocytes, using gene expression as an indicator. As shown in Table 1, 1,747 genes with increased expression (gene expression rates of 2.0 or higher) were shown to be involved in the biological process of cytokine induction by GO-BP (the relevant portion is outlined in a bold box; the same applies below). Pathway analysis demonstrated the induction of cytokine and complement pathway activation and inflammatory responses. Meanwhile, as shown in Table 2, 970 genes with suppressed expression (gene expression rates of 0.5 or lower) were shown to inhibit cell division by GO-BP, and pathway analysis also demonstrated inhibition of the cell cycle. In other words, the cellular events identified by GO-BP and pathway analysis directly reflect the symptoms of HFS, namely, the induction of dermatitis and the inhibition of keratinocyte proliferation.

[0055]

[0056]

[0057] Assuming that keratinocyte protection would be achieved with a single agent, we investigated the biological processes mediated by gene expression in the antipruritic drug (nalfurafine) and the analgesic drug (pregabalin). Itching and pain are symptoms associated with skin disorders, and their suppression is clinically required. GO-BP analysis of the top 300 genes whose expression levels were upregulated by both drugs surprisingly revealed that nalfurafine promotes epithelial cell proliferation (Table 3). This result suggests that comparative transcriptome analysis could suppress HFS, a side effect of sorafenib. Furthermore, the gene clusters annotated by this GO-BP analysis were identified, including PPARD, ZEB1, and IFT172, as shown in Table 4. These three genes have been shown in the literature to support keratinocyte proliferation and are thought to be target molecules for nalfurafine's keratinocyte-protecting effect.

[0058]

[0059]

[0060] Next, the protective effect of nalfurafine against sorafenib-induced cell proliferation inhibition was examined in an in vitro cell proliferation assay. Bucladesine sodium (BD, commercialized as Actosin ointment containing bucladesine sodium as an 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 comparative drugs. As a result, as shown in Figure 2, in the presence of 10 μM sorafenib, keratinocyte viability was significantly reduced to 82.1% (p<0.01), but recovered to 90.7% with pretreatment with nalfurafine and 91.7% with pretreatment with BD. TMPS, on the other hand, was reduced to 70.3% under these conditions. Nalfurafine and BD showed a protective effect on keratinocytes, with no statistically significant difference from the control group (no sorafenib treatment). The protective effect of κ-opioid agonists such as nalfurafine on keratinocytes has not been reported to date.

[0061] When used clinically as therapeutic or preventive drugs, events leading to undesirable symptoms must be avoided. Therefore, we investigated the gene expression of factors exacerbating keratinocyte skin damage caused by nalfurafine, BD, and TMPS. These skin damage events are known to significantly impair patients' quality of life (Non-Patent Document 19). Here, we examined the gene expression profiles of each drug during sorafenib treatment in three categories: dermatitis-inducing factors (IL4, IL6, IL13, IL17A, IL23A, IL24, IL31, IL33), mechanical pain-sensing factors (PIEZO1, PIEZO2, TRPA1, TRPV1), and melanin synthesis / pigmentation-inducing factors (IL37, MITF, TYR, TYRP1, DCT, MC1R, POMC). Table 5 summarizes the gene expression rates (fold) relative to the untreated condition. Gene induction by sorafenib (Sor) treatment was modified by each drug. The conditions under which gene expression rates relative to untreated conditions were lower than those observed with sorafenib alone (inhibiting exacerbation in each category) are shown in bold outlines. 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, demonstrating a favorable profile (gene expression rates smaller than Sor (bold outlined area) = higher, and numbers larger than Sor (underlined area) = lower are preferred as protective drugs).

[0062] Regarding mechanical pain sensitivity factors, BD, a treatment for pressure ulcers and skin ulcers, increases PIEZO2, which may be one of the factors contributing to the undesirable side effect of Actosin ointment, which causes pain (as stated in the interview form for the drug).Nalfurafine, an antipruritic drug, not only tends to inhibit PIEZO2, but also has analgesic properties as a kappa agonist, so it is expected to be clinically effective in suppressing itching and pain.

[0063] Furthermore, in the treatment of skin diseases, pigmentation induction is a major cosmetic concern for patients. While BD is thought to exacerbate pigmentation induction, nalfurafine also exhibits a favorable profile, showing a tendency to suppress it. The inhibitory effect of such a kappa opioid agonist on keratinocytes has not been reported to date.

[0064] From the above, nalfurafine has a favorable effect on protecting keratinocytes in sorafenib-induced skin damage, and therefore can be used as a therapeutic and preventive drug for HFS.

[0065]

[0066] Furthermore, because the keratinocyte protective effect 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 cytoprotective effect.

[0067] [Evaluation of the keratinocyte protective effect of combination drugs] The incidence of skin disorders, a side effect of multikinase inhibitors, was 47-59% for sorafenib and 50-81% for regorafenib, with regorafenib showing a slightly higher incidence (all data excerpted from drug interview forms). We investigated the gene expression that reflects the skin disorder effects of regorafenib as an event on keratinocytes. As a result, as shown in Table 6, 2,149 genes with increased expression (gene expression rate of 2.0 or higher) 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, epithelial development was strongly suppressed by GO-BP for 659 genes with suppressed expression (gene expression rate of 0.5 or lower) (the event GO:0008544 "epidermal formation and development" was Log １０(The P value was -28.27, indicating a very strong characteristic event.) Pathway analysis also showed that cell cycle inhibition leads to cell proliferation inhibition. In other words, the cellular events revealed by GO-BP and pathway analysis directly reflect the symptoms of HFS, namely, the induction of dermatitis and the strong inhibition of keratinocyte proliferation.

[0068] 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 of herbal medicine.To date, there have been no reports of the application of oral herbal medicines used as decoctions or extracts for local administration (external application) for treatment or prevention.

[0069]

[0070]

[0071] Five herbal medicines with potential anti-inflammatory and analgesic effects were selected based on their intended use, and selection was based on GO-BP events (keratinization, cell proliferation, cell-cell adhesion, wound healing, etc.) that contribute to keratinocyte cell proliferation and maintenance of barrier function. Table 8 shows the GO-BPs of 300 top genes whose expression is induced in keratinocytes upon treatment with five herbal medicines (Jumihaidokuto (JHT), Shofusan (SFS), Keishikajutsubuto (KKT), Keishigoshuyuto (GYT), and Yokukansan (YKS). Among these, multiple events related to cell proliferation and maintenance of barrier function (bold outlined area) were detected in JHT and SFS, indicating their strong effects.

[0072]

[0073] The GO-BP most suppressed in keratinocytes by regorafenib treatment was epidermis development (GO:0008544), and 50 genes involved in this biological process were identified (Table 9). Most of these genes are involved in skin keratinization, consistent with the pathology of HFS. It is believed that derepression of these genes will lead to the treatment of HFS. Treatment with JHT or SFS significantly increased the expression of 48 of the 50 genes, excluding IL17A and WHRN, and was found to work to reverse the inhibition of epidermal development (14 genes with JHT and 7 with SFS showed a two-fold or greater increase in expression, as indicated by the underlined parts).

[0074]

[0075] Next, keratinocytes were cultured for 2 hours in the presence of JHT or SFS, and then regorafenib was added and cell viability was examined in vitro. As shown in Figure 4, without the herbal medicine, cell viability was reduced to 12.4% due to cell proliferation inhibition by regorafenib, whereas in the presence of JHT, it was 23.4%, and in the presence of SFS, it was 23.0%, demonstrating a recovery of cell viability by approximately 2-fold, demonstrating a protective effect on keratinocytes. The cell viability results were in good agreement with the GO-BP analysis results, demonstrating the validity of comparative transcriptome analysis.

[0076] Furthermore, to examine the influence of herbal composition, we evaluated Unseiin (USI), a dermatological treatment containing different herbal ingredients than JHT and SFS, as a comparative example. USI had a cell viability rate of 15.3%, a weaker effect than JHT and SFS. The difference in effect was due to the difference in herbal composition. The herbal ingredients common to JHT and SFS but not USI were licorice (Glycyrrhiza Root), Bofu (Boufu), and Keigai (Single-leaf Prickly Pear) (Table 10). While not all herbal medicines for dermatological use are effective, these three herbal medicines appear to contribute significantly to the keratinocyte protective effect. Herbal medicines are generally administered orally as decoctions, and no studies have been found to date that specifically evaluate the keratinocyte protective effect of topical medications.

[0077]

[0078] [Protective Effect of κ-Opioid Agonists on Keratinocytes Against Inhibition of Epithelial Development] Comparative transcriptome analysis is also useful for identifying drug target molecules (genes). Once a notable GO-BP has been identified, genes involved in that process can be extracted from public databases (e.g., GENEONTOLYOGY), allowing us to determine which genes are affected by the drug based on their gene expression ratios. We found that exposure of keratinocytes to regorafenib strongly suppressed the expression of 50 genes associated with the GO-BP "epithelial development (GO:0008544)" (Table 9). Of these, five genes (KLK14, KRT25, LCE2A, LCE2C, and NOTCH1) showed enhanced expression in JHT and SFS, which exerted protective effects. In particular, KRT25, LCE2A, and LCE2C are known to be genes specifically expressed in the skin and constituting the cornified envelope necessary for forming the skin's barrier structure. This suggests that regorafenib broadly suppresses gene expression related to the cornified envelope of keratinocytes, inhibiting keratinization and damaging the skin barrier structure, leading to the onset of HFS. Furthermore, it was shown that JHT and SFS act to restore the cornified envelope, exerting a protective effect on keratinocytes.

[0079] When the effect of single 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 times 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.

[0080] Furthermore, when we looked at the expression of genes related to inflammation, pain, and pigmentation, which are undesirable symptoms of HFS, we found that kappa opioid agonists tended to suppress all of these, as shown in Figure 6, and that they can also be expected to have an effect of alleviating HFS symptoms. In Figure 6, PIEZO02 is a receptor that senses mechanical pain, and while it is undesirably strongly expressed with regorafenib, it is strongly suppressed with nalfurafine, making it particularly preferable.

[0081] [Melanin Production-Promoting Effect of Traditional Chinese Medicines on Vitiligo] While pigmentation associated with inflammation reduces quality of life, skin pigment loss, such as in vitiligo vulgaris, also reduces quality of life, necessitating the development of therapeutic drugs. Vitiligo caused by immune checkpoint inhibitors is also a problem in cancer treatment, but no such drugs are currently available. Comparative transcriptome analysis focusing on traditional Chinese medicines revealed that three traditional Chinese medicines (KKB, GYT, and KDB) activate melanin production, as shown in Figure 7. Furthermore, the herbal ingredients of these three traditional Chinese medicines are listed in Table 11. These three medicines share the herbal ingredients jujube (Daiso) and ginger (Shokyo). Therefore, formulations containing either or both of these herbal ingredients can be used to treat vitiligo. Traditional Chinese medicines are generally administered orally as decoctions, and no studies have evaluated their therapeutic effects on vitiligo, specifically for topical use. This discovery was the first of its kind.

[0082]

[0083] As described above, comparative transcriptome analysis is effective in identifying drug target genes and searching for compounds to treat side effects, and is also an extremely useful technique in drug discovery research.

Claims

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.

2. The topical preparation according to claim 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- ceutical acceptable salts thereof.

3. The topical preparation according to claim 1, wherein the protective effect of keratinocytes is at least one of the promotion of proliferation during cell damage, the suppression of expression of inflammatory cytokine genes, and the suppression of expression of pigmentation-inducing genes.

4. The topical preparation according to claim 1, wherein the herbal ingredient is at least one of licorice, bougainvillea root, and keigai.

5. The topical preparation according to any one of claims 1 to 4, for use in treating a disease including 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, acneiform reaction, epiphora, pigmentation, radiation dermatitis which is a disorder of the skin and tissue mucosal epithelium caused by radiation therapy, mucositis, stomatitis, angular cheilitis, pigmentation, or skin disorder of atopic dermatitis caused by scratching, for which keratinocyte protection is applied.

6. A topical preparation for the treatment of vitiligo, vitiligo vulgaris, and skin hypopigmentation, comprising at least one herbal ingredient selected from the group consisting of Daiso and Zingiber officinale.

7. A method for exploring new uses of pharmaceutical compositions by extracting opposing intracellular processes from the results of transcriptome analysis of multiple pharmaceutical compositions, comprising the steps of: obtaining a gene expression profile (GO-BP) reflecting a specific 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.

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.