FPR1 inhibitor, drug, severe drug eruption therapeutic agent, and FPR1 inhibitor screening method

Abstract

Provided are: an FPR1 inhibitor that can be used in a therapeutic agent based on cell death pathology specific to SJS / TEN, and that that can ameliorate the death rate of SJS / TEN; a drug; and a severe drug eruption therapeutic agent. Moreover, provided is an FPR1 inhibitor screening method for seeking these therapeutic agents. The FPR1 inhibitor, the drug, and the severe drug eruption therapeutic agent contain chenodeoxycholic acid or sulfinpyrazone as an active ingredient, and are used for treating severe drug eruption. The FPR1 inhibitor screening method is for seeking these therapeutic agents.

Description

FPR1 inhibitors, pharmaceuticals, treatments for severe drug eruptions, and screening methods for FPR1 inhibitors

[0001] This invention relates to an FPR1 inhibitor, a pharmaceutical product using the same, and a treatment for severe drug rash. It also relates to a screening method for the FPR1 inhibitor. This application claims priority based on Japanese Patent Application No. 2024-188058, filed on October 25, 2024, the contents of which are incorporated herein by reference.

[0002] Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), severe drug eruptions, are conditions characterized by fever, widespread vesicles, erosions, and mucosal damage. These diseases are caused by drug administration. They have high mortality rates and can leave long-term sequelae such as blindness even after recovery, posing a significant social problem. The pathogenesis of SJS / TEN, characterized by programmed cell death such as apoptosis and necroptosis of epidermal cells, is gradually being elucidated. However, drug development based on these mechanisms has not yet been achieved, leading to poor prognosis and high mortality rates. It is hoped that elucidating the pathogenesis and developing novel therapeutic drugs specific to keratinocyte cell death will improve survival rates.

[0003] Currently, the first-line drug treatment for SJS and TEN is early systemic corticosteroid therapy, with plasmapheresis or high-dose intravenous human immunoglobulin (IVIg) therapy being used in combination if steroid refractory. These treatments are described, for example, in Non-Patent Document 1.

[0004] SJS / TEN is a disease characterized by widespread skin erosion due to cell death in epidermal cells resulting from an immune response triggered by the administration of a causative drug. While this cell death was previously thought to be due to apoptosis, another type of cell death called necroptosis (programmed necrosis) is also considered important, as reported by the inventors in Non-Patent Documents 2 and 3. Necroptosis is a form of programmed cell death characterized by its morphological similarity to necrosis. Unlike apoptosis, cells undergoing necroptosis release damage-associated molecular patterns (DAMPs) containing various inflammatory cytokines, inducing inflammation in the surrounding area. Furthermore, the inventors have confirmed that SJS / TEN-like symptoms do not appear when a necroptosis inhibitor is administered to SJS / TEN model mice. Necroptosis is caused by the interaction between annexin A1 released by monocytes and Formyl peptide receptor 1 (FPR1), which is specifically expressed in skin cells of patients with severe drug rash.

[0005] Furthermore, in Patent Document 1, the present inventors disclose a pharmaceutical composition for the prevention or treatment of FPR1-induced necroptosis-related diseases, comprising as an active ingredient a substance that suppresses necroptosis induced by the binding of FPR1 and Annexin A1, and a method for screening substances that suppress necroptosis induced by the binding of FPR1 and Annexin A1. This technology aims to analyze the mechanism by which severe mucosal lesions and skin erythema, erosions, blisters, and epidermal peeling occur, and to provide a pharmaceutical composition for the fundamental treatment of these symptoms, as well as a method for screening the active ingredients of the said pharmaceutical composition.

[0006] International Publication No. 2014 / 126127

[0007] Sunaga Y, Kurosawa M, Ochiai H, et al. The nationwide epidemiological survey of Stevens-Johnson syndrome and toxic epidermal necrolysis in Japan, 2016-2018. J Dermatol Sci. 2020 Dec;100(3):175-182. Saito N, Qiao H, Yanagi T, et al. An annexin A1-FPR1 interaction contributes to Necroptosis of keratinocytes in severe cutaneous adverse drug reactions. Sci Transl Med. 2014 Jul 16;6(245):245ra95.Kinoshita M, Ogawa Y, Hama N, et al. Neutrophils initiate and worsen Stevens-Johnson syndrome and toxic epidermal necrolysis. Sci Transl Med. 2021 Jun 30;13(600):eaax2398.

[0008] As shown in Non-Patent Document 1, there has been little development of treatments for SJS / TEN. In particular, despite IVIg therapy being covered by insurance in 2014, a worsening of the mortality rate has been observed, and there is a strong desire for the development of more effective treatment methods.

[0009] This invention has been made in view of the above circumstances, and its purpose is to provide an FPR1 inhibitor, a pharmaceutical, and a treatment for severe drug rash that can be used as a therapeutic agent based on the pathological state of cell death specific to SJS / TEN and can improve the mortality rate of SJS / TEN. It also aims to provide a screening method for FPR1 inhibitors, which is a method for searching for these therapeutic agents.

[0010] To solve the above problems, the present invention has the following embodiments: [1] An FPR1 inhibitor for use in the treatment of severe drug rash, comprising chenodeoxycholic acid or sulfinpyrazone as an active ingredient. [2] The FPR1 inhibitor according to [1], comprising chenodeoxycholic acid. [3] The FPR1 inhibitor according to [1], comprising sulfinpyrazone. [4] The FPR1 inhibitor according to any one of [1] to [3], having cell death inhibitory activity. [5] A pharmaceutical product comprising the FPR1 inhibitor according to [1] to [4]. [6] A therapeutic agent for severe drug rash comprising the FPR1 inhibitor according to [1] to [4]. [7] The therapeutic agent for severe drug rash according to [6], wherein the severe drug rash is Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). [8] A method for screening FPR1 inhibitors, comprising: one or more screening steps of selecting one or more G protein-coupled receptors (GPCRs), quantitatively measuring the inhibitory activity of candidate compounds against the GPCRs in a compound library, and selecting candidate compounds with one or more of the inhibitory activities higher as suitable compounds; and measuring the cell death inhibitory activity of the suitable compounds and selecting an FPR1 inhibitor from the suitable compounds. [9] The method for screening FPR1 inhibitors according to [8], further comprising a screening step of selecting FPR1 as the GPCR.

[10] The method for screening FPR1 inhibitors according to [8] or [9], comprising a primary screening step of selecting FPR1 as the GPCR, and a secondary screening step of selecting SSTR2 as the GPCR.

[11] The method for screening FPR1 inhibitors according to any one of [8] to

[10] , wherein the quantitative measurement of the inhibitory activity of the candidate compounds against the GPCR is performed by a calcium assay or a β-arrestin assay.

[12] The method for screening FPR1 inhibitors according to either [8] or

[11] , wherein the measurement of the cell death inhibitory activity involves inducing FPR1 expression in epidermal cells to induce cell death activity, administering the compatible compound, and evaluating the inhibitory activity of the cell death activity.

[13] The method for screening FPR1 inhibitors according to

[12] , wherein an in vitro assay is performed using HaCaT cells as the epidermal cells.

[0011] According to the present invention, it is possible to provide an FPR1 inhibitor, a pharmaceutical, and a treatment for severe drug rash that can be used in therapeutics based on the pathological state of cell death specific to SJS / TEN, and that can improve the mortality rate of SJS / TEN. Furthermore, it is possible to provide a screening method for FPR1 inhibitors, which is a method for searching for these therapeutics.

[0012] This is a schematic diagram showing an overview of the screening of effective compounds in this embodiment. This is a graph showing the measurement of the FPR1 inhibition rate of the compound library validated as a primary screening by the β-arrestin assay. This is a graph showing the results of measuring the FPR1 and SSTR2 inhibition rates of hit compounds by the β-arrestin assay. This is a graph showing the inhibition rates of hit compounds at each concentration in the β-arrestin assay. This is a schematic diagram showing an overview of the screening of the core library for identifying FPR1 inhibitors in this embodiment. This is a graph showing the distribution of inhibition rates of compounds in the core library as a histogram. This is a graph showing the results of the confirmation assay for hit compounds in the core library screening. This is a graph showing the inhibition rates of 41 library compounds at five concentrations in the G protein assay. This is a graph showing the results of measuring the FPR1 and SSTR2 inhibition rates of compounds that hit in the G protein assay by the β-arrestin assay. This is a graph showing the results of measuring the FPR1 inhibition rate of existing drugs by the β-arrestin assay. This is a photograph showing the immunofluorescence image of FPR1 in HaCaT treated with Bz-ATP or LL-37. This is a graph showing the percentage of FPR1-positive cells treated with Bz-ATP and LL-37. This is a graph showing the flow cytometry results of FPR1 expression in HaCaT treated with Bz-ATP or LL-37. This is a graph showing the results of measuring the toxicity of LL-37 and Ac2-26 to HaCaT by viability staining. This is a figure showing the results of immunofluorescence staining of LL-37 and Ac2-26 to HaCaT with each marker. This is a graph showing the cell death suppression rate of each FPR1 inhibitor candidate compound. This is a figure showing the effect on conjunctivitis symptoms in a CDCA-administered mouse model. This is a graph showing symptom suppression in the model mouse. This is a photograph showing the effect on conjunctivitis symptoms in a CDCA-administered mouse model (male). This is a graph showing symptom suppression in the model mouse. This is a photograph showing the effect of CDCA administration on conjunctivitis symptoms in a mouse model (ST combination drug model). This is a graph showing symptom suppression in the model mouse (ST combination drug model).

[0013] The following describes embodiments of the FPR1 inhibitor, pharmaceutical, treatment for severe drug rash, and screening method for FPR1 inhibitors according to the present invention. However, the present invention is not limited to the following embodiments.

[0014] (FPR1 inhibitor) The FPR1 inhibitor of this embodiment contains chenodeoxycholic acid or sulfinpyrazone as an active ingredient. Chenodeoxycholic acid (CDCA) (C ２４ H ４０ O ４ CDCA (CAS registration number: 474-25-9) is a type of bile acid and is known as an organic acid synthesized from cholesterol in the liver. CDCA is also approved as a treatment for human gallstones. Sulfinpyrazone (C ２３ H ２０ N ２ O ３ S (CAS registration number: 57-96-5) is a compound known as a uric acid excretion promoter and has been used in the past to treat gout. The FPR1 inhibitor of this embodiment preferably contains chenodeoxycholic acid in particular.

[0015] The FPR1 inhibitor of this embodiment is preferably used as a treatment for severe drug eruptions. Here, a drug eruption refers to a rash caused by taking a drug, for example, an allergic drug eruption caused by an allergic reaction to a drug. A severe drug eruption specifically refers to a severe form of drug eruption. Known severe drug eruptions include Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). In this embodiment, the severe drug eruptions to be treated are not limited to these. In this embodiment, in particular, necroptosis occurs due to the interaction between annexin A1 released by monocytes and Formyl peptide receptor 1 (FPR1), which is specifically expressed in the skin cells of patients with severe drug eruptions, and the severe drug eruptions caused by this necroptosis are referred to. The severe drug eruptions to be treated in this embodiment may not include, for example, DIHS / DRESS.

[0016] The FPR1 inhibitor of this embodiment preferably has cell death inhibitory activity. Here, cell death preferably refers to necroptosis. As mentioned above, having necroptosis inhibitory activity allows for efficient treatment of the severe drug eruption.

[0017] (Pharmaceutical, Therapeutic for Severe Drug Eruption) The pharmaceutical of this embodiment contains the FPR1 inhibitor. The therapeutic for severe drug eruption of this embodiment contains the FPR1 inhibitor. The therapeutic for severe drug eruption includes the pharmaceutical. As described above, the therapeutic for severe drug eruption is preferable when the severe drug eruption is Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN).

[0018] The subjects exhibiting symptoms of severe drug rash, which are the target of this embodiment, can be appropriately selected from humans and other animals. Other animals mainly include mammals. Specifically, mammals include domestic animals (pigs, sheep, goats, cattle, horses, etc.), companion animals (dogs, cats, etc.), laboratory animals (mice, guinea pigs, hamsters, rats, other rodents, rabbits, etc.), and other animals (marmosets, chimpanzees, other monkeys). In this specification, the term "patient" mainly refers to human subjects, but is not limited to humans.

[0019] The purpose of administering the therapeutic agent for severe drug rash of this embodiment to patients with severe drug rash broadly includes medical treatment purposes such as treatment, prevention, and prevention of recurrence after treatment. Treatments include, but are not limited to, oral administration, topical application, and injection. The dosage, administration interval, method of administration, and route of administration are not particularly limited and can be appropriately selected depending on the conditions of the person being treated, such as age, weight, symptoms, and the treatment site. The dosage form and these administration conditions can be appropriately selected from conventional examples; for example, if the active ingredient is CDCA, conditions used for treating gallstones can be selected, and if sulfinpyrazone is used for treating gout can be selected.

[0020] (Method for Screening FPR1 Inhibitor) The method for screening an FPR1 inhibitor according to this embodiment selects one or more G protein-coupled receptors (GPCRs), quantitatively measures the inhibitory activity of a candidate compound in a compound library against the GPCR, and selects, as a suitable compound, one or more of the candidate compounds having high inhibitory activity in one or more screening steps, and measures the cell death inhibitory activity of the suitable compound, and selects an FPR1 inhibitor from the suitable compound.

[0021] First, in the screening step, a suitable compound is selected from candidate compounds contained in various compound libraries. The screening step is performed one or more times as described later.

[0022] In the screening step, one or more G protein-coupled receptors (GPCRs) are selected. Since FPR1 is one type of GPCR, by measuring the inhibitory activity of a candidate compound against the GPCR, a compound having high inhibitory activity and specificity against FPR1 can be selected. It is preferable to select FPR1 as the GPCR in at least one of the one or more screening steps. In addition, in the screening step, other GPCRs may be selected. Further, FPR1 and other GPCRs may be used in combination, and the inhibitory activity against these two types may be measured.

[0023] For example, in the primary screening step, FPR1 may be selected as the GPCR and screened, and then in the secondary screening step, another GPCR may be selected and screened. Other GPCRs may include, for example, somatostatin receptor 2 (SSTR2). Furthermore, two or more GPCRs may be used in combination in the secondary screening step. For example, FPR1 and another GPCR may be used in combination. In the secondary screening step, the inhibition rates for the two GPCRs may be used as appropriate for screening. For example, a compound with high inhibition rates for both GPCRs may be selected as a suitable compound from among the candidate compounds. Alternatively, a compound with a high inhibition rate for FPR1 and low specific inhibition for the other GPCR may be selected as a suitable compound from among the candidate compounds.

[0024] The quantitative measurement of the inhibitory activity of the candidate compounds against the GPCR may be performed by a calcium assay or a β-arrestin assay. For example, the β-arrestin assay can quantitatively measure the inhibitory activity against the GPCR in the following manner: Since β-arrestin binds to the activated GPCR as a scaffold protein, inhibition of the GPCR by other candidate compounds is reflected as a decrease in β-arrestin binding. By measuring this decrease in β-arrestin binding, the inhibitory activity can be quantitatively measured. For quantitative measurement of this decrease, luciferase can be used, for example. Specifically, the C-terminal fragment of luciferase, a luminescent protein, is tagged to the GPCR, and the N-terminal fluorescent fragment is tagged to β-arrestin, and the binding of β-arrestin can be measured using a bioluminescent probe consisting of these two proteins.

[0025] Regarding the quantitative measurement of inhibitory activity, the conditions for whether the quantitative inhibitory activity is above or below a certain standard can be appropriately selected. For example, when the inhibition rate against FPR1 exceeds 50% (or 75%, 80%, 90%, 95%, etc.), it can be evaluated that the inhibition rate against FPR1 is high; when the inhibition rate against other GPCRs is less than 25% (or 12.5%, 10%, 5%, etc.), it can be evaluated that the non-specific inhibition against other than FPR1 is low, and so on. These results can be used individually or in combination. For example, a compound with a high inhibition rate against FPR1 and a low inhibition rate against other GPCRs may be used as a suitable compound.

[0026] Next, in order to screen for FPR1 inhibitors from the above-mentioned suitable compounds, the measurement of cell death inhibitory activity may be performed. The measurement of cell death inhibitory activity may be to induce the expression of FPR1 in epidermal cells to induce cell death activity, and administer the above-mentioned suitable compound to evaluate the inhibitory activity of the cell death activity.

[0027] For example, as epidermal cells, those established as human epidermal keratinocyte strains can be used, and commercially available HaCaT cells, etc. can be appropriately used. Also, for example, an in vitro assay using HaCaT cells as epidermal cells may be performed. The expression of FPR1 in these cells is induced by P2X7R stimulation by LL-37, as reported by the present inventors in Non-Patent Document 2, so it may be performed by administering LL-37 or Bz-ATP, which is a 2X7R agonist, to the cells. These can be carried out by ordinary cell culture and detecting cell death by fluorescence immunostaining or flow cytometry.

[0028] (Effect of this embodiment) According to this embodiment, an FPR1 inhibitor, a medicament, and a therapeutic agent for severe drug eruption that can be used for therapeutic agents for the pathological condition of cell death specific to SJS / TEN and can improve the mortality rate of SJS / TEN can be provided. Also, a screening method for FPR1 inhibitors, which is a method for searching for these therapeutic agents, can be provided.

[0029] As shown in the examples described below, the inventors demonstrated that chenodeoxycholic acid (CDCA), an FPR1 inhibitor, suppresses cell death in an in vitro assay and effectively suppresses SJS / TEN-like symptoms in a mouse model. In SJS / TEN, FPR1 acts as an important mediator in the necroptosis signaling pathway during cell death. Therefore, inhibiting FPR1 can suppress necroptosis and may lead to the treatment of SJS / TEN symptoms.

[0030] To identify FPR1 inhibitors as therapeutic agents for SJS / TEN, the inventors conducted a two-step assay to evaluate the inhibitory effects on the FPR1 receptor and cell death. First, seven candidate compounds were identified, consisting of compounds extracted from a compound library screening and existing drugs that have been reported to have FPR1 inhibitory activity. Next, the cell death inhibitory effects of these candidate compounds were evaluated using an in vitro assay. The screening assay simply evaluated the decrease in FPR1 activation due to FPR1 inhibition. However, the in vitro assay reflects the extent to which cell death induced by downstream signaling of FPR1 ligands can be suppressed, thus providing more clinically relevant results. This is thought to explain the difference in results observed in the two experimental systems.

[0031] CDCA acts as an antagonist of FPR1 and binds to its receptor, but does not induce downstream signals that lead to ligand-induced cell death. Identifying effective therapeutic agents in clinical settings requires multiple validation steps using various approaches. Finally, the inventors confirmed the symptom-suppressing effect of CDCA in SJS / TEN model mice. The effect of CDCA was confirmed in both male and female mice, and in mice created using human peripheral blood mononuclear cells derived from patients with both acetaminophen and ST combination drugs as the causative agents of SJS / TEN. As a result, CDCA was found to be the most suitable drug for the treatment of SJS / TEN.

[0032] Considering the mechanisms of currently available therapeutic agents, only a limited number of drugs are effective in regulating the cell death process. Adrenocortical steroids regulate immune cells such as T cells and monocytes through immunosuppression, but do not directly inhibit cell death. Cys A has been suggested to suppress apoptosis by suppressing T cells and is known to also show an inhibitory effect on FPR1. In this example, it was considered as one of the candidates, but since cell death was only slightly suppressed in the in vitro assay, it is presumed that necroptosis in SJS / TEN is not suppressed by Cys A. IVIG is an anti-Fas antibody and is known to suppress apoptosis by inhibiting the Fas receptor. Plasma exchange therapy is a treatment that removes inflammatory cytokines, and Annexin A1 is also removed, potentially contributing to the suppression of necroptosis by reducing the ligand of FPR1. Since necroptosis is generally induced by TNFα, TNFα antagonists would be useful as inhibitors. However, since necroptosis in SJS / TEN is mediated by the Annexin A1-FPR1 interaction, it is suggested that there are limitations to the necroptosis-suppressing effect of TNFα antagonists. Given that conventional treatments have not shown improvement in mortality, a new approach is needed. Both apoptosis and necroptosis are involved in cell death in SJS / TEN, but it has been reported that SJS / TEN-specific cell death is suppressed by necroptosis inhibitors, and a therapeutic approach targeting necroptosis is desired.

[0033] CDCA is one of the major primary bile acids in humans, and its main role is to aid in the digestion and absorption of cholesterol through physiological emulsification. Furthermore, oral administration of CDCA has been used as a treatment for cholelithiasis because it has the ability to dissolve cholesterol gallstones. In addition, it has been suggested that CDCA has an anti-inflammatory effect by reducing the chemotaxis of neutrophils and monocytes by inhibiting the binding of N-formylmethionyl-leucyl-phenylalanine (fMLP), a ligand for FPR1, to FPR1. The inventors have revealed that CDCA suppresses epidermal cell necrosis and SJS / TEN-like symptoms through its inhibitory effect on FPR1. The inventors hypothesize that CDCA also acts as an antagonist of FPR1 in the cell death pathway, inhibiting the binding of Annexin A1 to FPR1 and blocking downstream signaling. Furthermore, it has been previously reported that CDCA suppresses immune function in rats and in vitro by inhibiting the release of IL-1, IL-6, and TNFα from monocytes. Considering that TNFα is involved in the development of SJS / TEN, suppressing TNFα secretion from monocytes, as previously reported, may yield additional benefits. From this perspective as well, CDCA could be a promising new therapeutic agent. Moreover, its therapeutic effect may be enhanced when administered in combination with other drugs whose efficacy has been proven. Since there are various pathways and factors in the pathogenesis of SJS / TEN, improvement in survival rates through a multifaceted approach is required. Therefore, not only CDCA monotherapy but also therapies in combination with other means should be considered for future applications.

[0034] (Another aspect of this embodiment) This embodiment also includes the following aspects as another aspect. Another aspect of this embodiment is a method for producing a pharmaceutical or a therapeutic agent for severe drug rash using the FRP1 inhibitor. Another aspect of this embodiment is the use of the FRP1 inhibitor for producing a pharmaceutical or a therapeutic agent for severe drug rash. Another aspect of this embodiment is the use of the FRP1 inhibitor for the treatment of severe drug rash. Another aspect of this embodiment is the use of the FRP1 inhibitor for use in the treatment of severe drug rash. Another aspect of this embodiment is a method for treating severe drug rash using the FRP1 inhibitor.

[0035] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be modified in various ways.

[0036] The effects of the present invention will be further clarified by the following examples and comparative examples. It should be noted that the present invention is not limited to the following examples, and can be implemented with appropriate modifications without altering its essence.

[0037] [Overview of the Tests] Each of these test examples was conducted for the purpose of finding an FPR1 inhibitor effective as a novel therapeutic agent for SJS / TEN. A high-throughput screening system was constructed to identify an FPR1 antagonist that inhibits signal transduction from a compound library owned by the Drug Discovery Initiative (DDI), Graduate School of Pharmaceutical Sciences, The University of Tokyo. Furthermore, CDCA having a high cell death inhibitory effect was identified as a candidate by an in vitro assay using HaCaT cells. Finally, the SJS / TEN-like symptom inhibitory effect of CDCA was confirmed in SJS / TEN model mice. Mouse samples were subjected to immunohistochemical analysis. This study was conducted in accordance with the Declaration of Helsinki after receiving approval from the Ethics Committee of Niigata University. SJS / TEN model mice were prepared by intravenous injection of PBMC from a patient after TEN recovery into immunodeficient mice (NOG mice) and oral administration of the causative drug. Six- to seven-week-old NOG mice were purchased from In-Vivo Science Inc. Human peripheral blood mononuclear cells (PBMC) were obtained from patients at Niigata University after obtaining informed consent. In mouse experiments, usually seven mice per group were placed in two cages, and a 12-hour light-dark cycle, temperature-controlled environment, and free access to food and water were provided. Mouse experiments were conducted based on a protocol approved by the Animal Experiment Ethics Committee of Niigata University.

[0038] [Equipment, Reagents, and Test Methods] (Compound Library) The core library and validation library were provided by the DDI of The University of Tokyo (https: / / www.ddi.f.u-tokyo.ac.jp / en / #5; Tokyo, Japan).

[0039] (β-Arrestin Assay) HEK293 cells (FPR1 or SSTR2 × ARRB1 cells) stably expressing FPR1 or SSTR2-ElucC with pcDNA4 and ElucN-ARR B1 with pcDNA3.1 were seeded in a 96-well white plate (2.0 × 10 ５ cells / ml, 100 μL / well), and incubated at 37°C in 5% CO ２The culture medium was incubated for 48 hours. The medium was replaced with 50 μL of phenol red-free medium containing 1% FBS and 8 μM of the library compound (final 4 μM) using a 12-channel pipette (Finnpipette, Thermo Scientific) or a 96-channel pipette (Liquidator, Mettler Toledo), and 5% CO2. ２ The cells were incubated at 37°C for 30 minutes. Using a multichannel pipette, the cells were stimulated with 50 μL of phenol red-free DMEM (containing 1% FBS) and 100 nM fMLP (50 nM final), and incubated at 37°C and 5% CO2. ２ The cells were incubated for 60 minutes. The reaction was quenched by removing the medium and freezing the plate at -30°C. Cells thawed by incubation at room temperature for 2 minutes were suspended in 100 μL of luminescence reagent (ELA). Luminescence was measured using a microtiter plate reader (TriStar LB941) (n=3 for each compound). After gentle shaking for 5 minutes, each well was monitored for 2 seconds. Wells treated with HBSS buffer were used as negative controls, and wells treated with fMLP only were used as positive controls. The maximum intensity of the three values ​​was used as the signal from the well. Based on the mean and standard deviation, the CV value and Z'- factor were calculated to evaluate the variability of the measurements within the plate and to verify whether the measurements were suitable for screening (C, D). The inhibitory activity of the compounds was evaluated based on InH (%) calculated using the following formula (G) (G). InH (%) = {1 - (A Ｓ -A Ｂ ) / ( A Ｐ -A Ｂ )}×100... (G) IC ５０ The values ​​were calculated by fitting the data to equations (H) and (I). x and y represent the inhibitor concentration (μM) and normalized emission intensity, respectively. y = 1 / (1 + exp(a + bln(x))) ... (H) IC ５０ (μM)=exp(-a / b)...(I)

[0040] Screening of the validation compound library showed that in the primary screening, many plates had Z-values ​​slightly below 0.5 for the Z'- factor. However, in the secondary screening, most plates settled around 0.5, and were judged to be acceptable for evaluation. The criterion for determining significant FPR1 inhibition was whether it exceeded the mean + 3 CV, and the criterion for concluding that there was no significant SSTR2 inhibition was whether it fell within the mean + 1 CV.

[0041] (G-protein assay) HEK293 cells were incubated in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin at 37°C and 5% CO2. ２ The cells were cultured. The cells were transfected with G16 plasmid and FPR1 plasmid to create an FPR1×G16 stable cell line. The FPR1×G16 stable cells were seeded into a 384-well black clear plate using a microplate dispenser (1.0 × 10⁶). ６ cells / ml, 10 μl / well). 5% CO2 ２After culturing at 37°C for 24 hours, calcium-sensitive dye (FLIPR 5, Molecular Devices) suspended in 10 μL of HBSS was introduced into each well using a dispenser. 384-well plates for the agonist plates were pre-treated at room temperature for 1 hour with HBSS buffer containing 20 mM HEPES, 0.5% BSA, and 200 nM fMLP. Pipette tips were also pre-treated by pipetting three times with the same fMLP solution. The agonist plates were refilled with fresh 200 nM fMLP solution (final 50 nM), and the 2 mM in DMSO compound plates were diluted to 30 μM with HBSS buffer (final 10 μM). After loading the dye for 1 hour, the cells, agonist, and compound plates were placed in a fluorescence microplate imager (FDSS 7000, Hamamatsu Photonics K.K.) and fluorescence measurements were performed. During measurement, fluorescence images of the assay plate were captured every second using blue excitation light (λ = 488 nm). After measuring the baseline fluorescence intensity for 20 seconds, 10 μL of library compound solution was automatically added to each well and incubated for 5 minutes. The increase in fluorescence after the injection of 10 μL of ligand solution was monitored for 3 minutes (n = 4 for each compound). Wells treated with HBSS buffer containing DMSO instead of the library compound were used as negative controls, and wells treated with fMLP alone were used as positive controls.

[0042] During all measurements, fluorescence intensity was recorded 500 times for each well of the assay plate. To extract information about the response to ligand stimulation, the fluorescence growth rate (FI) and relative fluorescence growth rate (RFI) were defined as follows: FI = (Peak intensity) - (Basal intensity) ... (A) PFI = ((Peak intensity) - (Basal intensity)) / ((Basal intensity)) ... (B) Peak intensity represents the maximum intensity after ligand stimulation, and basal intensity represents the average fluorescence intensity for 20 seconds prior to stimulation. Based on FI and RFI, the CV value, Z' factor, and inhibition rate (InH) were calculated as shown in equations (C) to (E). CV (%) = SD / Mean x 100... (C) Z' = 1 - (3 x SD ｐ +3xSD Ｂ ) / (Mean ｐ　 - 　 Mean Ｂ ) … (D) InH (%) = {1-(FI Ｓ　 - FI Ｂ ) / (FI Ｐ - FI Ｂ )} × 100 … (E) The subscripts are as follows: positive control (P), background = negative control (B), sample (S). Note that because the background results shown in formula (F) were not available, some results were analyzed using pseudo-inhibition rates. The subscript I indicates the internal standard. pseudo-InH (%) = {1 - (FI Ｓ - FI Ｉ ) / (FI Ｐ - FI Ｉ )}×100 … (F) EC ５０ (Maximum effective concentration at half value) and IC2 ５０ The (maximum half-value inhibitory concentration) value was roughly estimated as the concentration that induces a response midway between the baseline and the maximum value. ７０ This was estimated at the concentration that elicits a reaction at 70% of the maximum concentration.

[0043] Before evaluating the inhibition rate, the fluorescence intensity before ligand stimulation was examined. As a result, 26 chemicals showing a fluorescence increase of 0.5 or more before ligand stimulation were excluded. The inhibition rates of the remaining 9,574 compounds were calculated based on RFI. Based on primary and confirmatory screening of the core library, the Z'- factor was 0.5 or higher in most plates.

[0044] (SJS / TEN in virto assay) HaCaT cells were purchased from COSMO BIO. The cells were treated with LL-37 (ANASPEC) or Bz-ATP (Alomone Labs) for 24 hours to express FPR1, and then incubated with Ac2-26 (50 ng / ml) for 24 hours to induce necroptosis.

[0045] (Immunofluorescence) Immunofluorescence staining is performed by adding Bz-ATP or LL-37 to CnT-Prime, Epithelial Culture Medium (CnT-PR, CELLnTEC) and staining at 37°C in 5% CO2. ２ The analysis was performed on HaCaT cells incubated for 48 hours. FPR1 expression was confirmed by stimulating with FITC-labeled FPR1 ligand (1:100, Invitrogen) for 15 minutes. For cCasp3 and pMLKL analysis, cells were fixed with 4% paraformaldehyde for 10 minutes and blocked with 10% goat serum for 30 minutes at room temperature. Immunolabeling was performed with the following primary antibodies: anti-cCasp3 (1:100, Cell Signaling Technology) and anti-pMLKL (1:100, Signalway Antibody), overnight at 4°C. Cells were incubated with the following secondary antibodies in the dark at room temperature for 30 minutes: Alexa Fluor 488 and Alexa Fluor 594-labeled anti-rabbit and mouse IgG, respectively. Cell nuclei were counterstained with DAPI (1:100, Vector Laboratories). The images were acquired using a FLUOVIEW FV3000 confocal laser microscope (Olympus).

[0046] (Flow cytometry) HaCaT cells were incubated with Bz-ATP (300 μM) or LL-37 (30 μg / ml) in CnT-PR at 37°C and 5% CO2. ２ Cells were incubated for 24 hours. Dead cells were removed by 7-AAD staining (abcam). Cells were stained with FITC-labeled FPR1 ligand (1:4000, Invitrogen). Analysis was performed using FACSVerse and BD_FACSDiva software (BD Biosciences).

[0047] (Live-dead staining) Cell viability was evaluated using the Live / Dead Cell Staining Kit II (PromoKine), which uses two fluorescent dyes: calcein-AM for living cells and Ethidium homodymer III (EthD-III) for dead cells. HaCaT cells were kept in DMEM supplemented with 10% FBS and 100 μg / ml streptomycin at 37°C and 5% CO2. ２ Cells were cultured in LL-37 (10 μg / ml) and Ac2-26 (50 ng / ml). ２ The cells were incubated at 37°C for 24 hours. Images were acquired using a Keyence Bz-X710 all-in-one fluorescence microscope (Keyence). The number of viable and dead cells was determined using Image-J software.

[0048] (SJS / TEN model mice) The SJS / TEN model mice were created according to the protocol described in N. Saito, N. Yoshioka, R. Abe, H. Qiao, Y. Fujita, D. Hoshina, A. Suto, S. Kase, N. Kitaichi, M. Ozaki, H. Shimizu, Stevens-Johnson syndrome / toxic epidermal necrolysis mouse model generated by using PBMCs and the skin of patients. The Journal of Allergy and Clinical Immunology 131, 434-441.e431-439 (2013), etc. PBMCs were obtained from a 44-year-old male patient who recovered from TEN caused by acetaminophen. PBMCs (2 × 10⁻⁶) ６ NOG mice were intravenously injected with (1) the causative agent (acetaminophen, 1.5 mg / 100 μl), and from day 1, they were orally administered once daily. In addition, CDCA (200 mg / kg) or NS was orally administered once daily from day 1. Each group consisted of 7 mice, housed in two cages, under a 12-hour light-dark cycle, temperature control (22 ± 2°C), and humidity control (50 ± 10%), with ad libitum access to food and water. On day 14, general anesthesia was administered, and eye lesions were observed. Eye lesions were examined by histopathological examination.

[0049] (TUNEL assay) TUNEL is a method for detecting apoptotic cells with DNA fragmentation by labeling the ends of nucleic acids. The TUNEL assay was performed according to the manufacturer's protocol (Takara Bio). The conjunctiva was observed macroscopically, and the number of dead cells was counted microscopically in all four mice in each group.

[0050] (Statistical analysis) The p-value was calculated using Welch's t-test, and p < 0.05 was considered statistically significant.

[0051] [Test Example 1] (Search for compounds with FPR1 inhibitory activity) To find candidate FPR1 inhibitors, we first searched for drugs that inhibit specific signals mediated by FPR1. Two methodologies were used: comprehensive screening of a compound library and identification of existing drugs through drug repositioning. Figure 1 is a schematic diagram showing an overview of the screening of effective compounds in this example.

[0052] First, we screened the publicly available Japanese compound library held by DDI. This library consists of over 200,000 compounds and is meticulously curated, focusing on chemical diversity and potential protein affinity, including compounds from commercially available databases, unique scaffold structures from university laboratories, and natural compounds. The library is organized into subsets based on structure and purpose, providing high diversity for drug discovery research. In this example, for the purpose of developing therapeutic drugs, we first screened the "effective compound library," which consists of known active compounds and off-patent drugs, from the more than 200,000 compounds included in the aforementioned public compound library. Although this library contains only 3,337 compounds, their chemical and biological functions are being studied as chemical tools for pharmaceuticals and other targets.

[0053] Next, a primary screening (I in Figure 1) was performed to identify FPR1-specific inhibitors from an effective compound library. The inventors developed a high-throughput screening system. FPR1 is a G protein-coupled receptor (GPCR) that recognizes specific molecules or peptides, and its conformation changes upon reaction. Since β-arrestin binds to activated GPCRs as a scaffolding protein, a decrease in β-arrestin binding reflects the inhibition of GPCRs, which are effective targets for screening. To perform accurate screening, FPR1 was tagged with the C-terminal fragment of the luminescent protein luciferase (Eluc-C), and β-arrestin was tagged with the N-terminal fluorescent fragment (Eluc-N). The binding of β-arrestin was then measured using a bioluminescent probe consisting of these two proteins. It is known that when the luciferase fragment localizes to the vicinity through interaction with the tagged protein, complementarity between the two fragments occurs spontaneously, and the luminescence properties are restored. By utilizing this characteristic of split luciferase, β-arrestin binding can be quantitatively detected as an increase in luminescence.

[0054] Figure 2 is a graph showing the measurement of FPR1 inhibition rates of compound libraries validated as a primary screening using the β-arrestin assay. The validated compound library was screened mainly using the β-arrestin assay, and 345 compounds with high FPR1 inhibition rates (50% or higher) were identified.

[0055] Furthermore, to narrow down the candidates to compounds with even higher specificity, a secondary screening (II in Figure 1) was performed. To evaluate the specificity of FPR1 inhibition, the inhibitory activity against another GPCR, somatostatin receptor 2 (SSTR2), was also measured. Figure 3 is a graph showing the results of measuring the FPR1 and SSTR2 inhibition rates of the hit compounds using the β-arrestin assay. In the figure, the ranges where the FPR1 inhibition rate was 50% or higher and the SSTR2 inhibition rate was between -30% and 25% in the β-arrestin assay are enclosed by dotted lines. The 15 hit compounds are shown with light-colored, small circular markers, and SPD is represented by dark-colored, large circular markers. InH indicates the inhibition rate. As a result of secondary screening using β-arrestin assays for FPR1 and SSTR2, 15 compounds were selected from those with excessively low inhibition rates against SSTR2 (less than 30%) due to the possibility of SSTR2 activation. These 15 compounds showed high inhibition rates against FPR1 (50% or more) and no nonspecific inhibition against SSTR2 (less than 25%).

[0056] Next, to confirm the effect of the hit compounds, the concentrations of 12 of the 15 hit compounds that are readily available commercially were varied, and the number of repetitions was increased. Figure 4 is a graph showing the inhibition rate of the hit compounds at each concentration in the β-arrestin assay. The hit compounds showed a concentration-dependent increase in FPR1 inhibition rate. Of the 15 hit compounds, S-(+)-PD 123177 trifluoroacetate hydrate (SPD) was selected as a candidate compound because it is commercially available and easily obtainable, and it has the highest inhibitory activity against FPR1 (FPR1 inhibition rate of 84.4% and SSTR2 inhibition rate of -23.7% in the β-arrestin assay).

[0057] [Test Example 2] (Searching for compounds with FPR1 inhibitory activity from the core library) To further broaden the potential of candidate compounds as FPR1 inhibitors, a screening was performed on a medium-sized library (9,600 compounds) called the "core library," which is part of the entire DDI library and consists of drug-like compounds inferred from their molecular structure. This library provides diverse structural diversity, including not only compounds similar to those with reported activity but also compounds selected entirely randomly. Figure 5 is a schematic diagram showing an overview of the screening of the core library for identifying FPR1 inhibitors in this example. Because this library contains a large number of compounds, a G protein assay suitable for screening a vast number of compounds was performed first, before the β-arrestin assay, to narrow down the compounds. G protein signaling is a major downstream pathway of FPR1 and induces various biological effects such as inflammatory cytokines and chemotaxis. There is also a pathway in which Gq proteins cause calcium influx. To evaluate GPCR activation, expressing the promiscuous Gq protein G16 in cells allows all downstream GPCR signals to be evaluated through the increase in intracellular calcium. In this approach, intracellular Ca ２＋ The activation of FPR1 can be quantitatively monitored via a fluorescent dye that emits light in response to an increase in fluorescence intensity. This system enables rapid and high-throughput screening of large compound libraries for GPCR drug discovery, and provides inhibition rates based on the relative fluorescence intensity (RFI).

[0058] Figure 6 is a histogram showing the distribution of inhibition rates of compounds in the core library. The mean was 3.5%, and the variance was 16.1%pt. Screening of the core library using G protein assays revealed that compounds with inhibition rates of 50% or higher showed significant inhibition. As a result, 98 compounds were identified as hit compounds from the initial screening.

[0059] To confirm the reproducibility of the inhibitory properties and to quantitatively evaluate them, hit compounds were titrated four times with 10 μM. The average of these four measurements was compared with the primary screening results as a confirmation screening. Figure 7 is a graph showing the results of the confirmation assays for hit compounds in the core library screening. In the figure, each dot represents the results of the primary assay (n=1) and the confirmation assay (n=4). The inhibition rate was calculated using the confirmation assay on the vertical axis, and since negative control results were not obtained for the primary assay on the horizontal axis, a pseudo-inhibition rate was calculated. Compounds with an FPR1 inhibition rate of 50% or more in the primary screening and 40% or more in the confirmation assay are enclosed in a dotted line. 41 hit compounds are indicated by dark markers within the dotted line range. This comparison revealed that there were 41 compounds that showed an inhibition rate of 40% or more. Although the inhibition rate in the confirmation measurement was smaller due to differences in evaluation indices, most compounds showed similar trends in the two measurements.

[0060] Figure 8 is a graph showing the inhibition rates of 41 library compounds at five concentrations in a G protein assay. The dose-response curves for all 41 compounds that hit in the confirmatory screening showed dose-dependency. Of the 41 hit compounds, four compounds that showed a transient or sustained increase in fluorescence before ligand treatment were excluded. As a result of the primary and confirmatory screenings, 37 hit compounds were obtained from 9,600 compounds, and a high inhibition rate (>40%) against FPR1 was confirmed in the G protein assay.

[0061] Figure 9 is a graph showing the results of measuring the inhibition rates of FPR1 and SSTR2 in the β-arrestin assay for compounds that were hits in the G protein assay. The inhibition rates of FPR1 and SSTR2 were measured for 37 compounds (n=3). The range in which the FPR1 inhibition rate was 70% or higher and the SSTR2 inhibition rate was -20% < 20% in the β-arrestin assay is enclosed by a dotted line. Ten hit compounds are indicated by light-colored, small circular markers, and Z11 is indicated by a dark-colored, large circular marker. InH indicates the inhibition rate. When the 37 hit compounds were re-evaluated in the β-arrestin assay, 10 compounds were detected that showed high FPR1 inhibition rates (70% or higher) and negligible SSTR2 inhibition rates (-20% <, less than 20%), indicating highly specific FPR1 inhibitory activity. Of these 10 hit compounds, 6-amino-3-methyl-1-(2-methylpropyl)-5-(2-pyrrolidinyl(1,3-thiazole-4-yl))-1,3-dihydropyrimidine-2,4-dione (Z11), which has a relatively high FPR1-specific inhibitory rate (98.7% FPR1 inhibition rate and 3.3% SSTR2 inhibition rate in the β-arrestin assay), is commercially available and was identified as a candidate therapeutic agent.

[0062] [Example 3] (Searching for therapeutic candidates from existing FPR1 inhibitor compounds) We searched for therapeutic candidates from existing drugs that have been reported to have FPR1 inhibitory activity. We used the IUPHAR / BPS Guide to PHARMACOLOGY (https: / / www.guidetopharmacology.org). This is an open-access, expert-supervised resource on pharmacological targets and the substances that act on them. Of the 16 FPR1 antagonists listed in this database, only three are approved or have been approved by regulatory authorities for clinical use in humans: chenodeoxycholic acid (CDCA), cyclosporine A (Cys A), and sulfinpyrazone (SPZ). In addition, cyclosporine H (Cys H) and methionine benzimidazole 6 (MB6) are not approved drugs for specific human diseases, but they are known to have high FPR1 inhibitory activity and are frequently used in research papers. Before evaluating the effects of the five drugs, we examined their toxicity at the cellular level using viability staining. None of the drugs showed significant toxicity when used alone (not shown).

[0063] The FPR1 inhibition rates of these five drugs were measured using a β-arrestin assay. Figure 10 is a graph showing the results of measuring the FPR1 inhibition rates of existing drugs using a β-arrestin assay. The FPR1 inhibition rates of candidate compounds were measured using a β-arrestin assay. Dimethyl sulfoxide (DMSO) was used as the vehicle. As shown in the figure, Cys A did not show significant FPR1 inhibitory activity, but the other four drugs, CDCA, SPZ, Cys H, and MB6, showed FPR1 inhibitory activity. As a result, the inhibitory effect of seven candidate compounds—SPD, Z11, CDCA, Cys A, Cys H, SPZ, and MB6—on epidermal cell necroptosis was measured.

[0064] [Test Example 4] (In vitro assay of cell death in SJS / TEN) To evaluate the inhibitory effect of candidate compounds on necroptosis in keratinocytes, an in vitro assay method for detecting epidermal cell death in SJS / TEN was established. As reported by the inventors in Non-Patent Literature 2, FPR1 expression is induced on epidermal cells by P2X7R stimulation with LL-37, and necroptosis in SJS / TEN is caused by the binding of annexin A1 released from monocytes to FPR1. To induce FPR1 expression on immortalized epidermal cells, HaCaT, HaCaT cells were cultured with LL-37 or the P2X7R agonist Bz-ATP.

[0065] Figure 11 is a photograph showing immunofluorescence staining images of FPR1 in HaCaT treated with Bz-ATP or LL-37. Bz-ATP, a P2X7R ligand, was used at concentrations of 0 μM, 1 μM, 10 μM, and 100 μM, while LL-37 was used at concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, each for 48 hours. FPR1 was detected using the FPR1 fluorescent ligand. The scale bar in the figure represents 200 μm. Figure 12 is a graph showing the percentage of FPR1-positive cells treated with Bz-ATP and LL-37. (a) shows the results for LL-37, and (b) shows the results for Bz-ATP. The number of FPR1-positive cells was counted in a 400x field of view (n=5). Immunofluorescence staining using a fluorescent ligand for FPR1 showed that FPR1 is expressed in a concentration-dependent manner in HaCaT treated with Bz-ATP or LL-37.

[0066] Figure 13 is a graph showing the flow cytometry results of FPR1 expression in HaCaT cells treated with Bz-ATP or LL-37. Cells were treated with Bz-ATP (300 μM) or LL-37 (30 μg / ml) for 24 hours. Dead cells were removed by 7-AAD staining. FPR1 was detected using the FPR1 fluorescent ligand. FPR1-positive cells were 1.08% in the control group (-), 7.8% in the Bz-ATP group, and 13.96% in the LL-37 group. As shown in the figure, Bz-ATP and LL-37 stimulation induced FPR1 expression on HaCaT cells. These results confirm that P2X7R stimulation by BzATP or LL-37 induces FPR1 expression on HaCaT cells.

[0067] Next, we confirmed whether FPR1 stimulation reliably induces necroptosis in HaCaT cells. Flow cytometry results showed that LL-37 induced FPR1 expression more strongly than Bz-ATP. Therefore, a cell death assay was performed using only LL-37. After stimulating P2X7R with LL-37, Ac2-26, a peptide of annexin A1, was added, and cytotoxicity was measured. Figure 14 is a graph showing the results of measuring the toxicity of LL-37 and Ac2-26 to HaCaT cells by viable staining. The toxicity of HaCaT cells cultured for 24 hours with LL-37 (10 μg / ml), a P2X7R ligand, and Ac2-26 (50 ng / ml), an FPR1 ligand, was measured by viable staining. The difference was examined using Welch's t-test. Figure 15 shows the results of immunofluorescence staining of HaCaT cells with LL-37 and Ac2-26 to assess their toxicity. (a) is a photograph of the immunofluorescence image, showing the immunofluorescence images of the apoptosis marker cCasp3 and the necroptosis marker phosphorylated MLKL (pMLKL). The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Cultured HaCaT cells were stimulated with LL-37 (10 μg / ml) and Ac-2-26 (50 ng / ml) for 24 hours. The arrows indicate cCasp3 and pMLKL-positive cells, respectively. The scale bar is 50 μm. (b) shows the results of counting the ratio of cCasp3-positive cells to pMLKL-positive cells in a 400x field of view (n=5). The difference was examined using Welch's t-test.

[0068] Notably, pretreatment with LL-37 induced cell death upon subsequent Ac2-26 stimulation. Analysis of the morphology of cell death, using immunofluorescence staining, showed that Ac2-26 stimulation after LL-37 primarily induced necroptosis (7.25%), with less apoptosis (0.84%) (p = 0.009). These results allowed us to establish an in vitro assay for SJS / TEN using HaCaT.

[0069] [Test Example 5] (Verification of CDCA's cell death suppression by in vitro assay) The FPR1 inhibitory effects of seven candidate compounds were analyzed using the SJS / TEN in vitro assay. Figure 16 is a graph showing the cell death suppression rate by each FPR1 inhibitor candidate compound. The cell death suppression rate (%) is shown when HaCaT cultured in LL-37 (10 μg / ml) and Ac2-26 (50 ng / ml) was treated for 24 hours with various concentrations (0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM) of FPR1 inhibitor candidate compounds (n=3), including SPD, Z11, CDCA, CysA, CysH, SPZ, and MB6.

[0070] The rate of cell death suppression was examined by treating candidate compounds at various concentrations and using viability staining. Of the seven candidate compounds, the other compounds showed only slight suppression of cell death at high concentrations, but CDCA specifically and strongly suppressed cell death even at low concentrations. To verify whether CDCA inhibits necroptosis, immunofluorescence staining was performed in an SJS / TEN in vitro assay using pMLKL, a necroptosis marker. As a result, it was confirmed that CDCA reduced the number of pMLKL-positive cells, suggesting that CDCA inhibits HaCaT cell necroptosis (not shown). Based on these results, CDCA is considered a novel therapeutic agent for SJS / TEN.

[0071] [Test Example 6] (Verification of the symptom-suppressing effect of CDCA in a mouse model) Chenodeoxycholic acid (CDCA) is a major bile acid and is approved as a treatment for human cholelithiasis. To confirm the in vivo efficacy of CDCA, the inventors conducted a study of the SJS / TEN mouse model previously developed by the inventors (described in [Equipment, Reagents and Test Methods] above). These mice were generated from immunodeficient mice ((NOD) / Shi-scid, interleukin-1 receptor (IL-2R) γKO Jic (NOG) mice) by injecting PBMCs from patients who had recovered from TEN and orally administering the causative drug. In NOG mice, human cells engraft, allowing for evaluation of human drug-specific lymphocyte responses. This mouse model more closely mimics phenomena occurring in humans compared to other models, making it suitable for applying the results to human situations.

[0072] PBMCs were collected from patients who had recovered from TEN. PBMCs (2 x 10) ６NOG mice were intravenously injected with CDCA, and from day 1, the causative agent (acetaminophen, 1.5 mg / 100 μl) was administered orally once daily. Mice were also orally administered either CDCA or physiological saline (NS) once daily. Figure 17 shows the effect of CDCA administration on conjunctivitis symptoms in the mouse model. In the figure, from top to bottom, the clinical features of the conjunctiva on day 14 (1st column), H&E staining (2nd and 3rd columns), and TUNEL staining (4th and 5th columns) are shown. The arrow b indicates conjunctivitis. The arrow h indicates necrotic cells. The arrow n indicates TUNEL-positive cells. The scale bars are 500 μm (d, e, f, j, k, l) and 50 μm (g, h, i, m, n, o). The labels "Control" represent untreated (unadministered causative drug) mice (control group), "Vehicle" represent mice administered with the causative drug (drug-administered control group), and "CDCA" represent mice administered with CDCA in addition to the causative drug. Mice in the vehicle group, injected with PBMC and administered only with the causative drug, showed pronounced blepharoconjunctivitis as shown in figure b. However, no obvious conjunctivitis was observed in the CDCA-administered group, which received CDCA in addition to the causative drug. CDCA was able to completely suppress SJS / TEN-like conjunctivitis in the mouse model. Histopathological findings of the eyelids showed conjunctival deformation and damaged epithelial cells in the vehicle group, as shown in h, but no changes were observed in the CDCA-administered group, as shown in i.

[0073] Figure 18 is a graph showing symptom suppression in model mice. (a) shows the incidence of SJS / TEN-like disease in model mice (n=7). (b) shows the percentage (%) of TUNEL-positive cells and total conjunctival cells in a 400× field of view (n=4). Differences were examined using Welch's t-test. TUNEL staining, which detects cell death, showed abundant TUNEL-positive cells in the solvent group, as shown in Figure 17(n) and Figure 18(b). In contrast, the CDCA-administered group had fewer TUNEL-positive cells, as shown in Figure 17(o) and Figure 18(b). The percentage of TUNEL-positive cells was 35.74% in the solvent group and 2.17% in the CDCA group (p=0.00006). Furthermore, no toxicity was observed in any of the mice in the CDCA-administered group. These data clearly demonstrate the effectiveness of CDCA against cell death in preventing disease development in the SJS / TEN mouse model.

[0074] [Test Example 7] Further evaluation of the effectiveness of CDCA using SJS / TEN model mice was conducted. First, the effectiveness was evaluated using male mice. All the model mouse data in the above test example were from female immunodeficient mice. Therefore, in order to verify that there is no sex difference in effectiveness, the evaluation was conducted using male NOG immunodeficient mice.

[0075] Similar to Test Example 6, PBMC (2 × 10 ６ NOG mice were intravenously injected with CDCA, and from day 1, the causative agent (acetaminophen, 1.5 mg / 100 μl) was administered orally once daily. Mice were also administered orally once daily with either CDCA or physiological saline (NS). Figure 19 is a photographic diagram showing the effect on conjunctivitis symptoms in a CDCA-administered mouse model (male). In the figure, the clinical characteristics of the conjunctiva on day 14 are shown. In both the upper and lower rows, 1 to 5 refer to individual control mice. In the upper row, Vehicle is the control group, and CDCA is the CDCA-administered group.

[0076] Similar to Test Example 6, mice 1-5 in the control group each showed symptoms of conjunctivitis (indicated by + below each photograph). In contrast, none of the mice in the CDCA-administered group showed symptoms of conjunctivitis (indicated by - below each photograph). Furthermore, keratinocyte cell death was suppressed in the CDCA-administered group, as seen in pathological findings (not shown).

[0077] Figure 20 is a graph showing symptom suppression in male model mice. It shows the incidence of SJS / TEN-like disease (n=5) in the model mice. As shown in the figure, the incidence was 100% (5 / 5) in the control group, while it was 0% (0 / 5) in the CDCA-administered group.

[0078] These results indicate that conjunctivitis was suppressed by CDCA administration in male mouse models as well. In other words, these results were consistently observed in both male and female mice, suggesting that the therapeutic effect of CDCA is not sex-dependent.

[0079] [Test Example 8] The efficacy was evaluated using model mice with PBMCs from SJS / TEN patients with ST combination drug as the causative agent. To evaluate the generalizability of the findings from the above test example, a test was conducted using sulfamethoxazole / trimethoprim (SMX / TMP), i.e., ST combination drug, as a causative agent other than acetaminophen. Additional experiments were conducted using PBMCs from patients with SMX / TMP-induced SJS (a disease known to be associated with specific HLA class I alleles such as HLA-A 11:01 and HLA-B 15:02).

[0080] PBMCs were collected from an 85-year-old female patient with SMX / TMP-induced SJS, and SMX / TMP was orally administered as the causative agent. Other procedures were the same as those for the model mouse in the aforementioned study.

[0081] Figure 21 is a photographic diagram showing the effect of CDCA administration on conjunctivitis symptoms in a mouse model (ST combination drug model). Each image in the figure shows the clinical characteristics of the conjunctiva on day 14. The upper row 1-5 and lower row 1-4 refer to individual control mice. In the upper row, "Vehicle" is the control group, and "CDCA" is the CDCA-administered group.

[0082] Similar to Test Example 6, mice 1-5 in the control group each showed symptoms of conjunctivitis (indicated by + below each photograph). In contrast, mice 1-4 in the CDCA-administered group did not show symptoms of conjunctivitis (indicated by - below each photograph). Furthermore, keratinocyte cell death was suppressed in the CDCA-administered group, as seen in pathological findings (not shown).

[0083] Figure 22 is a graph showing symptom suppression in a model mouse (ST combination drug causative agent model). It shows the incidence of SJS / TEN-like disease (n=5) in the model mouse. As shown in the figure, the incidence was 100% (5 / 5) in the control group, while it was 0% (0 / 4) in the CDCA-administered group.

[0084] These results allowed us to evaluate the effectiveness of CDCA in a mouse model using PBMCs from patients with SJS / TEN caused by ST combination drugs.

[0085] Although embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims and their equivalents.

[0086] According to the present invention, it is possible to provide an FPR1 inhibitor, a pharmaceutical, and a treatment for severe drug rash that can be used in therapeutics based on the pathological state of cell death specific to SJS / TEN, and that can improve the mortality rate of SJS / TEN. Furthermore, it is possible to provide a screening method for FPR1 inhibitors, which is a method for searching for these therapeutics.

Claims

1. An FPR1 inhibitor containing chenodeoxycholic acid or sulfinpyrazone as the active ingredient, for use in the treatment of severe drug eruptions.

2. The FPR1 inhibitor according to claim 1, comprising chenodeoxycholic acid.

3. The FPR1 inhibitor according to claim 1, comprising sulfinpyrazone.

4. An FPR1 inhibitor according to any one of claims 1 to 3, which has cell death inhibitory activity.

5. A pharmaceutical product containing the FPR1 inhibitor according to any one of claims 1 to 3.

6. A therapeutic agent for severe drug rash containing the FPR1 inhibitor according to any one of claims 1 to 3.

7. The therapeutic agent for severe drug rash according to claim 6, wherein the severe drug rash is Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN).

8. A method for screening FPR1 inhibitors, comprising: one or more screening steps of selecting one or more G protein-coupled receptors (GPCRs), quantitatively measuring the inhibitory activity of candidate compounds against the GPCRs in a compound library, and selecting candidate compounds with one or more of the inhibitory activities higher as suitable compounds; and measuring the cell death inhibitory activity of the suitable compounds and selecting an FPR1 inhibitor from the suitable compounds.

9. A method for screening an FPR1 inhibitor according to claim 8, comprising a screening step of selecting FPR1 as the GPCR.

10. A method for screening an FPR1 inhibitor according to claim 8, comprising: a primary screening step of selecting FPR1 as the GPCR; and a secondary screening step of selecting SSTR2 as the GPCR.

11. A screening method for FPR1 inhibitors according to claim 8 or 9, wherein the quantitative measurement of the inhibitory activity of the candidate compound against the GPCR is performed by a calcium assay or a β-arrestin assay.

12. The method for screening an FPR1 inhibitor according to claim 8 or 9, wherein the measurement of the cell death inhibitory activity is performed by inducing the expression of FPR1 in epidermal cells to induce cell death activity, and then administering the suitable compound to evaluate the inhibitory activity of the cell death activity.

13. The method for screening an FPR1 inhibitor according to claim 12, wherein an in vitro assay is performed using HaCaT cells as the epidermal cells.

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