Therapeutic agents for idiopathic pulmonary fibrosis

Abstract

A therapeutic agent for idiopathic pulmonary fibrosis, which contains a PAK inhibitor as an effective ingredient.

Description

Technical Field

[0001] This invention relates to a therapeutic agent for idiopathic pulmonary fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown cause, characterized by refractory and progressive pulmonary fibrosis. The 5-year survival rate is approximately 20-40%, and the prognosis is very poor. IPF is caused by excessive extracellular matrix production by myofibroblasts induced by chronic inflammation or tissue damage, but the formation of immature fibroblast foci, in particular, is known to be closely associated with the pathological condition.

[0003] Sustained abnormal activation of pulmonary myofibroblasts is mediated by various proteins, including transforming growth factor (TGF), platelet-derived growth factor, and fibroblast growth factor. This sustained abnormal activation of pulmonary myofibroblasts is a major event in the occurrence and progression of IPF.

[0004] The IPF microenvironment is composed of highly heterogeneous cells with dynamic interactions. The ongoing interaction between fibrotic tissue and its microenvironment plays a crucial role in the occurrence, progression, and response to various treatments of fibrosis. The region between fibrotic tissue and morphologically normal lung typically contains fibroblasts of various sizes known as early fibroblastic foci (FFs). FFs are the primary pathogenic lesions in IPF, composed of activated fibroblasts and myofibroblasts, which are the main effector cells leading to dysregulated extracellular matrix (ECM) deposition in various fibrotic states. Consistent with the idea that fibrosis spreads from FFs to unaffected alveoli, these structures are sites of accumulation for myofibroblasts during pathological ECM deposition. Indeed, the greater the number of FFs, the stronger the association with symptom exacerbation in IPF patients.

[0005] The genomic landscape of IPF has been elucidated to some extent using traditional sequencing strategies such as batch whole exome sequencing and batch RNA sequencing (RNA-seq) (see, for example, non-patent literature 1).

[0006] Existing technical documents Non-patent literature Non-patent literature 1: Peyser R, MacDonnell S, Gao Y, Cheng L, Kim Y, Kaplan T, et al. Defining the Activated Fibroblast Population in Lung Fibrosis UsingSingle-Cell Sequencing. Am J Respir Cell Mol Biol. 2019;61(1):74-85. Summary of the Invention The problem the invention aims to solve However, computer simulations have historically only been performed using mixtures of normal tissues within a single tissue, excluding FF components. While single-cell RNA sequencing (scRNA-seq) is an unprecedented tool for analyzing the heterogeneity of IPF lungs at the single-cell level and developing comprehensive gene expression maps, it lacks spatial information specific to individual cells. Spatial information is indispensable for a comprehensive understanding of IPF progression. Localized tissue heterogeneity is a hallmark pathological feature of human pulmonary fibrosis.

[0007] Therefore, the urgent task is to develop sequencing technologies that can determine cell type-specific spatial maps. In this regard, digital spatial transcriptomics methods provide gene expression information through spatial resolution, contributing to a general understanding of tissue formation and disease etiology. Thus, this methodology could help in understanding fibrosis in IPF lungs under various conditions.

[0008] Currently, there is no fundamental treatment for IPF, making its development an urgent priority. Therefore, the object of this invention is to provide a therapeutic agent for idiopathic pulmonary fibrosis based on a novel mechanism of action, utilizing the aforementioned methodology.

[0009] Solution for solving the problem The present invention is described below.

[0010] [1] A therapeutic agent for idiopathic pulmonary fibrosis containing a PAK inhibitor as an active ingredient.

[0011] [2] A therapeutic agent for idiopathic pulmonary fibrosis containing a PAK2 inhibitor as the active ingredient.

[0012] [3] The idiopathic pulmonary fibrosis treatment agent described in [1] or [2] is applicable to patients with WNT5A+CTHRC1+ myofibroblasts in their lung tissue.

[0013] [4] A composition for the treatment of idiopathic pulmonary fibrosis, comprising the idiopathic pulmonary fibrosis treatment agent described in [1] or [2] and a pharmaceutically acceptable carrier.

[0014] [5] A diagnostic agent for diagnosing idiopathic pulmonary fibrosis, comprising a primer set for amplifying at least one molecule selected from the group consisting of POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1 and VCAN, and / or a probe binding to said molecule or its amplification product, and an antibody binding to said molecule.

[0015] [6] A method for detecting diagnostic markers for idiopathic pulmonary fibrosis, wherein the presence of at least one molecule selected from the group consisting of POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1 and VCAN is detected in a sample from a subject.

[0016] Invention Effects According to the present invention, a therapeutic agent for idiopathic pulmonary fibrosis based on a novel mechanism of action can be provided. Attached Figure Description

[0017] [ Figure 1 This describes the experimental workflow for spatial transcriptome and scRNA-seq analysis of human IPF lungs. The spatial transcriptome analysis used nine lung slices from three patients, and the scRNA-seq analysis used three IPF lungs and three control lungs.

[0018] [ Figure 2 [This is a spatial visualization image annotated with the expression levels of COL1A1, POSTN, and CTHRC1 shown in each panel. Representative magnified views of the delineated boxed areas show hematoxylin and eosin (H&E) staining, with FF in IPF lung tissue highlighted.]

[0019] [ Figure 3 [Image is a UMAP visualization of an labeled interstitial scRNA-seq dataset from three IPF lung samples and three control lung samples, with detailed cell type labeling.]

[0020] [ Figure 4 [ ] is a violin diagram showing the expression of selected marker genes in each cluster.

[0021] [ Figure 5 ] is to Figure 3 UMAP visualization images of the scRNA-seq dataset, categorized by lung origin (normal lung or IPF lung).

[0022] [ Figure 6 [This is a bar chart comparing lung interstitial cell types in healthy individuals and IPF patients.]

[0023] [ Figure 7 [This is the result of a pseudo-temporal trajectory analysis that predicts the different mesenchymal cell differentiation trajectories between healthy individuals and IPF patients.]

[0024] [ Figure 8 [Image] is an immunohistochemical image of PDGFRA, CTHRC1, and WNT5A in fibroblastic lesions of IPF lung tissue.

[0025] [ Figure 9 [This is a spatial visualization of FF and progressive fibrotic lesions (DF) based on inferred cell type distribution from Visium sections. FF and DF in IPF lung tissue are highlighted in a representative magnified view showing the bounding box regions stained with hematoxylin and eosin (H&E).]

[0026] [ Figure 10 The result is the result of 3D PCA mapping using the extracted 97 FF points and 412 DF points.

[0027] [ Figure 11 This is a dot plot showing the types of mesenchymal cells present in selected FF, DF, and SMC regions on Visium by integrating and analyzing single-cell data and spatial analysis data.

[0028] [ Figure 12 [] is a spatial transcriptomic point of the FF index (patient JKPF2-1).

[0029] [ Figure 13 [] is a bar chart representing the proportion of high FF index and low FF index populations in mesenchymal cell types.

[0030] [ Figure 14 The results are based on IPA predictions of upstream regulators for 746 differentially expressed genes between FF and DF. The figure shows the top 10 upstream regulators predicted to be "activated".

[0031] [ Figure 15 ] is the display Figure 14 A violin plot showing the average expression levels of the top 10 upstream regulators detected in the study across different cell types.

[0032] [ Figure 16[Image] is a visualization of PAK target gene characteristics in the mesenchymal population shown in UMAP.

[0033] [ Figure 17 [ ] is a visualization of the FF index characteristics in the interstitial population displayed in UMAP.

[0034] [ Figure 18 [Image] is a violin diagram showing PAK2 expression in high-FF index mesenchymal cells and low-FF index mesenchymal cells.

[0035] [ Figure 19 [This is a spatial visualization result based on FF / DF annotations on Visium slices, showing PAK target gene features and inferred cell type distribution.]

[0036] [ Figure 20 [ ] is a dot plot showing the expression of PAK family genes in interstitial cells compared to normal lung samples and IPF lung samples.

[0037] [ Figure 21 [The results are from hematoxylin and eosin (H&E) staining and immunohistochemical staining of PAK1, PAK2, and PAK3 in fibroblast lesions of IPF lung tissue.]

[0038] [ Figure 22 The results illustrate the effect of siRNA-mediated PAK1 / 2 knockdown on TGF-β-induced myofibroblast differentiation in lung fibroblasts. A schematic diagram of the protocol used for in vitro experiments is shown above. Representative immunoblotting results are shown below, illustrating the levels of PAK1, PAK2, type I collagen, and β-actin in lung fibroblasts after treatment with TGF-β1 (2 ng / mL) for 48 hours.

[0039] [ Figure 23 [Image shows the qPCR results of knockdown of WNT5A expression in human lung fibroblasts by PAK1 or PAK2 after treatment with TGF-β1 (2 ng / mL) for 48 hours.]

[0040] [ Figure 24The results illustrate the effects of the PAK inhibitors FRAX486 and FRAX597 on TGF-β-induced myofibroblast differentiation in lung fibroblasts. A schematic diagram of the protocol used for in vitro experiments is shown above. Representative immunoblotting results are shown below, illustrating the levels of α-smooth muscle actin (α-SMA), type I collagen, and β-actin in lung fibroblasts after 48 hours of treatment with FRAX486 (100 nM and 1,000 nM) and FRAX597 (100 nM and 1,000 nM) in the presence of TGF-β1 (2 ng / mL). Pirfenidone (PFD) and nintedanib (NTD) were used as positive controls to inhibit myofibroblast differentiation.

[0041] [ Figure 25 [This is a quantitative immunoblotting analysis showing the content of type I collagen and α-SMA.]

[0042] [ Figure 26 [Image shows the results of immunoblotting analysis of PAK2 and its downstream signal levels in TGF-β-induced myofibroblasts after treatment with FRAX486 (1,000 nM) and FRAX597 (1,000 nM) for 48 hours in the presence of TGF-β1.]

[0043] [ Figure 27 [Image 1] shows the effect of PAK2 inhibitors on IPF lung fibroblasts. [Image 2] shows representative immunoblotting results of the levels of α-SMA, type I collagen, and β-actin in IPF lung fibroblasts 48 hours after treatment with FRAX486 (1,000 nM) and FRAX597 (1,000 nM). Protein samples were recovered from cells 48 hours after the start of treatment.

[0044] [ Figure 28 This is a quantitative immunoblotting analysis showing the content of type I collagen and α-SMA in IPF lung fibroblasts.

[0045] [ Figure 29 [This is a schematic diagram of the FRAX486 treatment used in bleomycin-induced pulmonary fibrosis mice.]

[0046] [ Figure 30 [This is a violin diagram of Pak2 between bleomycin-induced lung fibroblasts and control fibroblasts.]

[0047] [ Figure 31 This is an immunoblotting analysis result confirming elevated PAK2 phosphorylation levels in lung tissue of a BLM-induced fibrosis model.

[0048] [ Figure 32[Figure 1] shows the changes in body weight observed after bleomycin administration. Body weight on day 0 before treatment began was set to 100. The values ​​in the figure represent mean ± SEM. P<0.01. Control: n=10; Bleomycin control: n=10; Bleomycin + FRAX486: n=10.

[0049] [ Figure 33 The results show the quantitative results of hydroxyproline in the left lung of each mouse. Each point represents data from one animal. P<0.001, P<0.0005.

[0050] [ Figure 34 [Images are H&E staining and Masson's trichrome staining images of representative lung sections from each group of treated mice.]

[0051] [ Figure 35 [This is a quantitative result of fibrosis using the Aschcroft score. Each point represents data from one animal.] P<0.0005.

[0052] [ Figure 36 [Image shows p-PAK2 IHC staining results from representative lung sections of treated mice from each group. Scale bar: 100 µm.] Detailed Implementation

[0053] [Therapeutic agents for idiopathic pulmonary fibrosis] As described in the following embodiments, the inventors discovered the anti-fibrotic properties of PAK2 inhibitors from the perspective of IPF treatment. In one embodiment, the present invention provides a therapeutic agent for idiopathic pulmonary fibrosis containing a PAK inhibitor as an active ingredient.

[0054] The PAK (p21-activated kinase) family consists of serine / threonine kinases that regulate various cellular activities. To date, six PAK subtypes (PAK1–6) have been identified.

[0055] PAK1 is known to be essential for the proliferation of solid tumors, and excessive activation or overexpression of PAK1 can cause diseases such as cancer, hypertension, diabetes, and Alzheimer's disease.

[0056] The amino acid sequence of PAK1 includes the sequence of human PAK1 registered in GenBank accession number AAA65441. The gene sequence of PAK1 includes the sequence of human PAK1 registered in GenBank accession number U24152.

[0057] PAK1 inhibitors are substances that inhibit signal transduction induced by PAK1. Any substance that inhibits the signal transduction pathway caused by PAK1 is acceptable and is not limited to any particular type, including nucleic acids, proteins, and small molecule compounds. Substances that directly act on PAK1 include small molecule compounds, proteins, antibodies, and aptamers. Substances that inhibit the expression of genes encoding PAK1 include siRNA and antisense oligonucleotides.

[0058] Small molecule compounds that inhibit the kinase activity of PAK1 include IPA-3 (1,1'-dithiodi-2-naphthol), AG-1478 (N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine), FRAX597 (6-[2-chloro-4-(1,3-thiazo-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one), FR AX486 (6-(2,4-dichlorophenyl)-8-ethyl-2-[[3-fluoro-4-(1-piperazinyl)phenyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one), PF-3758309 ((S)-N-(2-(dimethylamino)-1-phenylethyl)-6,6-dimethyl-3-((2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-formamide), etc. IPA-3 is a small molecule allosteric inhibitor of PAK1.

[0059] PAK2 is widely distributed throughout the body and is an important effector of the Rho family of small GTPases related to cytoskeleton remodeling.

[0060] In addition, PAK2 regulates various biological processes, including tumorigenesis, cellular senescence, and the promotion of overall aging. Specifically, PAK2 is an activating receptor for TGF-β and a downstream component of AKT. PAK2 activation is essential for the TGF-β-promoting fibrotic effect. Furthermore, PAK2 is a non-canonical SMAD effector of TGF-β in renal interstitial fibrosis, and its overexpression induces renal fibrosis. Some studies have elucidated the relationship between PAK2 and fibrosis in hepatic stellate cells and peritoneal mesothelial cells, but the role of PAK2 in the etiology of IPF remains insufficiently understood.

[0061] The amino acid sequence of PAK2 includes the sequence of human PAK2 registered in GenBank accession number AAA65442. The gene sequence of PAK2 includes the sequence of human PAK2 registered in GenBank accession number U24153.

[0062] PAK2 inhibitors are substances that inhibit signal transduction induced by PAK2. Any substance that inhibits the signal transduction pathway caused by PAK2 is acceptable and is not limited to any particular type, including nucleic acids, proteins, and small molecule compounds. Substances that directly act on PAK2 include small molecule compounds, proteins, antibodies, and aptamers. Substances that inhibit the expression of genes encoding PAK2 include siRNA and antisense oligonucleotides.

[0063] Small molecule compounds that inhibit the kinase activity of PAK2 include FRAX486 (6-(2,4-dichlorophenyl)-8-ethyl-2-[[3-fluoro-4-(1-piperazinyl)phenyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one), FRAX597 (6-[2-chloro-4-(1,3-thiazo-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one), FRAX1036, and PF-3758309 ((S)-N-(2-(dimethylamino)-1-phenylethyl)-6,6-dimethyl-3-((2-methylthiopheno[3-ethyl)-2-ethyl)-2-ethyl-3-((2-methylthiopheno[3-ethyl)-2-ethyl)-2-ethyl)-2-ethyl-3-((2-methylthiopheno[3-ethyl)-2-ethyl)-2-ethyl)-2-ethyl)-2-ethyl)-2-ethyl)-2-ethyl)-2-ethyl)-2-methylthiopheno[3 ... The following are listed as derivatives of N-(3-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine), 2-aminopyrido[2,3-d]pyrimidin-7(8H)-one, 1H-thieno[3,2-c]pyrazole, 3-amino-tetrahydropyrrolo[3,4-c]pyrazole, N4-(1H-pyrazol-3-yl)pyrimidin-2,4-diamine, N4-(1H-pyrazol-3-yl)pyrimidin-2,4-diamine, N2-bicyclic indole, indazole and benzimidazole derivatives, etc.

[0064] The PAK inhibitor used as a therapeutic agent for idiopathic pulmonary fibrosis in this embodiment may be a pan-PAK inhibitor, but is preferably a PAK2 selective inhibitor.

[0065] As a therapeutic agent for idiopathic pulmonary fibrosis in this embodiment, the aforementioned PAK inhibitor can be used in free form or in a pharmaceutically acceptable salt form. Alternatively, it can be used as a solvate of the free form or as a solvate of the salt.

[0066] Salts are pharmaceutically acceptable and are not particularly limited, including, for example, hydrochlorides, sulfates, hydrobromides, hydroiodides, phosphates, nitrates, benzoates, methanesulfonates, 2-hydroxyethanesulfonates, p-toluenesulfonates, acetates, propionates, oxalates, malonates, succinates, glutarates, adipates, tartrates, maleates, fumarates, malates, mandelates, etc. Solvents are pharmaceutically acceptable and are not particularly limited, including, for example, hydrates, organic solvates, etc.

[0067] As described in the following embodiments, since PAK2 is highly expressed in WNT5A+CTHRC1+ myofibroblasts, the idiopathic pulmonary fibrosis treatment agent of this embodiment is preferably applied to patients with WNT5A+CTHRC1+ myofibroblasts in their lung tissue.

[0068] [Composition for the Treatment of Idiopathic Pulmonary Fibrosis] In one embodiment, the present invention provides a composition for the treatment of idiopathic pulmonary fibrosis, comprising the above-mentioned idiopathic pulmonary fibrosis treatment agent and a pharmaceutically acceptable carrier.

[0069] The composition for treating idiopathic pulmonary fibrosis according to this embodiment can be administered orally in the form of tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or non-oral in the form of inhalation, injection, suppositories, topical skin preparations, etc.

[0070] As pharmaceutically acceptable carriers, carriers commonly used in pharmaceutical formulations may be used without particular restriction. More specifically, these include binders such as gelatin, corn starch, tragali gum, and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginate; solvents for injection such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives. Pharmaceutically acceptable carriers may be used alone or in combination of two or more.

[0071] The composition for treating idiopathic pulmonary fibrosis according to this embodiment may also include additives. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and wintergreen oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives. One additive may be used alone, or two or more additives may be used in combination.

[0072] (Administration method) There are no particular restrictions on the method of administration for idiopathic pulmonary fibrosis treatment agents or compositions, which can be determined based on the patient's symptoms, weight, age, and gender. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions are for oral administration. Injectable preparations are administered intravenously alone or mixed with routine rehydration solutions such as glucose and amino acids, and then further administered intra-arterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories are administered rectally. Topical preparations are applied, patched, or sprayed onto the affected area. Inhaled medications are administered using devices such as nebulizers, metered-dose inhalers, and dry powder inhalers.

[0073] (Dosage) The dosage of idiopathic pulmonary fibrosis treatment agents or compositions varies depending on the patient's symptoms, weight, age, and sex, and cannot be generalized. However, for oral administration, a dosage of 1 μg to 10 g or 0.01 to 2000 mg of active ingredient per day is sufficient. For injectable preparations, a dosage of 0.1 μg to 1 g or 0.001 to 200 mg of active ingredient per day is sufficient. For suppositories, a dosage of 1 μg to 10 g or 0.01 to 2000 mg of active ingredient per day is sufficient. For topical preparations, a dosage of 1 μg to 10 g or 0.01 to 2000 mg of active ingredient per day is sufficient. For inhaled preparations, a single inhalation of 1 μg to 10 g or 0.01 to 2000 mg of active ingredient per day is sufficient.

[0074] [Other Implementation Methods] In one embodiment, the present invention provides PAK inhibitors, pharmaceutically acceptable salts thereof, or solvates thereof for the treatment of idiopathic pulmonary fibrosis.

[0075] In one embodiment, the present invention provides a treatment for idiopathic pulmonary fibrosis, comprising administering an effective amount of a PAK inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof to a patient requiring treatment.

[0076] In one embodiment, the present invention provides the use of PAK inhibitors, pharmaceutically acceptable salts thereof, or solvates thereof in the preparation of idiopathic pulmonary fibrosis treatment agents or compositions for the treatment of idiopathic pulmonary fibrosis.

[0077] [Other Implementation Methods] In one embodiment, the present invention provides a therapeutic agent for idiopathic pulmonary fibrosis, comprising at least one inhibitor selected from the group consisting of PRKAA2 inhibitors, TWF1 inhibitors, PRKAA1 inhibitors, DDR2 inhibitors, PAK2 inhibitors, ABL2 inhibitors, DYRK1B inhibitors, FGFR4 inhibitors, CDK19 inhibitors, and ERBB4 inhibitors as an active ingredient.

[0078] [Diagnostic drugs for idiopathic pulmonary fibrosis diagnostic markers] In one embodiment, the present invention provides a diagnostic agent for diagnosing idiopathic pulmonary fibrosis, comprising a primer set for amplifying at least one molecule selected from the group consisting of POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1, and VCAN in a sample, and / or a probe binding to said molecule or its amplification product, and an antibody binding to said molecule.

[0079] Diagnostic drugs can detect molecules including nucleic acids such as DNA, RNA, and miRNA, as well as proteins. When the target is a nucleic acid, the diagnostic drug preferably contains a primer set for amplifying these molecules, and / or probes that bind to the molecule or its amplification product. When the target is a protein, the diagnostic drug preferably contains antibodies that bind to these molecules.

[0080] In this embodiment, the samples include cells, blood, urine, saliva, sweat, tissue exudate, etc. from tissue slices.

[0081] [Detection methods for diagnostic markers of idiopathic pulmonary fibrosis] In one embodiment, the present invention provides a method for detecting diagnostic markers for idiopathic pulmonary fibrosis, which detects the presence of at least one molecule selected from the group consisting of POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1, and VCAN in a sample from a subject.

[0082] The target molecules are at least one molecule selected from the group consisting of POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1, and VCAN. Preferably, all molecules from POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1, and VCAN are used as the target molecules. As described in the examples below, the average expression level of these genes can be used as the FF index to evaluate the sample.

[0083] Example The present invention will be further described in detail below through experimental examples, but the present invention is not limited to these examples.

[0084] [Experimental Example] Spatial transcriptome analysis of human IPF lung tissue according to Figure 1 The experimental flowchart shown illustrates spatial transcriptomic profiling of lung tissue (a total of 9 sections) from 3 IPF patients. Additionally, scRNA-seq analysis was performed on lung tissue from 3 IPF patients and 3 healthy individuals. As the first step in the spatial transcriptomic analysis of IPF lungs, standardized and visualization procedures based on UMAP (Uniform Manifold Approximation and Projection) were applied to each section.

[0085] In IPF lung, FF (fibrillary fibroblasts) contain activated fibroblasts and myofibroblasts and are considered the main pathogenic lesions of IPF. To characterize the cellular and molecular features of FF, histological identification was performed using H&E images (see [link to H&E image]). Figure 2 The Visium points that overlap with these regions were designated as FF points. The accuracy of these FF point selections was further verified by analyzing the spatial distribution of known FF markers such as COL1A1, POSTN, and CTHRC1 on each Visium slice.

[0086] [Detailed annotation of scRNA-seq data and integration of spatial Visium points] To further investigate the cell type composition within the fibrotic regions of IPF, the labeling of stromal cell types was optimized using major biomarkers associated with the etiology of IPF. Nine distinct clusters were identified using UMAP clustering, including the CTHRC1+ fibroblast population, which is known to play a crucial role in the pathogenesis of IPF (see [link to UMAP]). Figure 3 and Figure 4 In particular, an intermediate cell state between fibroblasts and smooth muscle cells (SMCs) / peripheral cell clusters was identified and named WNT5A+CTHRC1+ myofibroblasts. These cells exhibit fibroblast-like features expressing PDGFRA, PDGFRB, and RUNX1, lack DES expression, and possess specific markers such as WNT5A and WIF1 (see [link to study]). Figure 4 ).

[0087] The presence of this WNT5A+CTHRC1+ myofibroblast population was validated by analyzing publicly available single-cell datasets (GSE136831, GSE135893) from IPF patients. These WNT5A+CTHRC1+ cells were detected within myofibroblast clusters mapped between fibroblasts and SMC clusters on the UMAP. Furthermore, compared to controls, the proportions of myofibroblasts, CTHRC1 fibroblasts, and WNT5A+CTHRC1+ myofibroblasts were significantly increased in IPF patients (see [link to relevant documentation]). Figure 5 , 6 ).

[0088] Pseudo-temporal trajectory analysis revealed characteristic fibroblast differentiation pathways in IPF compared to controls. In healthy lung tissue, alveolar fibroblasts primarily differentiate into myofibroblasts. However, in IPF, alveolar fibroblasts follow two distinct trajectories: differentiating into myofibroblasts or pathogenic CTHRC1+ fibroblasts. The latter population further acquires smooth muscle-like features, including increased TAGLN and ACTA2 expression, and transforms into WNT5A+CTHRC1+ myofibroblasts (see [link to study]). Figure 7 Immunohistochemistry of human IPF lung tissue confirmed the presence of WNT5A+CTHRC1+PDGFRA+ fibroblasts in the FF and surrounding DF regions of IPF lung tissue (see [link]). Figure 8 ).

[0089] [Molecular and cellular differences between FF and DF in human IPF lungs] IPF lung tissue exhibits a heterogeneous pattern of fibrosis, similar to the DF region, which represents fibrotic remodeling. Mature scar tissue disrupts normal lung structure and impairs gas exchange. Comparing FF and DF is important for understanding the dynamic progression of fibrosis and identifying therapeutic targets in both the formation of active fibers and their transition to fibrotic remodeling. The DF region surrounding the FF was morphologically identified, characterized by abundant fibroblasts and the absence of SMCs and pericytes. For Visium points corresponding to the DF region, regions with significant contributions from SMCs or pericytes were manually reviewed using deconvolutiond scRNA-seq data (see [link to relevant documentation]). Figure 9 As a result, 97 FF points and 412 DF points were identified from the 9 slices. These spots were plotted on a principal component analysis (PCA) plot (see...). Figure 10 The results show a clear difference in transcriptomic profiles between FF and DF.

[0090] To elucidate the differences in cell type composition, mesenchymal scRNA-seq data were deconvolved with Visium data from FF and DF sites. The results revealed distinct cell distribution patterns in FF and DF. The FF region was primarily occupied by CTHRC1+ fibroblasts, proliferating fibroblasts, and aberrant basaloids, while the DF region, unlike the SMC / peripheral cell region, exhibited a high proportion of WNT5A+CTHRC1+ myofibroblasts. This further supports the idea that the transition to WNT5A+CTHRC1+ myofibroblasts may be partially involved in the mechanism of the transition from FF to DF (see [link to relevant documentation]). Figure 11 AberrantBasaloid showed high levels of intercellular interactions with both WNT5A+CTHRC1+ myofibroblasts and CTHRC1+ fibroblasts, suggesting the existence of a profibrotic circuit that promotes fibrosis progression.

[0091] To detect fibroblast populations within fibroblasts (FF) at the molecular level, an FF index was developed based on biomarkers identified through spatial analysis, followed by deconvolution in scRNA-seq data. While high-FF-index fibroblasts were also detected in control lung samples, their numbers were significantly higher in IPF samples. Spatially, the FF index effectively visualized fibroblast lesions in IPF lung sections (see [link to documentation]). Figure 12 Fibroblasts with a high FF index are mainly composed of pathogenic fibroblasts such as CTHRC1+ fibroblasts, proliferative fibroblasts, and WNT5A+CTHRC1+ myofibroblasts, while fibroblasts with a low FF index are mainly alveolar fibroblasts (see...). Figure 13These results suggest that fibroblasts with high FF indices may serve as therapeutic targets for IPF.

[0092] [Identification of PAK2 activation in fibroblastic lesions and surrounding DF regions and its therapeutic potential in IPF] To identify novel therapeutic target molecules for IPF, differentially expressed genes between FF and DF were analyzed. Ingenuity Pathway Analysis (IPA) results clearly confirmed the activation of the "pulmonary fibrosis idiopathic signaling pathway" at the FF site in IPF lung. Activation of several kinases at the FF site in IPF lung was identified through upstream regulatory factor prediction (see [link to original text]). Figure 14 Among these kinases, PRKAA2, as the catalytic subunit of AMPK, is expected to play a role in regulating TGF-β-induced myofibroblast differentiation and is therefore considered as a candidate. PAK2 has attracted attention as part of the exploration of novel therapeutic targets because it is highly expressed in pathogenic CTHRC1+ fibroblasts and WNT5A+CTHRC1+ myofibroblasts (see [link to article]). Figure 15 Furthermore, small molecule inhibitors targeting PAK2 are available.

[0093] To further investigate PAK2 activity at the single-cell level, a set of PAK target genes was established based on previously reported RNA-seq data. The mean expression level of PAK target features in each single cell was calculated using a module scoring algorithm and visualized using UMAP (see [link to UMAP]). Figure 16 Interestingly, the distribution of cells expressing high PAK target signatures was very similar to that of cells with high FF index (see [link to article]). Figure 17 This indicates that PAK target genes are primarily expressed in profibrotic fibroblasts. Furthermore, PAK2 expression was significantly higher in fibroblasts with high FF (fibrillation factor) index than in those with low FF index (see [link to relevant documentation]). Figure 18 Spatial distribution analysis confirmed that the expression of PAK target genes was concentrated around the FF and DF regions, closely corresponding to the distribution in CTHRC1+ fibroblasts and WNT5A+CTHRC1+ myofibroblasts, respectively (see [link to relevant documentation]). Figure 19 ).

[0094] Within the PAK family, PAK2 exhibits the highest expression level in mesenchymal cells compared to PAK1 and PAK3, and is significantly enriched in IPF lung tissue at both the transcriptomic and protein levels (see [link to relevant documentation]). Figure 20 , Figure 21 These results suggest the potential of PAK2 as a therapeutic target for IPF by modulating the activity of pathogenic fibroblasts.

[0095] [The role of PAK2 in TGF-β-induced fibrotic differentiation in human lung fibroblasts and its inhibition as a therapeutic target] To elucidate the roles of PAK1 and PAK2 in TGF-β-induced fibrosis, freshly isolated primary human lung fibroblasts were used to knock down PAK1 and PAK2 via siRNA. Knockdown of both PAK1 and PAK2 effectively reduced TGF-β-induced ECM production and WNT5A expression (see [link to study]). Figure 22 , Figure 23 This is consistent with the results of spatial transcriptome analysis.

[0096] Next, to characterize the pharmacological antifibrotic properties of PAK2-targeted drugs from the perspective of IPF treatment, FRAX486 and FRAX597, as PAK2 inhibitors, were selected and applied to a TGF-β-induced myofibroblast differentiation model of lung fibroblasts (see [link to article]). Figure 24 While FRAX486 and FRAX597 inhibit multiple PAK family members, including PAK1, PAK2, and PAK3, their primary effect is likely mediated by PAK2, given the expression pattern of PAK2 in IPF lung tissue (see [link to article]). Figure 20 Treatment with either FRAX486 or FRAX597 significantly inhibited TGF-β-induced myofibroblast differentiation based on protein levels of α-smooth muscle actin (α-SMA) and type I collagen (see [link to relevant documentation]). Figure 24 These inhibitory effects are dose-dependent (see [link to relevant documentation]). Figure 25 Furthermore, it significantly outperformed the two antifibrotic drugs commonly used in IPF treatment, namely pirfenidone (PFD) and nintedanib (NTD). Immunofluorescence labeling of α-SMA and type I collagen demonstrated the inhibitory effects of FRAX486 and FRAX597 on TGF-β-induced myofibroblast differentiation. The expression levels of PAK2 downstream proteins were analyzed using Western blotting (see [link to Western blotting]). Figure 26 The results showed that both inhibitors suppressed the expression and phosphorylation of PAK2, c-Abl, and LIMK1, but did not suppress the expression and phosphorylation of SMAD2 / 3. In particular, FRAX486 strongly inhibited the differentiation of lung fibroblasts into TGF-β-induced myofibroblasts. qPCR confirmed the transcriptional repression of downstream factors in the TGF-β-induced myofibroblast differentiation process.

[0097] Subsequently, the antifibrotic effects of FRAX486 and FRAX597 on lung fibroblasts isolated from IPF lungs were evaluated. Treatment with these inhibitors significantly reduced the expression of α-SMA and type I collagen in fibroblasts derived from IPF (see [link to study]). Figure 27 , Figure 28 Consistent with the TGF-β-induced myofibroblast differentiation model, the expression of PAK2 downstream proteins was significantly inhibited by FRAX486 and FRAX597 in fibroblasts derived from IPF. Furthermore, RT-PCR analysis confirmed the transcriptional repression of PAK2 downstream targets in IPF fibroblasts. In summary, the antifibrotic effects of FRAX486 and FRAX597 in both the TGF-β-induced model and in lung fibroblasts derived from IPF are mediated by the inhibition of pro-fibrotic differentiation.

[0098] [Therapeutic Potential of PAK2 Inhibitors in In vivo Fibrosis Models] To investigate the physiological antifibrotic effects of FRAX486 and FRAX597 during the progression of pulmonary fibrosis, a bleomycin (BLM)-induced pulmonary fibrosis mouse model was used to mimic the pathological characteristics of human IPF (see [link to study]. Figure 29 Prior to the experiments, the expression profiles of PAK family members in BLM-induced mouse lungs were validated using publicly available scRNA-seq datasets. Consistent with findings in IPF lungs, increased PAK2 expression was observed in BLM-induced lung fibroblasts (see [link to study]). Figure 30 On the other hand, PAK1 and PAK3 expression was not detected in these fibroblasts. Furthermore, elevated PAK2 phosphorylation levels were confirmed in lung tissue from the BLM-induced fibrosis model, indicating its activation in fibrosis progression (see [link to relevant documentation]). Figure 31 ).

[0099] Based on in vitro experimental results demonstrating superior efficacy of FRAX486 compared to FRAX597, FRAX486 was selected for further in vivo analysis. FRAX486 was administered intraperitoneally on day 7 following the initiation of BLM treatment at the corresponding fibrotic stage, and its therapeutic potential was assessed (see [reference]). Figure 29 ).like Figure 32 As shown, severe weight loss was observed in BLM-treated mice compared to the weight loss observed in control mice. Surprisingly, FRAX486-based treatment led to weight recovery in BLM-induced mice, with levels comparable to those in control mice by day 21.

[0100] Histological analysis confirmed the anti-fibrotic effect of FRAX486. As confirmed by hydroxyproline assay and Masson's trichrome staining, intraperitoneal injection of FRAX486 significantly reduced the incidence of BLM-induced pulmonary fibrosis (see reference). Figure 33 , Figure 34Further quantification using the Aschcroft score revealed a significant reduction in fibrotic lesions (see reference). Figure 35 Immunohistochemical analysis also showed that treatment with FRAX486 significantly inhibited the expression of phosphorylated PAK2 (p-PAK2) in the lungs of BLM-induced mice (see reference). Figure 36 ).

[0101] Based on the above findings, FRAX486 was shown to attenuate PAK2-mediated signaling and alleviate pathological symptoms in the BLM-induced pulmonary fibrosis model, thus demonstrating its potential as a therapeutic agent for IPF.

[0102] Industrial practicality According to the present invention, a therapeutic agent for idiopathic pulmonary fibrosis based on a novel mechanism of action can be provided.

Claims

1. An idiopathic pulmonary fibrosis therapeutic agent containing a PAK inhibitor as an effective ingredient.

2. An idiopathic pulmonary fibrosis therapeutic agent containing a PAK2 inhibitor as an effective ingredient.

3. The idiopathic pulmonary fibrosis therapeutic agent according to claim 1 or 2, which is for a patient having WNT5A+CTHRC1+ myofibroblasts in lung tissue as a suitable subject.

4. An idiopathic pulmonary fibrosis therapeutic composition containing the idiopathic pulmonary fibrosis therapeutic agent according to claim 1 or 2 and a pharmaceutically acceptable carrier.

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