Therapeutic agent for idiopathic pulmonary fibrosis

Abstract

This therapeutic agent for idiopathic pulmonary fibrosis contains a PAK inhibitor as an active ingredient.

Description

Idiopathic pulmonary fibrosis treatment

[0001] The present invention relates to a therapeutic agent for idiopathic pulmonary fibrosis.

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown cause characterized by intractable and progressive pulmonary fibrosis and a poor prognosis with a 5-year survival rate of approximately 20-40%. IPF is caused by excessive production of extracellular matrix by myofibroblasts induced by chronic inflammation and tissue injury, and the formation of immature fibroblast nests in particular is known to be deeply involved in the pathogenesis.

[0003] Sustained aberrant activation of pulmonary myofibroblasts is mediated by various proteins, including transforming growth factors (TGFs), platelet-derived growth factors, and fibroblast growth factors. Sustained aberrant activation of pulmonary myofibroblasts is a key event in the development and progression of IPF. The IPF microenvironment is composed of highly heterogeneous cells that dynamically interact. The constant interaction between fibrotic tissue and its microenvironment plays a key role in the development, progression, and response to various treatments of fibrosis. The area between fibrotic tissue and morphologically normal lung typically contains fibroblasts of various sizes, termed early fibroblast foci (FFs). FFs are the primary pathogenic lesions in IPF and are composed of activated fibroblasts and myofibroblasts, which are the primary effector cells responsible for dysregulated extracellular matrix (ECM) deposition in various fibrotic states. Consistent with the idea that fibrosis spreads from FFs to uninvolved alveoli, these structures are sites of myofibroblast accumulation during pathological ECM deposition. Indeed, a higher number of FFs is associated with worsening symptoms in IPF patients.

[0004] The genomic landscape of IPF has been elucidated to some extent using conventional sequencing strategies, such as bulk whole-exome sequencing and bulk RNA sequencing (RNA-seq) (see, for example, Non-Patent Document 1).

[0005] Peyser R, MacDonnell S, Gao Y, Cheng L, Kim Y, Kaplan T, et al. Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing. Am J Respir Cell Mol Biol. 2019;61(1):74-85.

[0006] However, in silico analysis has only been performed using a mixture of normal tissues and FF components within a single tissue. Single-cell RNA sequencing (scRNA-seq) is a novel tool for analyzing IPF lung heterogeneity at the single-cell level and developing a comprehensive gene expression atlas, but it lacks spatial information from single cells. Spatial information is essential for a comprehensive understanding of IPF progression. Regional tissue heterogeneity is a distinctive pathological feature of human lung fibrosis. Therefore, the development of sequencing technologies that determine cell-type-specific spatial profiles is urgently needed. In this regard, digital spatial transcriptomics approaches provide gene expression information with spatial resolution, helping to gain a general understanding of tissue formation and disease pathogenesis. Therefore, utilizing this methodology can aid in understanding IPF lung fibrosis in various conditions.

[0007] There is currently no fundamental treatment for IPF, and its development is urgently needed. Therefore, an object of the present invention is to provide a therapeutic agent for idiopathic pulmonary fibrosis based on a new mechanism of action by making full use of the above-mentioned methodology.

[0008] The present invention is as follows: [1] A therapeutic agent for idiopathic pulmonary fibrosis, comprising a PAK inhibitor as an active ingredient. [2] A therapeutic agent for idiopathic pulmonary fibrosis, comprising a PAK2 inhibitor as an active ingredient. [3] The therapeutic agent for idiopathic pulmonary fibrosis according to [1] or [2], which is intended for use in patients having WNT5A+ CTHRC1+ myofibroblasts in their lung tissue. [4] A composition for treating idiopathic pulmonary fibrosis, comprising the therapeutic agent for idiopathic pulmonary fibrosis according to [1] or [2] and a pharmaceutically acceptable carrier. [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 in a sample, and / or a probe that binds to the molecule or its amplification product, and an antibody that binds to the molecule. [6] A method for detecting a diagnostic marker for idiopathic pulmonary fibrosis, which comprises detecting the abundance 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 from a sample derived from a subject.

[0009] According to the present invention, it is possible to provide a therapeutic agent for idiopathic pulmonary fibrosis based on a new mechanism of action.

[0010] Experimental workflow for spatial transcriptome analysis and scRNA-seq analysis of human IPF lungs. Nine lung sections from three patients were used for spatial transcriptome analysis, and three IPF lungs and three control lungs were used for scRNA-seq analysis. Spatial visualization of fibroblastic foci (FF) annotation by expression levels of COL1A1, POSTN, and CTHRC1, as indicated in each panel. Representative magnified images of the delimited boxed regions show hematoxylin and eosin (H&E) staining highlighting FF in IPF lung tissue. UMAP visualization of annotated interstitial scRNA-seq datasets from three IPF and three control lung samples, labeled with detailed cell types. Violin plots showing the expression of selected marker genes in each cluster. UMAP visualization of the scRNA-seq dataset from Figure 3, categorized by origin from normal or IPF lungs. Bar graphs comparing the lung interstitial cell type composition between healthy controls and IPF patients. Pseudo-temporal developmental trajectory analysis predicts different stromal cell differentiation trajectories between healthy individuals and IPF patients. Immunohistochemical images of PDGFRA, CTHRC1, and WNT5A in fibroblastic foci in IPF lung tissue. Spatial visualization of FF and advanced fibrotic lesion (DF) annotation along the estimated cell type distribution on a Visium section. Representative zoomed-in image of a bounding box region showing hematoxylin and eosin (H&E) staining highlights FF and DF in IPF lung tissue. 3D PCA mapping results using 97 extracted FF spots and 412 extracted DF spots. Dot plots show the presence of interstitial cell types in FF, DF, and SMC regions selected in Visium by integrating single-cell data and spatial analysis data. Spatial transcriptome spots of FF index (patient JKPF2-1). Bar graphs showing the proportion of high and low FF index populations in interstitial cell types. Figure 1 shows the results of upstream regulator prediction for 746 differentially expressed genes between FF and DF by IPA. The top 10 upstream regulators predicted as "activated" are shown.Violin plots showing the average expression levels of the top 10 upstream regulators detected in Figure 14 in each cell type. UMAP visualization of PAK target gene signatures in stromal populations. UMAP visualization of FF index signatures in stromal populations. Violin plots showing PAK2 expression in high and low FF index stromal cells. Spatial visualization of PAK target gene signatures and predicted cell type distribution along the FF / DF annotation on the Visium section. Dot plots showing the expression of PAK family genes in stromal cells of normal and IPF lung samples. Hematoxylin and eosin (H&E) staining and immunohistochemical staining of PAK1, PAK2, and PAK3 in fibroblastic foci of IPF lung tissue. Results showing the effect of siRNA-mediated PAK1 / 2 knockdown on TGF-β-induced myofibroblast differentiation of lung fibroblasts. The top panel shows a schematic diagram of the protocol used for in vitro experiments. The bottom panel shows representative immunoblot analysis results showing the levels of PAK1, PAK2, type I collagen, and β-actin in PAK1- or PAK2-knockdown lung fibroblasts after 48 hours of TGF-β1 (2 ng / mL) treatment. qPCR results showing WNT5A expression in PAK1- or PAK2-knockdown human lung fibroblasts after 48 hours of TGF-β1 (2 ng / mL) treatment. The top panel shows the effects of the PAK inhibitors FRAX486 and FRAX597 on TGF-β-induced myofibroblast differentiation of lung fibroblasts. The top panel shows a schematic diagram of the protocol used for in vitro experiments. The bottom panel shows representative immunoblot analysis results showing the levels of α-smooth muscle actin (α-SMA), type I collagen, and β-actin in lung fibroblasts treated with FRAX486 (100 nM and 1,000 nM) and FRAX597 (100 nM and 1,000 nM) for 48 hours in the presence of TGF-β1 (2 ng / mL). Pirfenidone (PFD) and nintedanib (NTD) were used as positive controls to inhibit myofibroblast differentiation. Quantitative immunoblot analysis shows the levels of type I collagen and α-SMA.Immunoblot analysis showing the levels of PAK2 and its downstream signaling in TGF-β-induced myofibroblasts treated with FRAX486 (1,000 nM) and FRAX597 (1,000 nM) for 48 hours in the presence of TGF-β1. Results showing the effect of a PAK2 inhibitor on IPF lung fibroblasts. Representative immunoblot analysis showing the levels of α-SMA, type I collagen, and β-actin in IPF lung fibroblasts treated with FRAX486 (1,000 nM) and FRAX597 (1,000 nM) for 48 hours. Protein samples were harvested from cells 48 hours after the start of treatment. Quantitative immunoblot analysis showing the levels of type I collagen and α-SMA in IPF lung fibroblasts. Schematic protocol used for FRAX486 treatment of mice with bleomycin-induced pulmonary fibrosis. Figure 1 shows a violin plot of Pak2 expression between bleomycin-induced and control lung fibroblasts. Figure 2 shows immunoblot analysis results confirming increased PAK2 phosphorylation in lung tissue from a BLM-induced fibrosis model. Figure 3 shows a graph showing changes in body weight observed after bleomycin administration. Body weight on day 0, before treatment, was set at 100. Values ​​represent the mean ± SEM. **P < 0.01. Control: n = 10; Bleomycin control: n = 10; Bleomycin + FRAX486: n = 10. Hydroxyproline quantification in the left lung of each mouse is shown. Each dot represents data from one animal. ***P < 0.001, ****P < 0.0005. Figure 4 shows H&E-stained and Masson's trichrome-stained images of representative lung sections from each group of treated mice. Quantification of fibrosis using the Aschcroft score is shown. Each dot represents data from one animal. ****P < 0.0005. p-PAK2 IHC staining results of representative lung sections from each group of treated mice. Scale bar: 100 μm.

[0011] [Therapeutic Agent for Idiopathic Pulmonary Fibrosis] As described below in the Examples, the inventors have discovered the anti-fibrotic properties of PAK2 inhibitors from the perspective of treating IPF. In one embodiment, the present invention provides a therapeutic agent for idiopathic pulmonary fibrosis comprising a PAK inhibitor as an active ingredient.

[0012] The PAK (p21-activated kinase) family consists of serine / threonine kinases that regulate a variety of cellular activities. To date, six PAK isoforms (PAK1-6) have been identified.

[0013] PAK1 is essential for the growth of solid tumors, and its overactivation or overexpression is known to cause diseases such as cancer, hypertension, diabetes, and Alzheimer's disease.

[0014] An example of the amino acid sequence of PAK1 is the sequence of human PAK1 registered under GenBank accession number AAA65441. An example of the gene sequence of PAK1 is the sequence of human PAK1 registered under GenBank accession number U24152.

[0015] PAK1 inhibitors are substances that inhibit signal transduction by PAK1, and are not limited as long as they inhibit the resulting signal transduction pathway, and examples include nucleic acids, proteins, low molecular weight compounds, etc. Substances that act directly on PAK1 include low molecular weight compounds, proteins, antibodies, aptamers, etc. Substances that suppress the expression of the gene encoding PAK1 include siRNA, antisense oligonucleotides, etc.

[0016] 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-thiazol-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one), and FRAX4 (6-[2-chloro-4-(1,3-thiazol-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one). Examples include 86 (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)-carboxamide), and IPA-3, a small molecule allosteric inhibitor of PAK1.

[0017] PAK2 is widely distributed throughout the body and is a key effector of the Rho family of small GTPases involved in cytoskeletal reorganization. Furthermore, PAK2 regulates various biological processes, including tumorigenesis, cellular senescence, and the promotion of organismal aging. In particular, PAK2 is an activating receptor for TGF-β and a downstream component of AKT. PAK2 activation is essential for the profibrotic effects of TGF-β. Furthermore, PAK2 is a non-canonical SMAD effector of TGF-β in renal interstitial fibrogenesis, and its overexpression induces renal fibrogenesis. Although several studies have demonstrated the relationship between PAK2 and fibrosis in hepatic stellate cells and peritoneal mesothelial cells, the involvement of PAK2 in the pathogenesis of IPF has not been fully investigated.

[0018] An example of the amino acid sequence of PAK2 is the sequence registered under GenBank accession number AAA65442 for human PAK2. An example of the gene sequence of PAK2 is the sequence registered under GenBank accession number U24153 for human PAK2.

[0019] PAK2 inhibitors are substances that inhibit signal transduction by PAK2, and are not limited as long as they inhibit the resulting signal transduction pathway, and examples include nucleic acids, proteins, low molecular weight compounds, etc. Substances that act directly on PAK2 include low molecular weight compounds, proteins, antibodies, aptamers, etc. Substances that suppress the expression of the gene encoding PAK2 include siRNA, antisense oligonucleotides, etc.

[0020] 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-thiazol-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-methylthieno[3,2-d]pyrimidin-4-yl)pyrimidin-4-yl). )amino)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide), IPA-3 (1,1'-dithiodi-2-naphthol), AG-1478 (N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine), 2-aminopyrido[2,3-d]pyrimidin-7(8H)-one, 1H-thieno[3,2-c]pyrazole, 3-amino-tetrahydropyrrole[3,4-c]pyrazole, N4-(1H-pyrazol-3-yl)pyrimidine-2,4-diamine, N2-bicyclic indolyl, indazolyl, and benzimidazolyl derivatives of N4-(1H-pyrazol-3-yl)pyrimidine-2,4-diamine, and the like.

[0021] The PAK inhibitor used in the therapeutic agent for idiopathic pulmonary fibrosis of this embodiment may be a pan-PAK inhibitor, but is preferably a PAK2 selective inhibitor.

[0022] As the therapeutic agent for idiopathic pulmonary fibrosis of this embodiment, the PAK inhibitor may be used in the form of a free form or a pharmaceutically acceptable salt, or may be used in the form of a solvate of the free form or a solvate of the salt.

[0023] The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfonate, 2-hydroxyethanesulfonate, p-toluenesulfonate, acetate, propanoate, oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate, fumarate, malate, mandelate, etc. The solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate, and examples thereof include hydrates, organic solvents, etc.

[0024] As described later in the Examples, PAK2 is highly expressed in WNT5A+ CTHRC1+ myofibroblasts, and therefore, the therapeutic agent for idiopathic pulmonary fibrosis of this embodiment is preferably applied to patients who have WNT5A+ CTHRC1+ myofibroblasts in their lung tissue.

[0025] [Composition for treating idiopathic pulmonary fibrosis] In one embodiment, the present invention provides a composition for treating idiopathic pulmonary fibrosis, comprising the above-mentioned therapeutic agent for idiopathic pulmonary fibrosis and a pharmaceutically acceptable carrier.

[0026] The composition for treating idiopathic pulmonary fibrosis of this embodiment can be administered orally in the form of, for example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or parenterally in the form of inhalants, injections, suppositories, topical skin preparations, etc.

[0027] As the pharmaceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, etc.; solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. The pharmaceutically acceptable carriers can be used alone or in combination of two or more.

[0028] The composition for treating idiopathic pulmonary fibrosis of this embodiment may further contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and rhizome oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc. The additives can be used alone or in combination of two or more.

[0029] (Administration Method) The administration method of the therapeutic agent for idiopathic pulmonary fibrosis or the therapeutic composition for idiopathic pulmonary fibrosis is not particularly limited and may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. are administered orally. Injections are administered intravenously alone or mixed with common replacement fluids such as glucose and amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories are administered rectally. Topical skin preparations are applied, pasted, or sprayed onto the affected area. Inhalants are administered using devices such as nebulizers, metered-dose inhalers, and dry powder inhalers.

[0030] (Dosage) The dosage of the therapeutic agent for idiopathic pulmonary fibrosis or the therapeutic composition for idiopathic pulmonary fibrosis varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined in general. However, in the case of oral administration, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of injections, for example, 0.1 μg to 1 g of the active ingredient may be administered per day, for example, 0.001 to 200 mg per day. In the case of suppositories, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of topical skin preparations, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of inhalants, for example, 1 μg to 10 g of active ingredient may be administered in a single inhalation, for example, 0.01 to 2000 mg in a single inhalation.

[0031] Other Embodiments In one embodiment, the present invention provides a PAK inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof for the treatment of idiopathic pulmonary fibrosis.

[0032] In one embodiment, the present invention provides a method for treating idiopathic pulmonary fibrosis, comprising administering to a patient in need thereof an effective amount of a PAK inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0033] In one embodiment, the present invention provides use of a PAK inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof for producing a therapeutic agent for idiopathic pulmonary fibrosis or a composition for treating idiopathic pulmonary fibrosis.

[0034] [Other Embodiments] In one embodiment, the present invention provides a therapeutic agent for idiopathic pulmonary fibrosis, comprising as an active ingredient at least one inhibitor selected from the group consisting of a PRKAA2 inhibitor, a TWF1 inhibitor, a PRKAA1 inhibitor, a DDR2 inhibitor, a PAK2 inhibitor, an ABL2 inhibitor, a DYRK1B inhibitor, an FGFR4 inhibitor, a CDK19 inhibitor, and an ERBB4 inhibitor.

[0035] [Diagnostic agent for diagnostic markers for idiopathic pulmonary fibrosis] 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 that binds to the molecule or its amplification product, and an antibody that binds to the molecule.

[0036] Molecules to be detected by diagnostic agents include nucleic acids such as DNA, RNA, and miRNA, and proteins. When the target molecules are nucleic acids, the diagnostic agent preferably contains a primer set for amplifying these molecules and / or a probe that binds to the molecules or their amplification products. When the target molecules are proteins, the diagnostic agent preferably contains an antibody that binds to these molecules.

[0037] In this embodiment, the sample may be cells derived from a tissue fragment, blood, urine, saliva, sweat, tissue exudate, or the like.

[0038] [Method for detecting a diagnostic marker for idiopathic pulmonary fibrosis] In one embodiment, the present invention provides a method for detecting a diagnostic marker for idiopathic pulmonary fibrosis, which comprises detecting the abundance 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 from a sample derived from a subject.

[0039] The molecule to be detected is 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 it is preferable to detect all of the molecules POSTN, THBS2, MMP11, CTHRC1, COL5A2, MMP14, MXRA5, ENC1, COL12A1, MMP7, COL8A1, ASPN, TGFBI, COL5A1, SPON2, COL7A1, ADAM12, CREB3L1, PMEPA1, and VCAN. As described below in the Examples, the average expression levels of these genes can be used as the FF index to evaluate the sample.

[0040] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0041] Experimental Example: Spatial Transcriptome Analysis of Human IPF Lung Tissues. Using the experimental flowchart shown in Figure 1, we performed spatial transcriptome profiling of lung tissues (a total of nine sections) from three IPF patients. Furthermore, we performed scRNA-seq analysis of lung tissues from three IPF patients and three healthy controls. As the first step in spatial transcriptome analysis of IPF lungs, we applied a standardized procedure for normalization and visualization using UMAP (Uniform Manifold Approximation and Projection) of each section. In IPF lungs, FFs contain activated fibroblasts and myofibroblasts and are considered the primary pathogenic lesion of IPF. To characterize the cellular and molecular features of FFs, we histologically identified them in H&E images (see Figure 2), and Visium spots overlapping these regions were designated as FF spots. The accuracy of these FF spot selections was further verified by examining the spatial distribution of known FF markers, such as COL1A1, POSTN, and CTHRC1, on each Visium section.

[0042] Detailed annotation of scRNA-seq data and integration with spatial Visium spots. To further explore the cell type composition within fibrotic regions of IPF, we refined the annotation of stromal cell types using key markers involved in IPF pathogenesis. UMAP clustering identified nine distinct clusters, including a population of CTHRC1+ fibroblasts, known to play an important role in IPF pathogenesis (Figures 3 and 4). In particular, we identified an intermediate cell state bridging the fibroblast and smooth muscle cell (SMC) / pericyte clusters, which we termed WNT5A+ CTHRC1+ myofibroblasts. These cells exhibited fibroblast-like characteristics, expressing PDGFRA, PDGFRB, and RUNX1, lacking DES expression, and possessing unique markers such as WNT5A and WIF1 (Figure 4). The existence of this WNT5A+ CTHRC1+ myofibroblast population was verified by analyzing publicly available single-cell datasets (GSE136831, GSE135893) derived from IPF patients. These WNT5A+ CTHRC1+ cells were detected within myofibroblast clusters, which were mapped between fibroblast and SMC clusters on the UMAP. Furthermore, the proportions of myofibroblasts, CTHRC1+ fibroblasts, and WNT5A+ CTHRC1+ myofibroblasts were significantly increased in IPF patients compared with controls (Figures 5 and 6). Pseudotime trajectory analysis revealed fibroblast differentiation pathways characteristic of IPF compared with controls. In healthy lung tissue, alveolar fibroblasts primarily differentiate into myofibroblasts. However, in IPF, alveolar fibroblasts follow two distinct trajectories: differentiation into myofibroblasts or pathogenic CTHRC1+ fibroblasts. The latter population further acquires smooth muscle-like characteristics, including increased expression of TAGLN and ACTA2, and transitions into WNT5A+ CTHRC1+ myofibroblasts (Figure 7). Immunohistochemistry of human IPF lung tissue confirmed the presence of WNT5A+ CTHRC1+ PDGFRA+ fibroblasts in the FF and peripheral DF regions of IPF lung tissue (Figure 8).

[0043] Molecular and Cellular Differences between FF and DF in Human IPF Lungs IPF lung tissue exhibits heterogeneous patterns of fibrosis, as well as DF regions, representing fibrotic remodeling, where mature scar tissue disrupts normal lung architecture and impairs gas exchange. Comparing FF and DF is important for understanding the dynamic progression of fibrosis and identifying therapeutic targets for both active fibrosis and its transition to fibrotic remodeling. DF regions surrounding FF were morphologically identified, characterized by a predominance of fibroblasts and the absence of SMCs and pericytes. Visium spots corresponding to DF regions were manually curated using scRNA-seq deconvolution data, excluding regions with significant contributions from SMCs or pericytes (see Figure 9). As a result, 97 FF spots and 412 DF spots were identified across nine sections. These spots were plotted on a principal component analysis (PCA) map (see Figure 10), revealing distinct transcriptome profile differences between FF and DF. To elucidate differences in cell type composition, we deconvolved the interstitial scRNA-seq data with Visium data for FF and DF spots, revealing distinct patterns of cellular distribution in FF and DF. While FF regions were primarily populated by CTHRC1+ fibroblasts, proliferative fibroblasts, and aberrant basaloids, DF regions, unlike SMC / pericyte regions, contained a higher proportion of WNT5A+ CTHRC1+ myofibroblasts, further supporting the idea that the transition to WNT5A+ CTHRC1+ myofibroblasts may be partially involved in the mechanism of FF-DF transition (see Figure 11). Aberrant basaloids showed high levels of cell-cell interaction with both WNT5A+ CTHRC1+ myofibroblasts and CTHRC1+ fibroblasts, suggesting the existence of a pro-fibrotic loop that promotes fibrosis progression. To molecularly detect fibroblast populations within FF, we developed an FF index based on markers identified by spatial analysis and subsequently deconvolved it with scRNA-seq data. Although fibroblasts with a high FF index were also detected in control lung samples, the amount of fibroblasts with a high FF index was higher in IPF samples.Spatially, the FF index effectively visualized fibroblast foci in IPF lung sections (see Figure 12). Fibroblasts with a high FF index were mainly composed of pathogenic fibroblasts, such as CTHRC1+ fibroblasts, proliferative fibroblasts, and WNT5A+ CTHRC1+ myofibroblasts, whereas fibroblasts with a low FF index were alveolar fibroblasts (see Figure 13). These findings suggest that fibroblasts with a high FF index may serve as therapeutic targets for IPF.

[0044] Identification of PAK2 Activation in Fibroblast Foci and Surrounding DF Areas and Its Therapeutic Potential in IPF: To identify novel therapeutic targets for IPF, we analyzed differentially expressed genes between FF and DF. Ingenuity Pathway Analysis (IPA) results clearly demonstrated an activated "pulmonary fibrosis idiopathic signaling pathway" in FF spots of IPF lungs. Prediction of upstream regulators revealed activation of several kinases in FF spots of IPF lungs (Figure 14). Among these kinases, PRKAA2, the catalytic subunit of AMPK, was considered as a candidate because of its potential role in regulating TGF-β-induced myofibroblast differentiation. As part of our search for novel therapeutic targets, we focused on PAK2 because it is highly expressed in pathogenic CTHRC1+ fibroblasts and WNT5A+ CTHRC1+ myofibroblasts (Figure 15) and because small molecule inhibitors targeting PAK2 are available.

[0045] To further investigate PAK2 activity at the single-cell level, we established a PAK target gene set based on previously published RNA-seq data. The average expression level of the PAK target signature in each single cell was calculated using the module score algorithm and visualized using UMAP (Figure 16). Interestingly, the distribution of cells expressing a high PAK target signature closely resembled that of cells with a high FF index (Figure 17), suggesting that PAK target genes are primarily expressed in profibrotic fibroblasts. Furthermore, PAK2 expression was significantly higher in fibroblasts with a high FF index than in fibroblasts with a low FF index (Figure 18). Spatial distribution analysis confirmed that PAK target gene expression was concentrated around FF and DF regions, closely matching the distribution of CTHRC1+ fibroblasts and WNT5A+ CTHRC1+ myofibroblasts, respectively (Figure 19). Among the PAK family, PAK2 showed the highest expression in interstitial cells compared with PAK1 and PAK3, and was significantly enriched in IPF lung tissue at both the transcriptome and protein levels (Fig. 20 and Fig. 21). These findings suggest that PAK2 may be a potential therapeutic target for IPF by regulating the activity of pathogenic fibroblasts.

[0046] The Role of PAK2 in TGF-β-Induced Profibrotic Differentiation of Human Lung Fibroblasts and Its Inhibition as a Therapeutic Target: To elucidate the role of PAK1 and PAK2 in TGF-β-induced fibrotic properties, we performed siRNA-mediated knockdown of PAK1 and PAK2 in freshly isolated primary human lung fibroblasts. Knockdown of both PAK1 and PAK2 effectively reduced TGF-β-induced ECM production and WNT5A expression (Figures 22 and 23). This was consistent with the results of spatial transcriptome analysis. To characterize the pharmacological antifibrotic properties of targeting PAK2 for the treatment of IPF, we selected the PAK2 inhibitors FRAX486 and FRAX597 and applied them to a TGF-β-induced myofibroblast differentiation model of lung fibroblasts (Figure 24). FRAX486 and FRAX597 inhibit multiple PAK family members, including PAK1, PAK2, and PAK3. However, given the expression pattern of PAK2 in IPF lung tissue, their primary effect may be mediated through PAK2 (Figure 20). Treatment with either FRAX486 or FRAX597 strongly suppressed TGF-β-induced myofibroblast differentiation, as measured by α-smooth muscle actin (α-SMA) and type I collagen protein levels (Figure 24). These inhibitory effects were dose-dependent (Figure 25) and significantly greater than those of pirfenidone (PFD) and nintedanib (NTD), two antifibrotic drugs commonly used to treat IPF. Immunofluorescent labeling of α-SMA and type I collagen demonstrated the inhibitory effects of FRAX486 and FRAX597 on TGF-β-induced myofibroblast differentiation. Western blotting analysis of the expression levels of downstream proteins of PAK2 (see Figure 26) revealed that both inhibitors suppressed the expression and phosphorylation of PAK2, c-Abl, and LIMK1, but not SMAD2 / 3. Notably, FRAX486 potently inhibited TGF-β-induced myofibroblast differentiation of lung fibroblasts. Transcriptional inhibition of downstream factors during TGF-β-induced myofibroblast differentiation was confirmed by qPCR.Next, we evaluated the anti-fibrotic effects of FRAX486 and FRAX597 on lung fibroblasts isolated from IPF lungs. Treatment with these inhibitors significantly reduced the expression of α-SMA and type I collagen in IPF-derived fibroblasts (Figures 27 and 28). Consistent with the TGF-β-induced myofibroblast differentiation model, FRAX486 and FRAX597 significantly inhibited the expression of PAK2 downstream proteins in IPF-derived fibroblasts. Furthermore, RT-PCR analysis confirmed the transcriptional inhibition of PAK2 downstream targets in IPF fibroblasts. These results suggest that the anti-fibrotic effects of FRAX486 and FRAX597 are mediated through the suppression of pro-fibrotic differentiation in both the TGF-β-induced model and IPF-derived lung fibroblasts.

[0047] Therapeutic Potential of PAK2 Inhibitors in an In Vivo Fibrosis Model To investigate the physiological anti-fibrotic effects of FRAX486 and FRAX597 during the progression of pulmonary fibrosis, we used a bleomycin (BLM)-induced pulmonary fibrosis mouse model, which mimics the pathological characteristics of human IPF (see Figure 29). Prior to initiating the experiment, we verified the expression profile of PAK family members in BLM-induced mouse lungs using publicly available scRNA-seq datasets. Consistent with the findings in IPF lungs, we observed elevated PAK2 expression in fibroblasts from BLM-induced lungs (see Figure 30). Meanwhile, expression of PAK1 and PAK3 was undetectable in these fibroblasts. Furthermore, we confirmed increased PAK2 phosphorylation in lung tissue from the BLM-induced fibrosis model, indicating its activation during fibrosis progression (see Figure 31). Based on the in vitro results showing superior efficacy of FRAX486 compared with FRAX597, we selected FRAX486 for further in vivo analysis. FRAX486 was administered intraperitoneally on day 7 after BLM treatment, corresponding to the onset of fibrosis, to evaluate its therapeutic potential (see Figure 29). As shown in Figure 32, severe weight loss was observed in BLM-treated mice compared to that observed in control mice. Surprisingly, treatment with FRAX486 restored body weight in BLM-induced mice, with body weight levels at day 21 comparable to those in control mice. Histological analysis confirmed the anti-fibrotic effect of FRAX486. As confirmed by the hydroxyproline assay and Masson's trichrome staining results, intraperitoneal injection of FRAX486 significantly attenuated the development of BLM-induced pulmonary fibrosis (see Figures 33 and 34). Further quantification using the Aschcroft score demonstrated a significant reduction in fibrotic lesions (see Figure 35). Immunohistochemical analysis also demonstrated that FRAX486 treatment significantly inhibited the expression of phosphorylated PAK2 (p-PAK2) in the lungs of BLM-induced mice (Figure 36). Collectively, these findings demonstrate that FRAX486 attenuates PAK2-mediated signaling and alleviates pathological symptoms in a BLM-induced pulmonary fibrosis model, supporting its potential as a therapeutic agent for IPF.

[0048] According to the present invention, it is possible to provide a therapeutic agent for idiopathic pulmonary fibrosis based on a new mechanism of action.

Claims

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

2. A therapeutic agent for idiopathic pulmonary fibrosis, containing a PAK2 inhibitor as an active ingredient.

3. The therapeutic agent for idiopathic pulmonary fibrosis according to claim 1 or 2, which is applicable to patients having WNT5A+ CTHRC1+ myofibroblasts in lung tissue.

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

Images