Extracellular matrix modulating agent

Abstract

To provide an agent for modulating expression of extracellular matrix by corneal endothelial cells.SOLUTION: The present invention pertains to an agent for modulating expression of extracellular matrix by corneal endothelial cells, containing 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof, or a solvate thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for regulating the expression of an extracellular matrix by a corneal endothelial cell. More specifically, the present invention relates to an agent for regulating the expression of an extracellular matrix by a corneal endothelial cell, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof, or a solvate thereof as an active ingredient. [Background technology]

[0002] The cornea is an important tissue that not only constitutes the wall of the eyeball together with the sclera, but also acts as an entrance for taking in external images into the eye because it is a transparent tissue. The thickness of the cornea is approximately 500 μm at the center, and it consists of five layers, starting from the outside: the corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, and corneal endothelium.

[0003] Corneal endothelial cells produce various extracellular matrix components to form a specialized basement membrane, Descemet's membrane. Descemet's membrane is composed of two layers: an anterior striation layer (fetal layer) and a posterior nonstriation layer (postnatal layer) that grows continuously and thickens throughout life due to the secretory activity of endothelial cells. Normal Descemet's membrane contains collagen types VIII, IV (chains α1-α2), and fibronectin on the stromal side, and entactin, laminin, perlecan, and collagen type IV (chains α3-α6) on the endothelial side. Virtually all corneal endothelial disorders, such as pseudophakic bullous keratopathy, are associated with abnormal extracellular matrix accumulation. In these conditions, corneal endothelial cells undergo stress-induced transdifferentiation into myofibroblasts, which produce excessive amounts of collagen (mostly type I collagen), resulting in the formation of an abnormal posterior fibrillar layer on the surface of Descemet's membrane. Such a posterior fibrotic layer is thought to be the result of a common final pathway in endothelial dysfunction (see Non-Patent Document 1).

[0004] Alterations in the matrix composition of Descemet's membrane, together with the formation of wart-like protrusions, so-called guttae, are also characteristic early clinical features of Fuchs endothelial corneal dystrophy (FECD) (Figure 1). These matrix changes lead to loss of contrast sensitivity and increased glare due to light scattering in affected patients (see Non-Patent Document 2), thereby severely affecting the patient's quality of vision. Corneal endothelial cell dysfunction further leads to corneal edema, loss of corneal transparency, and irreversible blindness. FECD is a slowly progressive bilateral corneal disease that becomes clinically evident in adults over the age of 40. In 2015, FECD accounted for 39% of corneal grafts performed worldwide. Currently, there is no treatment for FECD other than corneal transplantation.

[0005] A recent publication showed that FECD is characterized by specific changes in the expression pattern of matrix proteins that are different from those of patients with pseudophakic bullous keratopathy and normal subjects (see Non-Patent Document 3). In FECD specimens, several matrix proteins were identified to be selectively upregulated at both the mRNA and protein levels, including collagen types I, III, and XVI, fibronectin, agrin, laminin α1, TGFBI, and clusterin. Immunohistochemical examination confirmed that these matrix proteins, especially collagen type III, are involved in Descemet's membrane thickening and guttai formation (Figure 2). Abnormal matrix deposition in FECD may be induced by elevated levels of TGF-β2 in the aqueous humor of FECD patients. Upregulation of collagen type VI, laminin α3, and fibronectin was recently confirmed in cultured endothelial cells from FECD patients (see Non-Patent Document 4).

[0006] Since Descemet's membrane thickening and Guttei formation are known to be the first clinical signs of FECD, inhibiting abnormal matrix deposition can slow the progression of the disease and ultimately avoid corneal transplantation.The inventors hypothesized that stress-induced or TGFβ-induced Rho kinase (Rho-associated, coiled-coil-containing protein kinase: ROCK) signaling activation is involved in fibrotic response and abnormal matrix production by corneal endothelial cells, and that ROCK inhibition can attenuate, prevent, or even reverse these changes, thereby normalizing visual function in FECD patients.

[0007] Rho is a small GTPase that, when activated by guanine nucleotide exchange factors, activates ROCK, which phosphorylates a variety of substrates, including myosin light chain and LIM kinase. ROCK signaling is activated by wounding, integrin stimulation, cytokines and growth factors, and controls a wide range of fundamental cellular events, including adhesion, migration, proliferation, differentiation and apoptosis. These processes are primarily mediated by regulation of the cytoskeleton.

[0008] A large body of evidence has been accumulating regarding the involvement of the Rho-ROCK signaling pathway in the development of fibrotic lesions in multiple organ systems, including the kidney and lung (see Non-Patent Documents 5-8). ROCK signaling activation appears to be involved in the profibrotic response of epithelial, endothelial and mesenchymal cells in response to stress or injury, thereby driving the transition of fibroblasts into myofibroblasts that produce altered collagen-rich extracellular matrix. Consistently, ROCK inhibitors have been used to suppress or prevent fibrosis in animal models, and more importantly, induce recovery from already established fibrosis (see Non-Patent Document 6). These anti-fibrotic effects may be mediated by the prevention of myofibroblast transformation and excessive matrix production induced by TGFβ (see Non-Patent Documents 9-12). Fasudil, a small molecule inhibitor of ROCK, has been shown to have antifibrotic effects in various fibrotic diseases, for example, fasudil attenuated the expression of α-SMA, MLCP, LIMK1, p-cofilin, type I collagen, and type III collagen proteins in human fibroblasts (see Non-Patent Document 12). Thus, a body of evidence indicates that ROCK inhibition has great potential as a powerful therapeutic tool in the treatment of fibrosis.

[0009] The transforming growth factor-β (TGF-β) signaling pathway is a key mediator of fibroblast activation that drives abnormal synthesis of extracellular matrix in fibrotic diseases. Moreover, activation of canonical Wnt signaling is required for TGF-β-mediated fibrosis, highlighting the important role of the interaction of both pathways in the pathogenesis of fibrotic diseases (see Non-Patent Document 13). Wnt signaling and TGF-β signaling have also been suggested to activate endothelial-mesenchymal transition (EMT).

[0010] Previous studies have shown that inhibition of ROCK signaling using Y-27632, H-1152 and thiazovivin stimulates cell adhesion, migration, proliferation and wound healing, and suppresses corneal endothelial cell apoptosis and endothelial-mesenchymal transition (EMT) in in vitro and in vivo animal models (see Non-Patent Documents 14-25). Similar findings have been reported for K-115, stimulating endothelial cell proliferation and wound healing, upregulating functional endothelial markers and downregulating EMT markers (see Non-Patent Documents 26 and 27). These studies provided evidence for the usefulness of ROCK inhibitors as adjuvant drugs in cell injection therapy for corneal endothelial cell cultivation (see Non-Patent Document 28) and as eye drops to rescue failed Descemetorhexis (Descemet's membrane removal) strategies without corneal donor transplantation, the so-called DWEK (Descemet's membrane stripping without endothelial keratoplasty) or DSO (Descemet's membrane stripping only) procedures for FECD (see Non-Patent Document 29). [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Ljubimov A. et al., Invest. Ophthalmol. Vis. Sci., 1996, 37, 997-1007 [Non-Patent Document 2] Watanabe S. et al., Ophthalmology, 2015, 122(10), 2013-2019 [Non-Patent Document 3] Weller J. et al., Invest. Ophthalmol. Vis. Sci., 2014, 55, 3700-3708 [Non-Patent Document 4] Goyer B. et al., Tissue Engineering, 2018, 24(7&8), 607-615

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[0012] The present invention relates to an agent that regulates the expression of extracellular matrix by corneal endothelial cells, and an agent that suppresses guttate formation in FECD patients. [Means for solving the problem]

[0013] As a result of intensive testing carried out by the present inventors to solve the above problems, it has been found that 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or its salts or solvates thereof can suppress the expression of extracellular matrices such as agrin, type I and type III collagen, and fibronectin by corneal endothelial cells, thereby providing a medicament for treating corneal diseases characterized by abnormal matrix production. Furthermore, 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or its salts or solvates thereof can regulate the TGF-β signaling pathway and the transformation of myofibroblasts, providing a medicament for treating fibrosis in patients with early FECD. Furthermore, it has been found that the substance can also be instilled into the eye, thereby enabling a formulation that imposes only a slight burden on the patient.

[0014] In other words, the present invention relates to an agent for regulating the expression of extracellular matrix by corneal endothelial cells, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof. In addition, the present invention relates to an agent for preventing and / or suppressing guttachiosis, which contains 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof. Furthermore, the present invention relates to a drug for preventing and / or suppressing the recurrence of Guttee formation, which contains 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof.

[0015] A more detailed description of the invention follows. (1) An agent for regulating the expression of extracellular matrix by corneal endothelial cells, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof. (2) The agent for regulating the expression of extracellular matrix by corneal endothelial cells according to (1) above, wherein 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine is (S)-(-)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine. (3) A drug for regulating the expression of extracellular matrix by corneal endothelial cells of (1) or (2) above, which is a drug for preventing and / or suppressing guttachia formation. (4) A drug for preventing and / or suppressing guttae formation in FECD patients, which is the drug for preventing and / or suppressing guttae formation of the above-mentioned (3). (5) A drug for preventing and / or suppressing guttata formation in FECD patients as described in (4) above, which is a drug for preventing and / or suppressing guttata formation in early FECD patients. (6) A drug for regulating the expression of extracellular matrix by corneal endothelial cells of (1) or (2) above, which is a drug for preventing and / or suppressing the recurrence of Guttieri formation. (7) A drug for preventing and / or suppressing the recurrence of guttae formation in FECD patients, which is the drug for preventing and / or suppressing the recurrence of guttae formation of the above-mentioned (6). (8) A drug for preventing and / or suppressing the recurrence of Gutti formation in FECD patients as described in (7) above, which is a drug for preventing and / or suppressing the recurrence of Gutti formation in FECD patients after Descemet's membrane peeling without corneal donor transplantation (DWEK or DSO). (9) Any one of the above-mentioned (1) to (8) drugs, which is a liquid formulation. (10) A drug for regulating the expression of extracellular matrix by corneal endothelial cells of (1) or (2) above, which is a drug for preventing and / or treating corneal endothelial disorders. (11) The drug for regulating the expression of an extracellular matrix by a corneal endothelial cell according to (10) above, wherein the corneal endothelial disorder is a corneal endothelial disease such as bullous keratopathy or corneal endotheliitis. Agent. (12) A drug for regulating the expression of an extracellular matrix by corneal endothelial cells according to any one of the above (1) to (11), which is an eye drop. (13) A method for preparing a pharmaceutical formulation for regulating the expression of an extracellular matrix by a corneal endothelial cell according to any one of (1) to (12) above, the method comprising mixing 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof with a pharma- ceutically acceptable carrier. (14) A drug for regulating the TGF-β signaling pathway and myofibroblast transformation in patients with early FECD, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof, or a solvate thereof. (15) A drug for regulating the TGF-β signaling pathway in a patient with early FECD, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof, or a solvate thereof. (16) A drug for regulating myofibroblast transformation in a patient with early FECD, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, or a salt thereof, or a solvate thereof. (17) Any one of the above (14) to (16) for treating fibrosis in patients with early FECD. (18) Any one of the drugs (14) to (17) above, which is a liquid formulation. Effect of the Invention

[0016] The present invention provides an agent for regulating the expression of an extracellular matrix by corneal endothelial cells in order to prevent and / or treat corneal disorders caused by abnormalities in the expression of an extracellular matrix by corneal endothelial cells. The agent for regulating the expression of an extracellular matrix by corneal endothelial cells of the present invention can prevent and / or treat various types of corneal endothelial disorders, for example, corneal endothelial diseases such as bullous keratopathy or corneal endotheliitis, or corneal endothelial abnormalities caused by corneal transplantation, etc. Furthermore, the agent for regulating the expression of an extracellular matrix by corneal endothelial cells of the present invention is effective even when the active ingredient is at a low concentration, and therefore the agent for regulating the expression of an extracellular matrix by corneal endothelial cells of the present invention can be used as a highly effective and safe pharmaceutical composition with almost no side effects. Furthermore, the agent for regulating the expression of an extracellular matrix by corneal endothelial cells of the present invention can provide an eye drop that imposes only a slight burden on patients. [Brief description of the drawings]

[0017] [Figure 1] Schematic representation of the structure of Descemet's membrane in a healthy control patient and a patient with FECD. [Diagram 2] 1 shows immunohistochemical staining of type III collagen in Descemet's membrane and Guttei's membrane of a patient with FECD. [Diagram 3] Relative expression of FECD-associated matrix genes in corneal epithelial cells from normal donors (n=6) with or without incubation with 10-100 μM K-115 for 24 h. Gene expression was analyzed by quantitative real-time PCR and normalized to GAPDH (*p<0.01). [Figure 4] Relative expression of FECD-associated matrix genes by corneal endothelial cells from FECD patients (n=20) with or without 24-hour incubation with 30 μM K-115. Gene expression was analyzed by quantitative real-time PCR and normalized to GAPDH (*p<0.01, **p<0.001). [Diagram 5] Western blot analysis of fibronectin in corneal epithelial cells from FECD patients (n=4); equal sample loading was confirmed by β-actin. Densitometric analysis of band intensity represents the mean ± SD of four independent experiments (*p<0.01). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The invention is described in more detail below. 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine in the present invention has one asymmetric carbon atom, which leads to (R) and (S) isomers. In the present invention, both (R) and (S) isomers and their mixtures can be used. As the pharmaceutical active ingredient, high purity optically active material of (R) or (S) isomer is preferred. In terms of desired activity, (S) isomer is more preferred than (R) isomer.

[0019] The active ingredient of the present invention, 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, is known as a compound having a Rho kinase inhibitory effect, and can be prepared by a known method, for example, the method disclosed in WO99 / 20620A1. The (S) isomer of 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine is also known as K-115 or Ripasudil, which is manufactured and sold as a therapeutic agent for glaucoma and ocular hypertension. In Japan, Glanatec (trade name; 0.4% Ripasudil hydrochloride hydrate) eye drop is clinically available as an eye drop preparation.

[0020] Examples of salts of 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine include salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, and hydrobromic acid, and salts formed with organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, and camphorsulfonic acid. Particularly preferred is the hydrochloride salt. 1-(4-Fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof may exist not only in unsolvated form but also as a hydrate or solvate. Although the hydrate is preferred, the present invention includes all crystalline forms and hydrates or solvates.

[0021] As a Rho kinase inhibitor, AR-13324 (chemical name: 4-[(2S)-3-amino-1-(6-isoquinolinylamino)-1-oxo-2-propanyl]benzyl 2,4-dimethylbenzoate; Netarsudil (trade name); Rhopressor (trademark)) can also be used in the present invention.

[0022] In the present invention, the "agent for regulating the expression of extracellular matrix by corneal endothelial cells" refers to an agent that regulates the expression of extracellular matrix by corneal endothelial cells so as to suppress an increase in the function of corneal endothelial cells in a pathological condition such as FECD.

[0023] Since guttch formation in patients with early or late stage FECD mainly depends on increased expression of extracellular matrix elements, such as agrin, types I and III collagen, and fibronectin, in corneal endothelial cells, it is believed that the "agent for regulating the expression of extracellular matrix elements by corneal endothelial cells" of the present invention can prevent or suppress the progression of FECD.

[0024] By administering the "agent for regulating the expression of extracellular matrix by corneal endothelial cells" of the present invention, it becomes possible to regulate the function of the corneal endothelium, and therefore the "agent for regulating the expression of extracellular matrix by corneal endothelial cells" of the present invention can also be used as an agent for preventing or treating corneal edema and / or an agent for preventing or treating damage to the corneal endothelium.

[0025] The "regulating expression of extracellular matrix by corneal endothelial cells" of the present invention is formulated into a dosage form suitable for local administration to the eye using a common formulation technique frequently used in the art. As the dosage form, for example, but not limited to, a liquid formulation such as anterior chamber injection, ocular irrigation, or eye drops is preferred. As a preferred formulation, anterior chamber injection or ocular irrigation is a preferred dosage form from the viewpoint of therapeutic effect, but these impose a significant burden on patients. Therefore, eye drops are included in the preferred formulation from the viewpoint of ease of administration.

[0026] Preparation of eye drops can be realized, for example, by dissolving or suspending the desired above-mentioned components in an aqueous solvent such as sterilized pure water or physiological saline, or in a non-aqueous solvent such as vegetable oil including cottonseed oil, soybean oil, sesame oil or peanut oil, adjusting the pressure of the solution or suspension to a predetermined osmotic pressure, and carrying out sterilization such as filtration sterilization. It should be noted that when preparing eye ointments, an ointment base can be included in addition to the above-mentioned various components. The ointment base is preferably, but not limited to, an oil base such as petrolatum, liquid paraffin or polyethylene; an emulsion base in which an oil phase and an aqueous phase are emulsified by a surfactant; a water-soluble base consisting of hydroxypropylmethylcellulose, carboxymethylcellulose, polyethylene glycol, etc.

[0027] When 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, preferably (S)-(−)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof, or a solvate thereof is used for “regulating the expression of extracellular matrix by corneal endothelial cells” of the present invention, the dosage depends on the body weight, age, sex, and symptoms of the patient, the dosage form, the number of doses, etc., but generally, 1- The dosage of (4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, preferably (S)-(-)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine for an adult is in the range of 0.025 to 10,000 μg, preferably 0.025 to 2,000 μg, more preferably 0.1 to 2,000 μg, still more preferably 0.025 to 200 μg, or 0.025 to 100 μg per day. When 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, preferably (S)-(-)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine is used as an eye drop, the concentration of the active ingredient may be approximately 0.0001 to 5 w / v%, preferably approximately 0.01 to 4 w / v%.

[0028] Furthermore, the number of doses is not particularly limited, but it is preferable to administer the drug once or several times. In the case of a liquid eye drop, one to several drops can be instilled into the eye in one administration. EXAMPLES

[0029] Endothelium-Descemet's membrane (EDM)-complexes were prepared from normal donor corneas and from FECD patients undergoing DMEK surgery. EDM scrolls were bisected and incubated for 24–72 h in CorneaMax storage medium (Eurobio) with or without K-115 (Selleck Chemicals). Gene expression analysis was performed using specific real-time PCR arrays for FECD-related candidate genes as well as RT2 Profiler PCR Arrays (Quiagen). Array results were again validated using gene-specific real-time PCR assays. Protein expression analysis was performed by Western blotting. First, the expression levels of matrix genes pathologically upregulated in endothelial cells from FECD patients were analyzed in normal EDM specimens using various concentrations of K-115. It was found that the majority of matrix proteins (agrin, type I and III collagen, fibronectin) as well as α-smooth muscle actin (α-SMA) and β-actin were significantly downregulated, and that a concentration of 30 μM K-115 was the most effective (Figure 3). Gene expression levels were then analyzed in FECD specimens incubated in 30 μM K-115 for 24 hours. Upon K-115 treatment, a significant decrease in the expression levels of agrin, collagen types I and III, fibronectin, as well as α-SMA was observed compared to untreated controls (Figure 4). Collagen XVI, integrin α4 (ITGA4) and TGFBI were upregulated, although these proteins are not involved in guttate formation but are primarily involved in cell-matrix interactions and cell adhesion. Selected candidate genes, such as fibronectin and type III collagen, were also tested at the protein level and were found to be significantly downregulated when FECD specimens were treated with 30 μM K-115 after 72 h of incubation (Figure 5). To identify additional matrix genes that can be transcriptionally regulated by K-115, gene expression profiling was performed using Human Extracellular Matrix and Adhesion Molecule PCR arrays. Using this approach, downregulation of fibronectin and collagen types I and III was confirmed in corneal endothelial cells from FECD patients, as well as additional downregulation (data not shown) of vitronectin and collagen types V and XIV was detected. Essentially, these data support the notion that ROCK inhibition with K-115 positively impacts abnormal corneal endothelial matrix metabolism by downregulating mRNA and protein expression of FECD-associated matrix genes that contribute to Descemet's membrane thickening and guttachia formation, thus suggesting that K-115 represents a novel therapeutic modality and antifibrotic strategy for corneal endothelial cell "rejuvenation" in FECD and other corneal endothelial diseases.

Claims

1. An agent for regulating the expression of an extracellular matrix by a corneal endothelial cell, comprising 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof.

2. The agent for regulating expression of an extracellular matrix by a corneal endothelial cell according to claim 1, wherein the 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine is (S)-(-)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine.

3. The agent for regulating the expression of an extracellular matrix by a corneal endothelial cell according to claim 1 or 2, which is an agent for preventing and / or suppressing guttachia formation.

4. The drug for preventing and / or suppressing guttae formation according to claim 3, which is a drug for preventing and / or suppressing guttae formation in FECD patients.

5. The agent for regulating the expression of an extracellular matrix by corneal endothelial cells according to claim 1 or 2, which is an agent for preventing and / or suppressing the recurrence of guttachiosis.

6. The drug for preventing and / or suppressing the recurrence of guttae formation according to claim 5, which is a drug for preventing and / or suppressing the recurrence of guttae formation in FECD patients.

7. The pharmaceutical preparation according to any one of claims 1 to 6, which is a liquid formulation.

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