Medicines for the prevention or treatment of Fuchs' corneal endothelial dystrophy

Abstract

The present invention provides a pharmaceutical product comprising a low-molecular-weight compound such as erythromycin or a pharmaceutically acceptable salt thereof, or a solvate thereof, for the prevention or treatment of Fuchs' corneal endothelial dystrophy. [Solution] A low molecular weight compound such as erythromycin, or a pharmaceutically acceptable salt thereof, or a solvate thereof, which has an inhibitory effect on RNA foci formation.

Description

Technical Field

[0001] The present invention relates to a method, a compound, a medicament containing the compound, and uses thereof for preventing or treating Fuchs corneal endothelial dystrophy.

Background Art

[0002] Visual information is recognized when light taken in from the cornea, which is the transparent tissue at the forefront of the eyeball, reaches the retina, excites the nerve cells of the retina, and the generated electrical signal is transmitted to the visual cortex of the brain via the optic nerve. In order to obtain good eyesight, it is necessary for the cornea to be transparent.

[0003] Human corneal endothelial cells exist at a density of about 3,000 or more per square millimeter in youth, but once damaged, their regenerative ability in the living body is extremely limited. Fuchs corneal endothelial dystrophy (FECD) is a disease in which endothelial cells are damaged due to the occurrence of guttata cornea in both eyes, resulting in a progressive decrease in the number of endothelial cells and corneal clouding and edema. In normal people, the corneal endothelium gradually decreases with aging, but in Fuchs disease, along with a decrease in endothelial cell density, there is also abnormal cell function. As it progresses, it becomes bullous keratopathy, and vision decreases to hand motion or light perception.

[0004] To date, no therapeutic agent with established efficacy against Fuchs corneal endothelial dystrophy has been reported, and corneal transplantation is widely used as a surgical treatment. However, since the supply of donor corneas is insufficient in many countries including Japan, it is necessary to develop a treatment for suppressing the progression of the disease in early FECD patients. In addition, as treatment methods other than corneal transplantation, cultured corneal endothelial injection therapy and ROCK inhibitors have begun to be clinically applied, but only corneal transplantation has established safety and efficacy.

[0005] FECD is thought to be caused in part by widespread transcriptional abnormalities resulting from the formation of pathogenic RNA foci. The present invention is expected to reduce corneal endothelial cell death by decreasing RNA foci formation, depletion of splicing regulators including MBNL1 by RNA foci, and splicing abnormalities (S. Ong Tone, et al., Prog Retin Eye Res 2021 Vol. 80 Pages 100863; Accession Number: 32438095; PMCID: PMC7648733; DOI: 10.1016 / j.preteyeres.2020.100863). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2018 / 230713 [Patent Document 2] Japanese Patent Publication No. 2022-188056 [Patent Document 3] International Publication No. 2017 / 141926 [Patent Document 4] Japanese Patent Publication No. 2018-138621 [Patent Document 5] International Publication No. 2018 / 230711 [Non-patent literature]

[0007] [Non-Patent Document 1] S. Kinoshita, N. Koizumi, M. Ueno, N. Okumura, K. Imai, H. Tanaka, et al., N Engl J Med 2018 Vol. 378 Issue 11 Pages 995-1003 [Non-Patent Document 2] S. Kinoshita, KA Colby and FE Kruse, Cornea 2021 Vol. 40 Issue 10 Pages 1225-1228 [Non-Patent Document 3] S. Ong Tone, et al., Prog Retin Eye Res 2021 Vol. 80 Pages 100863 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The primary object of the present invention is to provide methods, compounds, and uses thereof for the prevention or treatment of Fuchs' corneal endothelial dystrophy. The present invention also aims to provide a pharmaceutical for the prevention or treatment of Fuchs' corneal endothelial dystrophy comprising the compound as an active ingredient. [Means for solving the problem]

[0009] The inventors have discovered that low-molecular-weight compounds such as erythromycin may be useful in the prevention or treatment of Fuchs' corneal endothelial dystrophy.

[0010] In other words, embodiments of the present invention include the following [1] to [5]. [1] A pharmaceutical product comprising erythromycin or a pharmaceutically acceptable salt or solvate thereof for the prevention or treatment of Fuchs' corneal endothelial dystrophy. [2] A pharmaceutical composition comprising erythromycin or a pharmaceutically acceptable salt thereof or a solvate for the prevention or treatment of Fuchs' corneal endothelial dystrophy. [3] A method for the prevention or treatment of Fuchs' corneal endothelial dystrophy, comprising administering erythromycin or a pharmaceutically acceptable salt thereof, or a solvate thereof, to a subject in need thereof. [4] Erythromycin or a pharmaceutically acceptable salt thereof, or a solvate thereof, for use in the prevention or treatment of Fuchs' corneal endothelial dystrophy. [5] Use of erythromycin or a pharmaceutically acceptable salt thereof, or a solvate thereof, in the manufacture of a medicament for the prevention or treatment of Fuchs corneal endothelial dystrophy.

Advantages of the Invention

[0011] Low-molecular-weight compounds such as erythromycin used in the present invention may be useful for the prevention or treatment of Fuchs corneal endothelial dystrophy.

Brief Description of the Drawings

[0012] [Figure 1] Shows the results of quantitative analysis of the RNA foci positive cell rate. [Figure 2] Shows the effect of erythromycin on the RNA foci positive cell rate. Administration of 50 μl of erythromycin showed a significant inhibitory effect on the RNA foci positive cell rate (Whitney U test, all p < 0.05).

Modes for Carrying Out the Invention

[0013] Low-molecular-weight compounds such as erythromycin used in the present invention can be used as a medicament as they are, or can also be used in the form of a pharmaceutically acceptable salt by a known method. Such salts include salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, etc., and salts of organic acids such as acetic acid, citric acid, tartaric acid, maleic acid, succinic acid, fumaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, etc.

[0014] Low-molecular-weight compounds such as erythromycin used in the present invention or a pharmaceutically acceptable salt thereof may incorporate solvent molecules to form a solvate, and such solvates are also included in the present invention. Such solvates include solvates with solvent molecules such as methanol, ethanol, isopropyl alcohol, butanol, dimethyl sulfoxide, acetonitrile, etc., and hydrates.

[0015] The low-molecular-weight compounds such as erythromycin used in the present invention have asymmetric carbons, and any of the optical isomers and mixtures thereof are included in the present invention.

[0016] The low-molecular-weight compounds such as erythromycin used in the present invention or pharmaceutically acceptable salts thereof have RNA foci formation inhibitory activity as shown in the test examples described below.

[0017] The "subject" refers to a human or non-human animal having or suspected of having Fuchs endothelial corneal dystrophy. In one embodiment of the present invention, the subject is a mammal. In one embodiment of the present invention, the subject is a human.

[0018] "RNA foci" refers to abnormal aggregation of RNA formed in cells when RNA molecules adopt abnormal conformations or accumulate at high concentrations. These aggregates disrupt normal cellular functions by sequestering RNA-binding proteins and cause dysregulation of RNA metabolism. In one embodiment of the present invention, a medicament containing a low-molecular-weight compound such as erythromycin is provided for treating diseases related to RNA foci formation. In a certain embodiment, the disease related to RNA foci formation is Fuchs endothelial corneal dystrophy.

[0019] The erythromycin (CAS number: 114-07-8) used in the present invention is a macrolide compound known as an antibacterial agent and is represented by the following structure.

Chemical formula

[0020] When the low-molecular-weight compounds such as erythromycin used in the present invention or pharmaceutically acceptable salts thereof are administered as a medicament, the compound of the present invention or a pharmaceutically acceptable salt thereof is used as it is or in a pharmaceutically acceptable non-toxic and inert carrier, for example, in a pharmaceutical composition containing 0.001% to 99.5%, preferably 0.1% to 90%, and is administered to mammals including humans.

[0021] As a carrier, one or more conventionally used solid, semi-solid, or liquid diluents, fillers, and other formulation aids that are pharmaceutically acceptable are used. The pharmaceutical composition according to the present invention is preferably administered in dose unit form. The pharmaceutical composition can be administered intra-tissue, orally, intravenously, topically (transdermally, ophthalmoscopy, intraperitoneally, intrathoracically, etc.), or rectally. The pharmaceutical composition according to the present invention is administered in dosage forms suitable for these administration methods, such as tablets, capsules, granules, fine granules, syrups, enteric-coated preparations, sustained-release preparations, injections, lotions, tapes, eye drops, eye drop ointments, inhalants, etc.

[0022] The dosage for pharmaceutical use should preferably be adjusted considering the patient's condition, such as age, weight, type and severity of the disease, the route of administration, the type of compound of the present invention, whether it is a salt or not, and the type of salt. [Examples]

[0023] German-derived immortalized FECD disease model cells (iFECD cells) and normal corneal endothelial cell model cells (iHCEC cells), established at Doshisha University, were introduced to Osaka University and used in each study.

[0024] TCF4 Repeat Extension Genotyping DNA was extracted from iFECD and iHCEC cells using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The quantity and quality of each isolated DNA sample were analyzed using a UV spectrophotometer (NanoDrop; NanoDrop Technologies, Wilmington, DE, USA). A short tandem repeat (STR) assay was performed to measure the CTG repeat length in the TCF4 third intron of cell-derived DNA. Specifically, for PCR, genomic DNA was amplified using 5'FAM-binding primer P1 (5'-CAGATGAGTTTGGTGTAAGATG-3' (SEQ ID NO: 1)) and unlabeled reverse primer P2 (5'-ACAAGCAGAAAGGGGGCTGCAA-3' (SEQ ID NO: 2)). For Triplet Repeat Primed (TP-)PCR, the 5'FAM-binding forward primer P1 and reverse primer P3 (5'-TACGCATCCCAGTTTGAGACG-3' (SEQ ID NO: 3)) were used. The reverse primer (P4) consists of a 5-unit CTG repeat and a 5' tail that functions as an anchor for reverse primer P3 (SEQ ID NO: 4). PCR and TP-PCR methods were performed, and fragment analysis of their products was conducted. PCR product sequencing was performed using a 3730XL model DNA Analyser (Applied Biosystems, Grand Island, NY, USA), and data analysis was performed using Peak Scanner 2 (Applied Biosystems).

[0025] Fluorescence in situ hybridization(FISH) iFECD cells and iHCEC cells were seeded on chamber slides, followed by 24-hour cell culture and subsequent administration of erythromycin. After an additional 48-hour cell culture, fluorescence in situ hybridization (FISH) was performed, and RNA foci were observed. Specifically, after aspirating the culture medium, it was washed once with phosphate-buffered saline (PBS). 3% PFA dissolved in PBS was added and fixed at room temperature for 15 minutes, after which the cells were washed twice with PBS for 5 minutes each. After washing with 0.5% Triton X-100 dissolved in PBS for 5 minutes, the cells were immersed for 10 minutes in a pre-hybridization solution containing 30% formamide and 2×SSC at room temperature. Next, the cells were incubated in a darkroom for 2 hours in a hybridization solution containing 2% BSA 10 mg / mL, 1 mg / mL yeast tRNA, 100 ng / μL 5'-Texas Red-(CAG)6CA-3' probe, 1% ribonucleoside-vanadyl complex 200 mM, 33% formamide, and 2×SSC. Subsequently, the cells were incubated in a darkroom for 30 minutes at 42°C in a post-hybridization solution containing 30% formamide and 2×SSC. After staining with 1×SSC + Hoechst 33324 for 30 min RT, the cells were washed once with PBS, and the coverslips were mounted on slides and embedded.

[0026] Quantitative RNA Foci analysis Multicolor imaging of iFECD and iHCEC cells was performed using a Keyence BZ-X700 all-in-one fluorescence microscope. Still images were taken using 100× oil immersion, and images were captured for each independent endothelial cell line in a chamber slide using the same image acquisition parameters. Nuclei were detected using an excitation filter of 360 / 40nm, a diclock mirror of 400nm, and an absorption filter of 460 / 50nm (DAPI) channel. RNA foci were defined using an excitation filter of 560 / 40nm, a diclock mirror of 595nm, and an absorption filter of 630 / 60nm (Texas Red) channel. Nuclei whose peripheral regions at the edge of the field of view were not fully captured were discarded to reduce segmentation errors. After acquisition, Z-stacks of approximately 5-6 μm of 0.1 μm thickness were processed as maximum intensity projection images using Keyence software. At least 100 nuclei were analyzed for each independent condition and cell line to verify the RNA foci-positive cell rate, thereby investigating the inhibitory effect of erythromycin on RNA foci formation.

[0027] result TCF4 Repeat Extension Genotyping We investigated the number of CTG repeats and the presence of abnormal repeat elongation in cell-derived DNA. For iFECD cells, an allele with 11 CTG repeats was detected by PCR, and TP-PCR positivity was observed. For iHCEC cells, two alleles with CTG repeat counts of 14 / 19 were identified by PCR.

[0028] Erythromycin's inhibitory effect on RNA foci formation To verify the inhibitory effect on RNA foci formation, a quantitative analysis of the RNA foci-positive cell rate was performed. The results are shown in Figure 1.

[0029] The percentage of RNA foci-positive cells decreased with erythromycin administration, as shown in Figure 2. RNA foci formation was significantly suppressed by erythromycin 50 μM compared to before administration (Figure 2, Mann-Whitney U test, all p<0.05). [Industrial applicability]

[0030] Low molecular weight compounds such as erythromycin used in the present invention may be useful for the prevention or treatment of Fuchs' corneal endothelial dystrophy and can be used in the manufacture of pharmaceuticals for the prevention or treatment of Fuchs' corneal endothelial dystrophy.

Claims

[Claim 1] A pharmaceutical product comprising erythromycin or a pharmaceutically acceptable salt or solvate thereof for the prevention or treatment of Fuchs' corneal endothelial dystrophy.

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