KR20240016895A - Prevention and treatment of macular degeneration through suppression of cathepsin S expression - Google Patents

Abstract

The present invention relates to the prevention and treatment of macular degeneration through inhibition of cathepsin S expression. Retinal pigment epithelial cells in which cathepsin S is knocked down have reduced expression levels of inflammatory cytokines and complement activity even under oxidative stress conditions, and NF- Not only does it have a superior complement activity inhibition effect compared to κB inhibitors, but it can also be useful as a substance for the prevention or treatment of dry and wet macular degeneration in that it inhibits intracellular new blood vessel formation and tube formation.

Description

Prevention and treatment of macular degeneration through suppression of cathepsin S expression}

The present invention relates to the prevention and treatment of macular degeneration through inhibition of cathepsin S expression.

Age-related Macular Degeneration (AMD) is an eye disease characterized by progressive retinal degeneration of the macula, and is the most common cause of irreversible central vision loss in older adults in developed countries. The global prevalence of AMD is approximately 8.7% between the ages of 45 and 85, with an early AMD prevalence of 8.0% and a late-stage AMD prevalence of 0.4%.

Age-related macular degeneration is classified into two types: dry and wet. Dry macular degeneration refers to a case where waste accumulates in the retina and appears in the form of white dots (drusen) or the retina atrophies as a result. It accounts for 3/4 of age-related macular degeneration. Dry macular degeneration does not cause sudden vision loss, but it causes chronic vision loss and can develop into a wet form.

Wet macular degeneration refers to macular degeneration in which abnormal blood vessels (new blood vessels) develop under the macula. These new blood vessels easily rupture and leak blood or fluid accumulates, causing exudate, bleeding, or drusen formation in the macular area. Destruction of the macula occurs relatively quickly, causing vision to deteriorate rapidly and eventually leading to blindness.

In both forms of macular degeneration, the optic cells in the macula are gradually destroyed, so over time, the function of the macula deteriorates and central vision begins to decrease. Initially, it may only occur in one eye, but the other eye is often affected. . Currently, treatments for macular degeneration are mainly treatments for wet macular degeneration, and there are no commercially available treatments for dry macular degeneration.

Cathepsin S is a cysteine-type protease that exists in lysosomes and induces the degradation of damaged proteins through the endosome-lysosome pathway. In immune cells, it is known to regulate immune responses through MHC class II activation, which is important for antigen presentation, by decomposing invariant chains, and in cancer cells, it is known to play a role in increasing the growth of cancer cells by controlling metastasis and angiogenesis. Although research has been conducted as a target related to various cancers, little is known about the therapeutic use of cathepsin S for macular degeneration.

(Korea Open Patent) No. 10-2010-0012295

In order to solve the above problems, the purpose of the present invention is to provide a composition for preventing or treating macular degeneration, an ophthalmic composition, a kit, and a health functional food composition for preventing or improving macular degeneration as follows.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S (CTSS).

Another object of the present invention is to provide an ophthalmic composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S (CTSS).

Another object of the present invention is to provide a health functional food composition for preventing or improving macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S.

Another object of the present invention is to provide a kit for preventing or treating macular degeneration, including a composition containing as an active ingredient a substance that reduces the expression or activity of cathepsin S, and instructions.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S (CTSS).

According to one embodiment of the present invention, the material includes siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), and CRISPR/Cas (Clustered Regularly Interspaced Short It may be one or more selected from the group consisting of Palindromic Repeats, natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

According to one embodiment of the present invention, the macular degeneration may be macular degeneration under oxidative stress conditions, but is not limited thereto.

According to one embodiment of the present invention, the macular degeneration may be dry macular degeneration or wet macular degeneration, but is not limited thereto.

According to one embodiment of the present invention, the composition may be characterized by one or more of the following, but is not limited thereto:

Inhibition of inflammatory cytokines and complement activity in the retina;

Inhibition of intravascular membrane formation; and

Inhibits new blood vessels.

According to one embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of TNF-α, IL-1β, and MCP-1, but is not limited thereto.

According to one embodiment of the present invention, the complement may be any one or more selected from the group consisting of C3, C5, C3a, C5a, and C5b-9, but is not limited thereto.

The present invention provides an ophthalmic composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S.

According to one embodiment of the present invention, the composition may be an eye drop composition, but is not limited thereto.

The present invention provides a health functional food composition for preventing or improving macular degeneration containing as an active ingredient a substance that reduces the expression or activity of cathepsin S.

According to one embodiment of the present invention, the material includes siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), and CRISPR/Cas (Clustered Regularly Interspaced Short It may be one or more selected from the group consisting of Palindromic Repeats, natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

The present invention provides a kit for preventing or treating macular degeneration, including a composition containing as an active ingredient a substance that reduces the expression or activity of cathepsin S, and instructions.

In addition, the present invention provides a method for preventing, improving, or treating macular degeneration, comprising administering a composition containing as an active ingredient a substance that reduces the expression or activity of the cathepsin S.

In addition, the present invention provides a use for preventing, improving, or treating macular degeneration of a composition containing as an active ingredient a substance that reduces the expression or activity of the cathepsin S.

In addition, the present invention provides a use for preparing an agent for preventing, improving, or treating macular degeneration using a composition containing as an active ingredient a substance that reduces the expression or activity of the cathepsin S.

In addition, the present invention provides the use of a composition containing as an active ingredient a substance that reduces the expression or activity of the cathepsin S for preparing an ophthalmic preparation for preventing or treating macular degeneration.

According to the prevention and treatment of macular degeneration through inhibition of cathepsin S expression, retinal pigment epithelial cells in which cathepsin S has been knocked down have reduced expression levels of inflammatory cytokines and complement activity even under oxidative stress conditions, and have decreased complement activity compared to NF-κB inhibitors. Not only does it have an excellent activity inhibition effect, but it also inhibits intracellular new blood vessel formation and tube formation, so it can be useful as a substance for the prevention or treatment of dry and wet macular degeneration.

Figure 1a is a graph showing the survival rate of RPE cells according to H ₂ O ₂ treatment.
Figures 1b and 1c are graphs showing iNOS expression levels in RPE cells according to H ₂ O ₂ treatment and photographs showing Western blot results.
Figures 2a and 2b are graphs showing the expression level of cathepsin S in RPE cells according to H ₂ O ₂ treatment and photographs showing Western blot results.
Figures 3a and 3b are graphs showing the expression level of cathepsin S in RPE cells and cathepsin S knockdown RPE cells according to H ₂ O ₂ treatment, and photographs showing immunofluorescence staining results.
Figures 4a to 4c are graphs showing the expression levels of inflammatory cytokines in RPE cells and cathepsin S knockdown RPE cells according to H ₂ O ₂ treatment, and Figure 4d shows the results of analyzing each expression level by Western blot experiment.
Figures 5A to 5B are graphs showing complement factor expression levels in RPE cells and cathepsin S knockdown RPE cells according to H ₂ O ₂ treatment, and Figures 5C and 5D show the results of analyzing each expression level by Western blot experiment. am.
Figures 6a and 6b are immunofluorescence staining results showing the effect of suppressing complement activity when treating RPE cells with CTSS siRNA.
Figure 7a is a photograph showing the results of Western blot on the expression level of angiogenic factors in RPE cells treated with H ₂ O ₂ and in cathepsin S knockdown RPE cells by CTSS siRNA.
Figure 7b is an inverted fluorescence micrograph showing the effect of reducing tube formation in RPE cells by H ₂ O ₂ treatment and in cathepsin S knockdown RPE cells by CTSS siRNA treatment.
Figure 8 is a photograph showing the effect of reducing choroidal neovascularization when treating CTSS siRNA in a choroidal neovascularization mouse model.
Figure 9 is a schematic diagram showing the mechanism of cathepsin S in age-related macular degeneration.

The present inventors completed the present invention by confirming the effect of preventing or treating macular degeneration by cathepsin S knockdown cells and cathepsin S siRNA treatment in one embodiment according to the present invention. Therefore, the present invention is cathepsin S (cathepsin S). Provided is a pharmaceutical composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or activity of (S, CTSS).

In the present invention, “reduced expression” refers to any action that reduces the expression of a gene, specifically gene knock-out, knock-down, gene interference, deletion, duplication, or deletion in the gene DNA sequence. This can be achieved by introducing mutations such as inversion or replacement, but is not limited to this.

In the present invention, “activity reduction” refers to the inhibition of one or more functions generated by cathepsin S, which causes macular degeneration, progresses or worsens the symptoms of macular degeneration, or slows the progression of macular degeneration. It may be a function related to the occurrence/worsening of signs of macular degeneration, such as increasing, but is not limited to this.

In one embodiment of the present invention, the material includes siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), and CRISPR/Cas (Clustered Regularly Interspaced Short It may be one or more selected from the group consisting of Palindromic Repeats, natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

In the present invention, “polynucleotide” or “nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of a single or double strand. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

In the present invention, “siRNA” is an RNA molecule, generally 17-24 bp in length, that can interfere with and inhibit the expression of a gene containing a nucleic acid sequence complementary to the sequence of the siRNA in a process called RNA interference. It means, but is not limited to this.

In the present invention, siRNA may include both single-stranded siRNA and double-stranded siRNA. In this case, double-stranded siRNA is derived from a longer double-stranded RNA molecule (dsRNA), which is cleaved by an endo-ribonuclease (Dicer) to form double-stranded siRNA, which is then activated by the nuclear protein complex (RISC). Single-stranded siRNA may be formed, but is not limited thereto.

In the present invention, methods commonly used in the art can be applied to the method for synthesizing or separating and purifying siRNA, and the method for delivering siRNA, for example, non-viral methods such as various plasmids and liposomes that can be expressed in eukaryotic cells. It may be a sexual vector, but is not limited thereto.

In the present invention, “shRNA” refers to a short hairpin RNA with a loop structure that induces the NAi phenomenon and induces mRNA degradation by complementary binding to the target mRNA. shRNA may be microRNA-adapted shRNAs, or simple hairpin shRNAs, but is not limited thereto, and includes all forms modified to improve the function of shRNA.

In the present invention, “miRNA” is a single-stranded RNA molecule that is generally ~22 nucleotides (nt) in length, and forms a complex with Argonaute protein (AGO) to bind to the 3'UTR of the target mRNA and inhibit the expression of the gene. It means a fine adjuster.

In the present invention, “antisense oligonucleotide” is a drug with the potential ability to realize personalized medicine along with RNAi therapeutics. It is a single-stranded nucleic acid polymer synthesized with an average of 12 to 25 nucleotides and is specific for target RNA. It has sequence complementarity and can regulate protein expression through several mechanisms. In addition, antisense oligonucleotides, single-stranded ASOs, can regulate gene expression chemically or depending on the binding site and target, or affect mRNA splicing through several mechanisms. When ASO complements the pre-mRNA target site, Ribonuclease H (RNase H), which hydrolyzes the RNA strand of the RNA-DNA heteroduplex, is activated and can inhibit target mRNA degradation and protein expression, but is not limited to this. , ASOs can directly inhibit the target site, or ASOs can induce steric hindrance by binding to mRNA, thereby interfering with the interaction with RNA-binding proteins. In the present invention, the antisense oligonucleotide may be modified and used as phosphorothioate (PS), PMO (Phosphorodiamidate morpholino oligonucleotide), PNA (Peptide nucleic acid), Locked nucleic acid (LNA), Ribose Modifications, etc., but is not limited thereto. , includes all modifications using techniques that can be generally applied in the art to improve functionality.

In the present invention, “CRISPR/Cas” may refer to ‘guide RNA (gRNA)’ made of RNA and ‘Cas’, an enzyme that cuts DNA, for example, Cas9, Cas11, or Cas12. It is not limited thereto, and Cas may have a broad meaning including all types of enzymes that can be used in the CRIPR system.

In the present invention, “Cas9” refers to “CRISPR-Cas9,” and CRISPR-Cas9 is a third-generation gene scissors that recognizes, cuts, and edits specific base sequences to be used, and inserts or edits specific genes at the target site in the genome. It can be useful for simple, quick, and efficient operation of stopping the activity of a specific gene.

Cas9 protein or gene information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto. Additionally, a person skilled in the art can appropriately connect additional domains to the Cas9 protein depending on its purpose.

In the present invention, the Cas9 protein may include not only wild-type Cas9 but also all variants of Cas9 as long as it has the function of a nuclease for gene editing. The origin of the Cas9 protein is not limited, and a non-limiting example is Streptococcus. Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, or Streptococcus mutans mutans) and can be appropriately selected and used by those skilled in the art, but is not limited thereto.

In the present invention, “gRNA” is a single-stranded RNA that finds the location of the specific DNA to be edited and guides the Cas protein to the target DNA. The gRNA is adjacent to the PAM (protospacer adjacent motif) site and is used for editing. It may contain a sequence complementary to a 10 to 25 bp base sequence of the desired DNA. The guide RNA may have 1 to 3 additional nucleotides, more specifically 2 or 3 nucleotides, before the 5' region of the sequence that can form base pairs with the complementary strand of the target DNA sequence. In addition, the guide RNA may include a portion having a sequence complementary to the sequence in the target DNA (also called spacer region, target DNA recognition sequence, base pairing region, etc.) and a hairpin structure for Cas protein binding. More specifically, it may include, but is not limited to, a portion having a sequence complementary to the sequence in the target DNA, a hairpin structure for Cas protein binding, and a terminator sequence.

The gRNA of the present invention may target the cathepsin S gene and may be composed of pairs to simultaneously target different regions of the gene, but is not limited thereto.

In the present invention, “natural product” refers to a product originating from living organisms, such as living organisms such as animals and plants living on land and in the ocean, and products of cell or tissue culture of living organisms, and can mean products in their natural form without any artificial addition, and their origin. There is no particular limitation as long as it is an extract of a natural product, and for example, it may be a plant extract, an animal extract, a microbial extract, or a mineral extract. In addition, the natural product may refer to the natural product itself, but is not limited thereto, and may refer to any modified form containing the pharmacologically active ingredients of the natural product. For example, it may be an extract, or a fraction thereof, a fermented product, juice, etc., but is not limited thereto.

In the present invention, conditions such as the solvent for the extract are not particularly limited, and any conditions generally available to those skilled in the art may be applied depending on the characteristics of the natural product or the inclusion of pharmacologically active ingredients.

In the present invention, the method of fractionation into fractions is not particularly limited as long as it is a commonly used method of fractionating the extract. Non-limiting examples include chromatographic separation, separation method using solubility difference, separation method using specific gravity difference, and centrifugation using density difference. It can be fractionated using one or more methods selected from the group consisting of separation methods, solid sample extraction methods, and separation methods using color differences.

In the present invention, “protein” is used interchangeably with “polypeptide” or “peptide” and refers to a polymer of amino acid residues, e.g., as commonly found in proteins in their native state, and in recombinant forms. It may include, but is not limited to, proteins produced by expression vectors.

In the present invention, “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector. Broadly speaking, any plasmid and vector can be used as long as it can replicate and stabilize within the host. The recombinant expression vector of the present invention preferably includes a promoter, which is a transcription initiation factor to which RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may contain a sequence that regulates, a terminator, etc., and the expression vector containing the recombinant expression vector and an appropriate transcription/translation control signal can be constructed by methods well known to those skilled in the art.

In addition, tag genes to increase the production of recombinant proteins, tag genes to maintain the structural stability of recombinant proteins, tag genes to easily separate recombinant proteins, antibiotic resistance genes to select transformants, etc. It may additionally include marker genes for selection, etc., and tags for easy separation are not limited thereto, but are Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, etc., and the marker gene for selection may include Representative examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, and The same antibiotic resistance genes, aadA genes, etc. may be included, and representative examples of the promoters include pEMU promoter, MAS promoter, histone promoter, Clp promoter, 35S promoter derived from cauliflower mosaic virus, and cauliflower mosaic virus. virus) derived 19S RNA promoter, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, EF-1α (Elongation factor-1 alpha) promoter The terminator may include nopaline synthase (NOS), rice amylase RAmy1 A terminator, phaseolin terminator, terminator of the Octopine gene of Agrobacterium tumefaciens, and rrnB1/ of E. coli. There is no limit to the type of gene to be added, such as B2 terminator, as long as it is a type that is already used in the production of recombinant proteins.

In the present invention, the method of transformation with a recombinant vector, the method of producing a target protein from a transformant transformed with a recombinant vector, etc. are all possible using any method known in the art, and at this time, the transformation is performed according to the characteristics of the recombinant vector. The transformant may be an animal or a plant, but is not limited thereto, and any transformant may be used as long as it is intended to obtain a highly functional protein in high yield.

In the present invention, “peptidomimetic” means designed to mimic biologically active peptides and may be, but is limited to, unnatural amino acids or other unusual compounds to stabilize the structure or alter biological activity. That is not the case. Peptidomimetics can be modified to improve the target specificity and stability of the peptide and reduce the possibility of degradation. For example, bending may be restricted to limit the conformational mobility of the peptide, or non-natural compounds may be included in the structure to reduce the possibility of enzymatic degradation, but are not limited thereto.

In the present invention, “antibody” is a term interchangeable with “antibody fragment” and refers to an antibody naturally present in the body, but is not limited thereto, and is produced by advances in genetic engineering, cell engineering, and developmental engineering technology. This includes both human antibody phage libraries and antibodies obtained from human antibody-producing transgenic animals. Additionally, an antibody fragment refers to a fragment that includes a portion of an antibody and can perform the same function as an antibody, for example, Fab, Fab', F (ab')2, scFv, diabody, dsFv. and peptides containing CDRs, etc., but are not limited thereto. The antibody fragment can be prepared by applying methods commonly performed in the art.

In the present invention, “exosome” refers to neurospheres, fibroblasts, epithelial cells, muscle cells, heart cells, kidney cells, nerve cells, hair cells, hair follicle cells, epithelial cells, beta cells, This may mean discharge from somatic cells derived from human tissue, including gastric mucosal cells, goblet cells, G cells, immune cells, pericytes, mast cells, and endothelial cells, but is not limited thereto. In addition, exosomes are released from cells extracted from solutions discharged from the human body, such as urine, saliva, sweat, and blood, or from pluripotent stem cells such as bone marrow-derived stem cells such as nerve cord blood, adipose-derived stem cells, adult stem cells, embryonic stem cells, and iPSCs. This may mean discharge from stem cells, placental stem cells, etc., but is not limited thereto. In addition, in order to improve exosome secretion or functionality, such as facilitating exosome release or increasing the number of exosomes released, physical stimulation such as electrical stimulation, heat, sound waves, laser, oxidative stress, LED light, and chemical substances are used. Chemical stimulation through processing, etc., biological stimulation through processing of substances such as enzymes that act on in vivo mechanisms, etc. may be applied, but are not limited thereto. In the present invention, the size of exosomes may be the size of a typical exosome, but is not limited thereto, and may vary depending on the discharge method, stimulation method, or culture method, and may vary depending on the amount and type of loaded material. there is.

In the present invention, any method for stimulating, secreting, and obtaining exosomes can be applied as long as it is obvious to those skilled in the art, and is not limited thereto.

In the present invention, a “compound” is a substance made by chemically combining two or more different elements, and includes both organic compounds and inorganic compounds. The compound of the present invention may refer to any chemical substance that can inhibit the expression or activity of cathepsin S.

In the present invention, “substances that reduce the expression or activity of cathepsin S” can be delivered to target cells through methods or delivery vehicles commonly used in the art. For example, it may be transmitted by a vector, but is not limited thereto. In addition, the substance of the present invention may be delivered through a generally applicable method or delivery system depending on the dosage, cycle, user's age, gender, clinical symptoms, disease progression, etc., but is not limited thereto. .

In the present invention, “macular degeneration” refers to a degenerative disease that occurs in the macula of eye tissue and causes decreased vision.

The macular degeneration can be classified into dry and wet. There is no special treatment for dry macular degeneration (non-exudative or atrophic), but treatment methods for wet macular degeneration (neoplastic macular degeneration and exudation) include intraocular injection and photodynamic therapy. , laser light coagulation, etc., and taking antioxidants or wearing sunglasses may be recommended to lower the risk of macular disease progression, but is not limited to this.

All age-related macular degeneration (AMD) begins with dry macular degeneration, some patients (approximately 10%) progress to wet macular degeneration, and approximately 85% of AMD patients have dry macular degeneration alone. Although relatively few patients develop wet macular degeneration, 80 to 90 percent of severe vision loss due to AMD may be caused by wet macular degeneration.

In dry macular degeneration, macular tissue becomes thinner, cells disappear, and accumulated waste from rods and cones can form deposits called drusen (yellow spots) on the retina (the transparent structure at the back of the eye that is sensitive to light). , dry macular degeneration may affect both eyes at the same time, and there may be no scarring or signs of bleeding or other fluid leakage into the macula.

Wet macular degeneration occurs when abnormal blood vessels grow in the choroid (the layer of blood vessels between the retina and the outer white layer of the eye called the sclera) beneath the macula, leaking blood and fluid (hence the term “wet”) and causing scar tissue. A lump may develop under the macula, and wet macular degeneration may initially occur in one eye but eventually affect both eyes.

In the present invention, macular degeneration includes all macular degeneration caused by endogenous or exogenous factors. In one embodiment of the present invention, the macular degeneration may be macular degeneration under oxidative stress conditions, but is not limited thereto. , In one embodiment of the present invention, the macular degeneration may be dry macular degeneration or wet macular degeneration, but is not limited thereto.

In one embodiment of the present invention, the composition may be characterized by one or more of the following, but is not limited thereto:

Inhibition of inflammatory cytokines and complement activity in the retina;

Inhibition of intravascular membrane formation; and

Inhibits new blood vessels.

In one embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of TNF-α, IL-1β, and MCP-1, but is not limited thereto.

When examining the eyes of patients with macular degeneration, higher-than-normal levels of complement may be detected. In one embodiment of the present invention, the complement may be any one or more selected from the group consisting of C3, C5, C3a, C5a, and C5b-9, but is not limited thereto.

The composition for preventing or treating macular degeneration according to the present invention may be used in combination with the existing treatment or treatment method for macular degeneration, but is not limited thereto.

The pharmaceutical composition for prevention or treatment according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives to the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners may be used in the syrup according to the present invention, and, if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, or sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

The present invention provides an ophthalmic composition for preventing or treating macular degeneration containing as an active ingredient a substance that reduces the expression or expression of cathepsin S.

The ophthalmic composition of the present invention is for use in the prevention or treatment of macular degeneration. In the present invention, the ophthalmic composition for prevention or treatment may be used in the same way as the ophthalmic pharmaceutical composition. Additionally, the ophthalmic composition can be used for the treatment and/or prevention of microbial infections. Microorganisms can be bacteria, viruses, fungi, or amoebas, parasites, or combinations thereof. The bacteria may be, but are not limited to, mycobacteria. In addition, it can be used for the prevention or treatment of diseases such as conjunctivitis, corneal abrasion, inflammatory ulcerative keratitis, epithelial keratitis, stromal keratitis, and herpesvirus-related keratitis, and the treatment is used to increase the efficacy of the treatment for the disease. It can be used in combination in addition to.

It may contain additives commonly contained in ophthalmic preparations, for example, buffering agents, isotonic agents, pH adjusters, etc.

The buffering agent may be sodium hydrogen phosphate (or its hydrate), sodium dihydrogen phosphate (or its hydrate), etc., but is not limited thereto. The amount of the buffer used can be appropriately selected by a person skilled in the art, for example, sodium hydrogen phosphate (or its hydrate) and sodium dihydrogen phosphate (or its hydrate) in the range of 0.80 w/v% to 3.00 w/v%, respectively. You can use it.

Isotonic agents include sodium chloride, potassium chloride, boric acid, and borax, but are not limited thereto. The amount of isotonic agent used can be appropriately selected by a person skilled in the art. For example, when using sodium chloride, it can be used in the range of 0.75 w/v% to 0.95 w/v%. When using sodium chloride, boric acid, and borax, they can be used in the range of 0.05 w/v% to 1.00 w/v%, respectively.

The pH adjuster can be added to adjust the pH of the resulting ophthalmic liquid composition to an appropriate range, for example, pH 5.0 to 8.0, preferably about pH 7.0, and hydrochloric acid or sodium hydroxide can usually be used. The pH adjuster may be used in the amount necessary to obtain pH in an appropriate range, but is not limited thereto.

The ophthalmic composition of the present invention can be prepared by being included in a general ophthalmic preparation at an optimal concentration that can maximize the effect while not causing side effects. At this time, the ophthalmic preparation can be mixed in an aqueous medium, and the aqueous medium can be sterilized purified water, water for injection, etc. suitable for the production of ophthalmic preparations, but is not limited thereto.

Additionally, the ophthalmic composition of the present invention may be in the form of a solution, suspension, emulsion, ointment, cream, gel, or controlled/sustained release medium, for example, the composition may be in the form of a contact lens solution, eye drops, tears, etc. , Preferably, in one embodiment of the present invention, the composition may be an eye drop composition, but is not limited thereto.

In addition, the ophthalmic composition of the present invention provides treatment cycle, amount, concentration, and formulation according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, administration route, patient's age, gender, weight, and severity of the disease. The shape, etc. may be determined.

The present invention provides a health functional food composition for preventing or improving macular degeneration containing as an active ingredient a substance that reduces the expression or expression of cathepsin S.

In one embodiment of the present invention, the material includes siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), and CRISPR/Cas (Clustered Regularly Interspaced Short It may be one or more selected from the group consisting of Palindromic Repeats), natural products, proteins, antibodies, exosomes, and compounds, but is not limited thereto.

Health food and health functional food compositions containing the active substances according to the present invention can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to general methods. Additionally, the mixing amount of the active substance can be appropriately determined depending on the purpose of use (for example, prevention or improvement).

In general, the amount of the composition in health foods and health functional foods may be 0.1 to 90 parts by weight of the total weight of the food. However, in the case of long-term intake for the purpose of maintaining or controlling health, the amount may be below the above range, and from the viewpoint of safety, the active ingredient according to the present invention may be used in an amount above the above range.

The health food and health functional food composition of the present invention has no particular restrictions on other ingredients other than containing the active substance of the present invention as an essential ingredient in the indicated ratio, and like ordinary beverages, various flavoring agents or natural carbohydrates are added as additional ingredients. may be included. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. The ratio of natural carbohydrates may generally be about 1 to 20 parts by weight, preferably about 5 to 12 parts by weight, per 100 parts by weight of the health functional food composition of the present invention, but is not limited thereto.

In addition to the above, health food and health functional food compositions containing the active substances of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants, and thickening agents (cheese, chocolate, etc.) ), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be included. In addition, the health food and health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

These ingredients can be used independently or in combination, and are generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the active substance of the present invention, but are not limited thereto. .

The present invention provides a kit for preventing or treating macular degeneration, including a composition containing as an active ingredient a substance that reduces the expression or activity of cathepsin S, and instructions.

In addition to the above components, the “kit” of the present invention may include other components, devices, materials, etc. commonly required for storage, management, and enhancement of the effect of the composition of the present invention. In addition, all components included in the kit can be used one or more times without limitation, there is no restriction on the order or subsequent use of each substance, and the application of each substance may be carried out simultaneously or in small steps.

The kit of the present invention may include a container in addition to the above agent and instructions. The container may serve to package the component, and may also serve to store and secure the component. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partially or entirely made of plastic, glass, paper, or foil. , wax, etc. The container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also provide access to the contents by needle. A stopper can be installed. The kit may include an external package, and the external package may include, but is not limited to, instructions for use of the components.

The terms used in the present invention are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

Throughout the specification of the present invention, when a part is said to “include” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary. The terms “about”, “substantially”, etc. used throughout the specification of the present invention are used to mean at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention Precise or absolute figures are used to aid understanding and to prevent unscrupulous infringers from taking unfair advantage of the disclosure.

Throughout the specification of the present invention, the term “combination thereof” included in the Markushi format expression refers to a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of constituent elements.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. H ₂ O ₂ Confirmation of decreased survival rate of RPE cells following treatment

MTT analysis was performed to confirm the decrease in viability of retinal pigment epithelium cells (RPE cells) following H ₂ O ₂ treatment, and the survival rate of RPE cells and iNOS according to H ₂ O ₂ treatment. Expression was confirmed.

First, human retinal-pigmented epithelial cell line (hereinafter referred to as ARPE-19 cells (ATCC, Manassas, VA, USA)) was used to inoculate a 96-well plate and culture for 24 hours. Afterwards, the cells were treated with 6.25μM, 12.5μM, 25μM, 50μM, and 100μM H ₂ O ₂ for 48 hours, and 3- (4,5-dimethylthiazol-2-yl)-2,5- Diphenyl-tetrazolium bromide (MTT) solution was added to the medium and cultured at 37°C for 4 hours. MTT-containing medium was removed and cells were lysed in 50 μL of DMSO. The optical density at 540 nm was confirmed using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA), and the cell viability was measured by repeating the experiment three times and calculating the average value.

As a result, the cell survival rate in the H ₂ O ₂ treated group was found to decrease compared to the control group, confirming that H ₂ O ₂ acts as a stress that can affect RPE cell survival (Figure 1a).

In addition, the expression level of iNOS expressed in RPE cells according to H ₂ O ₂ treatment was analyzed. Specifically, after treating RPE cells with 10μM, 50μM, and 100μM of H ₂ O ₂ , the relative iNOS mRNA expression level was confirmed, and a western blot experiment was performed. Total RNA was prepared using the Ribospin™ IIkit from GeneAll Biotechnology (Seoul, South Korea) according to the manufacturer's recommendations. Total RNA (1 μg) was reverse transcribed into first-strand cDNA using iScript™ cDNA Synthesiskits (Bio-RadLaboratories, Inc., California, USA) according to the manufacturer's instructions, and the Thermal Cycler CFX96TM Real-Time PCR System (Bio-RadLaboratories, Inc., California, USA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using RadLaboratories, USA), and each experiment was repeated three times to obtain the results. RPE cells were washed with ice-cold PBS and extracted in protein lysis buffer (Intron Biotechnology, Seoul, South Korea). Protein samples from cell lysates were mixed with an equal volume of 2×SDS sample buffer, denatured by heating for 5 minutes, and then separated on a 10% SDS-PAGE gel. After electrophoresis, the protein was transferred to a polyvinylidene difluoride membrane, and the membrane was blocked and washed using 5% BSA for 40 minutes. Afterwards, the cells were incubated overnight at 4°C with specific antibodies against iNOS and β-actin in Tris-buffered saline (TBS) containing Tween-20 (TBS-T, 0.1%). At this time, β-actin was used as a loading control. The primary antibody was washed from the membrane using TBS-T (Х3), and the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:1000-2000) for 2 hours. After three washes in TBS-T, immunopositive bands were visualized using the ChemiDocTMXRS+ imaging system with Image Lab ^™ software (Bio-Rad Laboratories, USA), and the results from three replicates each were averaged. .

As a result, when treated with 50μM and 100μM H ₂ O ₂ , the relative iNOS mRNA level in RPE cells appeared to increase (Figure 1b). Additionally, Western blot experiments confirmed that iNOS protein increased in a H ₂ O ₂ concentration-dependent manner (Figure 1c). In other words, it was confirmed that H ₂ O ₂ acts as oxidative stress on RPE cells.

Example 2. Confirmation of increased expression of cathepsin S (CTSS) in RPE cells under oxidative stress conditions

To determine the expression of cathepsin S (CTSS) in RPE cells under oxidative stress conditions, RPE cells were treated with H ₂ O ₂ and the expression of cathepsin S (CTSS) was measured. Specifically, H ₂ O ₂ was treated at 10 μM and 50 μM, and in order to confirm the expression of cathepsin S, analysis of the mRNA expression level of cathepsin S and Western blot experiments were performed, and cathepsin S-specific antibodies were used. The same experimental methods and conditions as in Example 1 were applied except that they were used.

As a result, when treated with H ₂ O ₂ , the relative mRNA level of cathepsin S was high compared to the control group (Figure 2a), and as a result of the Western blot experiment, the cathepsin S protein band was also thick and dark (Figure 2b), indicating that RPE cells It was confirmed that the expression of cathepsin S increases when oxidative stress acts on.

Example 3. Confirmation of cathepsin S (CTSS) non-response in cathepsin S knockdown RPE cells under oxidative stress conditions

We also analyzed whether cathepsin S expression increased due to oxidative stress in cathepsin S knockdown RPE cells.

Example 3-1. H ₂ O ₂ Determination of CTSS mRNA expression levels in cathepsin S knockdown RPE cells

To prepare cathepsin S knockdown RPE cells, siRNA transfection was performed. Specifically, cells were cultured at 50 to 60% confluence, and the transfection mixture was cultured with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), OPTI-MEM, and CTSS siRNA (Santa cruz sc-29940). After incubation for 10 minutes, cells were treated according to the manufacturer's instructions and cultured for 24 hours before transfection. Cells were cultured to 70 to 80% confluence and then treated with the indicated 50 μM H ₂ O ₂ for an additional 48 hours. Cultured. Afterwards, RT-PCR was performed according to the given protocol, and the average of the results from three repeated experiments was derived. At this time, β-actin was used as a loading control, and cathepsin S knockdown RPE cell line was prepared by infecting ARPE-19 cells with control siRNA (control) or CTSS siRNA for 24 hours. Control and cathepsin S knockdown RPE cell lines were treated with H ₂ O ₂ and the relative mRNA expression levels of cathepsin S were analyzed. The mRNA expression level was measured using the same conditions and methods as in Example 2.

As a result, the relative expression level of cathepsin S mRNA in cathepsin S knockdown RPE cells was found to be approximately 1/100 of that in the control group, and despite H ₂ O ₂ treatment, cathepsin S expression in RPE cells was significantly reduced. It was confirmed to be inhibited (Figure 3a).

Example 3-2. H ₂ O ₂ Immunofluorescence analysis of cathepsin S knockdown RPE cells.

Inhibition of cathepsin S in the cathepsin S knockdown RPE cells of Example 3-1 was confirmed through immunofluorescence analysis. Specifically, cells prepared in the same way as the mRNA expression level analysis were washed twice with sterile PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then the cells were washed three times with PBS to stop fixation. Cells were permeabilized with PBS containing 0.01% Triton X-100 for 10 min. After blocking for 1 hour, the insert membrane was removed using a scalpel and placed on Parafilm before incubation with primary antibodies. All primary antibodies were used diluted 1:50 in 5% BSA and PBS and incubated overnight at 4°C. Afterwards, the cells were washed three times with PBS, and secondary FITC- or TRITC-conjugated antibodies were diluted 1:50 and applied for 1 hour at room temperature. After adding 1 mg/ml DAPI 10 minutes before removing the secondary Ab to stain the nucleus, cells were coated with mounting medium and a glass coverslip was placed on top. The membrane/coverslip was mounted on a glass slide using mounting medium, and the cells were observed under a fluorescence microscope (Leica DFC7000T).

As a result, when using a fluorescent probe, H ₂ O ₂ treatment significantly increased cathepsin S expression, which was confirmed by bright red fluorescence (Figure 3b). As a result of analyzing RPE cells exposed to H ₂ O ₂ (50 μM, 48 h) or control after transfection with control siRNA or CTSS siRNA for 24 hours, cathepsin S was significantly higher in CTSS siRNA + H compared to control siRNA + H ₂ O ₂ It decreased significantly in ₂ O ₂ (p<0.05).

According to these results, it was confirmed that when cathepsin S is knocked down, the expression of cathepsin S induced by this is greatly reduced despite H ₂ O ₂ treatment, thereby reducing the red fluorescence signal.

Example 4. Confirmation of reduction of inflammatory cytokines in cathepsin S knockdown RPE cells

The effect of reducing inflammatory cytokines in cathepsin S knockdown RPE cells was confirmed. Specifically, after treating RPE cells with H ₂ O ₂ , the expression of p-NF-κB (active form of NF-κB) and inflammatory cytokines was analyzed by RT-PCR. The same experimental group cells (cathepsin S knockdown RPE cells) and control cells as in Example 3 were used, and each cell was treated with H ₂ O ₂ under the same conditions as in Example 3 (H ₂ O ₂ 50 μM, 48 h). Afterwards, RT-PCR and Western blot experiments were performed to confirm the expression of p-NF-κB and inflammatory cytokines in the same manner as Example 1 or 2. At this time, NF-κB and β-actin were loaded. It was used as a control.

As a result, when treated with H ₂ O ₂ , the relative expression levels of TNF-α, IL-1β, and MCP-1 in the control group were found to be significantly higher than those in the cathepsin S knockdown RPE cells (FIGS. 4A to 4C). Additionally, p-NF-κB, TNF-α, IL-1β, and MCP-1 protein expression induced by H ₂ O ₂ was reduced by cathepsin S knockdown (Figure 4d). According to these results, it was analyzed that cathepsin S suppresses the expression of inflammatory cytokines TNF-α, IL-1β, and MCP-1 in RPE cells at the transcription level.

Example 5. Confirmation of decreased complement factor expression in cathepsin S knockdown RPE cells

The effect of reducing complement factor expression in cathepsin S knockdown RPE cells was confirmed. Specifically, the expression level of complement factors was examined after treating RPE cells with H ₂ O ₂ . Among complement factors, the expression of C3aR and C5aR, receptors for C3a and C5a, was analyzed by RT-PCR, and the protein expression of C3 (C3a), C5 (C5a), C3aR, C5aR, and the active form of the membrane attack complex C5b-9 was analyzed by Western blot. It was analyzed through experiment. At this time, the same experimental group cells (cathepsin S knockdown RPE cells) and control cells as in Example 3 were used, and each cell was treated with H ₂ O ₂ under the same conditions as in Example 3 (50 μM, 48 h). In addition, the experimental methods and conditions for RT-PCR and Western blot were applied the same as Example 1 or 2.

As a result, when treated with H ₂ O ₂ , the relative expression levels of C3aR and C5aR in the control group were found to be significantly higher than those in the cathepsin S knockdown RPE cells (Figures 5a and 5b). In addition, H ₂ O ₂ -induced C3 (C3a), C5 (C5a), C3aR, C5aR and C5b-9 protein expression appeared to be reduced by cathepsin S knockdown (Figures 5c and 5d), indicating that cathepsin It was confirmed that S knockdown resulted in a statistically significant complement inhibition effect (p<0.05). According to these results, it was analyzed that cathepsin S inhibits the complement system in RPE cells.

According to Examples 1 to 5, the correlation between H ₂ O ₂ and cathepsin S expression was confirmed as the survival rate of RPE cells decreased and the expression of cathepsin S increased during H ₂ O ₂ treatment. In addition, it was confirmed that by knocking down cathepsin S in RPE cells, the expression of cathepsin S was suppressed despite H ₂ O ₂ treatment, and inflammatory cytokines and complement activity were suppressed, thereby reducing the inflammatory response. Accordingly, it was confirmed that inhibiting cathepsin S is important in macular degeneration, and it was analyzed whether the same results were obtained by treating cathepsin S expression or inhibitors.

Example 6. Confirmation of excellent complement activity inhibition effect of cathepsin S siRNA

In order to confirm the inhibitory activity of complement activity when treated with an inhibitor of cathepsin S expression or activity, cathepsin S siRNA (CTSS siRNA, CTSSiRNA) was treated on RPE cells and the effect of inhibiting complement activity was analyzed. In this case, the NF-κB inhibitor was treated as a comparison group. Specifically, RPE cells were treated with control siRNA or CTSS siRNA for 24 hours in the presence or absence of Bay 1170-82 (10 μM), an NF-κB inhibitor. Immunofluorescence analysis was then performed on C3a and C5a expression in RPE cells. The experimental method and conditions were applied the same as in Example 3.

As a result, when treated with Bay 1170-82, the red fluorescence signal decreased compared to the control DMSO, showing that C3a and C5a expression was suppressed. However, the effect of CTSS siRNA on suppressing C3a and C5a expression was significantly superior to that of Bay 1170-82. confirmed (Figures 6a and 6b). Additionally, simultaneous treatment with CTSS siRNA and NF-κB inhibitor appeared to reduce C3a and C5a expression more than CTSS siRNA or NF-κB inhibitor alone, suggesting that CTSS siRNA acts synergistically with NF-κB inhibitor in inhibiting complement activity. It was confirmed to have an effect. According to these results, it is analyzed that cathepsin S acts in the same pathway as NF-κB and may act as a regulator of complement activation.

Example 7. Confirmation of excellent reduction of angiogenesis by cathepsin S siRNA

It was determined whether treatment with an inhibitor of cathepsin S expression or activity reduces blood vessel formation within RPE cells induced by oxidative stress. Specifically, the expression levels of PPARγ and VEGFA/AKT, which play key roles in angiogenesis, differentiation, migration, and proliferation, were confirmed through Western blot experiments. The experimental method and conditions were applied the same as in Example 2. In addition, in order to check whether the cells formed tubes, HUVEC (1 × 10 ⁵ ) were treated in a 96 well-plate coated with growth factor-reduced Matrigel, and then RPE cells were incubated with CTSS siRNA and H ₂ O. Cells were treated for ₂ hours with conditioned media (CM) prepared by treating them at 37°C for 48 hours, and images were analyzed using an inverted fluorescence microscope.

As a result, the expression levels of PPARγ and VEGFA/AKT (p-AKT) increased upon H ₂ O ₂ treatment, whereas when treated with CTSS siRNA, the expression levels of PPARγ and VEGFA/AKT (p-AKT) increased compared to the control group despite oxidative stress conditions. ) was confirmed to be significantly reduced (Figure 7a). In addition, unlike the significant increase in tube formation in the control group when treated with H ₂ O ₂ , when treated with CTSS siRNA, tube formation was confirmed to be significantly suppressed even more than in the untreated control group with H ₂ O ₂ (FIG. 7b).

According to these results, in RPE cells treated with CTSS siRNA, not only new blood vessels but also intracellular tube formation was significantly suppressed, and CTSS siRNA was found to be effective in preventing and treating macular degeneration, the main cause of which is new blood vessel hyperplasia. .

Example 8. Confirmation of the excellent effect of cathepsin S siRNA in reducing neovascularization in a choroidal neovascularization (CNV) model

The effect of cathepsin S siRNA on choroidal neovascularization in vivo was confirmed. First, to confirm the in vivo effect, a choroidal neovascularization (CNV) model was created. Animal research protocols were approved by the Chung-Ang University Animal Care and Use Committee (approval number, A2021042). All animal experiments were conducted in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmological and Vision Research. Choroidal neovascularization in the mouse model was induced by laser. Six-week-old male C57BL/6J mice (Orient Co., Sungnam, Korea) were anesthetized with 240 mg/kg avertin (2-2-2-tribromoethanol 1.2%) by intraperitoneal injection (Papioannou and Fox, 1993). Using a diode laser at the 12 o'clock and 6 o'clock positions, a 2 to 3 disk diameter on the optical disc (75 um spot size, 120 mW, 100 ms), and a slit lamp delivery system (OcuLight Tx, IRIDEX, CA, USA) with a round coverslip. Three retinal burn lesions were induced in each mouse's right eye (n=10). Bubble formation was considered a sign of valid burns and Bruch membrane rupture. After 36 hours, siRNA diluted in RNAse-free water was injected into both eyes at a distance of 0.5 mm from the limbus using a 5uL syringe (Hamilton Co, Reno, NV, USA) and a 32-gauge needle ( 5 μL [15 nmol]/eye). Mouse Cts (mCts) siRNA (n=5) and control siRNA (n=5) (both Santa Cruz Biotechnology) were each injected into the right eye. Seven days after laser treatment, the eye was removed and fixed with 4% paraformaldehyde. The anterior segment was removed to obtain the retina/choroid area. The choroid/retinal pigment epithelium complex was stained immunohistochemically as follows. The choroid/retinal pigment epithelium complex was reacted with anti-CD31 antibody at a 1:100 dilution overnight, and choroidal neovascularization was visualized under a fluorescence microscope using secondary Alexa Fluor 594-conjugated goat anti-mouse IgG (provided by Invitrogen).

As a result, it was confirmed that the amount of choroidal neovascularization was significantly reduced after injection of CTSS siRNA compared to after injection of control siRNA in the choroidal neovascularization mouse model (FIG. 8).

Overall, cathepsin S is induced within RPE cells by H ₂ O ₂ (Example 2), and inflammatory cytokines (Examples 3 and 4) and complement activity (Example 5) in the cathepsin S knockdown RPE model. It was confirmed that this was significantly inhibited, and the effect of suppressing complement activity by CTSS siRNA was not only superior to that of the NF-κB inhibitor (Example 6), but also the effect of angiogenesis and tube formation was confirmed to be significantly superior (Example 7). These experimental results were also shown in the choroidal neovascularization mouse model (Example 8), and substances that reduce the expression or activity of cathepsin S, such as CTSS siRNA, can be used for both dry and wet macular degeneration. It is expected to be used as a substance for preventing or treating sexual problems.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (12)

A pharmaceutical composition for preventing or treating macular degeneration, comprising as an active ingredient a substance that reduces the expression or activity of cathepsin S (CTSS).
According to paragraph 1,
The materials include siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, and peptides. A pharmaceutical composition for preventing or treating macular degeneration, which is at least one selected from the group consisting of peptidomimetics, antibodies, exosomes, and compounds.
According to paragraph 1,
A pharmaceutical composition for preventing or treating macular degeneration, wherein the macular degeneration is macular degeneration under oxidative stress conditions.
According to paragraph 1,
A pharmaceutical composition for preventing or treating macular degeneration, wherein the macular degeneration is dry macular degeneration or wet macular degeneration.
According to paragraph 1,
A pharmaceutical composition for preventing or treating macular degeneration, characterized by one or more of the following:
Inhibition of inflammatory cytokines and complement activity in the retina;
Inhibition of intravascular membrane formation; and
Inhibits new blood vessels.
According to clause 5,
A pharmaceutical composition for preventing or treating macular degeneration, wherein the inflammatory cytokine is at least one selected from the group consisting of TNF-α, IL-1β, and MCP-1.
According to clause 5,
A pharmaceutical composition for preventing or treating macular degeneration, wherein the complement is at least one selected from the group consisting of C3, C5, C3a, C5a, and C5b-9.
An ophthalmic composition for preventing or treating macular degeneration, comprising as an active ingredient a substance that reduces the expression or activity of cathepsin S.
According to clause 8,
The composition is an ophthalmic composition for preventing or treating macular degeneration.
A health functional food composition for preventing or improving macular degeneration, comprising as an active ingredient a substance that reduces the expression or activity of cathepsin S.
According to clause 10,
The materials include siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, and peptides. A health functional food composition for preventing or improving macular degeneration, which is at least one selected from the group consisting of peptidomimetics, antibodies, exosomes, and compounds.
A kit for preventing or treating macular degeneration, comprising a composition containing as an active ingredient a substance that reduces the expression or activity of cathepsin S, and instructions.