Pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney diseases. When the cetirizine and L-5-hydroxytryptophan according to the present invention are administered alone or in combination, cyst formation is inhibited in an autosomal dominant polycystic kidney disease model induced by PKD1 or PKD2 gene mutation. Therefore, the cetirizine or L-5-hydroxytryptophan according to the present invention is expected to be advantageously used as a therapeutic agent for autosomal dominant polycystic kidney diseases.

Description

Pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease

[0001] The present invention relates to a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2023-0080951, filed June 23, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Autosomal dominant polycystic kidney disease (ADPKD) is the most common human genetic disorder of the kidney and is primarily caused by germline mutations in the PKD1 or PKD2 genes, which encode the ciliary cyst proteins polycystin 1 and 2 (PC1 and PC2). Defects in the PKD1 gene are known to be responsible for 85% of ADPKD cases, followed by defects in the PKD2 gene. The PC1 and PC2 proteins colocalize throughout the membrane of renal epithelial cells, the extracellular matrix, and primary cilia, and deficiency of the PC1-PC2 complex in cilia is known to play a role in cyst formation.

[0005] Patients with ADPKD develop multiple cysts in both kidneys, increasing in size and number until renal failure and death occur in their 50s or 60s. Cyst progression occurs as cyst epithelial cells proliferate, followed by the filling of the cyst with cystic fluid, leading to increased cyst size. ADPKD accounts for approximately 2% of dialysis cases in Korea, making it the fourth most common cause after diabetes, hypertension, and glomerulonephritis. ADPKD not only causes structural and functional defects in both kidneys but can also be accompanied by various renal complications, necessitating management from a relatively early stage of the disease.

[0006] Studies of the signaling pathways and pathological disorders underlying ADPKD have reported that many of the same metabolic pathways involved in epithelial proliferation, apoptosis, and extracellular matrix remodeling are shared between cancer and cystic diseases.

[0007] Despite efforts to pinpoint targetable ADPKD-related signals, there is no cure for ADPKD, and tolvaptan is the only FDA-approved drug that alleviates ADPKD symptoms. Therefore, there is an urgent need for more effective drugs that slow the progression of ADPKD. Drug repurposing is a strategy for identifying new clinical indications for approved or investigational drugs, and its time-effectiveness and known pharmacokinetic and safety profiles make it an increasingly attractive proposition.

[0008] Meanwhile, induced pluripotent stem cells (iPS cells) are cells that have pluripotency, obtained by dedifferentiation from differentiated cells such as somatic cells, and can differentiate into various organ cells. Since iPS cells can be obtained by reprogramming cells using dedifferentiation-inducing factors, it is possible to create pluripotent cell lines that are immune-compatible with patients without somatic cell transfer. All iPS cells have pluripotency, the ability to create all cells in our body, and the ability to self-renew, which allows them to create an unlimited number of cells that resemble themselves. While human embryonic stem cells also exhibit pluripotency, their scope of research and application is limited due to ethical issues. In contrast, iPS cells can be used from human somatic cells, so they have the advantage of being free from ethical issues.

[0009] Accordingly, the present inventors produced PKD1 or PKD2 gene mutant kidney organoids using human induced pluripotent stem cells and established a screening platform for FDA-approved drugs that suppress cyst formation in ADPKD patients, thereby aiming to select drugs with therapeutic effects for ADPKD, for which there is currently no effective treatment, and to utilize them as ADPKD treatments.

[0010]

[0011] The present inventors screened for a new therapeutic drug for autosomal dominant polycystic kidney disease, which has no existing effective treatment, by producing a PKD1 or PKD2 gene mutant kidney organoid model and observing cyst formation in the kidney organoid model. As a result, they confirmed that cetirizine and L-5-hydroxytryptophan inhibit cyst formation in a PKD1 or PKD2 gene mutant kidney organoid model, and completed the present invention based on this.

[0012] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof as an active ingredient.

[0013] Another object of the present invention is to provide a food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and food-wise acceptable salts thereof as an active ingredient.

[0014] Another object of the present invention is to provide a method for screening candidate drugs for treating autosomal dominant polycystic kidney disease.

[0015]

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0017]

[0018] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof as an active ingredient.

[0019] In addition, the present invention provides a food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising as an active ingredient at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and food-wise acceptable salts thereof.

[0020] In addition, the present invention provides a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising cetirizine or a pharmaceutically acceptable salt thereof and L-5-hydroxytryptophan or a pharmaceutically acceptable salt thereof as active ingredients.

[0021] In addition, the present invention provides a food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising cetirizine or a food-based acceptable salt thereof and L-5-hydroxytryptophan or a food-based acceptable salt thereof as active ingredients.

[0022] As one embodiment of the present invention, the composition can inhibit cyst formation in the kidney, but is not limited thereto.

[0023] As another embodiment of the present invention, the autosomal dominant polycystic kidney disease may be caused by a mutation in the PKD1 or PKD2 gene, but is not limited thereto.

[0024] As another embodiment of the present invention, the mutation of the PKD1 gene may be, but is not limited to, a deletion at position 36 of the exon of the PKD1 gene.

[0025] As another embodiment of the present invention, the mutation of the PKD2 gene may be, but is not limited to, a deletion at the exon 4 position of the PKD2 gene.

[0026] As another embodiment of the present invention, the cetirizine may have, but is not limited to, activity of inhibiting B-Raf or Raf-1 expression, or phosphorylation of p38-MAPK or ERK1 / 2.

[0027] As another embodiment of the present invention, the L-5-hydroxytryptophan may have, but is not limited to, an activity of inhibiting phosphorylation of mTOR.

[0028] In addition, the present invention provides a method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, comprising the following steps:

[0029] (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells;

[0030] (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells;

[0031] (c) a step of observing cyst formation after treating the candidate drug to the renal organoid; and

[0032] (d) A step of selecting a candidate drug for the treatment of autosomal dominant polycystic kidney disease when the above cyst formation is suppressed compared to before candidate drug treatment.

[0033] In addition, the present invention provides a method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, comprising the following steps:

[0034] (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells;

[0035] (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells;

[0036] (c) a step of measuring B-Raf or Raf-1 expression, or a step of measuring phosphorylation of p38-MAPK, ERK1 / 2, or mTOR after treating the candidate drug to the renal organoid; and

[0037] (d) A step of selecting a candidate drug for treating autosomal dominant polycystic kidney disease when the expression of B-Raf or Raf-1 is reduced compared to before candidate drug treatment, or when the phosphorylation of p38-MAPK, ERK1 / 2, or mTOR is suppressed compared to before candidate drug treatment.

[0038] As one embodiment of the present invention, the PKD gene may be, but is not limited to, the PKD1 or PKD2 gene.

[0039] As another embodiment of the present invention, the mutation of the PKD1 gene may be a deletion of exon 36 of the PKD1 gene using CRISPR / Cas9, but is not limited thereto.

[0040] As another embodiment of the present invention, the mutation of the PKD2 gene may be a deletion of exon 4 of the PKD2 gene using CRISPR / Cas9, but is not limited thereto.

[0041] In addition, the present invention provides a pharmaceutical composition comprising, as an active ingredient, at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof.

[0042] In addition, the present invention provides a method for preventing, treating, or improving autosomal dominant polycystic kidney disease, comprising administering to a subject in need thereof a therapeutically effective amount of at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or a composition containing the same as an active ingredient.

[0043] In addition, the present invention provides the use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or comprising the same as an active ingredient, for use in preventing, treating, or improving autosomal dominant polycystic kidney disease.

[0044] In addition, the present invention provides the use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or comprising the same as an active ingredient, for use in the manufacture of a medicament for preventing, treating, or improving autosomal dominant polycystic kidney disease.

[0045]

[0046] Cetirizine and L-5-hydroxytryptophan according to the present invention, when administered alone or in combination, have the effect of inhibiting cyst formation in a model of autosomal dominant polycystic kidney disease induced by mutations in the PKD1 or PKD2 gene. Therefore, cetirizine or L-5-hydroxytryptophan according to the present invention are expected to be useful as a therapeutic agent for autosomal dominant polycystic kidney disease.

[0047]

[0048] Figure 1a is PKD1 according to one embodiment of the present invention. - / - and PKD2 - / - This is a diagram schematically showing the mutation induction process.

[0049] Figure 1b is PKD1 according to one embodiment of the present invention. - / - This is a diagram confirming the level of PKD1 protein expression in mutants.

[0050] Figure 1c illustrates a protocol for renal organoid differentiation and cyst formation according to one embodiment of the present invention and PKD1 - / - and PKD2 - / - This is a drawing showing cyst formation in mutant kidney organoids confirmed by bright-field imaging (scale bar = 500 μm).

[0051] Figure 2a is PKD1 according to one embodiment of the present invention. - / - This is a drawing confirming the inhibitory effect of cetirizine and L-5-hydroxytryptophan treatment on cyst formation in mutant kidney organoids (scale bar = 500 μm, x10 magnification), and Fig. 2b is a drawing showing the quantification of Fig. 2a.

[0052] Figure 3a is PKD2 according to one embodiment of the present invention. - / -This is a drawing confirming the inhibitory effect of cetirizine and L-5-hydroxytryptophan treatment on cyst formation in mutant kidney organoids (scale bar = 500 μm, x10 magnification), and Fig. 3b is a drawing showing the quantification of Fig. 3a.

[0053] Figures 4a and 4b are PKD1 according to one embodiment of the present invention. - / - Concentration-dependent IC of cetirizine (Fig. 4a) and L-5-hydroxytryptophan (Fig. 4b) on cyst formation in mutant kidney organoids 50 This is a diagram showing the values.

[0054] Figures 5a and 5b are PKD1 according to one embodiment of the present invention. - / - Figure 5 shows immunoblot results for PI3K / Akt / B-Raf / Raf-1, MAPK / ERK, and mTOR after treatment with cetirizine (Fig. 5a) and L-5-hydroxytryptophan (Fig. 5b) in mutant kidney organoids.

[0055]

[0056] In one experimental example of the present invention, a deletion mutation at the exon 36 position of the PKD1 gene and a deletion mutation at the exon 4 position of the PKD2 gene were induced using CRISPR-Cas9 technology, and then human induced pluripotent stem cells were transfected with the mutation and renal organoids were cultured to generate a PKD1 autosomal dominant polycystic kidney disease model. - / - and PKD2 - / - Kidney organoids were prepared (see Experimental Examples 1 and 2).

[0057] In another experimental example of the present invention, an FDA approved drug library was screened to detect PKD1 - / - and PKD2 - / - Drugs that suppress increased cyst formation in renal organoids were screened, and as a result, it was confirmed that cetirizine, a histamine receptor antagonist, and L-5-hydroxytryptophan, a selective serotonin reuptake inhibitor, exhibited cyst formation suppressing effects (see Experimental Example 3).

[0058] In another experimental example of the present invention, the effects of cetirizine and L-5-hydroxytryptophan on intracellular signaling pathways were confirmed. PI3K / Akt, mTOR / c-Myc, Wnt, cAMP / PKA, and MAPK signals as well as Ca 2+ Several signaling pathways are regulated by cilia-dependent and -independent shear stress responses of renal epithelial cells, including those involved in cyst formation, and PKD was used to study shear stress-dependent intracellular signaling in cyst formation. - / - The effects of fluid flow were investigated in mutant renal organoids. As a result, it was confirmed that cetirizine exhibited inhibitory activity on B-Raf and Raf-1 expression and phosphorylation of p38-MAPK and ERK1 / 2, and L-5-hydroxytryptophan exhibited inhibitory activity on mTOR phosphorylation (see Experimental Example 4).

[0059]

[0060] Hereinafter, the present invention will be described in detail.

[0061]

[0062] The present invention provides a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising as an active ingredient at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof.

[0063] In the present invention, "cetirizine" can be represented by the following chemical formula 1 and is known as a second-generation antihistamine used to treat allergic rhinitis, dermatitis, and urticaria, and is administered orally. The effect usually begins within 1 hour and lasts for approximately 1 day. The degree of benefit of this drug is similar to that of other antihistamines such as diphenhydramine. In addition, the cetirizine can be obtained as a commercial product or prepared by a known method (Chinese Patent No. CN102321047B, Preparation method of cetirizine hydrochloride).

[0064] [Chemical Formula 1]

[0065]

[0066] In the present invention, “L-5-hydroxytryptophan” can be represented by the following chemical formula 2, and is not only a naturally occurring amino acid and chemical precursor, but also a metabolic intermediate in the biosynthesis process of serotonin, a neurotransmitter derived from tryptophan. L-5-hydroxytryptophan is sold as a general medicine for use as an antidepressant, appetite suppressant, and sleep aid in the United States, Canada, the Netherlands, and the United Kingdom, and can be obtained as a commercial product or manufactured by a known method (J.-G. Feng, Isolation and purification of 5-hydroxytryptophan in extract from seeds of Griffonia simplicifolia by ion-exchange resin, Chinese Traditional and Herbal Drugs 44(17):2410-2415.). The L-5-hydroxytryptophan can act as a selective serotonin reuptake inhibitor, but is not limited thereto.

[0067] [Chemical Formula 2]

[0068]

[0069] In the present invention, a "selective serotonin reuptake inhibitor (SSRI)" is a type of antidepressant used to treat depression, anxiety disorders, obsessive-compulsive disorder, and some personality disorders. SSRIs increase the extracellular level of the neurotransmitter serotonin by preventing its reuptake into presynaptic cells. This increases the level of serotonin available to bind to postsynaptic receptors.

[0070] In the present invention, "cyst" is a general term for a sac-shaped structure separated from the surrounding tissue within the body. The locations of their occurrence vary, and their impact on the body ranges from mild to severely painful. Consequently, the diagnosis and treatment methods also vary widely.

[0071] In the present invention, the composition can inhibit cyst formation in the kidney, for example, by reducing the size of cysts formed in the kidney by 10 to 100%, 10 to 90%, 10 to 80%, 10 to 70%, 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 100%, 20 to 80%, 20 to 60%, 20 to 40%, 30 to 100%, 30 to 80%, 30 to 60%, 30 to 40%, 40 to 100%, 40 to 80%, 40 to 60%, 50 to 100%, 50 to 80%, 50 to 60%, 60 to It can be reduced by, but is not limited to, 100%, 60 to 80%, 70 to 100%, 70 to 80%, 80 to 100%, or 90 to 100%.

[0072] In the present invention, the PKD (polycystic kidney disease) gene is a gene involved in polycystic kidney disease, and includes PKD1, PKD2, and PKD3.

[0073] In the present invention, the autosomal dominant polycystic kidney disease may be caused by a mutation in the PKD1 gene or the PKD2 gene, but is not limited thereto.

[0074] At this time, the mutation of the PKD1 gene may be a deletion at the exon 36 position of the PKD1 gene, and the mutation of the PKD2 gene may be a deletion at the exon 4 position of the PKD2 gene, but is not limited thereto.

[0075] In the present invention, the PKD1 gene may include or consist of the base sequence of NCBI Reference Sequence: NG_008617.1, but is not limited thereto.

[0076] In the present invention, the PKD2 gene may include or consist of the base sequence of NCBI Reference Sequence: NG_008604.1, but is not limited thereto.

[0077] In the present invention, the mutation of the PKD1 gene may be a deletion mutation at the exon 36 position of the PKD1 gene complementary to the guide RNA represented by SEQ ID NO: 1, and according to one embodiment of the present invention, it may be a deletion of two bases at the exon 36 position of the PKD1 gene complementary to the 15th and 16th bases from the 5' end in the base sequence of SEQ ID NO: 1, but is not limited thereto.

[0078] In the present invention, the mutation of the PKD2 gene may be a deletion mutation at the exon 4 position of the PKD2 gene complementary to the guide RNA represented by SEQ ID NO: 2, and according to one embodiment of the present invention, it may be a deletion of two bases at the exon 4 position of the PKD2 gene complementary to the 2nd and 3rd bases from the 5' end in the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0079] In the present invention, the cetirizine may have an activity of inhibiting B-Raf or Raf-1 expression, or inhibiting phosphorylation of p38-MAPK or ERK1 / 2, but is not limited thereto.

[0080] In the present invention, the L-5-hydroxytryptophan may have an activity of inhibiting phosphorylation of mTOR, but is not limited thereto.

[0081] In the present invention, the composition may contain cetirizine or a pharmaceutically / foodstuffally acceptable salt thereof, or L-5-hydroxytryptophan or a pharmaceutically / foodstuffally acceptable salt thereof, either singly or in combination, but is not limited thereto.

[0082] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising cetirizine or a pharmaceutically acceptable salt thereof and L-5-hydroxytryptophan or a pharmaceutically acceptable salt thereof as active ingredients.

[0083] In the present invention, the cetirizine or a pharmaceutically / foodstuffally acceptable salt thereof; or L-5-hydroxytryptophan or a pharmaceutically / foodstuffally acceptable salt thereof may be administered alone or in combination, but is not limited thereto.

[0084] In the present invention, cetirizine or a pharmaceutically / foodstuffally acceptable salt thereof; Or when L-5-hydroxytryptophan or a pharmaceutically / foodstuffally acceptable salt thereof is administered alone, the concentration thereof is 0.1 to 100 μM, 0.1 to 70 μM, 0.1 to 50 μM, 0.1 to 30 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 2.5 μM, 2.5 to 100 μM, 2.5 to 70 μM, 2.5 to 50 μM, 2.5 to 30 μM, 2.5 to 10 μM, 2.5 to 5 μM, 5 to 100 μM, 5 to 70 μM, 5 to 50 μM, 5 to 30 μM, 5 to 10 μM, 10 to 100 μM, 10 to It may be 70 μM, 10 to 50 μM, 10 to 30 μM, 30 to 100 μM, 30 to 70 μM, 30 to 50 μM, 50 to 100 μM, 50 to 70 μM, 2.5 μM, 5 μM, 10 μM, 30 μM, or 50 μM, and according to one embodiment of the present invention, each may be 30 μM, but is not limited thereto.

[0085] In the present invention, when cetirizine or a pharmaceutically / foodstuffally acceptable salt thereof and L-5-hydroxytryptophan or a pharmaceutically / foodstuffally acceptable salt thereof are administered in combination, there is no limitation on the order of administration of the drugs, and they may be administered sequentially or simultaneously, and there is no limitation on the number of times each drug is administered, and they may be administered singly or multiple times. At this time, the concentrations of these may be 0.1 to 50 μM, 0.1 to 30 μM, 0.1 to 20 μM, 0.1 to 10 μM, 1 to 50 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 5 to 50 μM, 5 to 30 μM, 5 to 20 μM, 5 to 10 μM, 7 to 50 μM, 7 to 30 μM, 7 to 20 μM, 7 to 10 μM, 10 to 50 μM, 10 to 30 μM, 10 to 20 μM, or 10 μM, respectively, with respect to the entire composition, and according to one embodiment of the present invention, may be 10 μM, respectively, but is not limited thereto.

[0086] In the present invention, “pharmaceutically acceptable salt” includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base.

[0087] In the present invention, “food-wise acceptable salt” includes a salt derived from a food-wise acceptable organic acid, inorganic acid, or base.

[0088] Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, they can be prepared by heating equimolar amounts of the compound and the acid or alcohol in water, followed by evaporation of the mixture to dryness, or by suction filtration of the precipitated salt.

[0089] Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and then evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare sodium, potassium, or calcium salts as the metal salt, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0090] The content of cetirizine, L-5-hydroxytryptophan, or a pharmaceutically acceptable salt thereof in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt%, or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0091] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0092] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0093] 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0094] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0095] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (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, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0096] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0097] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0098] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0099] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0100] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0101] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0102] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0103] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0104] 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. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0105] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0106] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0107] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0108] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0109] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0110] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0111]

[0112] In addition, the present invention provides a food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising as an active ingredient at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and food-wise acceptable salts thereof.

[0113] In addition, a food composition for preventing or improving autosomal dominant polycystic kidney disease is provided, comprising cetirizine or a food-based acceptable salt thereof and L-5-hydroxytryptophan or a food-based acceptable salt thereof as active ingredients.

[0114] In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

[0115] In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

[0116] In the present invention, the term “functional food” is the same as food for special health use (FoSHU), and refers to a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, “functionality” means regulating nutrients for the structure and functions of the human body or obtaining a useful effect for health purposes such as physiological actions.

[0117] When the cetirizine, L-5-hydroxytryptophan, or a food-wise acceptable salt thereof of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the cetirizine, L-5-hydroxytryptophan, or a food-wise acceptable salt thereof of the present invention can be added in an amount of 15 wt% or less, or 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0118] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense are included.

[0119] The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g, per 100 mL of the composition of the present invention.

[0120] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0121] In addition, the present invention provides a pharmaceutical composition comprising, as an active ingredient, at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof.

[0122] In addition, the present invention provides a method for preventing, treating, or improving autosomal dominant polycystic kidney disease, comprising administering to a subject in need thereof a therapeutically effective amount of at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or a composition containing the same as an active ingredient.

[0123] In addition, the present invention provides the use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or comprising the same as an active ingredient, for use in preventing, treating, or improving autosomal dominant polycystic kidney disease.

[0124] In addition, the present invention provides the use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or comprising the same as an active ingredient, for use in the manufacture of a medicament for preventing, treating, or improving autosomal dominant polycystic kidney disease.

[0125]

[0126] In addition, the present invention provides a method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, comprising the following steps:

[0127] (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells;

[0128] (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells;

[0129] (c) a step of observing cyst formation after treating the candidate drug to the renal organoid; and

[0130] (d) A step of selecting a candidate drug for the treatment of autosomal dominant polycystic kidney disease when the above cyst formation is suppressed compared to before candidate drug treatment.

[0131] In the present invention, the PKD gene may be a PKD1 or PKD2 gene, but is not limited thereto.

[0132] In the present invention, the pluripotent stem cell may be an induced pluripotent stem cell or an embryonic stem cell, and according to one embodiment of the present invention, may be a human induced pluripotent stem cell, but is not limited thereto.

[0133] In the present invention, there is no limitation on the method for inducing mutation of the gene, and according to one embodiment of the present invention, CRISPR / Cas9 can be used.

[0134] In the present invention, the mutation of the PKD1 gene is a deletion mutation at the exon 36 position of the PKD1 gene using CRISPR / Cas9, and may be a deletion mutation at the exon 36 position of the PKD1 gene complementary to the guide RNA (gRNA) represented by sequence number 1, but is not limited thereto.

[0135] In the present invention, the mutation of the PKD2 gene is a deletion mutation at the exon 4 position of the PKD2 gene using CRISPR / Cas9, and may be a deletion mutation at the exon 4 position of the PKD2 gene complementary to the guide RNA (gRNA) represented by sequence number 2, but is not limited thereto.

[0136] In the present invention, the "CRISPR / Cas9" system is a genome editing method called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) gene scissors, which consists of RNA (gRNA) that specifically binds to a specific base sequence and Cas9 nuclease, which acts as scissors to cut the specific base sequence. Using this CRISPR / CAS9 system, it is possible to knock-out, which suppresses the function of a specific gene, by introducing plasmid DNA into cells or animals.

[0137] In the present invention, there is no particular limitation on the method of transfection in the step (a), and for example, nucleofection, transient transfection, cell fusion, liposome-mediated transfection, polybrene-mediated transfection, transfection using calcium phosphate (Graham, FL et al., Virology, 52:456 (1973)), transfection using DEAE dextran, transfection using microinjection (Capecchi, MR, Cell, 22:479 (1980)), transfection using cationic lipid (Wong, TK et al., Gene, 10:87 (1980)), electroporation (Neumann E et al., EMBO J, 1:841 (1982)), transduction or transfection, as described in the literature (Basic methods in molecular biology, Davis et al., 1986 and Molecular Cloning: A laboratory manual, Davis et al., 1986) can be performed according to methods well known to those skilled in the art to which the present invention pertains.

[0138] In the present invention, "organoid" refers to a three-dimensional aggregate of one or more cell types that mimics the superficial appearance or actual structure or function of a tissue or organ, and can be formed through self-renewal and self-organization from adult stem cells, embryonic stem cells, or induced pluripotent stem cells. Organoids can similarly reproduce the physiological functions of the human body, and by constructing organoids from patient tissues, they enable disease modeling based on the patient's genetic information, drug screening through repeated testing, etc.

[0139] In the present invention, the method for culturing and producing the kidney organoid can be performed according to a method widely known in the art, but is not limited thereto.

[0140] In the present invention, “candidate drug” means an unknown substance used for screening to confirm whether or not cyst formation is inhibited in an autosomal dominant polycystic kidney disease model or the degree of cyst formation inhibition, and may be, for example, one or more selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts, but is not limited thereto.

[0141] In the present invention, treating the candidate drug may mean adding the candidate drug to an organoid culture medium and then culturing it for a certain period of time, but is not limited thereto.

[0142] In the present invention, the step of observing the cyst formation may be performed according to a method widely known in the art, and according to one embodiment of the present invention, the cyst size may be observed through ImageJ, but is not limited thereto.

[0143] In addition, the present invention provides a method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, comprising the following steps:

[0144] (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells;

[0145] (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells;

[0146] (c) a step of measuring B-Raf or Raf-1 expression, or a step of measuring phosphorylation of p38-MAPK, ERK1 / 2, or mTOR after treating the candidate drug to the renal organoid; and

[0147] (d) A step of selecting a candidate drug for treating autosomal dominant polycystic kidney disease when the expression of B-Raf or Raf-1 is reduced compared to before candidate drug treatment, or when the phosphorylation of p38-MAPK, ERK1 / 2, or mTOR is suppressed compared to before candidate drug treatment.

[0148] In the present invention, the step of measuring the expression of B-Raf or Raf-1 or the step of measuring the phosphorylation of p38-MAPK, ERK1 / 2, or mTOR may be measured using one or more methods selected from the group consisting of immunoblot, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chip, but is not limited thereto.

[0149] In addition, the present invention provides a method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, comprising the following steps:

[0150] (a) a step of treating a candidate drug to a PKD gene mutant cell or animal model;

[0151] (b) a step of observing the cyst size in a cell or animal model treated with the candidate drug; and

[0152] (c) A step of selecting a candidate drug for the treatment of autosomal dominant polycystic kidney disease when the cyst size is reduced compared to before treatment with the candidate drug.

[0153] In the present invention, the step of treating the animal model with the candidate drug in step (a) may be performed through parenteral or oral administration or stereotactic injection, but is not limited thereto, and a person skilled in the art will be able to select an appropriate method to test the candidate drug on an animal.

[0154] In the present invention, when the term "comprising" is used, it means that other components can be included rather than excluding other components unless specifically stated otherwise. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0155]

[0156] Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples and experimental examples.

[0157]

[0158] [Example]

[0159] Example 1. Renal organoid differentiation

[0160] Human induced pluripotent stem cells (iPSCs) WTC11 cell line obtained from The Catholic University of Korea were used, and cells between passages 30 and 60 were used. Renal organoid differentiation was performed as described in previous studies (BS Freedman et al., Nat Commun 6, 8715 (2015); JW Kim et al., Exp Mol Med 53, 1580-1591 (2021); JW Kim et al., Adv Sci (Weinh) 9, e2103526 (2022)). That is, human pluripotent stem cells (hPSCs) were plated at a density of 15,000 cells / well in 6-well plates in mTeSR1 medium (Stem Cell Technologies) + 10 μM Y27632 (LC Laboratories) on glass plates (LabTek) coated with 3% GelTrex (Thermo Fisher Scientific). The medium was exchanged with mTeSR1 containing 1.5% GelTrex (3 days), RPMI (Thermo Fisher Scientific) + 12 μM CHIR99021 (Tocris) (1.5 days), or RPMI + B27 supplement (Thermo Fisher Scientific) (1.5 days), and the cells were fed every 2–3 days to promote renal organoid differentiation.

[0161]

[0162] Example 2. CRISPR / Cas9 all-in-one plasmid structure and PKD1 - / - and PKD2 - / - Generation of human iPSC lines

[0163] For genetic modification, the all-in-one CRISPR / Cas9 was purchased from ThermoFisher Scientific (Cat. A21174, GeneArt CRISPR Nuclease Vector Kit) and used in the experiment. PKD1 and PKD2 gene oligos (PKD1 gRNA sequence: GTG GGT GCG AGC TTC CCC CC, SEQ ID NO: 1; PKD2 gRNA sequence: CGA ATA CGG CAA CTC CGA GT, SEQ ID NO: 2) were produced according to the ThermoFisher instructions and cloned into the all-in-one vector. The all-in-one vector contains CAS9, gRNA, and OPF labeling. After cloning, sequencing was performed to confirm that the desired gene was inserted into the vector, and the DNA was used in the experiment. The all-in-one Cas9 nuclease reporter vector DNA obtained by the clone was used for transfection into iPSCs (WTC11) by electroporation. The following day, only OPF-expressing cells were selectively harvested from 96-well plates using FAC, cultured for 7–10 days, and only cells with colonies were sequenced for DNA extraction and analysis. To verify the PKD1 mutant allele, single-cell-derived clones of the PKD1-targeted hPSC line were expanded, and genomic DNA was extracted using the AccuPrep Genomoic DNA Extraction Kit (Bioneer, K-3032). The CRISPR-Cas9 target region was amplified using a PCR master mix (PKD1 primers: 5'-AGGTTAACATGGGCTTGGCT-3' (forward), SEQ ID NO: 3; and 5'-GAGACAAGAGACGGAGGTGG -3' (reverse), SEQ ID NO: 4; PKD2 primers: 5'-GTAAAACGACGGCCAGT-3' (forward), SEQ ID NO: 5; and 5'-CAGGAAACAGCTATGAC-3' (reverse), SEQ ID NO: 6), and Sanger sequencing was performed to confirm the CRISPR mutation for each allele.

[0164]

[0165] Example 3. Cyst formation analysis

[0166] On day 16 of differentiation, differentiated kidney organoids were dissected from 6-well plates using a needle and transferred to 6-well culture plates containing RPMI + B27 supplemented medium. Briefly, PKD1 - / - and PKD2 - / - Mutant organoids were exposed to laminar fluid shear stress in a waving plate device. After shaking on the waving plate in an incubator for a week, RPMI + B27 supplemented medium was replaced every 3 days to allow cystogenesis. After 6 days, PKD1 - / - Mutant organoids were confirmed to exhibit renal tubular dilatation, forming balloon-like fluid-filled sacs known as cystogenesis. PKD2 - / - PKD1 also in mutant organoids - / - It was confirmed that cyst formation occurred in the same manner as the mutation.

[0167]

[0168] Example 4. PKD1 - / - and PKD2 - / - Screening FDA-approved drugs using human kidney organoid cultures

[0169] A library of 2,570 FDA-approved drugs was purchased from Selleckchem (Selleckchem, FDA-approved drug library, Cat. L1300) and diluted with DMSO or water to create a 10 mM stock. The FDA library of 2,570 drugs was designed to be used in the PKD1 - / - and PKD2 - / -In the initial screening of cysts, a final concentration of 30 μM was used. The cysts were shaken on a shaking plate in an incubator for 3 days, and the cyst sizes were quantified using ImageJ. Statistically significant changes in size were confirmed using a paired t-test.

[0170] Initially, FDA-approved drugs were screened for a 20-40% reduction in cyst size. The primary drug screen selected cetirizine, an antihistamine, and L-5-hydroxytryptophan, a selective serotonin reuptake inhibitor (SSRI).

[0171] To analyze the effects of cetirizine and L-5-hydroxytryptophan on cyst growth after drug treatment, PKD1 - / - Mutant cysts were treated with cetirizine and L-5-hydroxytryptophan (0, 2.5, 5, 10, 30, and 50 μM) on day 21 and imaged in real time for 3 days. IC 50 Values ​​were calculated by nonlinear regression and log(compound) vs. normalized response-variable slope using GraphPad Prism software.

[0172]

[0173] Example 5. Immunoblot analysis

[0174] Immunoblotting was performed from cyst formations on days 14–20 of culture in PKD1 kidney organoids. Total protein was isolated from cyst formations using a lysis buffer prepared by mixing T-PER tissue protein extraction reagent (Invitrogen, Cat no. #78510) and protease and phosphatase inhibitor tablets (Fisher, Cat no. #PIA32961) according to the manufacturer's instructions. Protein concentration was measured using the Bradford Assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were separated by dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were electrophoretically transferred from the unstained gel to a PVDF membrane (Bio-Rad, Hercules, CA, USA). PKD1 - / - NuPAGE samples for hPSCs detection TMThe gels were run on 3–8% Tris-Acetate protein gels (Invitrogen, EA03785) and a high molecular weight protein ladder (Invitrogen, Cat.no #LC5699) was used on each gel to track the 460 kDa protein. After completion of the transfer, the membranes were blocked with PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% skim milk for 1 h, and the membranes were probed overnight at 4°C with the following primary antibodies: rabbit monoclonal anti-pSer473-Akt (Cell signaling, Catalog no. 9271S), rabbit monoclonal anti-Akt (Cell signaling, Catalog no. 9272S), rabbit monoclonal anti-pThr180 / Tyr182-p38 MAPK (Cell signaling, Catalog no. 4511S), rabbit monoclonal anti-p38 MAPK (Cell signaling, Catalog no. 9212S), rabbit monoclonal Anti-pThr202 / Tyr204-Erk1 / 2 (Cell signaling, Catalog no. 9101S), rabbit monoclonal anti-Erk1 / 2 (Cell signaling, Catalog no. 4695S), rabbit polyclonal anti-pSer2448-mTOR (Cell signaling, Catalog no. 2971S), rabbit monoclonal anti-mTOR (Cell signaling, Catalog no. 2983), rabbit monoclonal anti-pThr41 / Ser45-beta catenin (Thermo Fisher, Catalog no. 702969), mouse monoclonal anti-beta catenin (BD Biosciences, Catalog no. 610154), mouse monoclonal anti-adenylate cyclase V / VI (Santa Cruz, Catalog no. 514785), rabbit monoclonal Anti-c-Myc (abcam, Catalog no.ab32072), rabbit polyclonal anti-PKD1 (MyBioSource, Catalog no. 173900), and mouse monoclonal anti-β-actin (Sigma Aldrich, Catalog no. A5441).

[0175] Secondary antibodies used were goat anti-rabbit (Catalog no. 111-035-003) and donkey anti-mouse IgG (Catalog no. 715-035-151) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Antibody-antigen reactive sites were visualized using enhanced chemiluminescence (WEST-ZOL ® The immunoblots were observed using a fluorescence microscope (Intron Biotechnology, Sungnam, Republic of Korea), and the band densities of the immunoblots were quantified by densitometry using a laser scanner and Quantity One software (Version 5.2, Bio-Rad Laboratory, Berkeley, CA).

[0176]

[0177] Example 6. Statistical Analysis

[0178] Data are expressed as mean ± standard deviation (SD). The Kruskal-Wallis nonparametric test was used to evaluate differences between groups, and the Mann-Whitney U test (GraphPad Software, La Jolla, CA) was used to compare the control and treatment groups, and the treatment and combination therapy groups. A P value < 0.05 was considered statistically significant.

[0179]

[0180] [Experimental Example]

[0181] Experimental Example 1. Modeling Polycystic Kidney Disease Using Kidney Organoids

[0182] To explore the utility of a scalable organoid platform for disease modeling and screening for phenotypic modifiers, we generated mutations in the PKD1 and PKD2 genes associated with autosomal dominant polycystic kidney disease (ADPKD). CRISPR-Cas9 technology has been widely used for precision genome editing in iPSCs to generate human disease models and develop novel therapies. To test PKD1 and PKD2 mutant iPSCs, we used CRISPR-Cas9 technology to generate clones of WTC11 human iPSCs harboring genetic deletions of PKD1 and PKD2. The coding sequences for PKD1 and PKD2 were targeted to generate PKD1 and PKD2 mutant human iPSCs, and directed double-stranded DNA breaks and non-homologous end joining repair were generated via CRISPR-Cas9 in the WTC11 human embryonic stem cell (hESC) line. To introduce deletion mutations in exon 36 of the PKD1 gene and exon 4 of the PKD2 gene, genomic sequence-specific single guide RNAs (sgRNAs) complementary to exon 36 of the PKD1 gene and exon 4 of the PKD2 gene, respectively, were prepared. Sanger sequencing identified clones containing deletions predicted to cause loss of function for both alleles of PKD1 and PKD2 in separate targeting events. As described above, PKD1 - / - and PKD2 - / - The mutation induction process is schematically shown in Figure 1a.

[0183] To demonstrate the expected loss of function for the PKD1 gene, a validated PKD1 protein expression level was used. As shown in Figure 1b, wild-type PKD1 has a predicted molecular weight of 462 kDa, indicating a large molecule. Furthermore, PKD1 mutant-induced cyst formation was confirmed by detecting no PKD1 protein in clone #19 compared to the control group. Therefore, a PKD1 mutant human iPSC cell line was successfully generated using the CRISPR-Cas9 genome editing system.

[0184]

[0185] Experimental Example 2. Generation of iPSC-derived kidney organoids recapitulating human ADPKD.

[0186] To test whether PKD1 and PKD2 could generate ADPKD kidney organoids and cyst-forming phenotypes associated with PKD1 and PKD2 in renal lines, kidney organoids derived from PKD1 and PKD2 mutant human iPSCs were cultured for several days. To study cyst formation under altered shear stress, PKD1 and PKD2 mutant organoids were exposed to fluid shear in a waving plate in an incubator. Quiescent control organoids were cultured with the same volume of medium but with fluid flow treatment. To test whether PKD mutant hESC lines could replicate cyst outgrowth, cyst formation was examined by high-resolution imaging of wild-type, PKD1, and PKD2 mutant organoids cultured for 3 days in mTeSR1 medium. The medium was exchanged in RPMI with 12 μM CHIR99021 for 1.5 days, and cells were fed every 2–3 days to promote renal organoid differentiation in RPMI + B27 supplement.

[0187] As a result, as shown in Figure 1c, in PKD human iPSC cultures, we observed the formation of large, translucent cyst-like structures alongside tubular organoids. The cysts continued to expand in expanded cultures and were eventually isolated from the culture plate after 2 weeks of culture, forming free-growing cystic structures that reached a diameter of more than 1 mm. These findings suggest that PKD1 and PKD2 mutant renal organoids recapitulate human renal ADPKD through abnormal cyst formation from tubular organoids.

[0188]

[0189] Experimental Example 3. Identification of an FDA Drug that Inhibits Cyst Initiation Through Phenotypic Screening

[0190] Kidney organoid technology offers a novel platform for investigating drug therapies for kidney diseases, including ADPKD and chronic kidney disease (CKD). - / - To determine whether mutant human iPSC lines can replicate cell formation, PKD1 - / - Kidney organoids derived from mutant hiPSCs were cultured for several weeks. Furthermore, these lines were PKD1-expressing in the renal lineage. - / - , and cyst formation by laminar fluid shear stress in a wave plate device was investigated. Cysts were recognizable from day 16 and were morphologically distinct from non-cystic structures.

[0191] With the creation of numerous cysts, we screened a library of FDA-approved drugs to identify potential inhibitors of cyst initiation. FDA-approved drugs can provide broader insights into the use of various organoid culture models in the early stages of drug discovery, and may uncover novel mechanisms or approaches for therapeutic approaches beyond the inhibition of cyst formation. Cyst formation was initially inhibited by PKD1 in DMSO (vehicle)-treated mice. - / - It was measured using mutant organoids.

[0192] The primary screening was performed using a single dose of 30 μM in the primary screening for cyst formation, using a library of 2,570 FDA-approved drugs selected from two clusters: antihistamines and SSRIs. Approximately 30 cysts were screened per well, and brightfield microscopic imaging of each cyst was performed daily for 3 days. Two drugs that were shown to inhibit cyst formation were identified: cetirizine, a histamine receptor antagonist, and L-5-hydroxytryptophan, a selective serotonin reuptake inhibitor. Tolvaptan, an AVPR2 inhibitor and the first FDA-approved pharmacological treatment for ADPKD, was included as a control. As shown in Figures 2a and 2b, cyst formation following cetirizine and L-5-hydroxytryptophan treatment was confirmed compared to tolvaptan. While tolvaptan-treated cysts were enlarged, cetirizine and L-5-hydroxytryptophan at 30 μM reduced cyst size. Consistent with the cyst inhibition assay for each drug, co-treatment with 10 μM each of cetirizine and L-5-hydroxytryptophan significantly inhibited PKD1 - / - Significantly reduced cyst growth in mutant cell lines (4 independent experiments, n=4 wells per condition). Values ​​quantified in Fig. 2b are means ± standard deviations (SD), and * indicates PKD1 by post hoc Mann-Whitney U test. - / - Significance for cetirizine, # indicates significance for cetirizine, † indicates significance for L-5-hydroxytryptophan. Consistent with the cyst inhibition assay for each drug, co-treatment with 10 μM each of cetirizine and L-5-hydroxytryptophan significantly reduced PKD1 - / - Significantly reduced cyst growth in mutant cell lines (4 independent experiments, n=4 wells per condition). Values ​​quantified in Fig. 2b are means ± standard deviations (SD), and * indicates PKD1 by post hoc Mann-Whitney U test.- / - Significance for , # indicates significance for cetirizine, † indicates significance for L-5-hydroxytryptophan.

[0193] Also, PKD2 - / - Similarly, in the mutant cell formation, as shown in Figures 3a and 3b, it was confirmed that the cysts treated with tolvaptan were enlarged, whereas 30 μM cetirizine and L-5-hydroxytryptophan reduced the cyst size.

[0194]

[0195] As above, cetirizine and L-5-hydroxytryptophan suppressed cyst size and PKD1 - / - For secondary screening against mutant cysts, each drug was selected and tested at six concentrations (2.5, 5, 10, 30, and 50 μM). Considering the efficacy of the secondary screening, the effective levels of cetirizine and L-5-hydroxytryptophan for inhibiting cyst formation were investigated. As a result, as shown in Figures 4a and 4b, a concentration-dependent inhibition of cyst formation was observed after treatment with cetirizine and L-5-hydroxytryptophan, and the IC 50 It was confirmed that the concentrations were 87.18 μM for cetirizine and 17.5 μM for L-5-hydroxytryptophan.

[0196]

[0197] Experimental Example 4. Confirmation of the Dual Raf-1-MEK-ERK / mTOR Pathway in Controlling Cyst Initiation and Progressive Cyst Growth in ADPKD

[0198] In normal renal epithelial cells, cAMP has been shown to inhibit the Raf-1 / MEK / ERK pathway. However, in ADPKD, cAMP has been shown to stimulate B-Raf (via cSrc-dependent phosphorylation) and activate the MEK / ERK pathway, thereby increasing cell proliferation. These phenotypic changes in response to cAMP are due to the interaction of cAMP and Ca in response to B-Raf, an upstream kinase of the MEK / ERK pathway.2+ It appears to involve crosstalk between PKD1 and cSrc signaling. In addition, the effects of cetirizine and L-5-hydroxytryptophan on intracellular signaling components, including ERK / AKT / B-Raf / Raf-1 and MAPK, which regulate cell proliferation in ADPKD were investigated. As a result, as shown in Figure 5a, PKD1 - / - In mutant cyst formation, the ratios of pAKT / AKT, pMAPK / MAPK, and pERK1 / 2 / ERK1 / 2 were increased, and the ratios were significantly reduced when treated with cetirizine as an antihistamine.

[0199] One of the key components is the mammalian target of rapamycin (mTOR) kinase, a master regulator of protein synthesis, and the PI3K / Akt / mTOR / c-Myc pathway is crucial for the abnormally activated proliferation during ADPKD. As shown in Figure 5a, PKD1 - / - We confirmed that B-Raf, Raf-1, and c-Myc protein expression levels were increased in mutant cyst formation, and that these levels were significantly reduced when treated with cetirizine. However, we confirmed that p-mTOR / mTOR expression, which was similarly increased in cyst formation, was not reduced by cetirizine treatment.

[0200] In addition, we investigated whether treatment with SSRI drugs that regulate the Raf-1 / MEK / ERK and PI3K / Akt / mTOR / c-Myc pathways affects cyst growth. As a result, as shown in Figure 5b, PKD1 - / -In mutant cyst formation, pAKT / AKT, pMAPK / MAPK were significantly increased by L-5-hydroxytryptophan and were associated with enhanced total mTOR expression levels, whereas the expression of c-Myc and phosphorylated mTOR (p-mTOR) was markedly decreased by L-5-hydroxytryptophan treatment. These results suggest that the increased p-mTOR / mTOR expression in cyst formation by cetirizine treatment is a synergistic effect of phosphorylated mTOR, which was markedly reduced by co-treatment with L-5-hydroxytryptophan. The quantified values ​​in Figures 5a and 5b are the mean ± standard deviation (SD), and * indicates PKD1 by Mann-Whitney U test. - / - It indicates significance for .

[0201]

[0202] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0203]

[0204] When cetirizine and L-5-hydroxytryptophan according to the present invention are administered alone or in combination, they have the effect of inhibiting cyst formation in a model of autosomal dominant polycystic kidney disease induced by mutations in the PKD1 or PKD2 gene. Therefore, cetirizine or L-5-hydroxytryptophan according to the present invention can be usefully used as a therapeutic agent for autosomal dominant polycystic kidney disease, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising at least one active ingredient selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof.

2. In paragraph 1, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the composition inhibits cyst formation in the kidney.

3. In paragraph 1 or 2, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the autosomal dominant polycystic kidney disease is caused by a mutation in the PKD1 or PKD2 gene.

4. In any one of paragraphs 1 to 3, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the mutation of the PKD1 gene is a deletion at position 36 of exon 36 of the PKD1 gene.

5. In any one of paragraphs 1 to 4, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the mutation of the PKD2 gene is a deletion at the exon 4 position of the PKD2 gene.

6. In any one of paragraphs 1 to 5, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the above cetirizine has an activity of inhibiting B-Raf or Raf-1 expression or inhibiting phosphorylation of p38-MAPK or ERK1 / 2.

7. In any one of paragraphs 1 to 6, A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, characterized in that the above L-5-hydroxytryptophan has an activity of inhibiting the phosphorylation of mTOR.

8. A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease, comprising cetirizine or a pharmaceutically acceptable salt thereof and L-5-hydroxytryptophan or a pharmaceutically acceptable salt thereof as active ingredients.

9. A food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising at least one active ingredient selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and food-related acceptable salts thereof.

10. A food composition for preventing or improving autosomal dominant polycystic kidney disease, comprising cetirizine or a food-based acceptable salt thereof and L-5-hydroxytryptophan or a food-based acceptable salt thereof as active ingredients.

11. A method for screening a candidate drug for the treatment of autosomal dominant polycystic kidney disease, comprising the following steps: (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells; (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells; (c) a step of observing cyst formation after treating the candidate drug to the renal organoid; and (d) A step of selecting a candidate drug for the treatment of autosomal dominant polycystic kidney disease when the above cyst formation is suppressed compared to before candidate drug treatment.

12. In paragraph 11, A method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, characterized in that the above PKD gene is a PKD1 or PKD2 gene.

13. In paragraph 11 or 12, A method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, characterized in that the mutation of the above PKD1 gene is caused by deleting the exon 36 position of the PKD1 gene using CRISPR / Cas9.

14. In any one of paragraphs 11 to 13, A method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, characterized in that the mutation of the above PKD2 gene is caused by deleting the exon 4 position of the PKD2 gene using CRISPR / Cas9.

15. A method for screening a candidate drug for the treatment of autosomal dominant polycystic kidney disease, comprising the following steps: (a) a step of inducing a mutation in the PKD gene and transfecting the isolated pluripotent stem cells; (b) a step of generating kidney organoids using PKD gene mutant pluripotent stem cells; (c) a step of measuring B-Raf or Raf-1 expression, or a step of measuring phosphorylation of p38-MAPK, ERK1 / 2, or mTOR after treating the candidate drug to the renal organoid; and (d) A step of selecting a candidate drug for treating autosomal dominant polycystic kidney disease when the expression of B-Raf or Raf-1 is reduced compared to before candidate drug treatment, or when the phosphorylation of p38-MAPK, ERK1 / 2, or mTOR is suppressed compared to before candidate drug treatment.

16. In paragraph 15, A method for screening a candidate drug for treating autosomal dominant polycystic kidney disease, characterized in that the above PKD gene is a PKD1 or PKD2 gene.

17. A pharmaceutical composition comprising, as an active ingredient, at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof.

18. A method for preventing, treating, or improving autosomal dominant polycystic kidney disease, comprising administering to a subject in need thereof a therapeutically effective amount of at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof; or a composition containing the same as an active ingredient.

19. Use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof, for use in preventing, treating, or improving autosomal dominant polycystic kidney disease; or comprising the same as an active ingredient.

20. Use of a composition comprising at least one selected from the group consisting of cetirizine, L-5-hydroxytryptophan, and pharmaceutically acceptable salts thereof, for use in the manufacture of a medicament for preventing, treating, or improving autosomal dominant polycystic kidney disease; or an active ingredient thereof.

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