Pharmaceutical composition for treating and / or preventing renal cystic fibrosis

Abstract

There is provided a pharmaceutical composition for use in the treatment and / or prevention of renal cystic fibrosis, the composition comprising a retinoic acid receptor (RAR) agonist.

Description

Technical Field

[0001] The present application relates to a pharmaceutical composition for treating and / or preventing renal cystic ciliopathy. Background Art

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is a refractory genetic disease in which numerous cysts progressively form in the kidneys and progress to end-stage renal failure in middle age or later. The causative genes of ADPKD are PKD1 in 85% of cases and PKD2 in 15% of cases. Studies have been conducted using experimental animals such as disease model mice and rats in which these genes have been altered, but the complete pathogenesis has not been elucidated, and a radical cure has not been developed. The only clinically approved and used vasopressin V2 receptor antagonist, tolvaptan, inhibits cyst enlargement and renal function decline, but its efficacy is limited and it is not a radical cure (Non-Patent Document 1). In addition, patients taking tolvaptan are subject to various behavioral restrictions, such as the need for frequent fluid intake to prevent the development of side effects (such as dehydration and hypernatremia due to its strong diuretic effect), so there is a need to develop a radical therapeutic agent that improves the quality of life of patients.

[0003] In recent years, research has been actively conducted to analyze detailed pathology and search for therapeutic agents by generating iPS cells from somatic cells of patients with refractory diseases, or generating disease-specific iPS cells by introducing mutations into the causative genes of iPS cells derived from healthy individuals, and inducing these cells to differentiate into diseased cell types in vitro to prepare a disease model that reproduces the pathology.

[0004] List of Cited Documents

[0005] Non-Patent Document

[0006] Non-Patent Document 1

[0007] Torres VE et al., N Engl J Med. Dec 20, 2012; 367(25): 2407-2418. Summary of the Invention

[0008] Technical Problem

[0009] An object of the present application is to provide a pharmaceutical composition for treating and / or preventing renal cystic ciliopathy.

[0010] Solution to the Problem

[0011] The present application provides a pharmaceutical composition for treating and / or preventing renal cystic fibrosis, said composition comprising a retinoic acid receptor (RAR) agonist.

[0012] Advantages of the present invention

[0013] The present application provides a pharmaceutical composition for treating and / or preventing renal cystic fibrosis, said composition comprising a retinoic acid receptor (RAR) agonist.

[0014] The inventors of the present invention have for the first time generated an in vitro renal collecting duct cyst model from iPS cells, and thus discovered a pharmaceutical composition for treating and / or preventing renal cystic fibrosis by using said model. Brief description of the drawings

[0015] Figure 1 Figure 1 Shows the shape of cysts cultured on day 5 in the absence (upper right) or presence (lower right) of 0.1 μM TTNPB.

[0016] Figure 2 Figure 2 Shows the cyst size after treatment with 2.5 μM AVP and 0.1% DMSO or 0.1 μM TTNPB. Data from three independent experiments are represented as mean ± s.d. (n = 3). A Student's t-test was performed.

[0017] Figure 3 Figure 3 Shows the cyst size after treatment with 2.5 μM AVP and 0.1% DMSO, 0.1 μM TTNPB or 0.1 μM ATRA. Data from three independent experiments are represented as mean ± s.d. (n = 3). One-way ANOVA with Tukey's test was performed, and * indicates p < 0.005.

[0018] Figure 4 Figure 4 Shows the shape of cysts cultured in the absence (left) or presence (right) of 0.1 μM AM80.

[0019] Figure 5 Figure 5 Shows the cyst size after treatment with 0.1 μM AM80. Data from three independent experiments are represented as mean ± s.d. (n = 3). A Student's t-test was performed.

[0020] Figure 6 Figure 6 ​​​​​​Cyst expansion of cysts cultured in the presence of each of various RAR agonists (CD271, fenretinide, AM580, etretinate, CD1530, Ch55, AGN-195183, and CD5789) at 1 μM is shown.

[0021] Figure 7 Figure 7 is a schematic diagram showing a microarray comparison of cysts cultured on day 5 in the absence (top panel) or presence (bottom panel) of 0.1 μM TTNPB.

[0022] Figure 8 Figure 8 is a volcano plot of DEGs in cysts cultured in the absence or presence of TTNPB (as evaluated by microarray).

[0023] Figure 9 Figure 9 is a GSEA plot of the TGFβ signaling pathway.

[0024] Figure 10 Figure 10 is a heat map of DEGs between cysts cultured in the absence or presence of TTNPB (as evaluated by microarray).

[0025] Figure 11 Figure 11 is a heat map showing that TTNPB increases the expression of cellular senescence genes (as evaluated by microarray).

[0026] Figure 12 Figure 12 is a schematic diagram of the administration of ATRA to ADPKD model mice by intraperitoneal injection.

[0027] Figure 13 Figure 13 Shows the body weights of mice with or without ATRA administration. Data are presented as mean ± s.d. (n = 8). One-way ANOVA with Tukey's test was performed, and there were no significant differences. Non-cystic: Pkd1 flox / - :Ksp-Cre, cystic: Pkd1 flox / flox :Ksp-Cre (ADPKD model).

[0028] Figure 14 Figure 14 Shows the ratio of the two kidney weights (2KW) to the body weight (BW). Data are presented as mean ± s.d. (n = 8). One-way ANOVA with Tukey's test was performed, and * indicates p < 0.01.

[0029] ​​​​​​​​Figure 15 Figure 15 Shows cross-sections of cystic kidneys from P9 Pkd1 flox / flox :Ksp-Cre mice with or without ATRA administration. Scale bar represents 1 mm.

[0030] Figure 16 Figure 16 Shows the cystic index, which indicates the severity of PKD in P9 Pkd1 flox / flox :Ksp-Cre mice after treatment with either vehicle alone (DMSO and sunflower oil) or vehicle containing ATRA. Data are represented as mean ± s.d. (n = 8). Student's t-test was performed, and * indicates p < 0.05.

[0031] Figure 17 Figure 17 Shows the BUN levels of P9 Pkd1 flox / flox :Ksp-Cre mice treated with or without ATRA. Data are represented as mean ± s.d. (n = 8). One-way ANOVA with Tukey's test was performed, and * indicates p < 0.005. Non-cystic: Pkd1 flox / + :Ksp-Cre mice, Cystic: Pkd1 flox / flox :Ksp-Cre mice.

[0032] Figure 18 Figure 18 Shows immunostaining images of cysts cultured in the absence (top panel) or presence (bottom panel) of doxycycline (DOX).

[0033] Figure 19 Figure 19 Shows the size of cysts after addition of doxycycline. Data from three independent experiments are represented as mean ± s.d. (n = 3). Student's t-test was performed.

[0034] Figure 20 Figure 20 Is a schematic diagram of administering AM80 (tamibarotene) to ADPKD model mice by intraperitoneal injection.

[0035] Figure 21 Figure 21 Shows planar images of kidneys from P9 Pkd1 flox / flox :Ksp-Cre mice with or without AM80 (tamibarotene) administration.

[0036] Figure 22 Figure 22 ​​​​​​​​The ratio of two kidney weights (2KW) to body weight (BW) is shown. Data are presented as mean ± s.d. One-way ANOVA with Dunnett's test was performed, and * indicates p < 0.05.

[0037] Figure 23 Figure 23 Shows cross-sections of cystic kidneys from P9 Pkd1 flox / flox :Ksp-Cre mice with or without administration of AM80 (tamibarotene).

[0038] Figure 24 Figure 24 Shows the cystic index, which indicates the severity of PKD in P9 Pkd1 flox / flox :Ksp-Cre mice after treatment with either vehicle alone (DMSO and sunflower oil) or vehicle containing AM80 (tamibarotene). Data are presented as mean ± s.d. One-way ANOVA with Dunnett's test was performed, and * indicates p < 0.05. DETAILED DESCRIPTION

[0039] In the present disclosure, when a numerical value is accompanied by the term "about", it is intended to include a range of ±10% of that value. For example, "about 20" shall include "18 to 22". A numerical range includes all values between the two endpoints and the values at the two endpoints. The term "about" for a range applies to both endpoints of the range. For example, "about 20 to 30" shall include "18 to 33".

[0040] Drug composition

[0041] The present application provides a pharmaceutical composition for treating and / or preventing renal cystic fibrosis, the composition containing a retinoic acid receptor (RAR) agonist.

[0042] ​​Ciliopathies are diseases caused by gene mutations in primary cilia and related structures. Renal cystic ciliopathies refer to ciliopathies complicated by renal cysts. Examples of renal cystic ciliopathies include polycystic kidney diseases, such as autosomal dominant polycystic kidney disease (ADPKD, also known as "autosomal manifest polycystic kidney disease") and autosomal recessive polycystic kidney disease (ARPKD, also known as "autosomal latent polycystic kidney disease"), nephronophthisis (renal consumption disease, nephronophthisis), Joubert syndrome, Barday-Biedl syndrome, Meckel-Gruber syndrome, type I oral-facial-digital syndrome, June syndrome, Senor-Lorken syndrome, and Alstrom syndrome. In one embodiment, the renal cystic ciliopathy is, for example, polycystic kidney disease, and preferably autosomal dominant polycystic kidney disease (ADPKD).

[0043] There is no particular limitation on the retinoic acid receptor (RAR) agonist, as long as it is a substance that binds to RAR and activates RAR. Examples of RAR agonists include 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB, CAS No.: 71441-28-6), all-trans retinoic acid (ATRA, CAS No.: 302-79-4), 9-cis retinoic acid (also known as: alitretinoin, CAS No.: 5300-03-8), 13-cis retinoic acid (also known as: isotretinoin, CAS No.: 4759-48-2), AM80 (also known as: tamibarotene, CAS No.: 94497-51-5), AM580 (CAS No.: 102121-60-8), AC261066 (CAS No.: 870773-76-5), AC55649 (CAS No.: 59662-49-6), AGN-190168 (also known as: tazarotene, CAS No.: 118292-40-3), tazarotenic acid (CAS No.: 118292-41-4), AGN-195183 (CAS No.: 367273-07-2), BMS641 (CAS No.: 369364-50-1), BMS753 (CAS No.: 215307-86-1), BMS961 (CAS No.: 185629-22-5), CD271 (also known as: adapalene, CAS No.: 106685-40-9), CD437 (CAS No.: 125316-60-1), CD1530 (CAS No.: 107430-66-0), CD2314 (CAS No.: 170355-37-0), CD5789 (also known as: tazarotene, CAS No.: 895542-09-3), Ch55 (CAS No.: 110368-33-7), etretinate (CAS No.: 54350-48-0), acitretin (CAS No.: 55079-83-9) and fenretinide (CAS No.: 65646-68-6), as well as their pharmaceutically acceptable salts and their hydrolysable pharmaceutically acceptable esters. In one embodiment, the RAR agonist is at least one selected from the group consisting of TTNPB, ATRA, AM80, AM580, AGN-195183, CD271, CD1530, CD5789, Ch55, etretinate and fenretinide, as well as their pharmaceutically acceptable salts and their hydrolysable pharmaceutically acceptable esters. In a further embodiment, the RAR agonist is at least one selected from the group consisting of AM80, its pharmaceutically acceptable salts and its hydrolysable pharmaceutically acceptable esters. In another embodiment, the RAR agonist is at least one selected from the group consisting of TTNPB, its pharmaceutically acceptable salts and its hydrolysable pharmaceutically acceptable esters.In another embodiment, the RAR agonist is at least one selected from the group consisting of ATRA, its pharmaceutically acceptable salts, and its hydrolysable pharmaceutically acceptable esters.

[0044] The term "pharmaceutically acceptable salt" refers to salts that do not have significant toxicity and can be used as drugs. Examples of pharmaceutically acceptable salts are base addition salts or acid addition salts. Examples of base addition salts include: alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts and iron salts; inorganic salts such as ammonium salts; organic amine salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, and aspartate salts. Examples of acid addition salts include: inorganic acid salts such as hydrochloride salts, sulfate salts, and nitrate salts; and organic acid salts such as mesylate salts, tosylate salts, citrate salts, and oxalate salts.

[0045] The term "hydrolysable pharmaceutically acceptable ester" refers to esters that are hydrolyzed in vivo and includes those that are readily decomposed in the human body to release the parent compound or its salt. Hydrolysable pharmaceutically acceptable esters can be cleaved by esterases in vivo to produce the active compound. Examples of hydrolysable pharmaceutically acceptable esters include lower alkyl esters, lower alkenyl esters, lower alkylamino lower alkyl esters, acylamino lower alkyl esters, acyloxy lower alkyl esters, aryl esters, and aryl lower alkyl esters. The term "lower" means, for example, 1 to 6 or 1 to 4 carbon atoms.

[0046] The hydrolysable pharmaceutically acceptable ester group can also be derived from, for example, pharmaceutically acceptable aliphatic carboxylic acids (including alkanoic acids, alkenoic acids, cycloalkanoic acids, and alkanedioic acids). The alkyl or alkenyl group of the pharmaceutically acceptable aliphatic carboxylic acid can have, for example, 6 or fewer carbon atoms. Specific examples of hydrolysable esters include formate esters, acetate esters, phosphate esters, propionate esters, butyrate esters, acrylate esters, and succinate esters.

[0047] This application also provides a pharmaceutical composition for treating and / or preventing renal cystic fibrosis, which composition contains a vector expressing the CDKN2B gene.

[0048] The vector expressing the CDKN2B gene can be a plasmid vector or a viral vector. When the vector expressing the CDKN2B gene is a plasmid vector, there is no particular limitation on the plasmid vector used, and any plasmid vector such as a cloning plasmid vector or an expression plasmid vector can be used. When the vector expressing the CDKN2B gene is a viral vector, examples of the viral vector used include but are not limited to adenovirus vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, retrovirus vectors, and Sendai virus vectors.

[0049] The vector expressing the CDKN2B gene may have regulatory sequences that regulate the expression of the CDKN2B gene. Examples of regulatory sequences include promoters, terminators, enhancers, polyadenylation signal sequences, and replication origin sequences. The regulatory sequences only need to be arranged to be able to functionally regulate the expression of the vector expressing the CDKN2B gene, and can be arranged based on known methods.

[0050] The pharmaceutical composition of the present application may contain a pharmaceutical carrier or additive. Examples of such carriers or additives include isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericides, antibacterial agents, pH regulators, stabilizers, chelating agents, oil base agents, gel base agents, wetting agents, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifying agents, buffering agents, solubilizing aids, and antioxidants. The pharmaceutical carrier or additive can be used alone or in a mixture of two or more.

[0051] The administration route of the pharmaceutical composition of the present application is not particularly limited, and includes oral or parenteral administration. Depending on the target disease, various known administration forms can be adopted. For example, parenteral administration can be systemic or local administration, and examples thereof include intratracheal administration, intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, and oral administration. In a preferred embodiment, when the subject is an animal, the pharmaceutical composition of the present application is administered intraperitoneally or orally. In a preferred embodiment, when the subject is a human, the pharmaceutical composition of the present application is administered orally.

[0052] Examples of oral dosage forms include granules, fine granules, powders, coated tablets, tablets, suppositories, dispersants, capsules, microcapsules, chewable tablets, liquids, suspensions, and emulsions. As dosage forms for injection administration, general dosage forms of pharmaceutical preparations can be adopted, such as preparations for intravenous injection, preparations for intracoronary administration, preparations for drip administration, and preparations for extended release of active substances. The injectable administration form can be provided in a sealed ampoule or vial, or can be provided as a lyophilized product that only needs to be added with a sterile liquid (such as water for injection) immediately before use. The injection solution or suspension can be prepared from powders, granules, or tablets. These dosage forms are produced by formulating according to common methods.

[0053] The dosage and frequency of administration of the pharmaceutical composition of the present application can be adequately set by those skilled in the art according to the animal species to be subjected to the administration, the health condition, age, body weight, administration route, administration form, etc. of the subject to be administered, so as to administer an effective amount of the active ingredient to the subject. For example, the pharmaceutical composition of the present application can be administered once to several times a day, or once to several times a day or every few days or every week or every few weeks, for example, such as once every one to four weeks. However, the examples are not limited thereto. The effective dose in a given case can be easily determined by routine experiments and is within the skills and judgment of an ordinary clinician. For example, if the RAR agonist is TTNPB, the RAR agonist can be orally administered at about 0.001 to about 100 mg / kg body weight, about 0.01 to about 100 mg / kg body weight, about 0.05 to about 10 mg / kg body weight, or about 0.1 to about 5 mg / kg body weight. For example, if the RAR agonist is ATRA, the RAR agonist can be orally administered at about 0.1 to about 1,000 mg, about 1 to about 1,000 mg, about 10 to about 500 mg, about 50 to about 100 mg, or about 60 to 80 mg per day. For example, if the RAR agonist is 9-cis retinoic acid, the RAR agonist can be orally administered at about 0.1 to about 1,000 mg, about 1 to about 1,000 mg, about 5 to about 100 mg, about 10 to about 50 mg, or about 30 mg per day. For example, if the RAR agonist is 13-cis retinoic acid, the RAR agonist can be orally administered at about 0.001 to about 50 mg / kg body weight, about 0.01 to about 50 mg / kg body weight, about 0.05 to about 10 mg / kg body weight, about 0.1 to about 5 mg / kg body weight, or about 0.5 to about 1 mg / kg body weight. For example, if the RAR agonist is AM80, the RAR agonist can be orally administered at about 0.01 to about 100 mg / m 2 2, about 0.1 to about 100 mg / m 2 2, about 0.5 to about 50 mg / m 2 2, about 1 to about 10 mg / m 2 2, or about 6 mg / m 2 2 of the RAR agonist.

[0054] The pharmaceutical composition of the present application can be used alone or in combination with one or more additional active ingredients. For example, the additional active ingredient is an active ingredient for treating and / or preventing renal cystic fibrosis. The expression "used in combination" not only means using a dosage form containing all ingredients and using dosage forms containing each ingredient alone in combination, but also means administering each ingredient simultaneously or sequentially, or delaying the administration of any ingredient, as long as they are used for treating and / or preventing renal cystic fibrosis. Two or more additional active ingredients can be used in combination.

[0055] As used herein, the term "treatment" means reducing or eliminating the cause of a disease, delaying or halting the progression of a disease, reducing, alleviating, mitigating or eliminating its symptoms, and / or inhibiting the worsening of its symptoms in a subject suffering from the disease.

[0056] As used herein, the term "prevention" means preventing the onset of a disease or reducing the likelihood of the development of a disease in a subject. Herein, the onset of a disease includes recurrence. A subject is, for example, a subject who may develop a disease but has not yet done so. Subjects who may develop polycystic kidney disease but have not yet done so include, for example, subjects with a genetic susceptibility to polycystic kidney disease. Examples of genetic susceptibility to polycystic kidney disease include gene mutations in the following: disease-causing genes for ADPKD (such as PKD1 and PKD2, etc.), disease-causing genes for ARPKD (such as PKHD1), disease-causing genes for nephronophthisis (such as NPHP1 to NPHP13, etc.), disease-causing genes for Joubert syndrome (such as JBTS1 to JBTS17, etc.), disease-causing genes for Bardet-Biedl syndrome (such as BBS1 to BBS15, etc.), disease-causing genes for Meckel-Gruber syndrome (such as MKS1 to MKS10, etc.), disease-causing genes for type I oro-facial digital syndrome (such as OFD1), disease-causing genes for Jeune syndrome (such as IFT80), disease-causing genes for Senior-Loken syndrome (such as NPHP1, NPHP3 to NPHP6, etc.), or disease-causing genes for Alstrom syndrome (such as ALMS1).

[0057] Examples of subjects for treating and / or preventing a disease include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and humans, preferably primates, and more preferably humans.

[0058] Treatment method

[0059] This application also provides a method for treating polycystic kidney disease, which comprises administering the pharmaceutical composition of this application to a subject in need thereof. This application also provides the use of an RAR agonist in the preparation of a pharmaceutical composition for treating polycystic kidney disease. This application also provides an RAR agonist for treating polycystic kidney disease. Examples of polycystic kidney disease and RAR agonists are as described above.

[0060] Prevention method

[0061] The present application also provides a method for preventing polycystic kidney disease, which comprises administering the pharmaceutical composition of the present application to a subject in need thereof. The present application also provides the use of an RAR agonist in the preparation of a pharmaceutical composition for preventing polycystic kidney disease. The present application also provides an RAR agonist for preventing polycystic kidney disease. Examples of polycystic kidney disease and RAR agonists are as described above.

[0062] Example

[0063] The following examples are provided for further description, but the present invention is not limited to these examples in any way.

[0064] [Materials and Methods]

[0065] Preparation of PKD1 knockout-induced pluripotent stem (iPS) cells

[0066] Experiments using human induced pluripotent stem cells were approved by the Ethics Committee of the Faculty of Medicine and Graduate School of Medicine, Kyoto University. The 1383D2 cell line was used as the human iPS cell line. PKD1 knockout iPS cells were prepared using the CRISPR-Cas9-regulated transcription and nuclear shuttle (CRONUS) system based on a previously reported DNA transposon (Ishida K. et al., Sci Rep. 8: 310 (2018); Shimizu T. et al., Biochem Biophys Res Commun. 529, 1186-1194 (2020)). Cells were sequentially transfected with the CRONUS-Puro vector (pPV-TetO-SphcCas9-GR-iC-A-EF1α-rtTA-iP, Addgene ID 100596) and the piggyBac vector for cloning sgRNA (pPV-H1-ccdB-mEF1α-RiH, Addgene ID 100598) together with the piggyBac transposase expression plasmid (pHL-EF1α-hcPBase-A, Addgene ID 100599) by lipid transfection using FuGENE6 (Promega). The CRONUS-Puro vector and the piggyBac vector for cloning sgRNA are compatible with puromycin selection and hygromycin selection, respectively. The following sequence was used: 5'-GAGACCACTTGGATCC GGGATCAGGTCTTCATCTAGGTTTTAGAGCTAGAAATAGCA-3' (SEQ ID NO:1, the target site is underlined), the gRNA sequence targeting the PKD1 exon 34 splice acceptor site was cloned into the pPV-H1-ccdB-mEF1α-RiH vector. RFP + colonies were manually harvested by drug selection of transfected cells and treated with doxycycline and dexamethasone to induce genome editing. Individual colonies were screened for genome editing efficiency by Sanger sequencing of bulk genomic DNA. Single cells were then isolated by flow cytometry and cloned and grown in 24-well culture plates (Corning) coated with iMatrix-511 (Nippi), and then genotyped by Sanger sequencing. Genome editing resulted in the generation of frameshifts and premature stop codons. Two bases (AG) at different sites within exon 34 served as the splice acceptor sequence, resulting in the generation of two types of PKD1 mutant mRNAs. The primer sequences used in this example are listed in Table 1.

[0067] [Table 1]

[0068]

[0069] Induction of PKD1 knockout iPS cells into ureteric bud organoids

[0070] As previously described, PKD1 knockout iPS cells were induced to differentiate into ureteric bud organoids (Mae, SI. & Ryosaka, M. et al. Cell Reports 32, 107963, 2020).

[0071] Preparation of ureteric bud tip cells

[0072] Ureteric bud organoids derived from PKD1 knockout iPS cells were treated with Accutase (Innovative Cell Technologies) at 37 °C for 5 minutes and then dissociated into single cells by pipetting. The cells were suspended in DMEM / F12 medium (Gibco) containing B27 supplement (without vitamin A) (Gibco), 3 μM CHIR99021 (StemRD), 0.1 μM TTNPB (Santa Cruz), 200 ng / ml FGF1 (R&D systems), 100 ng / ml GDNF (R&D systems), 10 μM Thiazovivin (Santa Cruz Biotechnology) and 1 μM A83-01 (Wako). The single cells were seeded at 1.0×10 5Cells were seeded at a density of [number of cells] / well into each well of a 48-well plate coated with 150 μL of hydrogel. The hydrogel was composed of DMEM / F12 medium containing 50% Matrigel (BD Biosciences) and was allowed to solidify at 37 °C for 1 hour before use. After 7 days, single cells formed ureteric bud tip cell colonies. The medium was changed every 2 to 3 days.

[0073] Subculture of ureteric bud tip cells

[0074] The hydrogel was dissolved in cell recovery solution (Corning) at 4 °C for 30 minutes, and then the ureteric bud tip cell colonies were isolated. After further washing with cell recovery solution at 4 °C for 30 minutes, the ureteric bud tip cell colonies were centrifuged at 500 g for 5 minutes at room temperature. The ureteric bud tip cell colonies were treated with Accutase at 37 °C for 5 minutes and then dissociated into single cells by pipetting. The cells were suspended in DMEM / F12 medium containing B27 supplement (without vitamin A), 3 μM CHIR99021, 0.1 μM TTNBP, 200 ng / ml FGF1, 100 ng / ml GDNF, 10 μM Thiazovivin, and 1 μM A83-01. The cells were seeded at a density of 1.0×10 5 Cells were seeded at a density of [number of cells] / well into each well of a 48-well plate coated with 150 μL of hydrogel. The hydrogel was composed of DMEM / F12 medium containing 50% Matrigel and was allowed to solidify at 37 °C for 1 hour before use. Ureteric bud tip cell colonies were prepared by culturing single cells at 37 °C for 7 days in 5% CO 2 The medium was changed every 2 to 3 days. The prepared ureteric bud tip cell colonies were passaged every 7 days in the same manner. The tip cell colonies cultured for 4 to 6 weeks or longer were used to reconstruct the following collecting duct organoids.

[0075] Reconstruction of collecting duct organoids from ureteric bud tip cells

[0076] Dissolve the hydrogel in cell recovery solution at 4°C for 30 minutes to isolate the ureteric bud tip cell colonies. After further washing with cell recovery solution at 4°C for 30 minutes, centrifuge the ureteric bud tip cell colonies at 500 g for 5 minutes at room temperature. Treat the ureteric bud tip cell colonies with Accutase at 37°C for 5 minutes, and then dissociate them into single cells by pipetting. Suspend the cells in Essential 6 medium (Gibco) containing 3 μM CHIR99021, 0.1 μM TTNPB, 200 ng / ml FGF1, 100 ng / ml GDNF, 10 μM Thiazovivin, and 1 μM A83-01. Seed the cells at 5.0×10 3 cells / well into a 96-well low-attachment plate (Sumitomo Bakelite). Culture the single cells at 37°C for 2 days in 5% CO 2 2 to prepare spheroids.

[0077] Suspend the spheroids in Essential 6 medium containing 10% Afamin / Wnt3a conditioned medium (MBL), 200 ng / ml R-spondin1 (R&D systems), 0.1 μM LDN193189 (Axon medchem), 200 ng / ml FGF1, 200 ng / ml FGF8 (Peprotech), 100 ng / ml GDNF, 0.1 μM TTNPB, 50 ng / ml EGF (R&D systems), 1 μM A83-01, and 10% Matrigel, and then dispense them at 2.5 to 3 mL / dish into 35 mm low-attachment dishes (Sumitomo Bakelite). Change the medium every 3 to 4 days. Culture the spheroids at 37°C for 14 to 21 days in 5% CO 2 2 to prepare artificial collecting duct organoids.

[0078] RNA sequencing analysis

[0079] RNA sequence library preparation, sequencing, mapping, and gene expression analysis were performed at DNAFORM. The quality of total RNA was evaluated using a Bioanalyzer (Agilent Technologies), and the RNA integrity number was confirmed to be above 7.0. After enriching poly(A)+ RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs), a double-stranded cDNA library (RNA-seq library) was prepared using the SMARTer Stranded Total RNA Seq Kit v2 PicoInput Mammalian (Clontech) according to the manufacturer's instructions. The RNA-seq library was sequenced on a NextSeq 500 system (Illumina) using paired-end reads (50 nt read 1 and 25 nt read 2). The obtained reads were mapped to the human GRCh38 genome using STAR (version 2.7.2b) or Hisat2 (version 2.1.0). The annotated gene reads were counted using featureCounts (version 1.6.1). FPKM and TPM values were calculated from the mapped reads by normalizing with the total counts. Differentially expressed genes (DEGs) were detected using the DESeq2 package (version 1.26.0). The list of DEGs detected by DESeq2 was used for GSEA. Heatmaps and volcano plots were created using the bioinfokit package (version 2.0.1).

[0080] Microarray analysis

[0081] For comprehensive profiling of transcripts encoding human proteins, microarray analysis was performed at Filgen using the human 8x60K LncRNA Expression Array (ArrayStar). Sample labeling and array hybridization were performed according to the Agilent one-color, microarray-based gene expression analysis protocol (Agilent Technology). Data were collected using Agilent's Feature Extraction software. The raw signal intensities were normalized using the quantile normalization method of GeneSpring GX v12.1. Statistically significantly differentially expressed mRNAs between two groups were extracted by volcano plot drawing, followed by heatmap drawing and GSEA analysis.

[0082] Animal model

[0083] Pkd1 as an ADPKD model mouse flox / flox: Ksp-cre mice progressively develop numerous cysts after birth and die of renal failure at approximately 14 days of age.

[0084] The mouse lineage was maintained with heterozygous Pkd1 flox / + : Ksp-Cre mice, and Pkd1 flox / flox : Ksp-cre mice were generated by mating mice of the same lineage.

[0085] ATRA treatment

[0086] On the third day after birth, ATRA (Sigma, #R2625) dissolved in sunflower oil (solvent) containing 2% DMSO was intraperitoneally administered to Pkd1 flox / flox : Ksp-cre mice (cystic) and Pkd1 flox / + : Ksp-Cre mice (non-cystic) at a dose of 10 mg / kg. A group of mice receiving only the solvent intraperitoneally served as the negative control. The mice were euthanized on the ninth day after birth, and kidney tissues and blood samples were collected.

[0087] AM80 treatment

[0088] On the third day after birth, AM80 (tamibarotene; Tocris, #3507) dissolved in sunflower oil (solvent) containing 2% DMSO was intraperitoneally administered to Pkd1 flox / flox : Ksp-cre mice (cystic) and Pkd1 flox / - : Ksp-Cre mice (non-cystic) at doses of 5 or 10 mg / kg. A group of mice receiving only the solvent intraperitoneally served as the negative control. The mice were euthanized on the ninth day after birth, and kidney tissue samples were collected.

[0089] Measurement of BUN (blood urea nitrogen)

[0090] BUN in mouse serum was measured using the UN-L kit (Serotec, #A667-00).

[0091] Hematoxylin-eosin (HE) staining and measurement of cystic index

[0092] The kidneys were fixed in 4% paraformaldehyde solution, and 4-μm thick paraffin sections were prepared. After dewaxing, HE staining was performed and the kidney tissues were photographed under an optical microscope. The cystic index was calculated by total cystic area / total kidney tissue area × 100 using image analysis and measurement software (WinROOF, Mitani Corporation).

[0093] [Results]

[0094] Examination of compounds that inhibit cyst expansion

[0095] Using the PKD1 knockout iPS cells of the 1383D2 cell line, artificial collecting duct organoids with cystic structures were induced by the above method. The following protocol was used to examine compounds that inhibit cyst expansion.

[0096] 1. Collect the organoids in which cyst formation has occurred in a test tube.

[0097] 2. Remove the medium and add 2 ml of cell recovery solution. (4°C, 30 minutes)

[0098] 3. Dissolve the gel by gently pipetting with a P-1000 pipetteman, then centrifuge. (500 g, 2 minutes)

[0099] 4. After removing the supernatant from 3 and adding 5 ml of medium containing FBS (STO medium), separate the cysts from the organoids by pipetting.

[0100] 5. Using a stereomicroscope, collect the cysts in a 15 ml test tube.

[0101] 6. Remove the supernatant, then add 2 ml of Accutase and incubate at 37°C for 5 minutes.

[0102] 7. Separate the cells into single cells by pipetting with a P-1000 pipetteman.

[0103] 8. After filtration, count the cells.

[0104] 9. After cell counting, transfer the cell suspension containing the required number of cells to a test tube, then add 5 ml of medium containing FBS (STO medium) to stop the Accutase reaction. To seed at 5×10 4 cells / well, calculate the amount of cell suspension required.

[0105] 10. Prepare a pellet by centrifugation (200 g, 5 minutes).

[0106] 11. Thoroughly remove the supernatant, then add DMEM / F12 + B27 w / o V.A. medium (10% Afamin / Wnt3a CM, 200 ng / ml R-Spondin 1, 200 ng / ml FGF1, 10 μM Forskolin, 2.5 μM AVP, 10 μM Y27632) for the suspension. To seed at 5×10 4 cells / well, calculate the amount of medium required.

[0107] 12. Seed the cell suspension from 11 at 5×10 4 cells / well into a pre-prepared 50% Matrigel plate. Slowly add the cell suspension to avoid disrupting the Matrigel.

[0108] 13. Prepare cyst structures by culturing the cells at 37 °C for 2 days in 5% CO 2 .

[0109] 14. Remove the medium and supplement the DMEM / F12 + B27 w / o V.A. medium with 200 ng / ml FGF1 and 2.5 μM AVP, then culture at 37 °C for 3 days in 5% CO 2 .

[0110] 15. Take nine (9) positions inside the well using a 4× objective lens under a fluorescence microscope (Keyence, BZ-X700), measure the cyst area using the BZ-X analyzer, and then calculate the average value.

[0111] Add the compounds that inhibit cyst expansion evaluated in step 14. Add TTNPB (Santa cruz, #sc-203303), AM80 (Cyman, #CAY-71770), and ATRA (Sigma, #R2625) to reach a dose of 0.1 μM. Add RAR agonists (CD271, fenretinide, AM580, etretinate, CD1530, Ch55, AGN-195183, and CD5789) to reach a dose of 1 μM.

[0112] Examine the effect of TTNPB, a potent retinoic acid receptor (RAR) agonist, as a compound that inhibits cyst expansion. Figure 1 Photographs showing the shape of cysts on day 5 of culture in the absence (upper right) or presence (lower right) of 0.1 μM TTNPB are shown. Figure 2 Results quantifying the size of these cysts are shown. TTNPB treatment demonstrated a significant reduction in cyst size.

[0113] TTNPB is an analogue of all-trans retinoic acid (ATRA). ATRA treatment also reduced cyst size ( Figure 3 ).

[0114] Next, examine the effect of AM80 (tacalcitol) as a compound that inhibits cyst expansion. Figure 4 Photographs showing the shape of cysts cultured in the absence (left) or presence (right) of 0.1 μM AM80 are shown. Figure 4Photographs of the cysts in [subject] demonstrated that AM80 reduced the cyst size. Figure 5 The results quantifying the sizes of these cysts were shown. It was demonstrated that AM80 significantly reduced the cyst size. These results indicated that the retinoic acid receptor (RAR) agonist had the effect of inhibiting cyst enlargement.

[0115] Next, the effects of various RAR agonists (CD271, fenretinide, AM580, etretinate, CD1530, Ch55, AGN-195183, and CD5789) were examined. When the cyst enlargement rate at 10 μM forskolin was 100% and the cyst enlargement rate at 0.1 μM rapamycin was 0%, the cyst enlargement rate ([ Figure 6 ) in the case of each RAR agonist (1 μM) was calculated. Compared with DMSO (control 1), it was confirmed that the eight RAR agonists exhibited the effect of inhibiting cyst enlargement. These results indicated that various RAR agonists had the effect of inhibiting cyst enlargement.

[0116] Mechanism of action of TTNPB

[0117] To clarify the mechanism of action of TTNPB, gene expression in cyst cells cultured with and without TTNPB was compared by microarray ([ Figure 7 and 8 ). In [figure], the gray graph on the right showed the genes differentially expressed more than 2-fold in cyst cells cultured in the presence of TTNPB, and the gray graph on the left showed the genes differentially expressed more than 2-fold in cyst cells cultured in the absence of TTNPB. The gene set enrichment analysis (GSEA) results indicated that the "TGFβ signaling pathway" (an important pathway for cyst enlargement and fibrosis) had been inhibited by TTNPB ([ Figure 8 ). Figure 9 )

[0118] The heat map indicated that TTNPB decreased the expression levels of MMP1, SLC2A1, CFTR, and HMGCR ([ Figure 10)。For each gene, the following is known: MMP1 is secreted at high levels in the blood of ADPKD patients (Nakamura T. et al., Am J Nephrol 2000; 20:32-36; Ameku T. et al., Sci Rep 6: 30013 (2016)); reducing glucose uptake in cells by SLC2A1, which decreases energy production in the glycolysis system, thus inhibiting cyst expansion (Rowe I. et al., Nat Med 2013: 488-493); CFTR-mediated cystic fluid secretion causes cyst expansion (Hanaoka K. & Guggino WB., J Am Soc Nephrol 11: 1179-1187, 2000); HMG-CoA reductase inhibitors (statins) inhibit cyst expansion (Zafar I. et al., Am J Physiol Renal Physiol 293: F854-F859, 2007; Cadnapaphornchai MA. et al., Clin J Am Soc Nephrol 9: 889-896, 2014). Thus, the results indicate that TTNPB inhibits the expression of these genes associated with cyst expansion.

[0119] In addition, the heatmap indicates that TTNPB promotes the expression of cell senescence marker genes such as CDKN2B (p15) ( Figure 11 ). This result is consistent with reports that cell senescence inhibits the progression of ADPKD.

[0120] These results suggest that retinoic acid agonists such as TTNPB and ATRA inhibit the expansion of collecting duct cysts. The results also suggest that retinoid signaling is one of the mechanisms for inhibiting cyst expansion.

[0121] Therapeutic effect of ATRA on ADPKD model mice

[0122] Since ATRA has been used as a therapeutic agent for patients with acute promyelocytic leukemia (APL), the efficacy of ATRA in ADPKD model mice was examined from the perspective of drug repositioning. To reflect the in vitro results of ATRA on the expansion of collecting duct cysts in vivo, Pkd1 was used flox / flox: Ksp-Cre mice in which the Pkd1 allele is conditionally inactivated under the expression of the kidney-specific Ksp-Cre recombinase in the distal nephron segment and the collecting duct. The mice develop renal failure from birth due to progressive cyst formation and die at about 2 weeks after birth. Since Ksp cadherin is specifically expressed in the collecting duct and the distal tubule, cysts originating from the collecting duct and the distal tubule are formed in these ADPKD model mice. ATRA was intraperitoneally administered to the mice at 10 mg / kg on the third day after birth (P3), and then the mice were sacrificed at P9 ( Figure 12 ).

[0123] No significant weight loss due to ATRA treatment was observed, indicating that ATRA has no significant side effects ( Figure 13 ). On the other hand, when ATRA has been administered to ADPKD model mice, the ratio of kidney weight to body weight (2KW / BW) was significantly reduced ( Figure 14 ).

[0124] Next, kidney tissue sections were evaluated by hematoxylin-eosin (HE) staining ( Figure 15 ). ATRA treatment inhibited the increase in kidney size and tended to inhibit cyst formation on the cortical side. ATRA treatment significantly reduced the proportion of the area occupied by cysts in the kidney tissue (cystic index) ( Figure 16 ).

[0125] ADPKD model mice showed elevated levels of BUN (blood urea nitrogen), an indicator of renal failure, and renal failure occurred. The BUN levels in the mice treated with ATRA were significantly lower than those in the control mice treated with the vehicle ( Figure 17 ). This result indicates that ATRA treatment inhibits the progression of renal failure.

[0126] These results indicate that ATRA inhibits cyst expansion in the collecting duct and the distal tubule in the in vivo ADPKD model. ATRA has been shown to exhibit a therapeutic effect on both in vitro and in vivo ADPKD models.

[0127] Effect of inhibiting cyst expansion caused by CDKN2B overexpression

[0128] The following protocol was used to examine the inhibitory effect of CDKN2B overexpression on cyst expansion.

[0129] 1. The CDKN2B ORF with a Kozak sequence added was introduced into the PB-TAG-ERN vector (addgene, #80476) by Gateway cloning.

[0130] 2. The above vector and the vector containing PBase were introduced into PKD1 homozygous mutant iPS cells by lipofection.

[0131] 3. Start drug selection with neomycin on the second day after introduction.

[0132] 4. Prepare single-cell-derived colonies from drug-resistant iPS cells by limiting dilution.

[0133] 5. Pick and establish six colonies to prepare renal collecting duct cyst organoids.

[0134] 6. Dissect cysts from the organoids and seed them on 50% Matrigel to form cyst structures in the same manner as in the protocol (step 12) for examining compounds that inhibit cyst expansion.

[0135] 7. After cyst formation (2 days later), add 2 mM doxycycline and culture the cells for another 3 days.

[0136] 8. After imaging under a fluorescence microscope (Keyence, BZ-X700), calculate the average area of the cysts.

[0137] Establish iPS cells in which CDKN2B expression can be conditionally induced by doxycycline to prepare renal collecting duct cysts. Figure 18 Immunostaining images of cysts cultured in the absence (top panel) or presence (bottom panel) of doxycycline are shown. Figure 19 Results quantifying the size of these cysts are shown. It is demonstrated that CDKN2B overexpression by addition of doxycycline significantly reduces cyst size. These results indicate that CDKN2B expression has an inhibitory effect on cyst expansion.

[0138] Therapeutic effect of AM80 on ADPKD model mice

[0139] The efficacy of AM80 was examined in ADPKD model mice. To reflect the in vitro results of AM80 on collecting duct cyst expansion in vivo, Pkd1 flox / flox :Ksp-Cre mice were used, in which the Pkd1 allele was conditionally inactivated under the expression of the renal-specific Ksp-Cre recombinase in the distal nephron segment and collecting duct. These mice develop renal failure from birth due to progressive cyst formation and die at approximately 2 weeks after birth. Since Ksp cadherin is specifically expressed in the collecting duct and distal tubule, cysts originating from the collecting duct and distal tubule are formed in these ADPKD model mice. AM80 was administered intraperitoneally to the mice at 5 or 10 mg / kg on the third day after birth (P3), and the mice were sacrificed at P9 ( Figure 20 ).

[0140] When 10 mg / kg AM80 was administered to ADPKD model mice, the kidney weight-to-body weight ratio (2KW / BW) was significantly reduced ( Figure 21 and22 )。

[0141] Next, renal tissue sections were evaluated by hematoxylin-eosin (HE) staining ( Figure 23 ). Treatment with 10 mg / kg AM80 inhibited the increase in renal size and tended to inhibit cyst formation on the cortical side. Treatment with 10 mg / kg AM80 significantly reduced the proportion of the area occupied by cysts in the renal tissue (cystic index)( Figure 24 )。

[0142] These results indicate that AM80 inhibits cyst expansion in the collecting ducts and distal tubules in the in vivo ADPKD model. It is demonstrated that AM80 exhibits a therapeutic effect on both in vitro and in vivo ADPKD models.

Claims

1. A pharmaceutical composition for treating and / or preventing renal cystic fibrosis, said pharmaceutical composition comprising a retinoic acid receptor (RAR) agonist.

2. The pharmaceutical composition according to claim 1, wherein the RAR agonist is at least one selected from the group consisting of TTNPB, ATRA, AM80, AM580, AGN-195183, CD271, CD1530, CD5789, Ch55, etretinate, and fenretinide, as well as their pharmaceutically acceptable salts and their hydrolysable pharmaceutically acceptable esters.

3. The pharmaceutical composition according to claim 2, wherein the RAR agonist is at least one selected from the group consisting of AM80, its pharmaceutically acceptable salts, and its hydrolysable pharmaceutically acceptable esters.

4. The pharmaceutical composition according to claim 2, wherein the RAR agonist is at least one selected from the group consisting of all-trans retinoic acid (ATRA), its pharmaceutically acceptable salts, and its hydrolysable pharmaceutically acceptable esters.

5. The pharmaceutical composition according to claim 2, wherein the RAR agonist is at least one selected from the group consisting of 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), its pharmaceutically acceptable salts, and its hydrolysable pharmaceutically acceptable esters.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the renal cystic fibrosis is polycystic kidney disease.

7. The pharmaceutical composition according to claim 6, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

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