Compounds for treatment of polycystic kidney disease

Abstract

The invention relates to an adenylate cyclase 1 inhibitor or a K+ ATP channel opener for use in preventing or treating a ciliopathy comprising the step of administering to a subject in need of preventing or treating a ciliopathy, a therapeutically effective amount of an adenylate cyclase 1 inhibitor or a K+ ATP channel opener.

Description

[0001] COMPOUNDS FOR TREATMENT OF POLYCYSTIC KIDNEY DISEASE

[0002] Field of the invention

[0003] The invention relates to an adenylate cyclase 1 inhibitor or a K“ ATP channel opener for use in preventing or treating a ciliopathy comprising the step of administering to a subject in need of preventing or treating a ciliopathy a therapeutically effective amount of the adenylate cyclase 1 inhibitor or a K+ATP channel opener.

[0004] Background of the invention

[0005] Cilia are microscopic, hair-like structures found on the surface of most vertebrate cells, playing crucial roles in various cellular processes. A ciliopathy is a genetic disorder that affects the structure, function, or formation of cellular cilia or their anchoring structures called basal bodies.

[0006] Ciliopathies are an emerging class of genetic multisystemic human disorders that are caused by a multitude of distinct genes that affect ci liary structurc / function. They arc unified by shared clinical features, such as mental retardation, cystic kidney, retinal defects and polydactyly, and by the common localization of the protein products of these genes at or near the primary cilium of cells. The primary cilium is now known to play an important role in the function of many human organs. The current scientific understanding of primary cilia views them as sensory cellular antennae that coordinate many cellular signaling pathways.

[0007] Polycystic kidney disease (PKD) is one of the most common ciliopathies and depicts a group of genetic kidney diseases that are characterized by progressive expansion of fluid-filled cysts in the kidney. Autosomal dominant PKD (ADPKD) and autosomal recessive PKD (ARPKD) represent the most common forms of PKD ADPKD, which typically manifests in adulthood, has an occurrence of 1 in 1000. While mutations of PKD1 and PKD2 account for 75-85% and 15%-20% of ADPKD cases, respectively, 7% of ADPKD pedigrees remain genetically unsolved. ARPKD, primarily caused by mutations in PKHD1 , has an estimated incidence of 1 :20,000, commonly manifests in utero or during neonatal period. Whole exon sequencing has identified a growing list of genes associated with PKD.

[0008] Most ADPKD patients will progress to end stage kidney disease in the fifties or sixties of life, requiring either dialysis or kidney transplantation, while those with ARPKD may face significant complications much earlier in life. Cystic kidney is one of the most prevalent features of ciliopathies attributing to the ciliary location of the affected genes. However, the role of primary cilium in human PKD is not fully understood Studies using animal models and clinical samples suggested that multiple metabolic pathways are dysregulated in PKD, including glycolysis and autophagy. Although primary cilium is an important regulator of autophagy, it is unclear whether the crosstalk between primary cilium and autophagy is dysregulated in PKD. Currently, there are limited treatment options for PKD.

[0009] Current therapeutic approaches for managing PKD are primarily restricted to the detection and treatment of complications. Tolvaptan, a vasopressin 2 receptor blocker, is the only therapy that can slow down cyst growth in ADPKD patients, though the adverse effects and cost may limit its clinical use. Rapamycin and everolimus failed clinical trials in ADPKD, despite their encouraging effects in pre-chnical animal models of ADPKD, highlighting the urgency to develop PKD models with higher pathophysiological relevance to PKD patients to assess suitable means to prevent or treat PKD.

[0010] For that reason, kidney organoids derived from human pluripotent stem (PSC) have been generated containing a genetic mutation in one of the genes of PKD1, PKD2 and PKHD1 and tubuloids, as well as PKD patient induced PSCs (iPSCs)-denved kidney organoids (Cruz et al. (2017), Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. Nat. Mater. 16, 1112-1119. 10.1038 / nmat4994; Hiratsuka et al. (2022), Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery. Sci. Adv. 8, eabq0866. 10.1126 / sciadv.abq0866; Tran et al. (2022), A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery. Cell Stem Cell 29, 1083-1101.e7. 10.1016 / j. stem.2022.06.005; Kuraoka et al. (2020), PKD 1 -Dependent Renal Cystogenesis in Human Induced Pluripotent Stem Cell-Derived Ureteric Bud / Collecting Duct Organoids. J. Am. Soc. Nephrol. JASN 31, 2355-2371; Xu, et al. (2022), Adult human kidney organoids originate from CD24+cells and represent an advanced model for adult polycystic kidney disease. Nat. Genet. 54, 1690-1701 . 10.1038 / s41588-022-01202-z; Shimizu et al. (2020), A novel ADPKD model using kidney organoids derived from disease-specific human iPSCs Biochem. Biophys. Res. Commun 529, 1186-1194. 10.1016 / j.bbrc.2020.06.141; Low et al. (2019), Cell Stem Cell 25, 373-387). Of note, the disclosure of Low et al. (2019) is incorporated by reference in its entirety.

[0011] Despite different causative genes, ADPKD and ARPKD share a multitude of pathogenetic characteristics, providing the foundation for developing therapeutic methods that can target both forms of PKD. Kidney organoids derived from PSCs ofpatients suffering from PKD (PKD organoids) show cyst formation dependent on dysregulated cyclic adenosine monophosphate (cAMP) and calcium signaling. Moreover, primary cilium is an important regulator of autophagy implying a contribution of autophagy to PKD. Studies using both animal models and PKD patient samples revealed that autophagy seems to be suppressed in the PKD kidney tissues. Recent study showed restoring autophagy can effectively inhibit cyst formation in organoid models of PKD (our study published as Liu M et al., “Kidney organoid models reveal cilium-autophagy metabolic axis as a therapeutic target for PKD both in vitro and in vivo . ” Cell Stem Cell. 2024 Jan 4;3 l(l):52-70). Interestingly, in the organoid model of PKD, genetic deletion of primary cilium can boost autophagy, alongside inhibition of kidney cyst formation (Liu et al., 2024). These results indicated that that autophagy represents a promising drug target for treating PKD.

[0012] PKD kidney organoids may also be engrafted into immunocompromised mice as in vivo organoid xenografts where they develop tubular cysts spontaneously corresponding to PKD in humans. This allows for evalution of drugs for an effective in vivo treatment of the disease.

[0013] LS 2019 / 0345494 Al relates to methods for the treatment of polycystic kidney disease, including autosomal dominant polycystic kidney disease, using modified oligonucleotides targeted to miR-17.

[0014] WO 2015 / 056805 Al relates to a combined drug for an injectable depot formulation for preventing and / or treating polycystic kidney disease. More specifically, it relates to an injectable depot formulation comprising a particle containing tolvaptan or a prodrug thereof and a somatostatin analogue. Moreover, a method for preventing and / or treating polycystic kidney disease using the drug is provided.

[0015] However, the adverse effects and costs of prior art may limit their clinical use

[0016] Hence, there is still a need for the efficient and safe treatment of ciliopathies like PKD. The objective of the present invention thus is to provide an improvement or an alternative to the prior art.

[0017] This problem is solved by provision of an adenylate cyclase 1 inhibitor or a K+ATP channel opener for use in the prevention or treatment of a ciliopathy, comprising the step of administering to a subject in need of preventing or treating a ciliopathy, a therapeutically effective amount of an adenylate cyclase 1 inhibitor or a K+ATP channel opener according to claim 1. Specific embodiments are subject matter of further dependent claims.

[0018] Summary of the invention:

[0019] In a first aspect the present invention provides an adenylate cyclase 1 inhibitor or a K+ATP channel opener for use in preventing or treating a ciliopathy, comprising the step of administering to a subject in need of preventing or treating a ciliopathy, a therapeutically effective amount of the adenylate cyclase 1 inhibitor or the K+ATP channel opener.

[0020] The invention prevents the initial development of ciliopathies and / or treats said diseases. In other words, the invention alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or signs of the disease, disorder, and / or condition.

[0021] Administering a therapeutically effective amount means at least the minimum amount of a compound (e.g., a compound of the invention, and / or fonnulations thereof) that elicits a desired biological response when administered as part of a therapeutic regimen. In other words, a therapeutically effective amount refers to the amount in which at least one of the above- mentioned effects of the invention is achieved.

[0022] The compound / s of the invention may be administered in any wax known to the skilled person such as local or systemic administration. Additionally, the compound / s may be administered in its / their pure form or as formulations containing said compound / s.

[0023] According to the invention, the compounds are administered to a subject in need of preventing or treating a ciliopathy. A subject in need of preventing a ciliopathy may refer to subjects which inherited genetic mutations that may result in the induction and progression of ciliopathies but where said ciliopathies have not yet developed. In other words, the compounds of the invention may be administered as a precautionary' measurement that would prevent the formation of ciliopathies. On the other hand, a subject in need of treating a ciliopathy7is a subject in which the ciliopathy has already7manifested which may go along with the detection of symptoms of the ciliopathy.

[0024] In ciliopathies, especially PKD, there is often an upregulation of cAMP signaling. Without wishing to be bound by any theory7, it is believed that, excessive cAMP signaling drives cystogenesis, the formation of fluid-filled cysts, by stimulating abnormal cell proliferation and promoting fluid secretion into cysts, thereby causing PKD progression. cAMP is produced intracellularly by adenylate cyclases, whereby ATP is enzymatically converted to cAMP and diphosphate. The inventors surprisingly found that the reduced cAMP levels mediated by adenylate cyclase inhibition counteracts the detrimental effects of elevated cAMP levels in ciliopathies, thereby providing means for the prevention or treatment of ciliopathies.

[0025] K+ATP channels have a plethora of important roles in cellular function and physiology such as cellular metabolism, its coupling to membrane excitability and cytoprotcction. Moreover, K+ATP channels play a significant role in controlling autophagy.

[0026] Autophagy is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. It allows the orderly degradation and recycling of cellular components. Defects in autophagy have been linked to various human diseases, including neurodegeneration and cancer. On the other hand, in disease, autophagy has been seen as an adaptive response to stress, promoting survival of the cell; but in other cases, it appears to promote cell death and morbidity. Without wishing to be bound by any theory, it is believed that, changes in cellular ion concentrations, particularly calcium, can significantly impact autophagocytosis processes.

[0027] The inventors surprisingly found that activating autophagy inhibited cystogenesis and by using K+ATP channel openers ciliopathies may be prevented or their severity may be reduced.

[0028] Specifically, the inventors found that upon applying either Minoxidil (a K+ATP channel opener) or ST034307 (an adenylate cyclase 1 inhibitor) to kidney organoids that form cysts (resembling the disease progression of PKD which is a ci I io path y ). said cyst formation is attenuated (Figs. 5 and 6). Moreover, the inventors show that systemic treatment of mice that have received a PKD-kidney organoid xenograft with a K+ATP channel opener causes reduced cyst formation (Fig. 10).

[0029] Detailed description of the invention

[0030] It may be provided that the subject of the invention is a mammal. In a preferred embodiment the subject is a human. It is further conceivable within the scope of the invention that the ciliopathy is selected from the group consisting of polycystic kidney disease (PKD), polycystic liver disease (PLD), von Hippel-Lindau disease, nephronophthisis, retinitis pigmentosa, Bardet-Biedl syndrome, Joubert syndrome and Meckel syndrome.

[0031] While there is a plethora of diseases associated with dysfunction of primary cilia or ciliary- related proteins, the above-mentioned disorders are among the most prevalent ciliopaihies. Hereby, PKD is a particularly prevalent ciliopathy.

[0032] In an embodiment of the invention the PKD is an autosomal recessive PKD (ARPKD) or an autosomal dominant PKD (ADPKD). These two forms are the most prevalent forms of PKD, wherein ADPKD is inherited in a dominant fashion, meaning that only one parent needs to be the bearer of the mutation to pass it to the child, whereas ARPKD is inherited in a recessive fashion, meaning that both parents must be the bearer of the mutation and both parents need to inherit it to their child, resulting in a much lower occurrence than ADPKD

[0033] It is further conceivable within the scope of the invention that the ADPKD is primarily associated with a genetic mutation in the gene PKD1 or the gene PKD2, although some cases may also result from mutations in other genes.

[0034] Mutations in the 2 different genes PKD1 and PKD2 cause a very similar disorder of the autosomal dominant form of polycystic kidney disease (ADPKD). PKD1 mutations account for approximately 75-85% of ADPKD cases and generally cause a more severe form of ADPKD compared to PKD2 mutations. Patients with PKD1 mutations typically reach end-stage renal disease at an average age of 54 years, compared to 74 years for PKD2 mutations. The PKD1 gene provides instructions for making polycystin-1 (PCI), a protein involved in kidney development, organization, and function.

[0035] In addition, ADPKD cases may also be associated with less common genes, such as GANAB and DNAJB11.

[0036] ARPKD on the other hand is mainly caused by mutations in the PKHD1 gene, whereas mutations in other genes such as DZIP1L arc also known to cause ARPKD.

[0037] It may be provided that the adenylate cyclase 1 inhibitor or the K+ATP channel opener is given in combination with rapamycin. Rapamycin, also known as sirolimus, is a compound that regulates, among others, cell growth and metabolism, and works primarily by inhibiting the mammalian target of rapamycin (mTOR).

[0038] In PKD kidney organoid models, the inventors could show that combination of either Minoxidil (a K+ATP channel opener) or ST034307 (an adenylate cyclase 1 inhibitor) with rapamycin shows synergistic effects in attenuating cyst formation (see Figs. 7 and 8), wherein the effect is dose-dependent. As such, rapamycin and the additional compound may be administered in an overall lower dose by still achieving an effect on cyst formation. This could allow to circumvent the disadvantage of the limited daily uptake of rapamycin in a human. In other words, whereas rapamycin itself may not be effective on preventing or treating a ciliopathy due to its limited daily uptake, the synergistic effect with either a K+ATP channel opener or an adenylate cyclase 1 inhibitor could potentiate the effect of the tolerated daily rapamycin dose.

[0039] Preferably, rapamycin is administered simultaneously with the other compound Tills can lead to an optimal efficacy and, thereby, maximum synergism so that the doses of each compound may be reduced. However, the invention also encompasses other treatment regimens besides simultaneous administration.

[0040] Nevertheless, the invention is not restricted to a combination of either of the two inventive compounds with rapamycin. For example, an adenylate cyclase 1 inhibitor may for example be combined with a K+ATP channel opener. As such a combinatory treatment with Minoxidil and ST034307 is conceivable within the scope of the invention.

[0041] Furthermore, adenylate cyclase 1 inhibitors may be used in combination with other treatments. While dmgs like tolvaptan (a V2 receptor antagonist) have shown efficacy, combining them with adenylate cyclase 1 inhibitors might provide synergistic effects. In addition, various kinase inhibitors could potentially be combined with adenylate cyclase 1 inhibitors for enhanced therapeutic effects. Moreover, also a combined treatment with 2-Deoxy-D-glucose (2-DG) or metformin may be a suitable approach to prevent or treat ciliopathies.

[0042] In line, a combination of a K+ATP channel opener with tolvaptan, 2-DG or metformin may be a suitable approach to prevent or treat ciliopathies. It is further conceivable within the scope of the invention that a K+ATP channel opener is combined with an adenylate cyclase inhibitor that inhibits other adenylate cyclase isoforms than adenylate cyclase 1. Also, a combination of the inventive compounds with rapamycin analogues (also called rapalogs) such as ridaforolimus, umirolimus, zotarolimus or DL001 is conceivable within the scope of the invention. Rapamycin analogues (rapalogs) are well known to the skilled person and consist of substances that share the basic macrolide ring structure of rapamycin.

[0043] Even more so, any combination of the compounds named hereinabove is conceivable within the scope of the invention in order to treat or prevent ciliopathies, preferably PKD. In other words, a combinatory treatment with a K+ATP channel opener, an adenylate cyclase 1 inhibitor, rapamycin and any of the above-mentioned compounds is conceivable within the scope of the invention.

[0044] According to a preferred embodiment of the invention, the K“ ATP channel opener is a compound having a 2,4-diaminopyrim idine-3-oxide core structure, being preferably selected from the group of compounds consisting of 2,4-diamino-6-pipcridinopyrimidinc-3-oxidc (Minoxidil), Minoxidil sulphate, 2,4-dianiino-6-pyrrolidinopyrimidine-3-oxide (Trimaninodil), Triaminodil sulphate, or a compound of the formula (I): wherein at least one of R1or R2is: and wherein the K+ATP channel opener is more preferable Minoxidil.

[0045] Minoxidil is an FDA-approved drug that is clinically prescribed for managing patterned hair loss and hypertension. Minoxidil is a K+ATP channel opener and activates autophagy by decreasing L-type calcium channel currents. The proven clinical safety of Minoxidil may greatly shorten the timeline of drug repurposing.

[0046] The inventors show that Minoxidil attenuates cyst formation in ARPKD and ADPKD organoids (see Figs. 5 and 6), wherein the effect is dose-dependent. Moreover, the inventors show that systemic administration of Minoxidil in mice that received an ARPKD- or ADPKD-kidncy xenograft results in significant reduction of cysts in the xenograft (see Fig. 10) underlining the efficacy of Minoxidil treatment on the prevention or treatment of ciliopathies, especially PKD.

[0047] According to another preferred embodiment of the invention, the adenylate cyclase 1 inhibitor is a compound selected from the group consisting of

[0048] NB001 SQ22536

[0049] AC10043 AC10048

[0050]

[0051] AC10068 AC10069

[0052]

[0053] The compounds mentioned hereinabove are potent and selective adenylyl cyclase 1 inhibitors. As such administration of either or several of said compounds may cause a reduction in intracellular cAMP levels thereby limiting cAMP signaling which is often elevated in ciliopathics. especially in PKD. Without wishing to be bound by any theory, it is believed that, excessive cAMP signaling drives cystogenesis by stimulating abnormal cell proliferation and promoting fluid secretion into cysts, thereby causing PKD progression.

[0054] In another preferred embodiment of the invention the adenylate cyclase 1 inhibitor is ST034307.

[0055] Compound ST034307 is an inhibitor of adenylate cyclases, with comparably high selectivity towards adenylate cyclase 1. So far, ST034307 has been known to relieve inflammatory pain. The inventors could show that ST034307 significantly attenuated cyst formation in kidney organoids that carry either ARPKD or ADPKD genetic lesion in addition to reducing intracellular cAMP levels (see Fig. 8). It may be provided that the K+ATP channel opener is Minoxidil and that Minoxidil is given in a combination with rapamycin, wherein the dose of rapamycin is between 0.001 and 0.5 mg / kg / day per human and the dose of Minoxidil is between 0.01 and 5 mg / kg / day per human. Of note, the unit describes that a certain amount of the compound, which is measured in mg, is administered per kg bodyweight of a human per day. For example, 0.2 mg / kg / day for a human having a bodyweight of 75 kg would result in a daily dose of 15 mg.

[0056] As described above, the inventors showed a synergistic effect on attenuating cyst formation upon combinatory treatment with Minoxidil and rapamycin (see also Fig. 7). The dose of rapamycin may be 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.50 mg / kg / day per human. The dose of Minoxidil maybe 0.01 , 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 mg / kg / day per human.

[0057] In a preferred embodiment the dose of rapamycin is between 0.01 and 0.03 mg / kg / day per human and the dose of Minoxidil is between 0.2 and 1 mg / kg / day per human.

[0058] In addition, it may be provided that the adenylate cyclase 1 inhibitor is ST034307 and that ST034307 is given in a combination with rapamycin, wherein the dose of rapamycin is between 0.001 and 0.5 mg / kg / day per human and the dose of ST034307 is betw een 0.01 and 5 mg / kg / day and preferably 0.25 and 1.0 mg / kg / day per human.

[0059] As described above, the inventors showed a synergistic effect on attenuating cyst formation upon combinatory treatment with ST034307 and rapamycin (see also Fig. 8). The dose of rapamycin may be 0.001 , 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.50 mg / kg / day per human. The dose of ST034307 may be 0.01, 0.50, 0.75 or 1.0 mg / kg / day per human. Of note, the unit describes that a certain amount of the compound, which is measured m mg, is administered per kg bodyw eight of a human per day. For example, 0.2 mg / kg / day for a human having a bodyweight of 75 kg would result in a dailydose of 15 mg. In a preferred embodiment the dose of rapamycin is between 0.01 and 0.03 mg / kg / day per human and the dose of ST034307 is between 0.25 and 1.0 mg / kg / day per human.

[0060] In a further aspect the invention relates to the Embodiments 1 to 10, as stated below:

[0061] Embodiment 1 : A method for the prevention or treatment of a ciliopathy, comprising the step of administering to a subject in need of preventing or treating a ciliopathy, a therapeutically effective amount of an adenylate cyclase 1 inhibitor or a K+ATP channel opener.

[0062] Embodiment 2: The method according to embodiment 1, wherein the subject is a mammal and preferably a human.

[0063] Embodiment 3 : The method according to embodiment 1 or 2, wherein the ciliopathy is selected from the group consisting of polycystic kidney disease (PKD), von Hippcl-Lindau disease, nephronophthisis, retinitis pigmentosa, Bardet-Biedl syndrome, Joubert syndrome and Meckel syndrome, whereby the ciliopathy is preferably PKD.

[0064] Embodiment 4: The method according to embodiment 3, wherein the PKD is an autosomal recessive PKD (ARPKD) or an autosomal dominant PKD (ADPKD).

[0065] Embodiment 5: The method according to embodiment 4, wherein the ADPKD is associated with a genetic mutation in the gene PKD1 or the gene PKD2.

[0066] Embodiment 6: The method according to one of the above embodiments, wherein the adenylate cyclase 1 inhibitor or the K+ATP channel opener is given in combination with rapamycin.

[0067] Embodiment 7 : The method according to one of the above embodiments, wherein the K+ATP channel opener is a compound having a 2,4-diaminopyrimidine-3-oxide core structure, being preferably selected from the group of compounds consisting of 2,4-diamino-6- piperidinopyrimidine-3-oxide (Minoxidil), Minoxidil sulphate, 2,4-diamino-6- pyrrolidinopyrimidine-3 -oxide (Trimaninodil), Triaminodil-sulphate, or a compound of the (Minoxidil), Minoxidil sulphate, 2,4-diamino-6-pyrrolidinopyrimidine-3-oxide (Trimaninodil), Triaminodil sulphate, or a compound of the formula (1): wherein at least one of R1or R2is: and wherein the K+ATP channel opener is more preferable Minoxidil.

[0068] Embodiment 8: The method according to one of the above embodiments, wherein the adenylate cyclase 1 inhibitor is a compound selected from the group consisting of NB001, SQ22536, NKY80, ST034307, ST072383, AC10042, AC10043, AC10048, AC10049, AC10050,

[0069] AC10051, AC10057, AC10058, AC10067, AC10068, AC10069, AC10070, AC10071,

[0070] AC10072, AC10073, AC10074, AC10075, AC10076, AC10077, AC10079, AC10080,

[0071] AC10081, AC10082 AC10084, AC10085, AC10087, AC10088, AC10089, AC10090,

[0072] AC 10091 and AC10092.

[0073] Embodiment 9: The method according to embodiment 7, wherein the adenylate cyclase 1 inhibitor is ST034307.

[0074] Embodiment 10: The method according to embodiment 6 or 7, wherein the K+ATP channel opener is Minoxidil and is given in a combination with rapamycin, wherein the dose of rapamycin is between 0.001 and 0.5 mg / kg / day per human and the dose of Minoxidil is between 0.01 and 5 mg / kg / day per human.

[0075] Further advantages, features and details of the invention w ill be apparent from the following description, in which embodiments of the invention are described in detail with reference to the figures.

[0076] The foregoing explanation of the embodiments describes the present invention exclusively in the context of examples. Of course, individual features of the embodiments can be freely combined with each other, provided that this is technically reasonable, without leaving the scope of the present invention.

[0077] Definitions: A / an: As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0078] Or / and / or: The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0079] Polycystic kidney disease / PKD: The term "polycystic kidney disease", abbreviated as "PKD", as used herein may refer to a genetic disorder in which the renal tubules become structurally abnormal, resulting in the development and grow th of multiple cysts within the kidney. These cysts may begin to develop in utero, in infancy, in childhood, or in adulthood. PKD generally may refer to any disease summarized under TCD-10 code Q61 , whereas it is preferably referred to 1CD-10 code Q61.1 (autosomal recessive PKD), Q61.2 (autosomal dominant PKD) or Q61.3 (unspecified PKD). Especially preferably it refers to a disease according to 1CD-10 code Q61.1 (autosomal recessive PKD) or Q61.2 (autosomal dominant PKD).

[0080] Adenylate cyclase: The term "adenylate cyclase" as used herein may interchangeably be used with "adenylyl cyclase" and "adenyl cyclase" and refers to enzymes of EC 4.6.1.1 that catalyze the reaction of adenosine triphosphate to 3 ',5 '-cyclic AMP and diphosphate.

[0081] Adenylate cyclase inhibitor: The term "adenylate cyclase inhibitor" as used herein refers to a compound that reduces or blocks the activity of adenylate cyclases (AC). Adenylate cyclase inhibitors may reduce or block the activity of a selective AC isoform, such as adenylate cyclase 1, or tire activity of multiple AC isoforms. The term "adenylate cyclase 1 inhibitor" as used herein refers to a compound that reduces or blocks the activity of adenylate cyclase 1 (ACT). Tire skilled person is aware that adenylate cyclase 1 inhibitors may also reduce or block the activity of other AC isoforms besides AC1. However, AC1 inhibitors show a greater selectivity for AC1 so that reduction of AC activity of other isoforms occurs to a lesser degree than reduction of AC1 activity. The higher degree of AC 1 mhibition is preferably shown by a lower pICjo -value (where pICso is calculated as -log(IC50)) in an in vitro assay for adenylate cyclase activity, such as disclosed in US 11,690,844 B2 which is performed as follows:

[0082] Membranes from Sf9 cells expressing AC1 to AC7 arc prepared as previously described (C. W. Dessauer, Methods Enzymol. 345, 112-126 (2002)). All activity assays are performed for 10 min at 30°C in a final volume of 50 pL. The final concentration of MgClz and Mg-ATP in the reaction are 10 mM and 200 pM, respectively. AC-containing membranes (10-20 pg) are premixed with guanosine 5'-3-O-(thio)triphosphate-Gas(50 nM final). Inhibitors are solubilized in DMSO and incubated with AC-containing membranes for 10 min on ice before the start of the reaction. The final concentration of DMSO in the reaction should not exceed 3% for either vehicle or inhibitors. Reactions are initiated upon addition of a reaction mix containing [a-32P]ATP. The reactions are terminated with stop solution (2.5% SDS, 50 mM ATP, and 1 .75 mM cAMP) and the products are then separated by sequential chromatography on Dowcx-50 and AI2O3 to isolate [32P]cAMP product, using [3H]cAMP to monitor column recovery rates by scintillation counting.

[0083] K+ATP channel: The term "K+ATP channel" as used herein may interchangeably be used with "ATP-scnsitivc potassium channel" or "KATP channel" and refers to a type of potassium channel that is gated by the intracellular nucleotides ATP and ADP.

[0084] K+ATP channel opener: The term "K+ATP channel opener" as used herein refers to compounds that increases the activity of K+ATP channels, i.e., by facilitating the opening of said channels. K+ATP channel opener cause an increased flux of K+ions.

[0085] Local administration / local delivery: As used herein, the term “local administration” or “local delivery”, in reference to delivery of compounds (such as therapeutic agents) described herein, refers to delivery that does not rely upon transport of said compounds to its intended target tissue or site via the vascular system. Said compounds described herein may be delivered directly to its intended target tissue or site, or in the vicinity thereof, e.g., in close proximity to the intended target tissue or site. For example, said compounds may be delivered by injection or by injection or implantation of a device containing the compounds. Following local administration in the vicinity of a target tissue or site, said compounds described herein, or one or more components thereof, may diffuse to the intended target tissue or site. It will be understood that once having been locally delivered a fraction of said compounds described herein (typically only a minor fraction of the administered dose) may enter the vascular system and be transported to another location, including back to its intended target tissue or site.

[0086] Systemic administration: As used herein, the term “systemic administration” and like terms are used herein consistently with their usage in the art to refer to administration of a compound (e.g., a therapeutic agent) such that the compound becomes widely distributed in the body in significant amounts and has a biological effect, e.g., its desired effect, in the blood and / or reaches its desired site of action via the vascular system. Typical systemic routes of administration include administration by (i) introducing the compound directly into the vascular system or (ii) subcutaneous, oral, pulmonary, intraperitoneal or intramuscular administration wherein the compound is absorbed, enters the vascular system, and is carried to one or more desired site(s) of action via the blood.

[0087] Subject: As used herein, the terms “subject”, “test subject” or “patient” refer to any organism to which a provided compound or composition is administered in accordance with the present invention e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and / or susceptible to a disease, disorder, and / or condition.

[0088] Effcctivc / thcrapcutically effective amount: As used herein, the term “effective” or “therapeutically effective amount” means at least the minimum amount of a compound (e.g., a therapeutic agent, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a compound is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound may vary depending on such factors as the desired biological endpoint, the compound to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or signs of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.

[0089] Treatment / treating: “Treatment” or “treating” includes (1) inhibiting a disease, disorder or condition in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and / or symptomatology), (2) ameliorating a disease, disorder or condition in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and / or symptomatology), and / or (3) effecting any measurable decrease in a disease, disorder or condition in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0090] Combination therapy / in combination with: The term “combination therapy”, as used herein, refers to those situations in which two or more different compounds (e.g., a therapeutic agent and / or formulation) are administered in overlapping regimens so that the subject is simultaneously exposed to both compounds. When used in combination therapy, two or more different compounds may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more compounds in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more compounds can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more compounds can be simultaneously administered, wherein the compounds are present in separate formulations. In another alternative, a first compound can be administered followed by one or more additional compounds. In the separate administration protocol, two or more compounds may be administered a few minutes apart, or a few hours apart, a few days apart, or a few weeks apart. In some embodiments, two or more compounds may be administered 1-2 weeks apart.

[0091] Analogues: As used herein the term “analogue” refers to a chemical compound that is structurally similar to another but differs slightly in composition, such as in the replacement of one atom by an atom of a different element or by a functional group.

[0092] Gene: As used herein, the term “gene” refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0093] Synergism / synergistic effect: The terms “synergism” or “synergistic effect” refer to the combined effect of two or more compounds that is greater than the sum of the separate effects of the cancer compounds / thcrapics alone.

[0094] Brief descriotion of the drawings

[0095] Figure 1, comprising Figures A - H, illustrate (A) a schematic of the differentiation protocol, wherein hPSCs were initially treated with CHIR99021 (defining CFUR) for 4 days to induce primitive streak cells, followed by an CFUR treatment in combination with FGF9 from days 7 to 9 (CHIR priming), again followed by continuous FGF9 treatment from days 9 to 20, wherein another CHIR pulse on day 14 was provided (CHIR patterning), eventually followed by incubation without prior treatment for up to 60 days; (B) immunofluorescence analysis for markers of primitive streak (T and MIXL1), nephrogenic intermediate mesoderm (WT1 and H0XD11), nephron progenitor (SALL1 and SIX2), pre-tubular aggregate (LHX1 and PAX8), podocyte (NPHS1), vascular progenitor (KDR), and endothelial cells (CD31) during indicated timepoints of differentiation, scale bars, 200 pm; (C) representative bright-field images of 3D kidney organoids (top panel, day 15 kidney organoid in liquid culture; bottom panel, day 24 kidney organoid in liquid-air interface culture), scale bars, 200 pm; (D and E) whole-mount immunofluorescence analysis (NPHS1, LTL [lotus tetragonolobus lectin], JAG1 [medial tubules], CDH1 [distal tubules], CD31, CD34, DAPI) of 3D kidney organoids (day 24) for epithelial (D), vascular, and epithelial compartments (E); (F) time course analysis of Ffc’GF gene expression (line) and VEGFA protein secretion (bars) during differentiation, data are represented as mean ± SEM (n = 2 independent experiments with 3 technical replicates); (G) comparison of VEGFA gene expression levels in PODOCALYXIN-positive and -negative cells ( PODXL and PODXL+) cells of kidney organoids (day 24), data are represented as mean ± SEM (n = 2 independent experiments with 3 technical replicates), statistical analysis was performed using unpaired Student’s t test; ****p < 0.0001; (H) whole-mount immunofluorescence analysis (NPHS1, CDH1, CD31, DAPI) of 3D kidney organoids (day 24) treated with VEGFR inhibitors for 3 days, scale bars, 200 pm.

[0096] Figure 2, comprising Figures A - B, illustrate (A) a schematic of the in vitro dextran uptake assay, wherein dextran is provided to kidney organoids 4 days after retrieving FGF9 for 4 hours (4 h pulse), live cell culture images were taken thereafter, kidney organoids were then cultured in the absence of dextran for another 24 hours (chase), before being fixed for immunofluorescence analysis; (B) left panels, live images of kidney organoids incubated with fluorescence-labeled dextran of various molecular weights, right panels, whole-mount immunofluorescence analysis (LTL, WT1) of kidney organoids following the dextran update assay, scale bars, 50 pm.

[0097] Figure 3, comprising Figures A - D, illustrate (A) whole-mount immunofluorescence analysis of 3D kidney organoids (day 24) derived from iPSCs of a human individuum having autosomal recessive polycystic kidney disease (ARPKD) and organoids derived from iPSCs where the genetic defect was corrected prior to organoid formation (corrected-ARPKD), scale bars, 500 pm; (B) representative bright-field images of 3D kidney organoids derived from H9 embryonic stem cells (ESCs), corrected-ARPKD iPSCs, and ARPKD iPSCs in the absence or presence of forskolin (FSK) or 8-Br-cAMP for 3 days (days 28-31), scale bars, 500 pm; (C) left panels, time course whole-mount immunofluorescence analysis of ARPKD kidney organoids treated with 10 pm FSK, rightmost panel, representative H&E staining image of ARPKD kidney organoid treated with 10 pm FSK for 7 days (days 28-35), scale bars, 500 pm; (D) immunofluorescence analysis of ARPKD kidney organoids in the absence or presence of 10 pm FSK for 3 days (days 28-31), followed by in vitro dextran uptake assay, white asterisks indicate cysts, scale bars, 50 pm.

[0098] Figure 4, comprising Figures A - F, illustrate (A and B) representative bright-field images (upper panel), intra-cellular cAMP concentration and H&E images (lower panel) of kidney organoids derived from iPSCs of a human individuum having autosomal recessive polycystic kidney' disease (ARPKD, panel A) and of a human individuum having autosomal dominant polycystic kidney' disease (ADPKD, panel B) treated with gradient concentrations of forskolin (FSK) for 3 days, scale bars, 200 pm, data are presented as means ± SD, n=3, **p < 0.01 , ****p < 0.0001, one-way ANOVA with Dunn’s multiple comparison test; (C and D) representative bright-field images of kidney organoids derived from corrected ARPKD iPSCs treated with gradient concentrations of forskolin (FSK) (C) or nifedipine (N1F) (D) for 3 days, scale bars, 200 pm; (E and F) representative bright-field images (upper panel), intra-cellular cAMP concentration and H&E images (lower panel) of kidney organoids derived from ARPKD iPSCs (E) and ADPKD iPSCs (F) treated with gradient concentrations of nifedipine (NIF) for 3 day s, scale bars, 200 pm, data are presented as means ± SD, n=3, **p < 0.01, ****p < 0.0001, oneway ANOVA with Dunn’s multiple comparison test.

[0099] Figure 5, illustrating representative bright-field and H&E images of kidney organoids denved from iPSCs of a human individuum having autosomal recessive polycystic kidney disease (ARPKD), of a human individuum having autosomal dominant polycystic kidney disease (ADPKD) and of kidney organoids derived from genetically engineered H9 embryonic stem cells (ESCs) with homozygous knockout of GANAB (Glucosidase II Alpha Subumt) with indicated drug treatment in the absence or presence of FSK for 3 day's, scale bars, 200 pm.

[0100] Figure 6, comprising Figures A - C, illustrate (A) representative bright-field images of kidney organoids derived from ARPKD iPSCs treated with gradient concentrations of rapamycin, 2- DG or metformin in the presence of FSK for 3 days, scale bars, 200 pm; (B) representative bright-field images of kidney organoids derived from ARPKD iPSCs treated with gradient concentrations of Minoxidil or ST034307 in the presence of FSK for 3 days, scale bars, 200 pm; (C) Western blot analysis of Cleaved Caspase-3 in kidney organoids derived from ARPKD iPSCs with indicated drug treatment in the presence of FSK for 3 days.

[0101] Figure 7, illustrates representative bright-field images of kidney organoids derived from ARPKD iPSCs treated with gradient concentrations of rapamycin and Minoxidil for in the presence of FSK for 3 days compared to a non-cyst-fonnmg non-treated control, scale bars 200 pm.

[0102] Figure 8, comprising Figures A - C, illustrate (A) representative bright-field images of kidney organoids derived from ARPKD iPSCs treated with gradient concentrations of rapamycin and ST034307 in the presence of FSK for 3 days compared to a non-cy st -forming non-treated control, scale bars 200 pm; (B and C) representative bright-field images (B) and intracellular cAMP concentration (C) of kidney organoids derived from ARPKD iPSCs and ADPKD iPSCs treated with FSK or NTF in the presence or absence of ST034307 for 3 days, scale bars, 200 pm, data arc presented as means ± SD, n=3. **p < 0.01, ***p < 0.001, Student’s t-test.

[0103] Figure 9, comprising Figures A - C, illustrate (A) a schematic of the kidney organoid implantation assay; (B and C) representative H&E images (B) and cystic index analysis (C) of ARPKD and corrected ARPKD organoid xenografts, scale bars, 500 pm, data are presented as means ± SD of n = 13 for ARPKD and n = 16 for corrected ARPKD, ****p < 0.0001, Student’s unpaired t-test.

[0104] Figure 10, comprising Figures A - E, illustrate (A) schematic of in vivo drug trial of Minoxidil on kidney organoid xenograft; (B and C) representative H&E images (B) and cystic index analysis (C) of ARPKD kidney organoid xenografts dissected from recipient mouse with 1 mg / kg / day or 5 mg / kg / day of Minoxidil intraperitoneal injection, scale bars, 500 pm, data arc presented as means ± SD, n > 17, *p < 0.05, one-way ANOVA with Dunn’s multiple comparison test; (D and E) representative H&E images (D) and cystic index analysis (E) of ADPKD kidney organoid xenografts dissected from recipient mouse with 1 mg / kg / day or 5 mg / kg / day of Minoxidil intraperitoneal injection, scale bars, 500 pm, data arc presented as means ± SD, n > 20, ****p < 0.0001, one-way ANOVA with Dunn’s multiple comparison test.

[0105] Examples

[0106] Example 1: Generating kidney organoids derived from human induced pluripotent stem cells In a first set of experiments 3D kidney organoids from hPSCs were generated through stepwise exposure to defined differentiation conditions.

[0107] First, hPSCs were treated with 10 mM CHIR99021 (defining CHIR) for 4 days to induce primitive streak cells (T“MIXL1+) with high efficiency (Figs. 1A and IB). To further differentiate primitive streak cells into intermediate mesoderm, a number of culture conditions were tested with the goal of inducing optimal levels of bone morphogenetic protein (BMP) signals because BMPs specify intermediate mesoderm in a dose-dependent manner. It was found that 3 days of factor-free cell culture most effectively drove primitive streak cells toward nephrogenic intermediate mesoderm (HOXDI I VVTI ) (Figs. 1A and IB). Thereafter, the nephrogenic intermediate mesoderm was exposed to 3 mM CHIR (priming CHIR) in the presence of FGF9 (50 ng / mL), leading to the generation of SIX2+SALLl+NPCs (Figs. 1A and I B). These cells self-assembled into clusters that morphologically resembled pre-tubular aggregates (PTAs) (Figs. 1A and IB). These transient, PTA-like structures not only expressed NPC markers (SIX2 and SALL1 ) but also acquired LHX1 and PAX8 expression, indicating the initiation of nephrogenesis (Fig. IB). Meanwhile, a small population of differentiating cells began to express the vascular progenitor marker kinase insert domain receptor (KDR) (Fig. IB). It was not until NPHS1+glomerulus-like structures appeared in the differentiation culture that KDR+cells acquired CD31 expression, indicating vascular maturation (Fig. IB). After 1 day of 1 mM CHIR (patterning CHIR) pulse on day 14, visually distinguishable epithelial structures began to emerge and gradually developed into highly complex tubule structures (Fig. 1C). Byday 24, each 3D kidney organoid became densely packed with multiple nephron components, including podocytes (NPHS I ), proximal tubules (lotus tetragonolobus lectin [LTL+]), medial tubules (JAG1+), and distal tubules (CDH1+) (Fig. I D). Alongside these nephron structures, CD31+CD34+endothelial cells formed a vascular network that spread throughout the entire kidney organoid (Fig. IE). Additionally, the levels of both VEGFA mRNA and secreted VEGFA protein were increased concurrently at the early stage of differentiation and maintained at high levels from day 14 onward (Fig. IF). VEGFA is highly expressed by podocytes, and dysrcgulation of VEGFA expression disrupts glomerular vascular endothelium phenotypes (Eremina et al. (2003) Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J. Clin. Invest. I l l, 707-716). Therefore, antibodies against PODOCALYXIN (PODXL) were used to label and sort podocytes from kidney organoids. Compared with PODXL" cells, PODXL+cells displayed more than 10-fold enrichment in VEGFA expression (Fig. 1G). When VEGFA signaling was disrupted using 3 different VEGF receptor (VEGFR) inhibitors, the established vascular network was severely compromised, whereas nephron structures were not affected (Fig. 1H).

[0108] These observations demonstrate that the protocol described hereinabove robustly differentiates hPSCs into vascularized 3D kidney organoids. Furthermore, differentiation and maturation of the resident vascular network are highly dependent upon autonomous VEGFA production by podocytes.

[0109] Example 2: Functional validation of hPSC-derived kidney organoids

[0110] An in vitro dextran uptake assay was employed to examine whether the kidney organoids described hereinabove exhibited physiologically relevant features (Fig. 2A).

[0111] After 4 h of dextran pulse, hPSC-derived kidney organoids took up 10-kDa and 70-kDa dextrans but excluded 2,000-kDa dextran (Fig. 2B, left panels). Likewise, 24 h later, both 10- kDa and 70-kDa dextrans, but not 2,000-kDa dextran, were retained within LTL+proximal tubule epithelial cells (Fig. 2B, right panels).

[0112] These observations highlight the functionality of hPSC-derived kidney organoids.

[0113] Example 3: ARPKD iPSC-derived kidney organoids recapitulate cystogenesis

[0114] In another set of experiments, it was tested whether the kidney organoids derived from hPSCs of subjects suffering from PKD resemble the disease progression, i.e., forming cysts. In particular, iPSCs from ARPKD patient-derived fibroblasts were used to generate a ARPKD kidney organoid. As a control an isogenic iPSC line was generated where the mutation in the PKHD1 gene was corrected and from which a kidney organoid was generated (corrected ARPKD kidney organoid).

[0115] Both types of iPSCs displayed similar kinetics and efficiency and gave rise to 3D kidneyorganoids comprised of nephron structures that were correctly patterned along the proximal- distal axis (Fig. 3A). ARPKD kidney organoids exhibited drastic cystogenesis upon forskolin- mediated upregulation of intracellular cAMP (Fig. 3B) in a dose-dependent manner. In contrast, corrected-ARPKD kidney organoids showed marginal cyst formation resembling that of wildtype hPSC-derived kidney organoids (Fig. 3B). Dunng cystogenesis, ARPKD kidney organoids showed a time -dep endent enlargement of the tubule lumen, first proximally and later extended to distal regions (Fig. 3C, left panels). Furthermore, there was a gradual reduction in the expression of segment-specific markers as cystogenesis progressed (Fig. 3C, left panels). After 1 week of forskolin treatment, tubule cysts covered more than 80% of the entire kidney organoid, with distorted glomeruli squeezed in between cysts (Fig. 3C, rightmost panel). An in vitro dextran uptake assay was conducted to evaluate the effect of cysts on kidney organoid function. Non-cystic ARPKD kidney organoids took up dextran in a size-selective manner (Fig. 3D, left panels), comparable with wild-type hPSC-derived kidney organoids (see also Fig. 2B). Upon cyst formation, LTL+proximal tubule epithelial cells completely lost the capability to take up dextran, regardless of the molecular weight (Fig. 3D, right panels).

[0116] Collectively, these findings validate the robustness of the differentiation protocol, allowing to extend these efforts to a wider range of genetic kidney diseases and to tailor patient-specific therapeutics.

[0117] Example 4: Kidney organoids derived from ARPKD- or ADPKD-patients develop cysts if treated with forskolin or nifedipine which can be reversed upon correction of the mutations

[0118] Tn another set of experiments the optimal conditions for cyst formation in either ARPKD- or ADPKD-kidney organoids have been tested.

[0119] Varying concentrations of forskolin (FSK) have been applied to ARPKD- (Fig. 4A) or ADPKD-kidney organoids (Fig. 4B) to induce cyst formation. FSK activates the enzyme adenylyl cyclase and increases intracellular levels of cAMP in either ARPKD- (Fig. 4A) or ADPKD-kidney organoids (Fig. 4B) causing the formation of cysts in either model.

[0120] In addition, a calcium channel blocker, nifedipine (NIF), was employed to stimulate cyst formation in both ADPKD- and ARPKD-kidney organoids (Figs. 4E and 4F). Both forskolin and nifedipine upregulated intracellular cAMP levels in a dose-dependent manner, in correlation with cyst severity in PKD kidney organoids (Figs 4A, 4B, 4E and 4F).

[0121] Furthermore, the concentrations applied in said experiments did not cause cyst formation in corrected- ARPKD kidney organoids (Figs. 4C and 4D).

[0122] These results demonstrate that PKD kidney organoid cyst formation relies on the core cystic signaling pathway, in agreement with various animal models of PKD.

[0123] Example 5: Minoxidil and ST034307 attenuate cyst formation in ARPKD and ADPKD kidney organoids In a next set of experiments different drugs were tested for their effect on cyst formation / repression.

[0124] To this end, ARPKD- or ADPKD-kidney organoids were generated as described above as well as kidney organoids derived from genetically engineered H9 embryonic stem cells (ESCs) with homozygous knockout of GANAB (Glucosidase II Alpha Subunit). Mutations in GANAB cause ADPKD. The organoids were treated with FSK for 3 days to induce cyst formation as well as 2-DG, metformin, rapamycin. Minoxidil or ST034307 given simultaneously for the 3 days with their respective doses indicated in the Figure (Fig. 5). Organoids of each PKD-model treated with FSK show clear cyst formation, whereas in control organoids that did not receive FSK cysts remained absent. Treatment with each of the above-described drugs attenuated cyst formation. Of note, treatment with Minoxidil or ST034307 suppressed cyst formation to a similar degree as treatment with 2-DG, metformin or rapamycin, each known to display cyst suppression effects.

[0125] These results demonstrate that cyst formation may be reduced by targeting adenylate cyclases or K+ATP channels

[0126] Example 6: Minoxidil and ST034307 attenuate cyst formation in ARPKD kidney organoids dose-dependently

[0127] In a next set of experiments the drugs of example 5 were applied in different doses to test for dose -dependency.

[0128] The experimental setup is similar to that of example 5 with the difference that solely kidney organoids derived from ARPKD iPSCs were used and that the drugs were applied in different doses as indicated in the Figure (Figs. 6A and 6B). Each of the tested drugs showed dosedependent reduction of cysts in the kidney organoids. Additionally, neither Minoxidil nor ST034307 induced cleaved caspase-3 expression, indicating that treatment with said drugs at the indicated doses does not induce apoptosis (Fig. 6C).

[0129] These results underline that Minoxidil and ST034307 attenuate cyst formation in a specific manner.

[0130] Example 7: Combinatory treatment of Minoxidil or ST034307 with rapamycin attenuates cyst formation in a synergistic manner In a next set of experiments different doses of rapamycin were applied in combination with different doses of either Minoxidil or ST034307 to ARPKD-kidney organoids. Additionally, ST034307 was applied in combination with either FSK or NIF to ARPKD- or ADPKD-kidney organoids.

[0131] The experimental setup is similar to that of example 6 with the difference that the drugs were applied in combination and in doses as indicated in the Figures (Figs. 7 and 8A). Attenuation of cyst formation in ARPKD-kidney organoids is achieved with lower doses of either Minoxidil or ST034307 upon combinatory treatment with rapamycin than by sole treatment with either drug.

[0132] These results demonstrate that Minoxidil as well as ST034307 display a synergism upon combination with rapamycin, wherein the doses needed to attenuate cyst formation are lower compared to sole treatment with either Minoxidil or ST034307. In addition, this indicates that upon combinatory treatment of PKD in vivo the overall doses of the drugs may be reduced, thereby lowering the risk of potential adverse side effects of the drugs.

[0133] In addition, ST034307 suppressed cyst formation in either ARPKD- or ADPKD-kidney organoids alongside significant reduction of intracellular cAMP levels (Figs. 8B and 8C).

[0134] These results imply, that ST034307 mediates attenuation of cyst formation by affecting intracellular cAMP levels.

[0135] Example 8: Implementation of an in vivo organoid xenograft model of ARPKD

[0136] A next set of experiments relates to the implementation of an in vivo organoid xenograft model of ARPKD in immune-compromised mice.

[0137] To this end, on day-28 of differentiation, both ARPKD and control kidney organoids (corrected ARPKD-kidney organoids, see also example 3 hereinabove) were engrafted into the sub-renal capsule space of immune compromised NSG mice (Fig. 9 A). Four weeks post engraftment, the grafts were recovered for histological analysis (Fig. 9A). This xenograft model facilitates the in vivo evaluation of the clinical potential of candidate drugs.

[0138] The model demonstrated specific cyst formation in ARPKD organoid xenograft but not in the gene-corrected organoid xenograft (Figs. 9B-9C). To quantify the amount of cyst formation the cystic index was applied. Cystic index (%) (Cl) refers to the cumulative area of the cysts divided by the total organoid area of the sample. H&E or brightfield images were used to measure the

[0139] CI by ImageJ. Reduction of cystic index was calculated as:

[0140] D,H f r, Reduction of Cystic index = 100%

[0141] These results underscore the feasibility of the model to examine treatment regimes for their efficacy in vivo.

[0142] Example 9: Systemic Minoxidil treatment attenuates cyst formation in an in vivo organoid xenograft model of ARPKD

[0143] In a final set of experiments the in vivo xenograft model of ARPKD described hereinabove was used to assess the efficacy of Minoxidil treatment in reducing cyst formation in the ARPKD xenograft.

[0144] To this end, mice received a xenograft as described above (see example 8). One week after transplantation, Minoxidil was injected intraperitoneally with cither Img / kg / day or 5mg / kg / day for 3 weeks (Fig. 10A). This reduced the cystic index by 18.85% and 33.93%, respectively (Figs. 10B and 10C). While the effect of Img / kg / day intraperitoneal injection of Mmoxidil on cyst formation didn’t reach statistical significance, the effect of 5mg / kg / day intraperitoneal injection of minoxidil significantly reduced cyst formation of ARPKD kidney organoid xenografts (Fig 10C).

[0145] These results strongly support the clinical potential of Minoxidil in the treatment of ARPKD.

[0146] Additionally, the experiment described herein was repeated with ADPKD xenografts. The results were similar to the ARPKD-experiment above, wherein already the Img / kg / day intraperitoneal injection resulted in significantly reduction of cyst formation of ADPKD kidney organoid xenografts (Fig 10D and 10E).

Claims

Claims1. An adenylate cyclase 1 inhibitor or a K+ATP channel opener for use in preventing or treating a ciliopathy comprising the step of administering to a subject in need of preventing or treating a ciliopathy a therapeutically effective amount of the adenylate cyclase 1 inhibitor or the K+ATP channel opener.

2. The adenylate cyclase 1 inhibitor or a K“ ATP channel opener for use according to claim 1 , characterized in that the subject is a mammal and preferably a human.

3. The adenylate cyclase 1 inhibitor or a K ATP channel opener for use according to claim 1 or 2, characterized in that the ciliopathy is selected from the group consisting of polycystic kidney disease (PKD), von Hippel-Lindau disease, nephronophthisis, retinitis pigmentosa, Bardet-Biedl syndrome, Joubert syndrome and Meckel syndrome, whereby the ciliopathy is preferably PKD.

4. The adenylate cyclase 1 inhibitor or a K“ ATP channel opener for use according to claim3, characterized in that the PKD is an autosomal recessive PKD (ARPKD) or an autosomal dominant PKD (ADPKD).

5. The adenylate cyclase 1 inhibitor or a K“ ATP channel opener for use according to claim4, characterized in that the ADPKD is associated with a genetic mutation in the gene PKD1 or the gene PKD2.

6. The adenylate cyclase 1 inhibitor or a K+ATP channel opener for use according to any of the above claims, characterized in that the adenylate cyclase 1 inhibitor or the K+ATP channel opener is given in combination with rapamycin.

7. The adenylate cyclase 1 inhibitor or a K+ATP channel opener for use according to any of the above claims, characterized in that the K+ATP channel opener is a compound having a 2,4-diaminopyrimidine-3-oxide core structure, being preferably selected from the group of compounds consisting of 2,4-diamino-6-piperidinopyrimidine-3-oxide (Minoxidil). Minoxidil sulphate, 2,4-diamino-6-pyrrolidinopyrimidine-3-oxide (Trimaninodil), Triaminodil-sulphate, or a compound of the formula (I):wherein at least one of R1or R2is:and wherein the K+ATP channel opener is more preferable Minoxidil.

8. The adenylate cyclase 1 inhibitor or a K+ATP channel opener for use according to any of the above claims, characterized in that the adenylate cyclase 1 inhibitor is a compound selected from the group consisting ofNBOO1 SQ22536ST072383 AC10042AC10058 AC10067AC10072 AC10073AC10079 AC10080AC10087 AC100889. The adenylate cyclase 1 inhibitor or a K+ATP channel opener foruse according to claim8. characterized in that the adenylate cyclase 1 inhibitor is ST034307.

10. The adenylate cyclase 1 inhibitor or a K+ATP channel opener for use according to claims 6 or 7, wherein the K+ATP channel opener is Minoxidil and is given in a combination with rapamycin. wherein the dose of rapamycin is between 0.001 and 0.5 mg / kg / day per human and the dose of Minoxidil is between 0.01 and 5 mg / kg / day per human.

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