Use of azathioprine for the treatment of cysts

Abstract

The invention relates to purine synthesis inhibitors for use in the treatment or prevention of kidney cysts and liver cysts in a patient with a kidney disease, wherein the patient did not underwent a kidney transplantation.

Description

[0001] USE OF AZATHIOPRINE FOR THE TREATMENT OF CYSTS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the treatment of cysts such as kidney and liver cysts.

[0004] The invention further relates to the repurposing of azathioprine and other purine synthesis inhibitors.

[0005] BACKGROUND OF THE INVENTION

[0006] Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, with a prevalence of 1 in ca. 1000 individuals and accounting for 10% of the European dialysis population. It is most frequently (75-80%) caused by mutations in the PKD1 gene encoding polycystin-1. Besides systemic manifestations such as liver cysts (in 80% of the patients), hypertension and cardiac abnormalities, the most prominent phenotype is the gradual development of kidney cysts, concomitant with kidney enlargement and a decline in kidney function. This leads to end-stage kidney disease at an average age of ca. 50 years. The cysts develop due to dedifferentiation and enhanced proliferation of mutated tubular epithelial cells in all segments of the nephron. cAMP-driven fluid secretion into the lumen further promotes cyst swelling. This process is molecularly regulated by changes in Ca2+, cAMP, PKA and PI3K-Akt-mTOR signalling, ciliary dysfunction, loss of polarity, apoptosis, mitochondrial abnormalities, metabolic reprogramming, inflammation, oxidative stress and epigenetic changes, among others. Despite the occurrence of all these alterations in cystic tissue or cells, their relative importance in the process of cyst formation remain unclear.

[0007] Azathioprine, is an immunosuppressant drug that is routinely used in autoimmune diseases (e.g. Crohn's disease, rheumatoid arthritis) and to prevent rejection after transplantation.

[0008] SUMMARY OF THE INVENTION

[0009] A transcriptomic profile of 40 individual cysts of 4 PKD1 patients together with tracer metabolomics experiments in different kidney cell lines derived from healthy individuals and ADPKD patients identified purine metabolism as a novel pathway that is similarly altered in all ADPKD tissues and cell types. Inhibition of purine metabolism with azathioprine reduced in vitro cyst formation in cystic cell lines derived from 3 different ADPKD patients. The present invention discloses that azathioprine can be used as purine antagonist, in non-transplanted ADPKD patients to attenuate kidney and liver cyst growth and slow down disease progression.

[0010] The invention is further summarised in the following statements:

[0011] 1. A purine synthesis inhibitor for use in the treatment or prevention of cysts in an organ in non-transplanted patients of said organ.

[0012] 2. The purine synthesis inhibitor for use according to statement 1, wherein the cysts are kidney or liver cysts.

[0013] 3. The purine synthesis inhibitor for use according to statement 1 or 2, wherein the cysts are kidney cysts and wherein the patients are individuals with a kidney disease.

[0014] 4. The purine synthesis inhibitor for use according to any one of statements 1 to 3, wherein the cysts are kidney cysts and wherein the patients are individuals with an inherited kidney disease.

[0015] 5. The purine synthesis inhibitor for use according to any one of statements 1 to 4, wherein the cysts are kidney cysts and wherein the patients are individuals with an autosomal dominant polycystic kidney disease.

[0016] 6. The purine synthesis inhibitor for use according to any one of statements 1 to 5, wherein the cysts are kidney cysts and wherein the patients are individuals with autosomal dominant polycystic kidney disease carrying a mutation in the PKD1 gene encoding polycystin-1.

[0017] 7. The purine synthesis inhibitor for use according to any one of statements 1 to 6, wherein the purine synthesis inhibitor inhibits adenosine synthesis.

[0018] 8. The purine synthesis inhibitor for use according to any one of statements 1 to 6, wherein the purine synthesis inhibitor is selected from the group consisting of mycophenolic acid, methotrexate, hadacidin pelitrexol, alanosine, adenine and thioinosine.

[0019] 9. The purine synthesis inhibitor for use according to any one of statements 1 to 6, wherein the purine synthesis inhibitor is a purine analogue such as azathioprine, cladribin, clofarabine, fludarabine, nelarabine, pentostatin and thioguanine.

[0020] 10. The purine synthesis inhibitor for use according to any one of statements 1 to 6, wherein the purine synthesis inhibitor is azathioprine.

[0021] 11. The purine synthesis inhibitor for use in the treatment of kidney cysts according any one of statements 1 to 6, wherein the purine synthesis inhibitor is azathioprine, wherein the patient has an autosomal dominant polycystic kidney disease, and wherein the patient did not underwent a kidney transplantation.

[0022] A method of treating or prevention of cysts in an organ in non-transplanted patients by a administering a purine synthesis inhibitor. Embodiments of the above second medical use claim are equally applicable to this method of treatment.

[0023] DETAILED DESCRIPTION

[0024] Fig. 1: Modulation of altered purine metabolism attenuates in vitro cyst formation.

[0025] A) Fractional m5 labelling in adenosine nucleotides (AMP, ADP, ATP and cAMP), guanosine nucleotides (GMP, GDP, GTP), uridine nucleotides (UMP, UDP, UTP) and cytidine nucleotides (CMP, CDP, CTP) in different control and ADPKD cell models: urine-derived early-stage PTECs (3 controls and 4 ADPKD cells; circle), CDECs (3 controls and 3 ADPKD cells; square), tissue-derived late-stage cells (2 controls and 2 ADPKD cells; triangle),. *p<0.05, **p<0.01 (repeated measurements ANOVA). B) In vitro cyst assay of 3 cystic cell lines (of 3 independent patients) in 50% Matrigel domes treated with DMSO, azathioprine (Aza) or rapamycin (RM). Left: representative images after 20 days of cyst growth; Right: quantification of total cyst area per image over time with different concentrations of azathioprine or 100 nM rapamycin.

[0026] C, D, E) Proliferation assay of 3 independent cystic cell lines treated with various concentrations of azathioprine (Aza) or 100 nM rapamycin (RM).

[0027] F,G) Azathioprine did not affect the outgrowth out of the Matrigel dome. F: representative images of the quantification of the outgrowth over time; G: quantification of the outgrowth in 3 cell lines from different ADPKD patients in the presence of various concentrations of azathioprine (Aza) or rapamycin (RM; 100 nM). H) Dose response curves for the 3 independent cell lines. Left: quantification of the cyst area after 21 days (n = 3 per cell line); Middle: quantification of the cell count after 7 days of proliferation (n = 4 per cell line); Right: quantification of the confluence after 21 days of proliferation and migration out of the Matrigel dome (n = 3 per cell line).

[0028] Fig. 2: Thiamiprine reduced cyst formation of human ADPKD cells in vitro without affecting cell proliferation.

[0029] Panel A:

[0030] Left: cells grown in 50% Matrigel for 21 days, while treated with 0.1% DMSO (control) or 6.25, 12.5 or 25 pM Thiamiprine (Thia).

[0031] Right: Quantification of the total cyst area per image over time. Panel B:

[0032] Left: quantification of cells derived from cystic tissues of ADPKD patients treated with increasing doses of Thiamiprine (Thia), rapamycin (RM) or azathioprine (Aza). Right: Dose-response curve for Thiamiprine and effect on in vitro cyst formation.

[0033] "Non-transplanted" in the context of the present invention is with respect to the organ where in the cyst occurs.

[0034] Thus in the context of kidney cysts the patient is a patient who did not underwent a kidney transplantation.

[0035] Thus in the context of liver cysts the patient is a patient who did not underwent a liver transplantation.

[0036] Treatment in the context of the present invention related to any improvement of one or more of the following parameters in kidney patients. Presence and development of kidney cysts, hypertension, cardiac abnormalities, kidney enlargement and decline in kidney function.

[0037] Azathioprine is a prodrug which is converted to 6-mercaptopurine and inhibits purine synthesis.

[0038] Other purine analogues which are suitable in the context of the present invention are cladribin, clofarabine, fludarabine, nelarabine, pentostatin, thioguanine and Thiamiprine.

[0039] Other compounds, suitable in the context of the present invention are the mmf (mycophenolic acid), methotrexate (indirect by inhibiting folic acid synthesis), hadacidin and analogues thereof, pelitrexol and l-alanosine, adenine (inhibits adenine phosphoribosyltransferase) and thioinosine (inhibits inosine monophosphate dehyd rogenase).

[0040] The present invention discloses RNA-Seq-based transcriptomics using samples from 40 individual kidney cysts from 4 ADPKD patients with PKD1 mutations. A transcriptomic heterogeneity in the cyst samples is described, which correlated with a decrease in tubular markers and an increase in interstitial markers. To evaluate potential targets to modulate cyst formation in ADPKD, focus was on changes occurring in all cysts and all subclusters in a similar rate and without any deviations along the trajectory. Furthermore, significant changes already observed in microcystic tissues are more likely primary events early in cyst development. A similar significant downregulation of purine degradation transcripts was found in all cysts and microcystic tissues, while no significant changes in purine synthesis were observed.

[0041] ADPKD-dependent alterations in purine metabolism were confirmed by tracer transcriptomics in human cell lines derived from urine or kidney tissue of healthy individuals or ADPKD patients. An enhanced m5 labelling was observed (reflecting labelling of ribose) in adenosine-derived nucleotides, and most prominently AMP and its derivate cAMP. As this enhanced labelling was not observed for pyrimidine nucleotides and less prominent in guanosine nucleotides, it indicates a change specific for adenosine nucleotides. Furthermore, the purine breakdown products xanthine and hypoxanthine were reduced in kidney tissues of the PKD1RC / RC mouse model [Hopp etal. (2022) Am J Physiol Rena! Physiol. 322, F258-F267]. Also urinary metabolomics in conditional PKD1 knockout mice confirmed altered purine-metabolism activity [Menezes et al. (2012) PLoS Genet. 8, el003053.], confirming earlier findings in other polycystic kidney models (cystic jck mice [Taylor et al. (2010) Am J Physiol Rena! Physiol. 298, F909-F922] and PCK rats [Abbiss et al. (2012) Nephrology 17, 104-110]). Azathioprine, a general purine-synthesis inhibitor, was able to inhibit cyst growth (Fig. 1) in human patient-derived cystic cell lines at clinically relevant concentrations [Tapner et al. (2004) J Hepatol. 40, 454-463]. In contrast to rapamycin, it did not inhibit proliferation, suggesting another, still elusive, mechanism of action. Based on these data, it is submitted that ADPKD cells have altered purine metabolism, already present in the early stages of the disease, which can be targeted to reduce cyst formation.

[0042] Azathioprine administration (under the brand name Imuran) is a common clinical therapy to treat autoimmune disorders (e.g. Crohn's disease, rheumatoid arthritis), to prevent rejection after transplantation [Dlaz-Villamann et al. (2023) Biomed Pharmacother. 168, 115706], and with potential towards cancer therapy. Although it is generally used as an immunosuppressant, it only affects rapidly proliferating (immune or cancer) cells that are dependent on de novo purine synthesis, leaving differentiated epithelial cells, who rely mostly on purine salvage, mostly undisturbed. However, as the present invention demonstrates, the effect of purine-synthesis inhibition by azathioprine on cyst cells appears to be independent of its effect on proliferation. As components of various molecules, purines have a central role in cell proliferation (DNA), metabolism (e.g. ATP, NAD(P)H, coenzyme A) and signalling (GDP, GTP, cAMP). Since these molecules and pathways are ubiquitously expressed and play central roles in cell physiology, independent of cell type, purine metabolism inhibition by azathioprine appears to affect a general mechanism needed for cystogenesis and thereby affect liver cysts and other cystic diseases as well.

[0043] Homogenous alterations that already occur in early-stage tissue and cells, identified altered purine metabolism as an important hallmark in ADPKD cells and tissues, which can be targeted with azathioprine to reduce cyst formation.

[0044] Example 1. Materials & methods

[0045] Tissue collection- Pieces of cystic membrane were dissected from polycystic kidneys from 4 nephrectomized patients (2 male and 2 female). From each patient, 9-11 cysts were analysed resulting in a total of 40 cysts dissected. From 7 cysts, a second piece was also analysed, resulting in 47 samples in total. Whenever possible, additional pieces were taken for histological analysis. Four microcystic tissues (MCT), defined as tissue from an ADPKD kidney that macroscopically appeared normal, were isolated from 2 other ADPKD patients. All patients had a confirmed PKD1 mutation. Healthy renal parenchymal samples were taken during renal transplantation preimplantation. For RNA extraction, all tissues were snap-frozen in liquid nitrogen and stored at -80°C. All collection of human material was approved by the Ethical Committee of the University Hospitals of Leuven.

[0046] RNA extraction and RNA-Seq- Following RNA extraction using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer's protocol, RNA concentration and quality were determined using DropSense96 (Perkin Elemer) and Bioanalyzer (Agilent). For RNA-Seq library preparation, the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina (Bioke) was used according to the manufacturer's protocol. After quality assessment of the obtained libraries, an equimolar pool was prepared. A qPCR was then carried out using the Kapa SYBR FAST Universal qPCR kit for Illumina (Roche). Depending on the number of reads / samples required, the libraries were sequenced on a HiSeq4000 or a NovaSeq (SI). Quality control of raw reads was performed with FastQC vO.11.7 Adapters were filtered with ea-utils fastq-mcf vl.05. Splice-aware alignment was performed with HISAT2 against the reference genome hg38 using the default parameters. Reads mapping to multiple loci in the reference genome were discarded. Resulting binary alignment map files were handled with Samtools vl.5. Quantification of reads per gene was performed with HT-seq Count vO.10.0, Python v2.7.14. The data was normalized by using the variance stabilisation transformation. Trajectory analysis- The reconstruction of cyst evolutionary trajectories was based on their similarity of transcriptomic information and was performed using Single-cell Trajectories Reconstruction, Exploration And Mapping (STREAM) following the standard published workflow with default parameters [Chen et al. (2019) Nat Commun. 10, 1903].

[0047] Statistical analysis- To analyse the differences between the different subclusters, a linear mixed-model analysis was performed on transformed data (inverse hyperbolic sine) with Tukey adjustments for multiple testing. The residual variability was allowed to differ between the subclusters (constant variance is an assumption in a classical AN OVA).

[0048] Human cell lines include proximal tubular epithelial cells (PTECs) and collecting duct epithelial cells (CDECs) generated from urine or tissue of healthy individuals or genotyped ADPKD patients with a heterozygous frameshift mutation in PKD1. Cystic cells were derived from cystic membranes of nephrectomized PKD1 ADPKD kidneys. Urine-derived cells were from young individuals and early-stage patients (age 5-29 y) and tissue-derived cells from older persons and late-stage patients (age 47-65 y). Informed consent was obtained from all participants and all study procedures were approved by the Ethical Committee of the University Hospitals of Leuven.

[0049] Urine-derived PTECs were immortalized, subcloned and expanded as disclosed in [Decuypere etal. (2021) Int J Mol Sci. 22, 13511]. In short, a pellet from centrifuged urine was grown in supplemented DMEM:F-12. Urine-derived CDECs were collected in the same manner, but the cell pellet was resuspended in a 1:1 mix of Renal Epithelial Cell Growth Medium 2 (PromoCell C26030) supplemented with Growth Medium Supplement Mix (C39606), 5% foetal bovine serum (FBS) and 100 U Pen / Strep, and DMEM high glucose (Gibco 31966-047) supplemented with 1% non-essential amino acids (Gibco 11140-035), 5 ng / ml bFGF (Peprotech 100-18B), 5 ng / ml PDGF-AB (Peprotech 100-00AB), 5 ng / ml EGF (Peprotech AF-100-15), 10% FBS and 100 U Pen / Strep. Primary CDECs were selected by fluorescence-activated cell sorting with an antibody against the collecting-duct marker L1CAM (Abeam ab95694).

[0050] Healthy tissue-derived PTECs and CDECs were isolated from biopsies during a living donor kidney transplantation and cystic tissue-derived cells from cystic membranes of ADPKD kidneys. Healthy tissue was cut and treated with collagenase D (0.67 mg / ml) for 1.5 h, while cystic membranes were subjected to trypsin treatment for 30 min. The treated tissues were then filtered using 125 pm (PTECs, cystic cells) or 90 pm (CDECs) sieves. After centrifugation at 260xg for 7 min, pellets were added to a Percoll gradient (density 1.07 g / ml and 1.04 g / ml) and centrifuged at 1620xg for 25 min with low acceleration and without brake. The layer between the Percoll fractions was then collected, washed and incubated in PTEC or CDEC medium.

[0051] Primary cells were conditionally immortalized with a retroviral construct containing SV40_LT_tsA58. This temperature-sensitive SV40 large T (LT) antigen makes SV40 LT unstable at 37°C. Hence, conditionally immortalized cells were cultured at 33°C. For experiments, monoclonal cell lines were incubated for 10 days at 37°C, which initiated cell differentiation. All cell lines were frequently tested for mycoplasma contamination.

[0052] Tracer metabolomics- DMEM:F12 without glucose, HEPES and glutamine (Biowest L0091) was supplemented withl5 mM HEPES, 2.5 mM glutamine and 17.5 mM D-glucose or13C-labeled D-glucose (Cambridge Isotopes CLM-1396). After 10 days at 37°C, differentiated cells were incubated with medium containing D-Glucose or13C-D-Glucose for 24 h. After washing with 0.9% ice-cold NaCI solution, cells were scraped in ice-cold cellular extraction buffer (80% methanol containing 2 pM d27-myristic acid), centrifuged at 20000xg for 15 min at 4°C and the supernatant was analysed with liquid chromatography-mass spectrometry using the LC Q-Exactive OrbiTRAP. To the pellet, 200 mM NaOH was added and incubated at 95°C for 30 min. After centrifugation (5000 rpm, 4°C, 10 min), the supernatant was used to determine the protein concentration with the bicinchoninic acid assay (Pierce). This protein concentration was used for normalization of the total abundance of the metabolites.

[0053] 3D Cyst assay- Cystic cells were seeded in a 10 pl dome of 50% Matrigel (Corning 356232) at a density of 10000 cells per dome, and the gel allowed to solidify for > 30 min. PTEC medium with azathioprine (0.37 - 10 pM), G6PDi-l (10 pM, Cayman Chemical) or DMSO (0.1%) was added on top. After 4 days at 33°C, 1 pM forskolin (FSK) and 10 pM IBMX were added additionally. Medium and compounds were refreshed twice a week. Images were acquired using the Organoid module in the IncuCyte S3 (Sartorius). Images were then analysed using an in-house developed machine-learning algorithm in NIS5.40 for the detection and measurement of cysts. Cell proliferation measurements- Cystic cells were seeded at 5000 cells per well and incubated at 33°C in the IncuCyte S3 to acquire images every 2 hours. Images were afterwards analysed using the Cell-by-Cell module in the IncuCyte software to determine the cell count.

[0054] EXAMPLE 2. Purine metabolism is similarly altered in ADPKD cystic tissues.

[0055] ADPKD cystic membrane samples, microcystic tissue samples and healthy tissues were analysed by RNA-Seq. The transcriptomic heterogeneity among the cystic tissues was further analysed in more depth via a trajectory reconstruction analysis (STREAM), defining 6 cystic clusters (C1-C6). These clusters and their trajectory are overall characterized by a gradual decrease in tubular markers and a gradual increase in interstitial markers. Among the pathways that were similarly affected in all clusters, purine metabolism was identified. Compared to healthy tissue, purine degradation transcripts were significantly downregulated, while purine synthesis transcripts did not differ significantly in the cyst samples.

[0056] EXAMPLE 3. Purine metabolism is altered in ADPKD cell lines- Metabolomics was performed with13C-labeled D-glucose in human kidney cell lines derived from the urine of young healthy individuals or early-stage ADPKD patients [Decuypere etal. (2014) cited above; Janssens et al. (2021) Kidney Int Rep. 6, 1687-98.]. By culturing urine-derived cells in different media and by sorting based on L1CAM presence, PTECs orCDECs were generated. Also tissue-derived cell lines from healthy kidney tissue or cystic membranes (late-stage ADPKD) were analysed. A significantly enhanced m5 labelling was observed specifically in the adenosine derivates AMP and cAMP in all human ADPKD tubular cell types (early-stage PTECs and CDECs, late-stage cystic cells)(Fig. 1A). The m5 labelling points to the labelling of the ribose in the nucleotide and indicates alterations in the pentose phosphate pathway (PPP) or purine metabolism. A change in PPP was unlikely, since the pyrimidine nucleotides CMP, CDP, CTP, UMP, UDP, UTP did not show enhanced m5 labelling. Additionally, changes in the PPP were not observed in the RNA-Seq of the cysts, labelling of PPP metabolites was not enhanced in ADPKD cell lines, and blocking the PPP with G6PDi-l did not affect in vitro cyst formation.

[0057] EXAMPLE 5. Modulation of purine metabolism attenuates in vitro cyst development

[0058] To analyse whether purine metabolism played a role in cyst development, purine-synthesis inhibitor azathioprine was used in an in vitro cyst assay, using 3 independent human cyst cell lines derived from kidney cystic tissue of 3 nephrectomized patients. Azathioprine attenuated FSK / IBMX-induced cyst formation, and was effective at concentrations around 1 - 3 pM in all 3 cell lines (Fig. IB & 1H). Interestingly, in contrast to the effect of rapamycin, azathioprine did not significantly affect 7-day cyst cell proliferation (Fig. 1C,D,E & 1H), nor did it significantly alter the migration and proliferation of the cells around the in vitro Matrigel dome after 20 days in the in vitro cyst assay (Fig. 1FG & 1H). This suggest that purine-metabolism inhibition attenuated cyst growth, but not via proliferation or migration. Example 6. Thiamiprine reduced cyst formation of human ADPKD cells in vitro without affecting cell proliferation

[0059] In addition to Azathriopine, Thiamiprine reduced in vitro cyst formation in human ADPKD cells derived from cystic tissues of ADPKD patients in a dose-dependent manner as shown in Figure 2. The left part of panel A shows representative images of the cells grown in 50% Matrigel for 21 days, while treated with 0.1% DMSO (control) or 6.25, 12.5 or 25 pM Thiamiprine (Thia). Cyst swelling was induced in all conditions with lOpM forskolin and lOOpM IBMX. The right part of panel A shows quantification of the total cyst area per image over time. A reduction in cyst formation is observed starting from 12.5pM Thia. Rapamycin (RM) and Azathioprine (Aza) are used as controls. Only at higher doses (50 and lOOpM), Thiamiprine affected proliferation as shown in figure 2B. Left panel shows the representative quantification of cells derived from cystic tissues of ADPKD patients treated with increasing doses of Thiamiprine (Thia), rapamycin (RM) or azathioprine (Aza). The right part of panel B shows the dose-response curve for Thiamiprine showing that a reducing affect is observed at 50 and lOOpM, but not at the lower doses that affect in vitro cyst formation.

[0060] EXAMPLE 7. In vivo experiments

[0061] To further justify the administration of azathioprine to ADPKD patients before transplantation, in vivo mice experiments are performed. Azathioprine is administered in an orthologous PKDlnl / nlmouse model, which show reduced expression of PKD1 and progressive kidney cyst formation in all tubular segments within 4 weeks of birth [Happe et al. (2013) Kidney Int. 83, 1099-1108]. This model also shows mild cystogenesis in the liver [Lantinga-van Leeuwen et al. (2004) Hum Mol Genet. 13, 3069-3077]. Other mouse models are Pkdl KI RC / RC model (Hopp et al. (2012) J Clin Invest 122, 4257-73), the Pkdlnl model (Lantinga-van Leeuwen et al. (2004) Hum Mol Genet 13, 3069-3077), and in the Cre / lox model (Lantinga-van Leeuwen et al. (2007) Hum Mol Genet 16, 3188-3196).

[0062] Azathioprine is administered orally via drinking water at a concentration of 80mg / L Given that a mouse weighs approximately 20-30 g and drinks approximately 4-8 mL of water per day, this corresponds to 12-40 mg / kg body weight(BW) / day. This is equivalent to a human estimated dose of 1-3.24 mg / kg, which is in line with the human oral daily dose of 1-3 mg / kg. Pilot experiments in wild-type and PKDlnl / nlmice (n = 5 per group) are first conducted to assess the animal's conditions (survival, weight, drinking behaviour) and the presence of azathioprine metabolites in (e.g. 6-MP) in the kidneys and livers. Based on these preliminary experiments, power analysis determines the number of animals needed to study the effect. End points to assess the effect of azathioprine are survival, kidney weight / BW, cystic index and kidney function. Secondary endpoints include kidney fibrosis and liver cysts and fibrosis. Blood, urine and kidney / liver tissues is collected for additional molecular analyses of biomarkers and purine metabolism.

[0063] These treatments are tested in 3 different PKD mouse strains, from mild progression to severe. Administration starts at p21, when the animals are weaned and can drink and eat independently. In the Pkdl KI RC / RC model (Hopp et al. (2012) J Clin Invest 122, 4257-73), animals are followed up for 1 year after first administration with bimonthly in vivo imaging; in the Pkdlnl model (Lantinga-van Leeuwen et al. (2004) Hum Mol Genet 13, 3069-3077), mice are followed up for 3 months with monthly imaging; and in the Cre / lox model (Lantinga-van Leeuwen et al. (2007) Hum Mol Genet 16, 3188-3196), cyst formation is induced by injecting tamoxifen 3 consecutive days before first administration and animals are followed up for 2 months, with an in vivo imaging session in between. At the end of the follow-up, mice are be sacrificed, kidneys are collected, weighed and processed for histological and molecular analysis.

[0064] EXAMPLE 7. Clinical trial study

[0065] In addition, a exploratory, randomized, double-blind, placebo-controlled, parallel group, multicentre, proof of concept study is initiated to evaluate orally administered azathioprine in ADPKD patients. 60 ADPKD patients with GFR>60 (n=40 for azathioprine and n=20 for placebo) that will be followed up for 6 months, are enrolled taking a standard daily dose of 1 mg / kg BW azathioprine (Imuran) orally. Posttransplantation patients are excluded. TPMT and NUDT15 deficiency is tested before enrolment. Primary endpoint is height-adjusted total kidney volume (htTKV), measured by MRI. Secondary endpoints include kidney function (eGFR) and liver cysts.

Claims

CLAIMS1. A purine synthesis inhibitor for use in the treatment or prevention of kidney cysts and liver cysts in a patient with a kidney disease, wherein the patient did not underwent a kidney transplantation.

2. The purine synthesis inhibitor for use according to claim 1, in the treatment or prevention of kidney wherein the patients are individuals with an inherited kidney disease.

3. The purine synthesis inhibitor for use according to claim 1 or 2, in the treatment or prevention of kidney cysts wherein the patients are individuals with an autosomal dominant polycystic kidney disease.

4. The purine synthesis inhibitor for use according to any one of claims 1 to 3, in the treatment or prevention of kidney cysts wherein the patients are individuals with autosomal dominant polycystic kidney disease carrying a mutation in the PKD1 gene encoding polycystin-1.

5. The purine synthesis inhibitor for use according to any one of claims 1 to 4, wherein the purine synthesis inhibitor is a purine analogue selected from the group consisting of azathioprine, cladribin, clofarabine, fludarabine, nelarabine, pentostatin and thioguanine.

6. The purine synthesis inhibitor for use according to any one of claims 1 to 5, wherein the purine synthesis inhibitor is azathioprine.

7. The purine synthesis inhibitor for use in the treatment of kidney cysts according any one of claims 1 to 6, wherein the purine synthesis inhibitor is azathioprine, wherein the patient has an autosomal dominant polycystic kidney disease, and wherein the patient did not underwent a kidney transplantation.

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