WO2025011999A1 - A3 adenosine receptor ligands for use in the treatment of diseases associated with ocrl gene disfunction - Google Patents

Abstract

ORCL deficiency, such as Lowe syndrome or Dent disease, is treated by an A₃ adenosine receptor ligand such as piclidenoson or namodenosone.

Description

A3 ADENOSINE RECEPTOR LIGANDS FOR USE IN THE TREATMENT OF DISEASES ASSOCIATED WITH OCRL GENE DISFUNCTION

FIELD OF THE INVENTION

This invention relates to methods and compositions comprising A3AR ligands for treating genetic diseases.

REFERENCES

The following is a list of some references believed to be relevant for the understanding of the state of the art in the field of this invention.

1. Attree, O. et al. The Lowe’s oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate- 5 -phosphatase. Nature 358, 239- 242 (1992).

2. Lowe, C., Terrey, M. & MaClachlan, E. A. Organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity. AMA Am J Dis Child 83, 164-184 (1952).

3. Zaniew, M. et al. Long-term renal outcome in children with OCRL mutations: Retrospective analysis of a large international cohort. Nephrology Dialysis Transplantation 33, 85-94 (2018).

4. de Matteis, M. A., Staiano, L., Emma, F. & Devuyst, O. The 5-phosphatase OCRL in Lowe syndrome and Dent disease 2. Nat Rev Nephrol 13, (2017).

5. Bbkenkamp, A. et al. Dent-2 disease: a mild variant of Lowe syndrome. J Pediatr 155, 94-99 (2009).

6. Hoopes, R. R. et al. Dent Disease with mutations in OCRL1. Am J Hum Genet 76, 260-267 (2005).

7. Shrimpton, A. E. et al. OCRL1 mutations in Dent 2 patients suggest a mechanism for phenotypic variability. Nephron Physiol 112, (2009).

8. ben El Kadhi, K., Emery, G. & Carreno, S. The unexpected role of Drosophila OCRL during cytokinesis. Comrnun Integr Biol 5, 291-293 (2012). Dambournet, D. et al. Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis. Nat Cell Biol 13, 981-988 (2011). de Leo, M. G. et al. Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL. Nat Cell Biol 18, (2016). Vicinanza, M. et al. OCRL controls trafficking through early endosomes via PtdIns4,5P(2)-dependent regulation of endosomal actin. EMBO J 30, 4970-4985 (2011). Reitblat et al. The efficacy and safety of Piclidenoson vs Methotrexate in early Rheumatoid Arthritis: Phase 3 randomized, double-blind, placebo-controlled Study. Arthritis Rheumatol. 73, suppl 10 (2021). David et al. Treatment of plaque-type psoriasis with oral CF101: Data from a Phase II/III multicenter, randomized, controlled trial. J. Drugs in Dermatology Vol. 15, Issue 8, 931-938 (2016). Bothwell, S. P. et al. Mouse model for Lowe syndrome/Dent Disease 2 renal tubulopathy. J Am Soc Nephrol 22, 443-448 (2011). Zhang, J. H., Chung, T. D. Y. & Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73 (1999). de Leo, M. G. et al. Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL. Nat Cell Biol 18, 839- 850 (2016). Bothwell, S. P., Farber, L. W., Hoagland, A. & Nussbaum, R. L. Speciesspecific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome. Mamm Genome 21, 458-466 (2010). Festa, B. P. et al. OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease. Hum Mol Genet 28, 1931-1946 (2019). Berquez, M. et al. The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease. Kidney Int 98, 883-896 (2020). 20. Stauffer, T. P., Ahn, S. & Meyer, T. Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Current Biology 8, 343-346 (1998).

21. Tall, E. G., Spector, I., Pentyala, S. N., Bitter, I. & Rebecchi, M. J. Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures. Current Biology 10, 743-746 (2000).

BACKGROUND

The Lowe syndrome (LS) is a rare genetic disease (1 in 500,000 births) caused by mutations in the OCRL (oculocerebrorenal syndrome of Lowe) gene on the X chromosome. The disease affects almost exclusively males even though cases of female patients have been reported. LS affects the eyes, central nervous system (CNS), and kidney. Eyes: congenital cataracts and glaucoma in 50% of cases; CNS: severe central hypotonia at birth, cognitive disability of different extent and behavioral abnormalities, which worsen with age, seizures in 30-50% of patients; Kidney: LS dysfunction of the proximal tubule (also referred to as proximal renal tubulopathy, or renal Fanconi syndrome) of variable severity, characterized by low-molecular-weight (LMW) proteinuria and inappropriate loss of solutes including amino acids, glucose, phosphate, and calcium. The dysfunction of the proximal tubule (PT) is often complicated by chronic kidney disease (CKD) and kidney failure. The life expectancy is approximately 40 years with death usually secondary to CKD and its related complications [1-4].

Presently there is no cure for LS: cataracts are surgically removed, the kidney manifestations are treated symptomatically, psychosocial and motor development is enhanced using behavioral approaches.

Dent disease 2, which is also caused by mutations in the OCRL gene, is characterized by renal Fanconi syndrome leading to CKD, without or with a very limited involvement of eye and CNS [5-7].

The OCRL gene encodes for a 5-phosphatase that acts on phosphoinositides (Pls) and huge progress has been achieved in the last decades on the comprehension of its cellular roles.

Pls are quantitatively minor lipids of cell membranes but fundamental regulators of key cell functions such as membrane trafficking, autophagy, cytoskeleton remodeling, signaling and cell division. OCRL, which hydrolyses PtdIns(4,5)P2 into PtdIns(4)P, resides on the Trans Golgi Network and early endosomes, but translocates to the plasma membrane (PM) upon growth factor stimulation, to the autolysosomes during starvation-induced autophagy and to the intercellular bridge during cytokinesis [8, 9]. When OCRL is inactivated, the homeostasis of PtdIns(4,5)P2 is deregulated and this PI accumulates on endosomes, autolysosomes, and PM with deleterious consequences [10, 11].

The highly selective A3AR (A3 adenosine Receptor) agonist piclidenoson, which is known chemically as 1 -deoxy- l-[6-[[(3-iodophenyl)methyl] amino] -9H-9-yl]- A-methyl-P-D-ribofuranuronamide (IB-MECA) has been demonstrated to be effective in treatment of rheumatoid arthritis [12], and psoriasis [13].

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a pharmaceutical composition comprising an A3 adenosine receptor (A3AR) ligand and a pharmaceutically acceptable carrier or diluent for use in the treatment of a disease associated with OCRL deficiency in a mammalian subject.

In one aspect, the present invention provides a method of treating a disease associated with OCRL deficiency, wherein said method comprises administering to a mammalian subject in need thereof a pharmaceutical composition comprising an A3 adenosine receptor (A3AR) ligand.

In one embodiment, said disease associated with OCRL deficiency is Lowe syndrome or Dent disease.

In one embodiment, said disease associated with OCRL deficiency is proximal renal tubulopathy, renal Fanconi syndrome, chronic kidney disease or kidney failure.

In one embodiment, said A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

In one embodiment, said A3AR agonist is selected from the group consisting of N⁶-2- (4-aminophenyl)ethyladenosine (APNEA), N⁶-(4-amino-3-iodobenzyl) adenosine- 5'-(N-methyluronamide) (AB-MECA), N⁶-(3-iodobcnzyl)-adcnosinc-5'-N- methyluronamide (IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)- adcnosinc-5'-N- methyluronamide (Cl-IB-MECA, having the WHO designation namodenoson). In one embodiment, said A3AR agonist is N⁶-(3-iodobenzyl)-adenosine-5'-N- methyluronamide (IB-MECA).

In one embodiment, said A3AR allosteric modulator is selected from the group consisting of:

N-(3,4-Dichloro-phenyl)-2-cyclopentyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3,4-Dichloro-phenyl)-2-cycloheptyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3 ,4-Dichloro-phenyl)-2-cyclobutyl- 1 H-imidazo [4,5-c] quinolin-4- amine ; and

N-(3 ,4-Dichloro-phenyl)-2-cyclohexyl- 1 H-imidazo [4,5-c] quinolin-4- amine .

In one embodiment, the pharmaceutical composition for use is in combination with an additional therapeutic agent or therapeutic procedure.

In one embodiment, said A3AR ligand is administered daily (e.g., once, or twice a day) or weekly (e.g., once, or twice a week).

In one embodiment, said mammalian subject is a human subject, a non-human primate, a farm animal (e.g., a horse, a cow, a goat, a sheep, a pig) or a pet (e.g., a dog, a cat, a rabbit, a Guinea pig).

In one embodiment, said A3AR ligand is administered at an amount of lOpg/kg- lOmg/kg body weight.

In one embodiment, said composition is administered orally.

BRIEF DESCRIPTION OF THE DRAWINGS

To better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1A shows a quantitative analysis of the mannose 6-phosphate receptor (M6PR) phenotype and its sensitivity to PI4KIIIb inhibitor (PIK93). The graph shows the number of cells (%) in Control (referred to as Mock) and OCRL-depleted (knockdown (KD)) Hela cells either untreated (OCRL KD) or treated with 250nM or 500nM of the PI4KIIIb inhibitor PIK93, presenting the distribution between the four possible morphological features of M6PR: perinuclear continuous, perinuclear continuous with peripheral spots, perinuclear fragmented, and perinuclear fragmented with peripheral spots. Fig. IB is a schematic representation of cell segmentation adopted to isolate the perinuclear (color-filled) and peripheral (cytoplasm outside the color-filled region) regions to analyze the distribution of M6PR.

Fig. 1C is a graph showing the level of cells with perinuclear M6PR (normalized to control (CTRL)). 0 represents the average percentage of OCRL-KD cells with perinuclear continuous M6PR and 1 represents the average percentage of CTRL cells with perinuclear continuous M6PR. Only the compounds inducing an increase of the percentage of OCRL-KD cells with perinuclear continuous M6PR above 60% (the 0.6 threshold line) were further tested as positive hits. The correction of M6PR redistribution induced by IB-MECA in OCRL KD cells is indicated.

Fig. 2A is a schematic representation of image segmentation and spots identification for the analysis of RAP-GST internalization in CTRL and OCRL KD cells in the secondary assay.

Fig. 2B is a graph showing results of the secondary assay. The positive hits were tested on Control (CTRL) and OCRL-KD HK2 cells. Intracellular RAP-GST fluorescence intensity was normalized assigning 1 and 0 to the mean fluorescence intensity of CTRL and OCRL KD cells respectively. IB-MECA is indicated.

Fig. 2C-2D are representative images of proximal tubule cells (PTCs) from Lowe syndrome patients either untreated or treated with IB-MECA (lOpM) that were subjected to a RAP-GST internalization assay and immunostained with anti-GST antibody.

Fig. 2E is a graph showing quantitative analysis of the RAP-GST staining intensity in endosomes. Significance was assessed by two tailed Student’s test t (p<0.001 n=150 from three independent experimental replicates).

Fig. 3A-3B are graphs reporting the urinary level of Clara cell protein 16 (CC16)/creatinine/body weight in untreated (3A) and IB-MECA treated (3B) mice, showing the basal level (BL) before the study commenced and the level after 5 months of treatment. Each dot represents 1 mouse. Significance was assessed by Wilcoxon test.

Fig. 3C is a graph showing body weight (in grams) of OcrlY/- mice either untreated (n=14) or treated with IB-MECA (n=14). Each dot represents 1 mouse. Significance was assessed by Wilcoxon test. Fig. 3D is a graph showing quantitative analysis of AF555-labelled P- lactoglobulin fluorescence in PTs of OCRL Y/+ (wildtype) mice, OcrlY/- mice untreated and OcrlY/- treated with IB-MECA. Significance was assessed by the Kruskal- Wallis (K-W) test (p<0.0001 n=960 PTs from 3 wildtype, 5 OcrlY/- mice untreated and 5 OcrlY/- treated with IB-MECA).

Fig. 4A-4B show SDS-PAGE and Western blot analysis of control HK2 cells (4A) and A3R (AD0RA3) KD HK2 cells (4B) which were untreated (-) or treated for 10 or 30 minutes with lOpM IB-MECA and stained with antibodies directed to phosphorylated Akt (p-Akt), Akt, phosphorylated- ERK1/2 (p-ERKl/2), ERK-1/2, phosphorylated-S6 (p-S6), and S6.

Fig. 4C is a graph showing normalized ADORA3 mRNA levels control (CTRL) and ADORA3-knockdown (ADORA3-KD) cells.

Fig. 4D shows live images taken by spinning disc confocal microscopy of control HK2 cells transfected with a plasmid encoding the Pleckstrin Homology (PH) domain of the PLC delta fused with GFP (PH-PLCd-GFP) and treated with IB-MECA (lOpM). The images were taken at time 0 (t=0), after 50 seconds (t=50s) and after 100 seconds (t=100). A Region of interest (ROI) spanning 10pm from the plasma membrane was selected in each cell and the fluorescence intensity was recorded within 100s from the administration of IB-MECA

Fig. 4E is a graph showing traces of the fluorescence intensity in the ROI (namely PH-PLCd at the plasma membrane) as a function of time in cells that were untreated or treated with IB-MECA (lOpM) either alone or in combination with the PLC inhibitor U73122 (5pM).

Fig. 5 is a graph showing quantitative analysis of PI4,5P2 levels on endosomes in control HK2 cells and in HK2 cells in which the OCRL was knocked down using siRNA (OCRLsiRNA). The levels are shown as arbitrary units (AU) referring to the ratio of PI4,5P2 fluorescence intensity over EEA1 fluorescence intensity. The cells were either untreated or treated for 2 , 4 or 16h with IB-MECA lOpM, or IpM. OCRL KD Cells were also treated for 16h with IB-MECA (lOpM or IpM) and U73122 (5pM). Significance was assessed by Kruskal- Wallis test (p<0,0001 n=20715 EEA1 spots in 150 cells/group from three independent replicates). DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is based on the identification of an A3 AR ligand, i.e., IB- MECA, that showed exceptional efficacy in reversing the deleterious effects of OCRL deficiency in cellular assays as well as in a mouse model.

Cellular assays suitable for testing libraries of compounds under High Content Screening (HCS) settings were developed to identify potential therapeutics for Lowe syndrome. To this aim an HCS pipeline, including cell based primary and secondary assays, was designed, and implemented, followed by validation of the hits in proximal tubule cells (PTCs) from Lowe syndrome patients and, assessment of the ability of the validated hits to correct the proteinuria in a mouse model of Lowe syndrome [14].

The HCS pipeline that was applied to find correctors for Lowe syndrome is described in detail in the Examples.

As used herein the term “correctors” refers to compounds that can reverse or compensate for the deleterious effects of an OCRL deficiency.

Generally, the pipeline included the following elements:

A primary assay was performed in a Hela cell line stably transduced with a cassette encoding a shRNA against OCRL under the control of a tetracycline/doxycycline responsive promoter. In this inducible model a precise and effective downregulation of OCRL expression (also referred to as OCRL knockdown, or OCLR-KD) can be achieved.

The primary assay that was used to test the efficacy of potential correctors was a fluorescence assay detecting redistribution of the mannose 6-phosphate receptor (M6PR). In control HeLa cells, M6PR distribution was predominantly perinuclear, while OCRL-KD HeLa cells displayed peripheral M6PR redistribution.

High content screening was performed with the LOPAC¹²⁸⁰ library of pharmacologically active compounds. Compounds inducing an increase of the percentage of OCRL-KD cells with perinuclear continuous M6PR above 60% were defined as positive hits (68 compounds). Compounds that tested positive in the primary assay were further validated in a secondary assay which followed the endocytosis of GST-tagged Receptor Associated Protein (RAP-GST), a megalin ligand, in HK-2 cells, a proximal tubular cell (PTC) human cell line.

Of the 68 hits which emerged from the primary screening, 20 were able to rescue RAP-GST to 60% of CTRL, with IB-MECA being the second most effective.

These results were further validated in proximal tubule cells (PTCs) from Lowe syndrome patients. IB-MECA was able to rescue the endocytic defect in these PTCs as well, and thus emerged as a most promising candidate for reversing the effects of OCRL deficiency.

Next, IB-MECA was tested in a mouse model of Lowe syndrome, showing a significant effect of reducing low molecular weight proteinuria in the affected mice.

Therefore, the invention is described in the following detailed description with reference to therapeutic methods employing an A3AR ligand (e.g., IB-MECA) for the treatment of a disease associated with OCRL deficiency in a subject in need of same.

As used in the specification and claims, the forms "a", "an” and "the” include singular as well as plural references unless the context clearly dictates otherwise. For example, the term "an A3AR ligand” includes one or more ligands.

Further, as used herein, the term "comprising” is intended to mean that the method or composition includes the recited elements but does not exclude others. Similarly, "consisting essentially of' is used to define methods and compositions that include the recited elements but exclude other elements that may have an essential significant therapeutic activity towards a disease associated with OCRL deficiency or disfunction. For example, a composition consisting essentially of an A3AR ligand will not include or include only insignificant amounts (amounts that will have an insignificant effect on a disease associated with OCRL deficiency or disfunction) of other active ingredients that have such an activity. Also, a composition consisting essentially of the A3AR ligand as defined herein would not exclude trace contaminants from the isolation and purification method, pharmaceutically acceptable carriers, such as phosphate buffered saline, excipients, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.

Further, all numerical values, e.g., concentration or dose or ranges thereof, are approximations which are varied (+) or (-) by up to 20%, at times by up to 10% of the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term "about" . It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known.

There is provided by the present invention a pharmaceutical composition comprising an A3 adenosine receptor (A3 AR) ligand and a pharmaceutically acceptable carrier or diluent for use in the treatment of a disease associated with OCRL deficiency in a mammalian subject.

A "pharmaceutical composition" in the context of the invention is intended to mean a combination of the active agent(s), together or separately, with a pharmaceutically acceptable carrier as well as other additives. The carrier may at times have the effect of improving the delivery or penetration of the active ingredient to the target tissue, improving the stability of the drug, slowing clearance rates, imparting slow-release properties, reducing undesired side effects etc. The carrier may also be a substance that stabilizes the formulation (e.g., a preservative). For examples of carriers, stabilizers, and adjuvants, see E.W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co (June 1990).

As used herein, the term "an A3 adenosine receptor (A3AR) ligand" encompasses A3AR agonists as well as A3AR allosteric modulators.

A3AR agonists are known in the art and are readily available. Generally, an A3AR agonist is any compound that is capable of specifically binding to the adenosine A3 receptor ("A, 7?"), thereby fully or partially activating said receptor thereby yielding a therapeutic effect (e.g., an antiproteinuric effect). The A3AR agonist is thus a molecule that exerts its prime effect through the binding and activation of the A3AR. This means that at the doses it is being administered it essentially binds to and activates only the A3R.

In an embodiment, the A3AR agonist has a binding affinity (Ki) to the human A3AR of less than 1000 nM, desirably less than 500 nM, advantageously less 200 nM and even less than 100 nM, typically less than 50 nM, preferably less than 20 nM, more preferably less than 10 nM and ideally less than 5 nM. The lower the Ki, the lower the dose of the A3AR agonist (that may be used) that will be effective in activating the A3R and thus achieving a therapeutic effect.

It should be noted that some A3AR agonists can also interact with and activate other receptors with lower affinities (namely a higher Ki). A molecule will be considered an A3AR agonist in the context of the invention (namely a molecule that exerts its prime effect through the binding and activation A3R) if its affinity to the A3R is at least 3 times (i.e., its Ki to the A3R is at least 3 times lower), preferably 10 times, desirably 20 times and most preferably at least 50 times larger than the affinity to any other of the adenosine receptors.

The affinity of A3AR agonists to the human A3R as well as its relative affinity to the other human adenosine receptors can be determined by various assays, such as a binding assay. Examples of binding assays include providing membranes or cells having the receptor and measuring the ability of the A3AR agonist to displace a bound radioactive agonist; utilizing cells that display the respective human adenosine receptor and measuring, in a functional assay, the ability of the A3AR agonist to activate or deactivate downstream signaling events such as the effect on adenylate cyclase measured through increase or decrease of the cAMP level; etc. Clearly, if the administered level of an A3 AR agonist is increased such that its blood level reaches a level approaching that of the Ki of the other adenosine receptors, activation of these receptors may occur following such administration, in addition to activation of the A3R. An A3AR agonist is thus preferably administered at a dose such that the blood level that will be attained will give rise to essentially only A3R activation.

The characteristics of some adenosine A3AR agonists and methods of their preparation are described in detail in, inter alia, US 5,688,774; US 5,773,423; US 5,573,772; US 5,443,836; US 6,048,865; WO 95/02604; WO 99/20284; WO 99/06053; WO 97/27173 and WO 01/19360, all of which are incorporated herein by reference.

A specific group of A3AR agonists are the N⁶-benzyladenosine-5'-uronamide derivatives. Some preferred N⁶-bcnzyladcnosinc-5'-uronamidc derivatives are N⁶-2-(4- aminophenyl)ethyladenosine (APNEA), N⁶-(4-amino-3- iodobenzyl) adenosine-5'-(N- methyluronamide) (AB-MECA) and l-deoxy-l-{6- [({3-iodophenyl} methyl)amino]- 9H- purine-9-yl}-N-methyl- P-D-ribofuranuronamide (IB-MECA) and 2-chloro-N⁶-(3- iodobenzyl)adenosine- 5'-N-mcthlyuronamidc (Cl-IB-MECA).

In a specific embodiment, the A3 AR agonist of the invention is l-deoxy-l-{6- [({ 3-iodophenyl} methyl)amino]- 9H-purine-9-yl}-N-methyl- P-D-ribofuranuronamide (IB-MECA).

When referring to an ”A <AR allosteric modulator” or ’A.,A/?A7” it is to be understood as referring to the positive regulation, activation or increase of the receptor activity by binding of the allosteric modulator at the receptor's allosteric site which may be different from the binding site of the endogenous ligand or agonist thereof.

In one example, "modulation” denotes an effect of the A3 AR ligand on the receptor exhibited by an increase of at least 15% in the efficacy of the A3 adenosine receptor by binding of the compound to the allosteric site of the receptor and/or by a decrease in dissociation rate of adenosine or an A3AR agonist to the orthosteric binding site.

In one example, the modulation is by an A3AR allosteric modulator (A3ARAM) that is an imidazoquinoline derivative.

Specific imidazoquinoline derivatives which can be used as allosteric modulators of the A3 AR are listed below:

N-(3,4-Dichloro-phenyl)-2-cyclopentyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3,4-Dichloro-phenyl)-2-cycloheptyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3 ,4-Dichloro-phenyl)-2-cyclobutyl- 1 H-imidazo [4,5-c] quinolin-4- amine ; and

N-(3 ,4-Dichloro-phenyl)-2-cyclohexyl- 1 H-imidazo [4,5-c] quinolin-4- amine . The above imidazoquinoline derivatives are regarded as allosteric modulators as they were shown to have, on the one hand, reduced affinity, if any, to the orthosteric binding sites of the Ai and AIA, AIB adenosine receptors and reduced affinity to the orthosteric binding site of the A3 adenosine receptor, and on the other hand, high affinity to the allosteric site of the A3 adenosine receptor [International Patent Application No. W007/089507, incorporated herein by reference].

A specifically preferred imidazoquinoline derivative in accordance with the present disclosure is N-(3,4-Dichloro-phenyl)-2-cyclohexyl-lH-imidazo[4,5-c]quinolin- 4-amine (also referred to at times by the abbreviation LUF6000 or CF602), being an A3AR allosteric modulator.

The present disclosure also makes use of physiologically acceptable salts of an A3AR selective ligand, such as the above-described compounds. A “physiologically acceptable salt” refers to any non-toxic alkali metal, alkaline earth metal, and ammonium salt commonly used in the pharmaceutical industry, including the sodium, potassium, lithium, calcium, magnesium, barium ammonium and protamine zinc salts, which are prepared by methods known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the ligand with a suitable organic or inorganic acid. The acid addition salts are those which retain the biological effectiveness and qualitative properties of the free bases, and which are not toxic or otherwise undesirable. Examples include, inter alia, acids derived from mineral acids, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, metaphosphoric and the like. Organic acids include, inter alia, tartaric, acetic, propionic, citric, malic, malonic, lactic, fumaric, benzoic, cinnamic, mandelic, glycolic, gluconic, pyruvic, succinic salicylic and arylsulphonic, e.g., p- toluenesulphonic, acids.

The term “pharmaceutically acceptable carrier” in the context of the present invention denotes any one of inert, non-toxic materials, which do not react with the A3AR agonist, and which can be added to formulations as diluents, carriers or to give form or consistency to the formulation.

In the context of the present invention the term ''treatment” comprises treating a disease associated with OCRL deficiency to reverse, attenuate, or ameliorate disease symptoms. Thus, treatment refers to administering a therapeutically effective amount of an A3AR ligand to achieve a desired therapeutic effect. The desired therapeutic effect may include, without being limited thereto, improving kidney disfunction and proteinuria (e.g., proximal renal tubulopathy, renal Fanconi syndrome, chronic kidney disease or kidney failure), but it may also include a reduction in the formation of cataracts or glaucoma, a reduction in seizures, as well as improving cognitive disabilities and behavioral difficulties.

The terms "disease associated with OCRL deficiency" and “disease associated with OCRL disfunction” are used interchangeably herein and refer to a disease, in particular a genetic disease, caused by mutations in the OCRL gene. This term particularly refers to Lowe syndrome and Dent disease. In an embodiment, the term refers to proximal renal tubulopathy, renal Fanconi syndrome, chronic kidney disease or kidney failure resulting from or associated with Lowe syndrome or Dent disease.

The A3AR ligand can be administered in a single dose (one time medication) or as a continuous treatment, for a period of days, weeks, months or even years.

Further in the context of some embodiments of the present disclosure, long term treatment encompasses chronic treatment, e.g., long term daily administration at times even without an envisaged end point for the treatment, throughout the patient’s life.

The composition of the present invention is administered and dosed in accordance with good medical practice, taking into consideration the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The choice of carrier will be determined in part by the specific active ingredient, as well as by the specific method used to administer the composition. Accordingly, there is a wide variety of suitable pharmaceutical compositions of the present invention.

The composition of the invention can be administered to the subject by a variety of delivery modes as known in the art, e.g., by oral, intraperitoneal, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, intranasal, or intraocular administration. In a preferred embodiment, the composition is administered orally. The carrier will be selected based on the desired form of the formulation. The A3AR ligand is administered in amounts which are sufficient to achieve a therapeutic effect, for example an anti-proteinuria effect. As will be appreciated, the amount of the A3AR ligand will depend on the severity of the disease, the intended therapeutic regimen, and the desired therapeutic dose. By way of example, where the dose is 1 mg per day and the desired administration regimen is once daily administration, the amount of the A3AR ligand in a pharmaceutical composition comprising same will be 1 mg. Where it is intended to divide this daily dose into 2 daily administrations, the amount of the active agent in the pharmaceutical composition will be 0.5 mg.

An amount effective to achieve the desired effect is determined by considerations known in the art. An "effective amount" for purposes herein must be effective to achieve a therapeutic effect, the therapeutic effect being as defined hereinbefore.

It is appreciated that the effective amount depends on a variety of factors including the affinity of the chosen A3AR agonist to the A3AR, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender of the subject to be treated, etc. The effective amount is typically tested in clinical studies having the aim of finding the effective dose range, the maximal tolerated dose, and the optimal dose. The manner of conducting such clinical studies is well known to a person versed in the art of clinical development.

An amount may also at times be determined based on amounts shown to be effective in animals. It is well known that an amount of X mg/Kg administered to animals (e.g., mice) can be converted to an equivalent amount in another species (notably humans) using one of possible conversion equations well known in the art.

In accordance with one embodiment of the invention, the administration of the A3AR agonist is preferably by daily administration, between once and a few times a day, preferably once or twice a day, the dose in each administration being in the range of between about 1 to about lOOOpg/kg body weight, preferably less than 400pg/kg body weight, and even less than 200pg/kg body weight. Typically, the dose of A3AR agonist is in a range of 1 to 100 pg/kg body weight.

In an embodiment, the injection is administered in an extended-release formulation.

The therapeutic use of an A3AR agonist may at times be in combination with other drugs or therapeutic procedures such as cataract extraction, glaucoma control, physical and speech therapy, drugs addressing behavioral problems, and correction of the tubular acidosis and bone disease using bicarbonate, phosphate, potassium, and water. In such a combination treatment the other drug and the A3AR agonist may be given to patients at the same time or at different times, depending on the dosing schedule of each of the drugs.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be a description rather than a limitation. Obviously, many modifications and variations of the present invention are possible in view of the above teaching. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described hereinafter.

DESCRIPTION OF NON-LIMITING EXAMPLES

METHODS

Mannose 6-phosphate receptor (M6PR) assay used in high content screening (HCS)

Hela-TRex cells were seeded in mClear black 96 well microplate (PerkinElmer) either in the absence (control cells) or the presence (OCRL-KD) of tetracycline (Ipg/mL). 72h post seeding, cells were treated, in quadruplicates, for 16h with the compounds of the LOPAC library (Sigma-Aldrich) at lOpM.

Cells were then fixed for lOmin with 4% paraformaldehyde (PFA) in PBS IX, permeabilized for 20min with Blocking Buffer (0.05% Saponin, 0.5% BSA and 50mM NH4C1 in PBS). Cells were then incubated for Ih at 25°C with anti-CLM6PR (Vicinanza et al., 2011) dissolved in Blocking buffer, washed three timed with PBS IX and incubated for 45min at 25°C with AlexaFluor-conjugated anti rabbit secondary antibody (1:400) and with DAPI (1:1000) dissolved in Blocking Buffer. The plates were imaged with Olympus ScanR and analyzed with ScanR 0.6 analysis software (at least 16 fields for well) were analyzed. In detail, nuclei were identified using the DAPI channel and the cytoplasm was obtained by expanding the nuclear region by 100 pixels in all the directions. The cytoplasmic area was further divided into two subregions, one within 40 pixels from the nucleus and the second from 40 to 100 pixels from the nucleus. The CLM6PR signal was then assigned to the inner ring (0-40 pixels) and labelled as perinuclear or to the outer ring (40-110 pixels) and labelled as peripheral. The two main classes were then further divided into perinuclear continuous, perinuclear fragmented, perinuclear fragmented with peripheral spots and peripheral to include all the possible morphological features of the CI-M6PR.

RAP-GST internalization assay

CTRL and OCRL-KD HK2 cells (6000 cells/well) were seeded in 96 well plates (mClear black microplate, PerkinElmer). Cells were then treated with 1, 3 and lOpM of the selected compounds (in quadruplicates) of the LOPAC library for 3h and 16h at 37°C. The cells were then serum starved for Ih and incubated for Ih at 4°C with 2.5pg/mL of RAP-GST to allow its binding and then incubated for 5min at 37°C to allow RAP-GST internalization. The cells were then washed with ice-cold complete culture medium and incubated for 45sec with Acid Wash Solution (150mM NaCl, 5mM Acetic Acid in water) to strip from cell surface the RAP-GST that was nonspecifically bound to the plasma membrane. The Cells were then fixed for lOmin with 4% PFA in PBS IX, permeabilized for 20min with Blocking Buffer (0.05% Saponin, 0.5% BSA and 50mM NH4CI in PBS) and then incubated for Ih at 25°C with a rabbit anti-GST (Vicinanza et al., 2011) and a mouse anti-EEAl antibody dissolved in Blocking buffer, washed three timed with PBS IX and incubated for 45min at 25°C with AlexaFluor568- conjugated anti rabbit and AlexaFluor 488 conjugated anti mouse secondary antibodies (1:400) and with DAPI (1:1000) dissolved in Blocking Buffer. For the acquisition of the images, at least 5 images fields were acquired per well of the 96- well plate by using confocal automated microscopy (Opera high-content system; Perkin-Elmer). A dedicated script was developed to perform the analysis of the intensity of RAP-GST spots within the EEA1 positive spots on the different images (Harmony and Acapella software; Perkin-Elmer). The script calculates the ratio value resulting from the average intensity of RAP-GST fluorescence divided by the average intensity of EEA1 fluorescence. Evaluation of Low Molecular Weight (Clara Cell Protein 16) Proteinuria

Mice were placed in metabolic cages for 24h with free access to food and drinking water. Urines were collected and body weight, water intake and diuresis were measured. Urinary Clara cell protein (CC16) concentration was measured in triplicate by enzyme-linked immunosorbent assay (ELISA; BIOMATIK EKU03200). The levels of urinary CC16 were normalized over urinary creatinine, measured by Jaffe method.

In vivo Endocytosis assays

Proximal Tubules endocytic capacity of untreated and IBMECA-treated OCRL- ko mice was examined by measuring P -lactoglobulin uptake. P -lactoglobulin was tagged with Alexa Fluor-555 (Alexa Fluor™ 555 Protein Labeling Kit Catalog number: A20174, Life technologies) in accordance with the manufacturer’s instructions. 25 minutes after retro-orbital intravenous injection of AF555-P-lactoglobulin (1 mg/kg B.W.) mice were anesthetized (with a sublethal dose of Ketamine and Medetomidine) and their kidneys were harvested after perfusion with sterile Phosphate-Buffer Saline. Kidneys were then fixed with 4% PFA in PBS for 8 hours, then incubated for 5 hours in 20% Sucrose in PBS and 16 hours in 30% Sucrose and then embedded in cryogenic Tissue-Tek OCT compound (Electron Microscopy Sciences, Hatfield, USA). The embedded tissues were cryosectioned at 10pm.

Evaluation of activation of A3R by IBMECA in HK2 cells - Western blotting

Total cell lysates of CTRL, OCRL-KD and ADORA3 KD cells either untreated or treated for 10 and 30 minutes with the Pan-PI3K inhibitor Wortmannin (lOOnM) or with the Phospholipase C inhibitor U73122 (5pM) alone or in combination with IBMECA (lOpM) were subjected to SDS-PAGE and Western blot to evaluate the activation of the PI3K-Akt-mTOR and MAPK pathways with the following antibodies: p-Akt, Akt, p-ERK, ERK, p-S6 and S6.

Mouse treatment with IBMECA

Experiments were conducted on age- and gender-matched OcrlY/- ;Inpp5b-/- mouse littermates harboring BAC-INPP5B expression in equal copies (BAC1) (129S/SvEv * 129S6/SvEvTac * FVB/N * C57BL/6 background), (Bothwell, 2011). Mice were housed under temperature- and humidity-controlled conditions with 12 h light/ 12 h dark cycles with free access to appropriate standard diet and water. Mice aged 8 weeks were treated with control diet (#4RF21 Mice and Rats, Mucedola, n=15 mice) or with IBMECA containing chow (#4RF21 added with IBMECA 0.75mg/Kg, Mucedola, n=15 mice) for 5 months. Urine samples were collected the day before the beginning of the treatment and then every month. The choice of the amount of IBMECA to be added to the chow was in line with the measured daily consumption of chow by single mice (3g/day) that resulted in the daily intake of lOOpg/Kg of IBMECA. siRNA transfection

HK2 cells were transfected using RNAiMAX (Invitrogen, USA) according to the manufacturer instructions. The cells were plated in 24-well plates (11,000/well) on glass coverslips, and in 6-well plates (130,000/well). A transfection mixture was prepared: for each well (24-well), 2pL of a 20 pM stock of OCRL siRNAs were diluted in 50pl of OptiMEM culture medium in a polypropylene tube. In a separate polypropylene tube, for each well, Ipl of RNAiMAX was diluted in 50pl of OptiMEM culture medium. The mixtures were incubated at RT for 5 min, then combined and kept at RT for 20 min, to allow the siRNA/RNAiMAX complexes to form. The cells were incubated with the transfection mixture at 37° C in the presence of 5% CO2 for 72 h. The same steps were used for the 6-well plates with different volumes according to the manufacturer instructions. The OCRL knockdown efficiency was checked by Western blot, as described in de Leo, et a/ 2016 and Vicinanza, et al (2011).

Immunofluorescence

Cells were grown on glass coverslips and treated according to the experimental procedure. They were fixed with 4% paraformaldehyde for 10 min at RT and washed three times in PBS. Cells were permeabilized and blocked in 0.05% (w/v) saponin, 0.5% (w/v) BSA, 50mM NH4C1, and 0.02% NaN3 in PBS (blocking buffer) for 45min, followed by a 1.5-2h incubation with the primary antibodies in blocking buffer in the dilution indicated in the Table 1. The cells were then washed with PBS and incubated with AlexaFluor conjugated-secondary antibodies (1:400) and DAPI (1:1000) diluted in blocking buffer. After immuno- staining, the cells were washed twice in PBS and once in sterile water, to remove salts. The coverslips were then mounted on glass microscope slides (Thermo Fisher Scientific, USA) with mowiol.

Table 1:

PI(4,5)P2 immunostaining

All steps were performed at room temperature. Cells in 300pl medium were fixed by the addition of 300pl of 4% paraformaldehyde in PBS to a final concentration of 2%, and incubated for 15 min at RT, followed by quenching with three rinses in PBS containing 50 mM NH4C1. Cells were permeabilized for 7 min by the addition of 20pM digitonin in buffer A (20 mM Na-PIPES, 137 mM NaCl, 2.7 mM KC1, pH 6.8). Digitonin was removed by three rinses in buffer A, and cells were blocked for 45 min with buffer A supplemented with 5% (v/v) of goat serum and 50 mM NH4C1. The cells were incubated with primary antibodies (see Table 1.1) in blocking buffer (buffer A supplemented with 5% (v/v) of goat serum at RT for Ih followed by three rinses in Buffer A. Secondary antibodies were applied in buffer A with 5% of goat serum at RT for Ih followed by three rinses in Buffer A. Then there is a post-fixation for 5 min in 2% PFA at RT, followed by quenching with one wash in PBS containing 50 mM NH4C1 and one wash in sterile water, to remove salts. The coverslips were then mounted on glass microscope slides (Thermo Fisher Scientific, USA) with mowiol.

Confocal fluorescence microscopy, image processing

Immunofluorescence samples were examined under a confocal laser microscope (Zeiss ESM800 and ESM700 confocal microscope systems; Carl Zeiss, Gottingen, Germany) equipped with 63x1.4 NA oil objective. Optical confocal sections were taken at 1 Airy unit with a resolution of 1024x1024 pixels and exported as TIFF files.

For quantification experiments, 10-15 fields that were randomly located on the coverslips and included 3-6 cells were scanned with the same microscope settings (i.e., laser power and detector amplification) below pixel saturation. Counting was performed either automatically or manually using the tool available at the NIH.Gov website, IJ user tools, chapter 19. Cell Lines

HeLa cells stably transfected with an expression cassette containing a shRNA- OCRL under a tetracycline (Tet) sensitive promoter were generated by using the BLOCK-IT Inducible Hl RNAi entry vector Kit (Life Technologies).

Tet administration (Ipg/mL) allows the expression of the shRNA and the subsequent depletion of the gene of interest. The expression cassette also contains a zeocin resistance gene that allows the use of zeocin to select and maintain the clones that contain the shRNA against OCRL. A single HeLa-shRNAOCRL clone (B2) was selected and used.

Human Proximal tubule epithelial (HK2) cell line was bought from ATCC. HK2 cells were cultured in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12, Gibco) supplemented with 5% FBS, 2mM L-glutamine, 1 U/ml antibiotics (penicillin/streptomycin) plus 1% insulin-transferrin-selenium (ITS-Sigma Aldrich). Cells were grown under a controlled atmosphere in the presence of 5% CO2 at 37°C. Cells were grown in a flask until 90% confluence and then detached with 0.05% Trypsin-EDTA.

PTCs isolation from Lowe syndrome patients and immortalization from the urine of control healthy subjects and from Lowe syndrome patients is reported in (Vicinanza et al. 2011).

Example 1: Identification of correctors in a Lowe Syndrome cell model by High Content Screening.

Primary assay: The first step in the definition of the HCS pipeline is the identification of a robust cellular phenotype. There are strict statistical parameters that a cellular phenotype must fit to be suitable for HCS. The screening window coefficient, or Z’ -factor, is accepted as the statistical parameter used to describe the quality and suitability of an assay for large-scale screening and represents a measure of the separation between the data variability of the positive controls and the negative controls [15]. A Hela cell line stably transduced with a cassette encoding a shRNA against OCRL under the control of a tetracycline/doxycycline responsive promoter was used. With this model a precise and effective downregulation of OCRL expression can be achieved simply by adding tetracycline and incubating the cells for 96 hours. In contrast to control HeLa cells, in which M6PR distribution was predominantly perinuclear, OCRL-KD HeLa cells displayed peripheral M6PR redistribution, which is a consequence of the impaired recycling from EE caused by PI4,5P2 accumulation (as it is rescued by the concomitant depletion of PI4,5P2 and PI4P producing enzymes) [11].

Based on the extent and distribution of M6PR structures four different sub populations, perinuclear continuous, perinuclear continuous with peripheral spots, perinuclear fragmented and perinuclear fragmented with peripheral spots were described [11]. According to this classification, OCRL KD determined a shift in the M6PR distribution from perinuclear continuous to the other three classes, highlighting that OCRL reduction/absence results in the appearance of dispersed M6PR.

In the M6PR distribution assay, the positive controls were Hela cells without tetracycline and the negative controls were Hela cells treated for 96h with tetracycline (OCRL-KD). The values of positive and negative controls were well separated with Z’ factor values between 0.5 and 0.8 (being a Z’ factor greater than 0.4 considered appropriately robust for compound screening in HCS).

The identification of the PI4KIIIb as possible target to ameliorate the endocytic dysfunctional phenotype arising when OCRL is absent led to the selection of a specific PI4KIIIb inhibitor (PIK93) as a positive control in the HC [11]. PIK93 was used at 250- 500nM on OCRL-KD cells and resulted effectively in increasing the number of OCRL- KD cells with perinuclear continuous M6PR up to 80% (Eig. 1A). OCRL KD cells display peripheral distribution of M6PR that is rescued to the perinuclear distribution, as in CTR cells, by PIK93 administration.

The 1280 compounds of LOPAC library from Sigma Aldrich were then tested on CTRL and OCRL-KD Hela cells at lOmM for 16 hours in 96 well plates. Images were segmented into two areas according to their distance from the nucleus: the perinuclear and peripheral area (Eig. IB). The cells were then assigned to the two areas and the percentage of CTRL and OCRL-KD cells with perinuclear continuous M6PR were normalized assigning 0 at the average percentage of OCRL-KD cells with perinuclear continuous MPR and 1 at the average percentage of CTRL cells with perinuclear continuous MPR. The percentage of OCRL-KD cells treated with the 1280 compounds and displaying perinuclear continuous M6PR were normalized as described above and only the compounds inducing an increase of the percentage of OCRL-KD cells with perinuclear continuous M6PR above 60% were further tested as positive hits (68 compounds) (Fig. 1C).

Secondary assay: The 68 hits were then tested in a secondary assay performed in a proximal tubule cell line (HK-2 cells) which is a cellular phenotype relevant for the pathological manifestations of kidney dysfunction in Lowe Syndrome: namely, the defective endocytosis of megalin ligands. Megalin trafficking impairment has been reported in HK2 cells lacking OCRL [11] and therefore this cell model and this assay were used to validate the activity of the 68 hits which emerged from the primary screening. The assay followed the uptake of GST-tagged Receptor Associated Protein (RAP-GST), a megalin ligand in HK-2 cells that was reported to be markedly impaired in the absence of OCRL [11]. The RAP-GST endocytosis assay was adapted to the 96 well plate format and CTRL and OCRL-depleted (OCRL-KD) HK-2 cells were incubated with the 68 compounds for 16 hours in serum- free medium. RAP-GST binding to cell surface was performed at 4°C and RAP-GST internalization was achieved by shifting the temperature to 37°C for 5 min. The cells were immunostained with anti-GST antibody. OCRL KD cells showed reduced RAP-GST binding to the plasma membrane (at 4°C) and a reduced amount of internalized RAP-GST after 5 minutes at 37°C.

The assay plates were imaged at the Opera (Perkin Elmer) and the image analysis was performed with Columbus software. The automatic image analysis consisted of single-cell identification and segmentation, thresholding to eliminate background and increase signal to noise ratio, RAP-GST spots identification and measurement of their fluorescence intensity (Fig. 2A). The values of all the cells analyzed were averaged and normalized by assigning 0 and 1 at the average values of RAP-GST fluorescence intensity in OCRL-KD and control HK2 cells respectively. The values of the OCRL-KD cells treated with the 68 hits were normalized (Fig. 2B), and out of the 68 hits which emerged from the primary screening, 20 were able to rescue RAP-GST to 60% of CTRL, with IB-MECA being the second most effective.

Validation in PTCs from Lowe syndrome patients: The dysfunctional endocytic phenotypes observed in cell lines transiently depleted of OCRL (by shRNAs or siRNAs) are present also in Lowe-PTCs [11, 16]. In accordance with its activity on cell lines, IB-MECA was also able to rescue the endocytic defect in PTCs derived from Lowe syndrome patients (Fig. 2C-2E).

Example 2: Evaluation of antiproteinuric effect of IB-MECA on the mouse model of Lowe Syndrome.

The available mouse model for Lowe syndrome was generated in 2011 by deleting Ocrl and lnpp5b (that compensate for the loss of Ocrl in mice) and by transgenic expression (BAC-mediated) of human INPP5B gene. This mouse model (Ocrl'^ InppSb'^ INPP5B mice), hereafter referred as Ocrl-KO, with a humanized INPP5B shows proximal tubular dysfunction (LMW proteinuria and aminoaciduria) and a mild reduction in growth, with no extrarenal pathological signs [14, 17]. Ocrl-KO mice show LMW proteinuria already at 21 days, as demonstrated by the high urinary content of CC16, NGAL, Transferrin and Albumin, with age-dependent worsening of the proteinuric phenotype. It was recently demonstrated that PTCs isolated from Ocrl- KO mice kidneys display PI4,5P2 accumulation and actin polymerization on endolysosomes, resulting in impaired endocytic trafficking of megalin and of other receptors such as M6PR and EGFR. Furthermore, these mice also show signs of muscle defects as evidenced by dysfunctional locomotion [18, 19].

IB-MECA was orally administered by feeding the mice with IB-MECA containing chow at the dose of lOOpg/Kg/day (Fig.3A). However, to determine the amount of IB-MECA to add to the chow the average daily food consumption of Ocrl- KO mice was first measured. The amount of food that each mouse consumes per day was determined (3±0,25 g/day/mouse) and hence each mouse received an IB-MECA containing chow in an amount of 0.75mg/kg. Next, the LMW proteinuria was evaluated by ELISA determination of CC16 urinary concentration (Basal level). 14 Ocrl-KO mice were fed with the standard chow (placebo, normal diet) and 14 Ocrl-KO mice with the IB-MECA containing chow for 5 months. One day before the sacrifice, mice were injected with AF555-labeled b-lactoglobulin. Ocrl-KO mice treated with IB-MECA display a significant reduction of the urinary LMW protein CC16 compared to untreated Ocrl-KO mice (Fig.3A-3B). IB-MECA had no observed effects on urinary volume and on growth rate as manifested by body weight (Fig.3C) suggesting that it does not affect postnatal development.

To evaluate if the IB-MECA dependent reduction in LMW proteinuria was due to increased apical receptor mediated endocytosis in Proximal tubule cells, the in vivo uptake of AlexaFluor(AF)-568-labelled b-lactoglobulin was followed in mouse kidney sections [18, 19]. Wildtype (Wt), and Ocrl-KO (also referred to as OCRLY/-) mice either untreated or treated with IB-MECA for 5 months were intravenously injected with AF568-P-lactoglobulin that reaches the kidneys, is filtered through the glomeruli and is endocytosed by PTCs. Ocrl-KO mice displayed a significant reduction of AF568- b-lactoglobulin PT endocytosis compared to Wt mice whereas IB-MECA-treated Ocrl- KO mice showed a significant rescue of AF568-P-lactoglobulin endocytosis like that observed in Wt mice (Fig.3D).

These data show that the A3 receptor agonist IB-MECA induces a significant improvement of PT endocytic activity and a subsequent reduction of LMW proteinuria in the mouse model of Lowe syndrome.

Example 3: Analysis of the mechanism of action of the A3 receptor agonist IB- MECA in proximal tubule cells.

To identify the mechanism of action through which IB-MECA exerts beneficial effects on OCRL-dependent dysfunctional phenotypes in a kidney cell line depleted of OCRL, in PTCs from Lowe patients and in the mouse model of Lowe syndrome as shown above, the signaling cascades activated in human kidney cells (HK2) upon IB- MECA treatment were investigated.

Control HK2 cells (which express A3R) and A3R (AD0RA3) knockdown HK2 cells were left untreated or treated for 10 or 30 minutes with 10[lM IB-MECA. Exposure of the control HK2 cells to IB-MECA resulted in the activation of PI3K/Akt/mT0R and PLC signaling cascades, as demonstrated by the increased phosphorylation of Akt, S6 and ERK1/2 (Fig.4A). In contrast, the IB-MECA dependent activation of the PI3K and PLC pathway was blunted in A3R knockdown HK2 cells (AD0RA3 KD) (Fig.4B), which apparently shows that IB-MECA signals via A3R. Fig. 4C shows AD0RA3 mRNA levels assessed by quantitative real-time PCR in control and AD0RA3-siRNA treated cells, showing a clear lower level of A3R receptor mRNA in the knockdown cells.

To validate that the efficacy of IB-MECA as corrector of the dysfunctional phenotypes associated with the absence of OCRL was through the PLC-mediated decrease of PI4,5P2 levels (mostly at the plasma membrane) the intracellular localization of the Pleckstrin-Homology (PH) domain of the PLC-d fused with the GFP (PH-PLCd-GFP) was followed. The PH domain binds specifically to PI4,5P2 localizing at the intracellular sites where PI4,5P2 is more abundant (e.g., the plasma membrane) [20, 21]. PH-PLCd-GFP expressing cells treated with IB-MECA (lOpM) showed a significant reduction of the plasma membrane targeting of PH-PLCd-GFP with the subsequent increase of the fluorescent signal in the cytosol. Interestingly, when PH- PLCd-GFP expressing cells are concomitantly treated with IB-MECA and U73122, no loss of plasma membrane PH-PLCd-GFP localization is observed, indicating that the reduction of PI4,5P2 after IB-MECA administration is through the A3R-dependent PLC activation (Fig. 4D, 4E) The IB-MECA-dependent decrease of PI4,5P2 at the plasma membrane is thus in line with the activation of PLC.

However, cells lacking OCRL display PI4,5P2 accumulation mostly at the early endosomes [11]. To assess whether the IB-MECA-induced reduction of PI4,5P2 at the plasma membrane results in a reduction of endosomal PI4,5P2 the effect of IB-MECA administration in three time points (2, 4, and 16h) and at two concentrations (1 and lOpM) on the total and endosomal PI4,5P2 levels was evaluated.

Control HK2 cells and OCRL KD HK2 cells were either untreated or treated for 2 , 4 or 16h with IB-MECA lOpM. OCRL KD cells were also treated for 16h with IB- MECA (lOpM) and U73122 (5pM). Cells were then fixed and immunostained with anti PI4,5P2 and anti EEA1 (to label the early endosomes) antibodies, as well as subjected to DAPI staining (revealing the cells’ nuclei). Briefly, early endosomes (EEA1 -positive spots) were identified, and a binary mask was drawn. The mask was then superimposed on PI4,5P2 immuno staining and the fluorescence intensity of PI4,5P2 within the EEA1- positive spots was measured. PI4,5P2 fluorescence on EEA1 spots was then normalized over the intensity of EEA1 spots.

IB-MECA induces a reduction of PI4,5P2 levels in a time and concentration dependent manner and its activity is completely blunted by the concomitant administration of U73122. Without wishing to be bound by theory, these results indicate that the mechanism of action of IB-MECA is through the A3R dependent activation of PLC that, in turn, lowers the levels of PI4,5P2 and rescues the aberrant phenotypes arising from OCRL absence and PI4,5P2 accumulation (Fig.5).

Claims

CLAIMS:

1. A pharmaceutical composition comprising an A3 adenosine receptor (A3AR) ligand and a pharmaceutically acceptable carrier or diluent for use in the treatment of a disease associated with OCRL deficiency in a mammalian subject.

2. The pharmaceutical composition for use according to claim 1, wherein said disease associated with OCRL deficiency is Lowe syndrome or Dent disease.

3. The pharmaceutical composition for use according to claim 1 or claim 2 wherein said disease associated with OCRL deficiency is proximal renal tubulopathy, renal Fanconi syndrome, chronic kidney disease or kidney failure.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein said A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

5. The pharmaceutical composition for use according to claim 4, wherein said A3AR agonist is selected from the group consisting of N⁶-2- (4- aminophenyljethyladenosine (APNEA), N⁶-(4-amino-3-iodobenzyl) adenosine- 5'-(N- methyluronamide) (AB-MECA), N⁶-(3-iodobenzyl)-adenosine-5'-N- methyluronamide (IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)- adcnosinc-5'-N-mcthyluronamidc (Cl-IB- MECA).

6. The pharmaceutical composition for use according to claim 5, wherein said A3AR agonist is N⁶-(3-iodobcnzyl)-adcnosinc-5'-N- methyluronamide (IB-MECA).

7. The pharmaceutical composition for use according to claim 4, wherein said A3AR allosteric modulator is selected from the group consisting of:

N-(3,4-Dichloro-phenyl)-2-cyclopentyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3,4-Dichloro-phenyl)-2-cycloheptyl-lH-imidazo[4,5-c]quinolin-4-amine;

N-(3 ,4-Dichloro-phenyl)-2-cyclobutyl- 1 H-imidazo [4,5-c] quinolin-4- amine ; and

N-(3 ,4-Dichloro-phenyl)-2-cyclohexyl- 1 H-imidazo [4,5-c] quinolin-4- amine .

8. The pharmaceutical composition for use according to any one of claims 1 to 7, in combination with an additional therapeutic agent or therapeutic procedure.

9. The pharmaceutical composition for use according to any one of claims 1 to 8 wherein said A3AR ligand is administered daily (e.g., once, or twice a day) or weekly (e.g., once, or twice a week).

10. The pharmaceutical composition for use according to any one of claims 1 to 9 wherein said composition is administered orally.