CALCIUM OXALATE CRYSTALLIZATION INHIBITORS FOR RENAL DISORDERS

Abstract

The present invention relates to inositol derivatives comprising two or more cyclohexanolpentakisester moieties linked by a common central linker and their use in therapy or prevention of a condition related to pathological crystallization. The invention further relates to useful intermediates in the synthesis of the compound of the invention.

Claims

1. A compound comprising, particularly consisting of, two or more cyclohexanolpentakisester moieties described by a general formula (I) linked by a common central linker L ##STR00028## wherein each X is independently of any other X is selected from OPO.sub.3.sup.2−, OPSO.sub.2.sup.2−, OSO.sub.3.sup.− or CO.sub.2.sup.−, L is a common central linker to which n individual moieties characterized by the formula in brackets are attached, and n is an integer from 2 to 10, particularly wherein n is selected from 2, 3, 4, 6 and 8, more particularly wherein n is selected from 2, 3 and 4, wherein the common central linker L has a molecular weight <1000 g/mol, particularly <700 g/mol, more particularly <500 g/mol, or even <400 g/mol and wherein L comprises or essentially consists of a linear or branched poly(ethylene glycol) or polyglycerol, and wherein optionally the linking moiety comprises a central core A selected from an oxygen, nitrogen and/or fluorine substituted or unsubstituted C.sub.1 to C.sub.4 alkyl, an oxygen, nitrogen and/or fluorine substituted or unsubstituted C.sub.4 to C.sub.7 cycloalkyl, or an oxygen, nitrogen and fluorine substituted or unsubstituted five- or six-membered aryl.

2. The compound according to claim 1, wherein any one of the cyclohexanolpentakisester moieties described by a general formula (I) is independently selected from a moiety described by general formulae (Ta) or (Ib), ##STR00029## wherein L is a common central linker and X has the meaning defined in claim 1.

3. The compound according to claim 1, wherein the compound is characterized by a general formula (II), (IIi), (IIii), or (IIiii): ##STR00030## wherein each X is selected from OPO.sub.3.sup.2−, OPSO.sub.2.sup.2−, OSO.sub.3.sup.− or CO.sub.2.sup.− and wherein L is selected from —(O—CH.sub.2—CH.sub.2).sub.m—O— or —(O—CH.sub.2—CH(OH)—CH.sub.2).sub.m—O—, with m having a value between 1 and 12, particularly with m having a value between 2 and 8.

4. The compound according to claim 1, wherein the compound is characterized by formula (IIa), (IIb), (IIc), IId), (IIe), (IIf), (IIg), (IIh), (IIi), (IIk), (IIm), or (IIn) ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## wherein k is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

5. The compound according to claim 1, wherein the compound is characterized by a general formula (III) ##STR00037## wherein each X has the meaning indicated above and wherein the linking moiety L is ##STR00038## and wherein o, p and q independently of each other have a value between 1 and 12 and the sum of o, p and q is ≤30, particularly o, p and q independently of each other have a value between 2 and 4 and the sum of o, p and q is ≤12; and A is selected from a carbon atom (C), a hydroxy-, amino-, halogen- or carboxy-substituted or an unsubstituted C.sub.4 to C.sub.7 cycloalkyl moiety or a hydroxy-, amino-, halogen- or carboxy-substituted or an unsubstituted aryl and R is selected from H and C.sub.1-C.sub.3 unsubstituted or N—, O and/or halogen substituted alkyl, particularly wherein R is H or unsubstituted alkyl or u, v and w independently of each other have a value between 1 and 12, and the sum of u, v and w is ≤30, and R is selected from H and C.sub.1-C.sub.3 unsubstituted or N—, O and/or halogen substituted alkyl, particularly wherein R is H or unsubstituted alkyl, particularly u, v and w independently of each other have a value between 2 and 4 and the sum u, v and w is ≤12.

6. The compound according to claim 5, wherein the compound is characterized by formula (IIId) or (IIIe): ##STR00039##

7. The compound according to claim 1, wherein the compound is characterized by a general formula (IV) ##STR00040## wherein each X has the meaning indicated above and wherein the linking moiety L is ##STR00041## wherein o, p, q and s independently of each other have a value between 1 and 12, and the sum of o, p, q and s is ≤40, particularly wherein o, p, q and s independently of each other have a value between 2 and 4 and the sum of o, p, q and s is ≤12 and A is a hydroxy-, amino-, halogen- or carboxy-substituted or an unsubstituted C.sub.4 to C.sub.7 cycloalkyl moiety or a hydroxy-, amino-, halogen- or carboxy-substituted or an unsubstituted aryl or u, v, w and y independently of each other have a value between 1 and 12 and the sum of u, v, w and y is ≤40, particularly u, v, w and y independently of each other have a value between 2 and 4 and the sum of u, v, w and y is ≤12.

8. The compound according to claim 7, wherein the compound is characterized by formula (IVd) or (IVe): ##STR00042##

9. A compound according to claim 1 for use as a medicament.

10. A method for treatment of a condition related to pathological crystallization comprising administering to a subject in need thereof, the compound according to claim 1, or a compound comprising, particularly consisting of, two or more cyclohexanolpentakisester moieties described by a general formula (I) linked by a common central linker L, ##STR00043## wherein X is selected from OPO.sub.3.sup.2−, OPSO.sub.2.sup.2−, or OSO.sub.3.sup.−, n is an integer from 2 to 10, particularly n is selected from 2, 3, 4, 6 and 8, particularly wherein n is selected from 2, 3 and 4, wherein L is a linking moiety comprising or consisting of a linear or branched alkyl, optionally substituted by oxygen, nitrogen, (fluorine), particularly wherein the linking moiety has a molecular weight <1000 g/mol, particularly <700 g/mol, more particularly <500 g/mol, or even <400 g/mol.

11. The method according to claim 10, wherein said condition related to pathological crystallization is nephrocalcinosis or nephrolithiasis due to pathological calcium oxalate or phosphate crystallization in a patient, particularly wherein the condition is primary and secondary hyperoxaluria, Dent disease, Bartter's syndrome, distal renal tubule acidosis, neurogenic bladder, autosomal dominant polycystic kidney, calcium oxalate kidney stone formation, struvite stones and renal calcification.

12. The method according to claim 10, wherein the condition related to pathological crystallization is associated with formation of oxalate, phosphate and/or calcium precipitates.

13. The method c according to claim 10, wherein the compound is formulated for intravenous, intraperitoneal, intramuscular, intra-arterial, topical, intravesical or, particularly, subcutaneous administration.

14. A compound described by general formula Va, Vb, Vc, VIa, VIb or VIc, ##STR00044## ##STR00045## ##STR00046## ##STR00047## wherein OPMB signifies an O-(bis-paramethoxybenzyl)phosphate moiety and k is an integer selected from 1 to 12.

15. The compound according to claim 13, wherein k is selected from 2, 3, 4, 5, 6 or 7.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0135] FIG. 1 shows the inhibition of CaOx crystallization by IIc (OEG.sub.4-(IP5).sub.2) and the comparison IP5-mono-PEG2 (OEG.sub.2-IP5) at pH 6.2, and at pH 5.5. CaOx crystallization was induced by adding 1 mM sodium oxalate to humane urine and characterized by light microscopy, followed by an automated image analysis to determine crystal number, hydrate forms (COM—CaOx monohydrate, COD—CaOx dihydrate, n.d.—not defined), and crystal area. Surface area of the different hydrate forms normalized to the total surface area of the control is plotted. IIc/OEG.sub.4-(IP5).sub.2 led to complete inhibition of COM crystals only at 300 nM, followed by complete CaOx (COM and COD) inhibition at 4 μM. In comparison, for the previous lead compound IP5-mono-PEG.sub.2 COM only inhibition was observed at 11 μM, while complete CaOx (COM and COD) inhibition was not observed in the tested range up to 100 μM.

[0136] FIG. 2 shows the inhibition of CaOx crystallization by compounds of the present invention: IIb (OEG.sub.2-(IP5).sub.2), IIc (OEG.sub.4-(IP5).sub.2) and IId (OEG.sub.8-(IP5).sub.2) at pH 6.2.

[0137] FIG. 3 shows the inhibition of CaOx crystallization by IP6 analogues previously described in PCT/EP2016/080657 at pH 6.2. CaOx crystallization was induced by adding 1 mM sodium oxalate to humane urine and characterized by light microscopy, followed by an automated image analysis to determine crystal number, hydrate forms (COM—CaOx monohydrate, COD—CaOx dihydrate, n.d.—not defined), and crystal area. Surface area of the different hydrate forms normalized to the total surface area of the control is plotted.

[0138] FIG. 4 shows the synthesis of compounds of the invention as described in example 1.

[0139] FIG. 5 shows images of CaOx crystal formation inhibition by a compound of the present invention OEG.sub.4-(IP5).sub.2 (scale bar: 50 μm).

[0140] FIG. 6 shows images of CaOx crystal formation inhibition by an IP6 analogue compound OEG.sub.2-IP5 previously described in PCT/EP2016/080657 (scale bar: 50 μm).

[0141] FIG. 7 shows the synthesis of compound (OEG.sub.4).sub.3-(IP5).sub.3.

[0142] FIG. 8 shows changes in the CaOx crystallization pattern induced by IP6 analogues. Effects of (OEG.sub.4).sub.3-(IP5).sub.3 on CaOx bulk crystallization in human urine spiked with 1 mM NaOx were assessed by light microscopy at t=7 h. COM—CaOx monohydrate, COD—CaOx dihydrate, n.d.—not defined; N=3, mean total area/field of view+SD for the respective crystal type normalized to the control (without inhibitor).

[0143] FIG. 9 shows that OEG.sub.4-(IP5).sub.2 stabilizes early precursor particles and leads to delayed COD growth. (A) Dose-dependent inhibition of CaOx crystallization by OEG.sub.4-(IP5).sub.2 in Bis-Tris buffer (pH 6.2) was characterized by SEM at t=7 h. Representative SEM images are shown (scale bar: 1 μm). (B) Crystallization of CaOx with 500 nM OEG.sub.4-(IP5).sub.2 and without inhibitor was sampled at t=10 min and imaged by SEM (scale bar: 1 μm). Crystallinity was characterized by TEM with SAED (inlets).

[0144] FIG. 10 shows inhibition of CaOx adhesion to renal epithelial cells by IP6 analogues. Anti-adhesive effects of (A) OEG.sub.4-(IP5).sub.2, (B) OEG.sub.2-IP5 and (C) (OEG.sub.2).sub.2-IP4 on CaOx attachment to RPTEC/TERT1 cells in vitro were assessed by differential interference contrast microscopy followed by quantification of the crystal occupied area. Cells at confluence were treated with 150 μg/cm.sup.2 CaOx premixed with compound for 30 minutes, before washing, fixation and imaging. (D) Example images of CaOx premixed with/without OEG.sub.4-(IP5).sub.2 adhering to the confluent cell layer (Leica CTR6000, 63× oil objective). (E) To assess the capacity of OEG.sub.4-(IP5).sub.2 to remove already bound CaOx of cell layers an adapted version of the assay described above was performed. RPTEC/TERT1 cells were incubated with CaOx for 30 minutes, unbound CaOx was removed by washing with PBS and cells were further incubated with OEG.sub.4-(IP5).sub.2 or medium control for 2 h. (N=3, mean+SD normalized to the CaOx ctrl, one-way ANOVA with Dunnett's multiple comparison, **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, compared to CaOx control.)

[0145] FIG. 11 shows that CaOx induced cellular injury of renal epithelial cells is prevented by OEG.sub.4-(IP5).sub.2 in vitro. (A) Heatmap and hierarchical clustering of relative gene expression levels determined by RNA sequencing (differentially expressed genes between CaOx versus CaOx-OEG.sub.4-(IP5).sub.2 with p<0.01, log 2 ratio=0.5, number of significant features 2000). (B) Gene set enrichment analysis of gene expression levels in the CaOx relative to the medium group and corresponding values in the CaOx-OEG.sub.4-(IP5).sub.2 group. Top gene ontology terms and corresponding normalized enrichment score for differentially up- or down-regulated genes in the CaOx group relative to the medium control group are plotted (top 10 upregulated gene ontology terms, all FDR≤0.05 and downregulated gene ontology terms with FDR≤0.05 are shown). Corresponding values for differentially expressed genes in the CaOx-OEG.sub.4-(IP5).sub.2 group are given (FDR≤0.05 is indicated by dark blue, FDR>0.05 by light blue). (C) Gene expression levels of genes involved in inflammatory and immune response, cellular signalling and ECM production in the different treatment groups. (D) Dose-dependent decrease in the number of dead cells by pre-incubation of CaOx with OEG.sub.4-(IP5).sub.2 before treatment of RPTEC cells in comparison to CaOx only treated cells was assessed by a viability stain (mean+SD normalized to CaOx ctrl, one-way ANOVA with Dunnett's multiple comparison, **** p<0.0001, ** p<0.01). (E) Example brightfield images showing CaOx deposition (black spots) and red fluorescence images indicating cell death of cells receiving no treatment, CaOx only or CaOx+10 μM OEG.sub.4-(IP5).sub.2 treatment (Leica CTR6000, 10× objective, scale bar: 100 μm). (N=3 for all experiments.)

[0146] Table 1 shows data from a screening of calcification inhibitors for activity in inhibiting growth of CaOx crystals. IP6 analogues previously described in PCT/EP2016/080657 and compounds OEG.sub.2-(IP5).sub.2, OEG.sub.4-(IP5).sub.2, OEG.sub.8-(IP5).sub.2 and (OEG.sub.4).sub.3-(IP5).sub.3 of the present invention are listed.

EXAMPLES

Example 1: Synthesis of IIc (OEG.SUB.4.-(IP5).SUB.2.)

[0147] Step 1:

[0148] Reagents: DMF, sodium hydride, p-methoxybenzyl chloride.

[0149] Myo-inositol-1,3,5-orthoformate is dissolved in dry DMF under nitrogen atmosphere, cooled to 0-5° C. and added portion-wise with sodium hydride (2.2 eq.). After stirring 1 h at room temperature, p-methoxybenzyl chloride (2.3 eq.) is added dropwise. The mixture is stirred at room temperature for 12 h, then it is added with water and extracted with ethyl acetate. Solvent removed under vacuum and residue crystallized with diethyl ether. Yield 70%.

[0150] Step 2:

[0151] Reagents: MeOH; HCl 2N.

[0152] The product of step 1 (1) is dissolved in 10 vol of methanol, added with 1 vol of HCl 2 N and heated at 50° C. for 3 hs. After cooling, the mixture is neutralized with ammonium hydroxide 30% and left overnight at room temperature. The solid formed, is filtered, washed with water and dried. After crystallization with diethyl ether compound 2 is obtained as whitish solid. Yield 80%.

[0153] Step 3:

[0154] Reagents: DMF, sodium hydride, p-methoxybenzyl chloride.

[0155] The product of step 2 (2) is dissolved in dry DMF under nitrogen atmosphere, cooled at 0-5° C. and added with sodium hydride (2 eq.) portion-wise. After 1 h at room temperature, the mixture is cooled at 0-5° C. and added with p-methoxybenzyl chloride (2 eq.). After 12 h at room temperature, the mixture is added with water and extracted with ethyl acetate. The residue is purified by column chromatography eluting with hexanes/ethyl acetate 6/4, then crystallized with diethyl ether. Yield 35%.

[0156] Step 4:

[0157] Reagents: dichloromethane, Tosyl chloride, triethylamine.

[0158] Tetraethylene glycol is dissolved in dry dichloromethane, added with triethylamine (3 eq.) and then with p-toluenesulfonyl chloride (2.5 eq.). After stirring for 12 h at room temperature, the solvent is removed under vacuum and the residue purified by column chromatography, eluting with ethyl acetate/hexanes 7/3. Yield 80%.

[0159] Step 5:

[0160] Reagents: DMF, sodium hydride, tetraethylene glycol bis tosylate (step 4).

[0161] The product of step 3 (3) is dissolved in dry DMF under nitrogen atmosphere, cooled to 0-5° C. and added with sodium hydride (1.1 eq.) portionwise. After stirring 1 h at room temperature, the mixture is again cooled to 0-5° C. and added with a solution of compound 4 (0.5 eq.) in DMF (3 vol). After stirring for 12 h at room temperature, the mixture is added with water and extracted with ethyl acetate. After removal of the solvent the mixture is purified by chromatography eluting with hexanes/ethyl acetate.

[0162] Yield 40%.

[0163] Step 6:

[0164] Reagents: Dichloromethane, trifluoroacetic acid. Not purified.

[0165] The product of step 5 (5) is dissolved in dichloromethane (10 vol) and added with trifluoroacetic acid (2 vol). After stirring for 3 h at room temperature, the solvent is removed under vacuum and the residue used for the next step without further purification. Yield quantitative.

[0166] Step 7:

[0167] Reagents: DMF, 5-methyl-tetrazole, dibenzyloxy-diisopropylphosphoroamide, then H.sub.2O.sub.2.

[0168] The product of step 6 (6) is dissolved in dry DMF (10 vol) under nitrogen atmosphere, added with 5-methyltetrazole (9 eq.) and cooled to −10° C., followed by drop-wise addition of dibenzyloxy-diisopropylphosphoroamide (6 eq.). After stirring for 12 h at room temperature, the mixture is cooled to 0-5° C. and added drop-wise with H.sub.2O.sub.2 30% (9 eq.). After stirring 1 h at room temperature, water is added and the mixture is extracted with ethyl acetate. After removal of the solvent, the residue is purified by column chromatography eluting with chloroform/methanol 96/4. Yield 40%.

[0169] Step 8:

[0170] Reagents: H.sub.2, Pd/C. Magnesium salt formation with Magnesium ethoxide.

[0171] The product of step 7 (7) is dissolved in methanol (15 vol), added with Pd/C 10% and hydrogenated at room temperature and room pressure for 24 hs. After filtration of the catalyst, the solvent is removed under reduced pressure and compound 8 as acid is obtained. Yield quantitative. The formation of the magnesium salt is performed by adding an aqueous solution of magnesium ethoxide to the aqueous solution of compound 8, followed by distillation of the solvent under vacuum.

Example 2: Synthesis of (OEG.SUB.4.).SUB.4.-(IP5).SUB.4

[0172] Compound 5 (PMB=p-methoxybenzyl; FIG. 7) was prepared in three steps starting from myoinositol-1,3,5-orthoformate, as described in the synthesis of OEG.sub.4-(IP5).sub.2 (Example 1; FIG. 4).

[0173] Reagents of step A (FIG. 7): P-Toluenesulfonyl chloride; dichloromethane, triethyl amine. Column chromatography. Yield 80%.

[0174] Reagents of step B (FIG. 7): Trimethylol propane, DMF, sodium hydride. Column chromatography. Yield 45%.

[0175] Reagents of step C (FIG. 7): Methanol, H.sub.2, Pd/C. Yield 80%.

[0176] Reagents of step D (FIG. 7): P-Toluenesulfonyl chloride; dichloromethane, triethyl amine. Column chromatography. Yield 70%.

[0177] Reagents of step E (FIG. 7): Compound 5 as shown in FIG. 7, DMF, sodium hydride. Column chromatography. Yield 20%.

[0178] Reagents of step F (FIG. 7): Dichloromethane, trifluoroacetic acid. Yield quantitative.

[0179] Reagents of step G (FIG. 7): DMF, dibenzyl-N,N-diisopropylphosphoramidite, 5-methyltetrazole. Column chromatography. Yield 30%.

[0180] Reagents of step H (FIG. 7): Methanol, H.sub.2, Pd/C. Yield 90%. Then sodium salt formation, quantitative.

Example 3: Inhibition of CaOx Crystallization

[0181] For comparison of the compounds' efficacy to inhibit calcium oxalate (CaOx) crystallization a simple in vitro assay in urine was used. In brief, human urine (pH 6.2) was mixed with compound, followed by the addition of 1 mM sodium oxalate inducing CaOx crystallization. After 7 hours of incubation at room temperature crystallization was measured by light microscopy, employing an automated image analysis to detect, classify and quantify the CaOx crystal types formed, namely CaOx monohydrate (COM) and CaOx dihydrate (COD).

[0182] The inventors found that all three novel divalent IP5 structures showed similar CaOx inhibitory efficacy in vitro with complete inhibition of CaOx crystallization at 4 μmol/L (FIG. 1). The length of the linker did not have an important impact on inhibitory efficacy. Compared to the previously developed, most potent compound IP5-mono-PEG2 (OEG.sub.2-IP5) (PCT/EP2012/004088 & PCT/EP2016/080657), OEG.sub.4-(IP5).sub.2 showed a 2500-fold increase in potency to inhibit CaOx crystallization to below 50% of the control (FIG. 1).

[0183] The inventors observed that first, the compounds at sub-inhibitory concentrations lead to a shift in crystallization from COM towards COD, further increasing concentrations of compound can then completely inhibit CaOx growth with an unexpected efficiency. All tested divalent IP5 structures showed complete CaOx inhibition in the in vitro assay at low micromolar concentrations, which are expected to be achieved in vivo (FIG. 2, Table 1).

[0184] The inventors showed that the efficacy to inhibit CaOx crystallization is dependent on the number of phosphate groups on the molecule and that complete inhibition of crystallization was observed with all OEG.sub.x-(IP5).sub.2 compounds at 4 μmol/L (Table 1). Furthermore, IP6 and other compounds tested up to 30 μM or 100 μM, respectively, did not lead to complete CaOx inhibition (FIG. 3, Table 1). Minimal impact of the length of the OEG chain or length of linker on total CaOx crystallization, was observed mostly affecting COM-COD formation dynamics. Moreover, decreasing the pH to 5.5 reduces the activity of OEG.sub.2-IP5 more drastically than of OEG.sub.4-(IP5).sub.2. The compound OEG.sub.2-IP5 at 100 μM did not result in <50% complete inhibition or COM inhibition. In addition, the compound OEG.sub.4-(IP5).sub.2 retained COM inhibition at 0.3 μM and 50% complete inhibition at 2.4 μM (FIG. 1, Table 1). The compound (OEG.sub.4).sub.3-(IP5).sub.3 displayed COM inhibition at 0.04 μM and complete inhibition at 2.4 μM (FIG. 8, Table 1).

TABLE-US-00001 TABLE 1 Complete 50% complete COM inhibition inhibition inhibition (i.e <5% total (<50% total (<5% of total Compound area) (μM) area) (μM) area) (μM) pH 6.2 IP6 >30 30 3 OEG.sub.2-IP5 >100 100 11 OEG.sub.11-IP5 >100 11 100 (OEG.sub.2).sub.2-IP4 >100 >100 >100 OEG.sub.2-(IP5).sub.2 4 0.3 0.3 OEG.sub.4-(IP5).sub.2 4 0.04 0.3 OEG.sub.8-(IP5).sub.2 4 2.4 2.4* (OEG.sub.4).sub.3-(IP5).sub.3 2.4 2.4 0.04 Citrate >100 >100 >100 pH 5.5 OEG.sub.2-IP5 >100 >100 >100 OEG.sub.4-(IP5).sub.2 >4 2.4 0.3 *High COD crystallization - possible misclassification as COM at 300 nM

Example 4: OEG.SUB.4.-(IP5).SUB.2 .Inhibits CaOx Crystallization by Early-Stage Precursor Stabilization

[0185] Scanning electron microscopy (SEM) experiments of early stage crystallization at t=10 min, revealed the presence of both, COM shaped- and round nanosized particles, while with 500 nM OEG.sub.4-(IP5).sub.2 only nanosized particles were detected (FIG. 9B). Nanosized particles were still present at t=1 h with 0.5-10 μM of inhibitor, in contrast to micrometer sized COM crystals being predominant in the control sample, suggesting the stabilization of early stage, nanosized CaOx particles by OEG.sub.4-(IP5).sub.2.

[0186] SEM experiments further confirmed a dose-dependent shift from predominately COM towards COD crystallization, and a COD face-specific growth inhibition with OEG.sub.4-(IP5).sub.2 (FIG. 9A). Taken together these results indicate that OEG.sub.4-(IP5).sub.2 alters the crystallization process at several stages, namely crystal nucleation and growth kinetics, as well as crystal polymorphism and shape.

Example 5: OEG.SUB.4.-(IP5).SUB.2 .Blocks CaOx Adhesion to Renal Epithelial Cells In Vitro More Efficiently than OEG.SUB.2.-4P5

[0187] Anti-adhesive effects of chosen IP6 analogues on CaOx crystals to renal proximal tubular epithelial cells (RPTECs) in vitro were investigated by differential interference contrast microscopy. Comparison of OEG.sub.4-(IP5).sub.2, OEG.sub.2-IP5 and (OEG.sub.2).sub.2-IP4 showed that concentrations of 200 nM, 4 μM and above 100 μM, respectively, lead to <25% of CaOx adhesion compared to the control (FIG. 10A-D). Citrate treatment did not lead to any detectable protection against CaOx adhesion up to 100 μM. Additionally, OEG.sub.4-(IP5).sub.2 not only inhibited CaOx adhesion, but also reversed the binding of prebound CaOx crystals at low micromolar concentrations (FIG. 10E).

Example 6: Cyto-Protective Effects of OEG.SUB.4.-(IP5).SUB.2 .on CaOx Induced Cellular Injury

[0188] RNA sequencing of RPTECs showed a similar gene expression profile between medium control, OEG.sub.4-(IP5).sub.2 control and cells treated with CaOx premixed with OEG.sub.4-(IP5).sub.2, while CaOx treatment alone induced drastic changes (FIG. 11A). Gene set enrichment analysis of differently expressed genes in the CaOx over medium samples revealed alteration of genes mainly involved in immune and inflammatory responses, which coincides with literature, as well as structural changes (e.g. microtubule organization) and protein modification and signaling effects (e.g. peptidyl-glutamic acid modification, ER-nucleus signaling pathway) (FIG. 11B). Comparing the top 10 enriched gene sets of differentially expressed genes in CaOx over medium samples versus CaOx over CaOx-OEG.sub.4-(IP5).sub.2 samples showed similar changes, indicating that premixing OEG.sub.4-(IP5).sub.2 with CaOx can prevent CaOx induced transcriptomic changes (FIG. 11B). Elevated expression of TNF alpha and TGF beta signaling pathway genes, cell surface glycoprotein, C-X-C motif chemokine ligand and complement cascade genes, in the CaOx group compared to all other treatment groups were detected (FIG. 11C). Further, CaOx crystal treatment triggered plasma membrane damage, as evidenced by ethidium homodimer-1 staining, which was prevented by OEG.sub.4-(IP5).sub.2 in a dose-dependent manner (FIG. 11 D,E).

[0189] Materials and Methods

[0190] Materials

[0191] Compounds were custom synthesized by Chimete Srl (Tortona, Italy). Phytic acid dodecasodium salt was purchased from Biosynth AG (Thal, Switzerland). Pooled human urine was purchased from Lee Biosolutions (Maryland Heights, Mo.). Oxalate assay kit (MAK315), calcium colorimetric assay kit (MAK022), Bis-Tris, sodium oxalate, formalin solution neutral buffered 10% and Mowiol 4-88 were purchased from Sigma-Aldrich (St. Louis, Mo.). Sodium chloride and calcium chloride dehydrate were obtained from Merck (Kenilworth, N.J.). Calcium oxalate monohydrate was purchased from abcr (Karlsruhe, Germany). 8-well glass bottom slides (80827) were purchased from ibidi (Martinsried, Germany). Trisodium citrate dihydrate analytical grade, Nunc Lab-Tek II chamber slides, cell culture plates, Live/Dead Viability/Cytotoxicity kit for mammalian cells and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific (Rochester, N.Y.). RPTEC/TERT1 cells, ProxUp basal medium and supplements were obtained from Evercyte (Vienna, Austria). RNeasy kit was purchased from Quiagen (Hilden, Germany) and TrueSeq RNA kit from Illumina (San Diego, Calif.).

[0192] CaOx Screening Assay and Analysis

[0193] Human urine was stored at −20° C. in 50 mL aliquots. After thawing, aliquots were centrifuged, filtered using a 0.45-μm syringe filter and pH set to 6.2. Oxalate concentrations of human urine samples were determined using the Oxalate Assay kit following the kit protocol, and found to be <100 μM for the samples used. Dilutions of sodium and compounds were prepared in Bis Tris buffer (50 mM, 150 mM NaCl, pH 6.2). Urine was mixed with compound in Eppendorf tubes, and subsequently sodium oxalate was added. The final assay mixture of 1 mL total volume contained 90% urine, 5% sodium oxalate (1 mM) and 5% compound dilution. Immediately upon sodium oxalate addition, the final assay mixture was vortexed and added to the imaging slide (8 well glass bottom chamber, ibidi). 400 μL per well were added and two wells for each sample were prepared. Crystallization was assessed after 7 h incubation at room temperature, using a Leica CTR6000 microscope (Leica Microsystems, Wetzlar, Germany) in brightfield mode. For visualization, images with a 40× objective were used. For quantification 2 wells/sample with five 20× objective images each were used. Without addition of sodium oxalate no crystallization was observed.

[0194] Inhibition of seeded crystals was evaluated in a similar manner. Crystallization was induced with 1 mM sodium oxalate in urine as above followed by compound addition after 1.5 h incubation at room temperature directly to each well. Samples were imaged before compound addition at t=1.5 h and afterwards at t=7 h total incubation time.

[0195] Quantification and classification of crystals was performed in Matlab (version R2016b or R2018b). In brief, crystals were segmented using edge detection and watershed algorithms and subsequently shape, intensity and texture features for each crystal were extracted. The extracted features served as input for a semi-supervised classification approach to distinguish COM (class 0), COD (class 1), not determined structures (class 2) and background noise (class 3). A cubic support vector machine (SVM) classifier was trained and subsequently used for classification of testing images.

[0196] All testing images were processed using the Batch Processing App. Efficacy of compounds to inhibit CaOx crystallization was compared by analysing the total area per field of view for each crystal type. The sum of the total area of the three crystal classes—COM, COD and unclassified, was normalized to the control within each experiment (N=3). Due to the inherent variability of the crystallization process itself, normalization within each experiment decreased the effect of inter-experimental variability.

[0197] Scanning Electron Microscopy

[0198] CaOx crystals for SEM were prepared in Bis Tris buffer (50 mM, 150 mM NaCl, pH 6.2) containing 1 mM NaOx, 2 mM CaCl.sub.2 and the indicated concentration of compound. 12 mm round glass coverslips were placed at the bottom of 24 well plates. First, stock solutions of 20× final concentrations of CaCl.sub.2 and inhibitor, and 10× final concentration of NaOx were prepared in the Bis Tris buffer. Assay mixture was prepared in Eppendorf tubes by first adding 800 μL of Bis Tris buffer, followed by addition of 50 μL CaCl.sub.2 (20× concentration) and 50 μL of inhibitor (20× concentration) and vortexing. Then 100 μL NaOx (10× final concentration) was added, the assay mixture was vortexed and immediately added to the prepared 24 well plate. 400 μL per well and 2 wells per sample were prepared. Samples were incubated at room temperature for the indicated time. Example images were taken using a Leica CTR6000 microscope (Leica Microsystems) in brightfield mode before samples were washed once with double distilled water (ddH.sub.2O) and drying at room temperature. Samples were imaged by brightfield microscopy again after drying to confirm little effects of the drying process on crystal morphology. After drying, glass coverslips were mounted on SEM stubs with silver paint and coated with a 6 nm layer of Platinum/Palladium using a CCU-010 Metal Sputter Coater (Safematic, Bad Ragaz, Switzerland). Samples were imaged using a Magellan 400 FEI SEM microscope (ThermoFisher Scientific, Rochester, N.Y.) in the secondary electron mode using the TLD detector.

[0199] Cell Experiments

[0200] RPTEC/TERT1 human proximal tubule cells (Evercyte) were cultured in ProxUp basal medium (Evercyte, DMEM/Ham's F12, Hepes buffer and GlutaMAX™) mixed with ProxUp supplements (Evercyte) at 37° C. at 5% CO.sub.2 according to the manufacturer's recommendations. Cells were regularly tested for Mycoplasma infection.

[0201] Cell viability was assessed using the Live/Dead Viability/Cytotoxicity kit (ThermoFisher Scientific). Cells were cultured in 24 well plates and 48 h after seeding (upon reaching confluence) treated with 150 μg/cm.sup.2 Calcium oxalate monohydrate (abcr) in ProxUp basal premixed with compound. After 48 h treatment, cell viability was determined by staining with the Live/Dead cytotoxicity kit (ThermoFisher Scientific), according to the kit protocol. Images were obtained by epifluorescence microscopy with a Leica CTR6000 microscope (Leica Microsystems, 3 wells/sample, 3 images/well) and the number of red fluorescence dots was quantified.

[0202] For quantification of CaOx adhesion to RPTEC cells, cells were cultured on 8-well Nunc Lab Tek chamber slides (ThermoFisher Scientific), and upon confluence at 48 h treated with COM (abcr) premixed with compound in ProxUp basal medium (pH set to 6.9) for 30 min at 37° C., 5% CO.sub.2. Cells were washed twice with phosphate buffered saline (PBS, pH 7.4), fixed with 10% neutral buffered formalin for 15 min, and rinsed with PBS, followed by a final rinse with ddH.sub.2O. The chamber of the slide was removed and cells mounted with Mowiol. Images were obtained with a Leica CTR6000 microscope (Leica Microsystems) in DIC mode, 63× oil objective (3 wells per sample; 8 images per well) and crystal occupied area quantified.

[0203] RNA Sequencing

[0204] Cells were cultured as described in the cell viability assay. Four treatment groups consisted of a medium control (ProxUp basal), 150 μg/cm.sup.2 Calcium oxalate monohydrate (abcr) only, 150 μg/cm.sup.2 Calcium oxalate monohydrate (abcr) premixed with 10 μM OEG.sub.4-(IP5).sub.2 and 10 μM OEG.sub.4-(IP5).sub.2 only. Total RNA was extracted using the RNeasy kit (Quiagen) according to the manufacturer's protocol. mRNA was purified and RNAseq library was prepared using the TrueSeq RNA kit. Sequencing was performed on a Novaseq 6000 (Illumina). Reads were aligned to the human reference genome GRCh38.p10 using the STAR tool and transcripts quantified using the Kallisto program. Ensembl release 91 was used for the gene model definitions. For the heatmap and hierarchical clustering of significantly different genes the log 2fold changes in comparison to the mean of all samples was calculated and log 2fold changes >4 were set to 4. Gene set enrichment analysis was performed on Webgestalt.org (v2019).sup.41. Input for GSEA were gene lists with the ensemble gene id and a ranking score (−log.sub.10*p-value*sign (log.sub.2 ratio)) as a metric for differential expression between two treatment groups. Differential expressed genes were compared to the gene ontology—biological process functional database and as a reference set the human genome—protein coding was used. For comparison of expression levels of selected genes the normalized gene count was used.

[0205] Data Analysis

[0206] Statistical analysis and graphs were prepared using GraphPad Prism (La Jolla, Calif.), unless otherwise stated. Data are expressed as mean+SD. Ordinary one-way ANOVA testing followed by post-hoc Dunnett's multiple comparison was used for comparison of treatment to control group. Image analysis was carried out using Matlab version R2018b and R2016b (MathWorks, Natick, Mass.) unless otherwise stated.