CN116917302A - Phytic acid analogues for the treatment of calcification-associated kidney disease - Google Patents

Abstract

The present invention relates to inositol polyphosphate oligoalkyl ether compounds, or pharmaceutically acceptable salts thereof, for use in the treatment or prevention of diseases associated with calcium salt crystal formation.

Description

Phytate analogues for the treatment of calcification-associated nephropathy

The present invention relates to compounds and compositions for the treatment of conditions associated with calcification of tissues, particularly renal tissue, caused by deposits of, or exposure to, calcium phosphate (CaP) and other calcium precipitates.

Background technique

WO2013045107 (A1) first disclosed the concept of using inositol polyphosphate polyalkyl ether derivatives as pharmaceutical agents. This compound was initially thought to be an agent with considerable potential to neutralize C. difficile toxins in the colon lumen, and subsequent analysis found that it was highly effective in reducing calcification when administered systemically, as shown in WO2017098047(A1), US10624909( B2) and US20200247837(A1) were first disclosed.

WO2020058321(A1) discloses other compounds based on the inositol polyphosphate backbone with improved pharmacological properties.

All patent documents mentioned in this specification are incorporated by reference in their entirety.

The object of the present invention is to provide further advantageous applications of inositol polyphosphate polyalkyl ether derivatives. This object is achieved by the subject-matter of the independent claims of the present description.

Contents of the invention

In one aspect, the present invention provides an inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, for treating or preventing diseases associated with calcium salt precipitation or calcium salt crystal formation. In the broadest sense, the disease targeted by the compounds of the invention is chronic kidney disease associated with the formation of calcium salt precipitates, in particular precipitates consisting of calcium phosphate and/or calcium oxalate.

Specific diseases whose treatment may benefit from administration of compounds described herein include renal fibrosis, particularly when associated with renal tissue calcification, or exposure to calcium phosphate, or calcium oxalate precipitation, renal inflammation, particularly when associated with renal tissue calcification or Nephritis, especially interstitial nephritis, glomerulonephritis, phosphate-induced renal fibrosis, phosphate-induced chronic kidney disease, chronic kidney disease associated with hyperphosphatemia, when associated with exposure to calcium phosphate or calcium oxalate precipitates, Development of chronic kidney disease, phosphate toxicity, hyperphosphaturia, hyperphosphatemia, and/or hyperFGF23emia.

According to an alternative to this aspect of the present invention, an inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, is provided for the treatment or prevention of diseases associated with calcium salt precipitation or calcium salt crystal formation, the The disease is selected from the group consisting of vascular calcification, coronary artery disease, vascular sclerosis, valvular calcification, nephrocalcinosis, cutaneous calcification, nephrolithiasis, and chondrocalcinosis.

One of the key findings derived from the data presented here, and which the inventors believe to be truly novel, is that a protective effect was observed in vivo even in the absence of overt calcification. The mouse model used in the examples is that within the time frame tested, CaP precipitates form in the renal tubules, but it is virtually impossible to detect calcification histologically in the kidney itself (von Kossa staining is negative). Calcification occurs if animals are maintained longer at the beginning of the experiment without inhibitor treatment or if nephrectomy is performed to speed up the process. A protective effect is of course also expected when calcification is present and measurable (which was the case in the cellular assay used in the previous examples).

Terms and definitions

For the purpose of interpreting this specification, the following definitions will apply and, wherever appropriate, terms used in the singular will also include the plural and vice versa. To the extent that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

As used herein, the terms "include," "have," "contains," and "include" and other similar forms, as well as their grammatical equivalents, are equivalent in meaning and are open-ended, that is, in these words Any following item or items is not meant to be an exhaustive enumeration of the item or items, nor is it meant to be limited to the listed item or items. For example, something "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain more than components A, B , and C, and may include one or more other components. Accordingly, it is intended and understood that "comprises" and similar forms and its grammatical equivalents include disclosures of embodiments that "consist essentially of" or "consist of."

Where a range of values is provided, it is to be understood that each intervening value between the upper and lower limits of the range to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise, and Any other stated in, or intervening, values are included within the invention subject to any specific exclusion in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

As used herein, and included in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, in cell culture, molecular genetics, nucleic acid chemistry, hybridization technology, and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

In the context of this specification, the term oligoalkyl ether refers to oligoethylene glycols and close chemical relatives such as oligopropylene glycols and oligoglycerols. The term "oligomeric" means the presence of more than one, in particular 2 to 20, more particularly 2 to 12, monomers (–CH ₂ –CH ₂ –O–), in the case of oligomeric glycols. Monomer (–CH(CH ₃ )–CH ₂ –O–) in the case of oligopropylene glycol.

In the context of this specification, the term myo-inositol polyphosphate refers to cyclohexane hexaol (myo-inositol, cyclohexane-1,2,3,4,5,6-hexaol), in which each OH is replaced by a phosphate moiety Substituted unless the OH is substituted by an oligomeric alkyl ether moiety according to the preceding definition. In a specific embodiment, the myo-inositol scaffold is myo-inositol ((1R,2S,3R,4R,5S,6S)-cyclohexane-1,2,3,4,5,6-hexanol).

In the context of this specification, the term inositol polyphosphate oligoalkyl ether compound relates to compounds comprising one or several inositol polyphosphate moieties as defined above, and at least one oligoalkyl ether.

In the context of this specification, the term calcification relates to the formation of calcium precipitates in affected tissue, in particular renal tissue.

Detailed ways

A first aspect of the present invention relates to an inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of diseases associated with calcium salt precipitation or calcium salt crystal formation. In the broadest sense, the disease is a chronic kidney disease associated with the formation of calcium salt precipitates, in particular those consisting of calcium phosphate and/or calcium oxalate.

Without wishing to be bound by theory, the inventors conclude from the results presented here that the pathophysiological mechanisms of the diseases mentioned here include, as a first step, the precipitation of calcium phosphate substances, which subsequently grow and adhere to cells ( and may grow further once attached to cells). The interaction of calcium phosphate precipitates with renal tubular cells, regardless of whether the precipitates are large or small, can cause damage to the cells.

The compounds and compositions provided herein apparently prevent new precipitates from forming/growing, but also prevent existing precipitates from adhering to cells, both of which confer a protective effect. There is therefore a dual mode of action.

Calcium precipitation

The chemical composition of calcium crystalline precipitates associated with renal and cardiovascular disease has been well studied (see Elliot, J Urology 100 (1968), 687–693; Xie et al., Cryst Growth Des. 2015 Jan 7;15(1) :204–211). In principle, the treatment according to the invention can prevent or ameliorate any situation in which the deposition of calcium phosphate, oxalate and mixed calcium phosphate-calcium oxalate crystals plays a role.

Calcium phosphate occurs in different crystalline forms in human pathological deposits; these include hydroxyapatite (HA, Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), brushite (CaHPO ₄ *2H ₂ O), monetite, and various amorphous calcium phosphates.

Calcium oxalate occurs in the form of mono-(calenite), di-(calenite), and trihydrate, and is often associated with other sediments, mainly phosphates.

The results in the examples show that any cellular sequelae of calcium salt crystal formation, even if associated with deposits preceding the macroscopically detectable calcification stage, can be prevented by the inventive treatment described in this specification.

Diseases related to calcium deposition that would benefit from treatment with this invention

"Renal fibrosis" or "tubulointerstitial fibrosis" associated with the formation of calcium salt crystals, and/or tissue exposure to calcium salt crystals, refers to the thickening and scarring of kidney tissue due to exposure to calcium phosphate , calcium oxalate or mixed CaP/CaOx precipitation leading to wound healing failure after chronic injury. During this process, fibrotic matrix deposition continues unchecked, leading to glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Patients with the disease can experience intense abdominal pain (with bleeding or heavy bleeding), swelling and discoloration of one or both legs, and eventually develop chronic kidney disease.

"Nephritis" or "nephritis" is defined as a complex network of interactions between renal parenchymal cells and innate immune cells such as macrophages and dendritic cells, with recruitment of circulating monocytes, lymphocytes, and neutrophils . Once stimulated, these cells activate specialized structures such as Toll-like receptors and Nod-like receptors (NLRs). By detecting danger-associated molecules, these receptors can initiate major innate immune pathways, such as nuclear factor kappa B (NF-κB) and the NLRP3 inflammasome, causing metabolic reprogramming and phenotypic changes in immune and parenchymal cells and triggering many Secretion of inflammatory mediators that can cause irreversible tissue damage and loss of function. In CKD, chronic inflammation leads to progressively lower glomerular filtration rate (GFR), which can eventually lead to kidney failure (end-stage renal disease, ESRD).

To the extent nephritis/nephritis is related to or caused by the interaction of renal tissue with calcium deposits, treatment according to the invention is expected to improve or prevent this condition. The examples presented herein demonstrate that calcium phosphate deposition/exposure leads to the upregulation of markers of inflammation and fibrosis in the kidney, and therefore any disease consisting of inflammation and fibrosis of the kidney should benefit from treatment that inhibits deposit formation. For each of these inflammatory conditions, there may also be other or even simultaneous causes of inflammation and fibrosis, as both are often multifactorial processes. However, elimination of a contributing factor is expected to improve overall clinical performance.

"Glomerulonephritis" refers to a group of diseases characterized by inflammatory changes in the glomerular capillaries. Patients with this disease may experience proteinuria, sometimes impaired kidney function with fluid retention, hypertension, and edema. Glomerulonephritis can occur as the primary renal disease or it can be indicative of a systemic disease process. When associated with the formation of calcium salt crystals, and/or tissue exposure to calcium salt crystals, glomerulonephritis is expected to benefit from treatment with the compounds described herein.

"Interstitial nephritis" refers to inflammation of the interstitium of the kidneys caused by an imbalance in calcium levels. Symptoms include increased urinary output, hematuria, mental status changes, and swelling. If associated with the formation of calcium salt crystals, and/or tissue exposure to calcium salt crystals, the condition is expected to benefit from treatment with a compound described herein.

"Phosphate-induced renal fibrosis" may be related to the range of calcium and phosphate concentrations that lead to renal tubular precipitation, see the data in Figure 2: calcium > 2 or 5 mmol/L and phosphate > 5 or 7 mmol/L. Of note, even if detectable plasma phosphate levels are normal (ie, in the absence of hyperphosphatemia), phosphate in the renal tubular fluid will be elevated (causing it to precipitate with calcium). Elevated plasma phosphate is the end result of end-stage renal disease that occurs when kidney function falls below the 20% threshold.

"Phosphate-induced chronic kidney disease" Phosphate-induced CKD may occur even if plasma phosphate levels are normal (i.e., in patients with early-stage CKD). Reference values may be tubular calcium >2 or 5 mmol/L and phosphate >5 or 7 mmol/L.

"Chronic kidney disease associated with hyperphosphatemia" refers to the hyperphosphatemia that occurs in progressive CKD due to increased GFR loss. This results in a blockade of tubular phosphate reabsorption and therefore increases phosphate retention, in other words, less phosphate clearance, impairing phosphate homeostasis (Sharon M. Moe, Prim Care. 2008 June; 35(2):215 –vi.). A useful reference value characterizing patients expected to benefit from treatment of the invention may be a plasma phosphate level of >1.46 mmol/L.

"Development of chronic kidney disease" refers to five stages, from stage one (mild impairment, eGFR of 90 or higher) to stage five (complete kidney failure, eGFR less than 15). Current guidelines identify a critical GFR of less than 60 mL/min per 1.73 ^m2 for CKD over 3 months.

"Phosphate toxicity" refers to disorders of renal phosphate excretion and reabsorption that impair phosphate homeostasis, which can cause severe damage to renal tissue (Razzaque, Clin Sci (Lond). 2011 Feb; 120(3):91–97) . In such cases, the condition causes tissue damage associated with the formation of crystals, and/or exposure of tissue to crystals, and/or deposition of crystals, which can be avoided by the treatment of the present invention, which treatment is suitable for patients with this condition. condition patients.

"Hyperphosphateuria" or "phosphateuria" refers to high levels of phosphate in the urine. In such cases, the condition causes tissue damage associated with the formation of crystals, and/or exposure of tissue to crystals, and/or deposition of crystals, which can be avoided by the treatment of the present invention, which treatment is suitable for patients with this condition. condition patients.

"Hyperphosphatemia" refers to elevated phosphate levels in the blood (>4.5 mg/dL; >1.46 mmol/L). In this case, the condition causes tissue damage associated with the formation of crystals, and/or exposure of the tissue to the crystals, and deposition of the crystals, which can be avoided by the treatment of the present invention, which treatment is suitable for patients suffering from the condition. patient.

"HyperFGF23emia" refers to an increase in local phosphate excretion due to elevated levels of fibroblast growth factor (FGF)-23, accompanied by a decrease in serum phosphate levels.

"Vascular calcification" refers to the pathological deposition of minerals in the vasculature frequently observed in patients with CDK or diabetes. Elevated calcium and/or phosphate levels may be the result of metabolic disorders caused by diabetes, dyslipidemia, oxidative stress, uremia, and hyperphosphatemia, which lead to the formation of osteoblast-like cells and the appearance of calcified deposits and hardening of blood vessel walls.

"Coronary artery disease" or "atherosclerotic heart disease" refers to the buildup of plaque in the damaged lining of the coronary arteries. Factors such as inflammatory cells, lipoproteins, and calcium attach to the plaque, causing further stenosis. Progression of the disease can eventually lead to myocardial infarction or stroke.

"Agiosclerosis" refers to the hardening of arterial walls due to calcification. Vascular sclerosis consists of lower elasticity of the vasculature leading to increased pulse wave pressure.

"Valvular calcification," particularly aortic valve calcification, refers to an active dysregulation of normal homeostatic processes and hemodynamic changes, such as ECM degradation, fibrosis, lipid accumulation, and neovascularization of valvular tissue that occur simultaneously with valvular, Especially calcification of the aortic and mitral valves.

"Nephrocalcinosis" refers to the deposition of calcium salts in the renal parenchyma, particularly in the medulla (medullary nephrocalcinosis) or cortex (cortical nephrocalcinosis) of the kidney.

"Calciphysis cutis" refers to the deposition of calcium phosphate in the skin, particularly in the extremities. If the dissolution point of calcium and phosphate is exceeded, precipitation of calcium salts occurs and is deposited as amorphous hydroxyapatite.

"Kidney stones", "nephrolithiasis", "nephrolithiasis", and "urolithiasis" refer to mineral deposits in kidney tissue due to accumulation and consequent supersaturation (hypercalciuria) of urine. To.

"Chondrocalcinosis" refers to the accumulation of calcium phosphate in the joints.

Based on the examples described herein, specific conditions whose treatment is expected to benefit from administration of the compounds described herein also include aortic stenosis, peripheral arterial disease, and cerebral calcification.

The data shown in Example 1 confirms that the compounds disclosed herein are effective in reducing or inhibiting calcium phosphate crystal formation, preventing and treating idiopathic calcium nephrolithiasis/idiopathic calcium kidney stones, especially those mainly composed of calcium phosphate and calcium oxalate. , or those composed of mixtures thereof, among which the utility is.

Compounds with an inositol scaffold

In certain embodiments, the inositol polyphosphate oligoalkyl ether compound used to treat or prevent the above diseases, or a pharmaceutically acceptable salt thereof, is described by general formula I, wherein one or two or three X is oligoethylene glycol, and the remaining X is OPO ₃ ^2- .

As shown in the examples provided herein, both mono- and di-oligoethylene glycol derivatives of different chain lengths inhibited calcium precipitate formation in cellular assays. Inositol scaffolds can have any stereochemistry. The inventors prefer to use myo-inositol.

It will be apparent to a person skilled in the art that the compounds mentioned herein are anionic and will be accompanied by cations when dissolved in an aqueous medium at physiological pH. The buffer used in the screening mainly included sodium (408mmol/L), and trace amounts of potassium (0.26mmol/L) and magnesium (4mmol/L).

In certain specific embodiments, two of the inositol substituents X shown in Formula I above are oligoethylene glycols and the remaining four X are OPO ₃ ^2- .

Examples of compounds that may be advantageously used in the treatment of the present invention include the general formulas I-1, I-2, I-3 and I-4, wherein in each case, X is phosphate and ^R1 is as described herein Oligoethylene glycol:

In a more specific embodiment thereof, the scaffold is myo-inositol and the oligoethylene glycol substituents are at positions 4 and 6, with the remaining substituents being phosphate. In an even more specific embodiment, the oligoethylene glycol substituent is O—(CH ₂ —CH ₂ O) ₂ —CH ₃ .

In other specific embodiments, one of the inositol substituents X shown in Formula I above is an oligoethylene glycol and the remaining five X are OPO ₃ ^2- . In a more specific embodiment thereof, the scaffold is myo-inositol and the oligoethylene glycol substituents are in position 4 or 6, especially in position 6, and the remaining substituents are phosphates. In an even more specific embodiment, the oligoethylene glycol substituent is O-(CH2-CH2O) ₂ -CH3.

In other specific embodiments, three of the inositol substituents X shown in Formula I above are oligoethylene glycols and the remaining three X are OPO32-. Examples thereof include the general formulas I-5 and I-6, where in each case X is phosphate and R1 is an oligoethylene glycol as described herein:

In certain embodiments, the oligoethylene glycol substituent of the inositol polyphosphate oligoalkyl ether compound provided by the present invention, or a pharmaceutically acceptable salt thereof, is composed of the formula O-(CH2-CH2-O) Description of nCH3, wherein n is selected from an integer from 2 to 20, in particular n is from 2 to 12. Different parameters in terms of physiological activity, pharmacological parameters and preparation of the compound will affect the optimal value of n.

In certain specific embodiments, the oligoethylene glycol substituent of the inositol polyphosphate oligoalkyl ether compound provided by the present invention, or a pharmaceutically acceptable salt thereof, is composed of the formula O-(CH2-CH2-O )nCH3 description, where n is 2. Table 1 of the examples shows the particular advantages of OEG ₂ -IP5 and (OEG ₂ ) ₂ -IP4, characterized in that n is 2.

Certain specific embodiments of the compounds used as specified herein are described by any of the following formulas:

Certain specific embodiments of the compounds used as specified herein are described by any of the following formulas:

Other embodiments of compounds for use according to aspects of the invention include:

Compounds with more than one myo-inositol scaffold

In certain embodiments, the inositol polyphosphate oligoalkyl ether compound used to treat or prevent the above-mentioned diseases, or a pharmaceutically acceptable salt thereof is described by general formula II, wherein each X is OPO32- and L is –(O–CH2–CH2)m–O–, where m has a value from 5 to 15, especially from 6 to 12.

In a specific embodiment, m is 7. In a specific embodiment, m is 9. In a specific embodiment, m is 10.

In a specific embodiment, m is 8.

Certain specific embodiments of the compounds designated for use herein are composed of any of Formula II-a (also referred to herein as OEG ₄ -(IP5) ₂ ) and II-b (also referred to herein as OEG ₈ -(IP5) ₂ ) describe:

In certain specific embodiments, the dinuclear myo-inositol polyphosphate oligoalkyl ether compound of general formula II used according to the present invention, or a pharmaceutically acceptable salt thereof, is characterized in that both myo-inositol moieties are myo-inositol .

Any inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, is provided for use in the treatment or prevention of diseases associated with the formation of calcium phosphate salts or other solid precipitates in the body, particularly in renal tissue.

Administration of compounds having two oligoethylene glycol moieties linked to the myo-inositol tetraphosphate scaffold (la) is particularly advantageous for the treatment or prevention of diseases associated with precipitation of calcium phosphate solid matter as described herein, and in It is particularly advantageous in preventing or treating mixed phosphate-calcium oxalate precipitates, or precipitates containing primarily oxalate but derived from the calcium phosphate core, as demonstrated by the data provided in Example 1, and as in calcium oxalate kidney stones. Exemplified by growth on calcium phosphate-based Randall plaques.

As shown in the examples, bis-polyethylene glycol compounds such as (OEG ₂ ) ₂ -IP4 also have a protective effect in mixed precipitation (CaP+CaOx).

Administration of compounds having two myo-inositol pentaphosphate skeletons (II) linked by oligoethylene glycol bridges, in particular compounds having eight –(O- _CH2 - _CH2 ) repeating units, in the treatment or prevention herein This is particularly advantageous in diseases associated with precipitation of calcium oxalate solid matter.

The skilled person is aware that any specifically mentioned pharmaceutical compound mentioned herein may exist as a pharmaceutically acceptable salt of the drug. Pharmaceutically acceptable salts include ionized drugs and oppositely charged counterions. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum salts, benzathine salts, calcium salts, ethylenediamine salts, lysine salts, magnesium salts, meglumine salts, potassium salts, procaine salts , sodium salt, tromethamine salt and zinc salt.

Manufacturing methods and treatment methods according to the invention

As an additional aspect, the present invention also includes inositol polyphosphate oligoalkyl ether compounds as detailed above, or pharmaceutically acceptable salts thereof, in preparations for the treatment or prevention of calcium salt precipitation or calcium salt crystal formation. Use in medicines for related diseases, in particular the disease is selected from the group consisting of renal fibrosis, especially when associated with calcification of renal tissue, nephritis, especially when associated with calcification of renal tissue, nephritis, especially interstitial Nephritis, glomerulonephritis, phosphate-induced renal fibrosis, phosphate-induced chronic kidney disease, chronic kidney disease associated with hyperphosphatemia, development of chronic kidney disease, phosphate toxicity, hyperphosphateuria, hyperphosphatemia disease, and/or hyperFGF23emia.

Similarly, there is provided the use of a compound of the present invention in a method for the preparation of a medicament for the treatment or prevention of a disease associated with calcium salt precipitation or calcium salt crystal formation, the disease being selected from the group consisting of vascular calcification, coronary artery disease, vascular sclerosis, valves Calcification, nephrocalcinosis, calcification cutis, nephrolithiasis, and chondrocalcinosis.

Similarly, the present invention includes a method of treating a patient who has been diagnosed with a disease associated with calcium salt precipitation or calcium salt crystal formation, in particular the disease is selected from the group consisting of renal fibrosis, especially when associated with calcification of renal tissue, nephritis especially when related to calcification of renal tissue, nephritis, especially interstitial nephritis, glomerulonephritis, phosphate-induced renal fibrosis, phosphate-induced chronic kidney disease, chronic kidney disease associated with hyperphosphatemia Renal disease, development of chronic kidney disease, phosphate toxicity, hyperphosphaturia, hyperphosphatemia, and/or hyperFGF23emia. The method requires administering to the patient an effective amount of an inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, as detailed herein.

Pharmaceutical compositions and administration

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition includes at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the compounds of the invention are generally formulated into pharmaceutical dosage forms to provide easily controllable dosages of the drug and to administer to patients a compact and easy-to-handle product.

In certain embodiments, pharmaceutical compositions for use in accordance with the present invention are formulated for administration by intradermal or subcutaneous injection.

Dosage regimens for the compounds of the present invention will vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the race, age, sex, health, medical condition, and weight of the recipient; the nature of the symptoms and extent; type of concurrent therapy; frequency of treatment; route of administration, patient's renal and hepatic function, and desired effect. In certain embodiments, the compounds of the present invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three, or four times daily.

The pharmaceutical composition used in the present invention can be subjected to conventional pharmaceutical operations, such as sterilization and/or can contain conventional inert diluents, lubricants, or buffers, as well as adjuvants, such as preservatives, stabilizers, wetting agents, Emulsifiers and buffers, etc. They can be produced by standard methods, for example by conventional mixing, granulating, dissolving or lyophilizing methods. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

The invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived. These examples are intended to illustrate the invention but not to limit its scope.

Description of the drawings

Figure 1 shows an overview of the developed calcium analysis platform. (A) Workflow outline. (B) Output overview of the analysis pipeline. Example images of brightfield (column 1), CellMask (column 2), Hoechst (column 4) and calcein (column 6) staining are shown along with RPTEC cells treated with 5/7mM Ca/P Two magnified regions of interest (ROI). CellMask and Hoechst channel images are used for single cell segmentation. The Hoechst local maximum (shown in dark blue, column 4) and the CellMask binary image are used as the seed and input image for the watershed algorithm, respectively. A comparison of the final cell segmentation (shown in blue) overlaid with the CellMask binary image (shown in red) is shown in column 3. The final segmentation results and Hoechst seeds are shown in column 5. Binary images of the calcein channel were generated by adaptive thresholding. The respective labeled CaP regions are shown in column 7.

Figure 2 shows the effect of increasing Ca/P concentration on RPTEC in vitro. (A) Heat map and hierarchical clustering of Ca/P treatment conditions and extracted image features. (B) Depicts some of the single features describing cellular changes as Ca/P concentration increases. The total number of cells, number of dead cells, single cell area, and single cell density (a measure of cell density) are shown. (C) Depicts some of the single features describing changes in CaP deposition and film patterning upon increasing Ca/P concentration. Shown are the total area of binary calcein staining, colorimetric quantification of calcium content extracted from the monolayer, maximum intensity of calcein fluorescence, structural similarity index measure (SSIM) and correlation of CellMask channel images. (D) shows an example brightfield, calcein, CellMask and EthD channel image and two magnified regions of interest (ROI) of RPTEC treated with 2/5mM Ca/P. The average scale value for each individual experiment is plotted in the colored circles, except that the absolute value of total calcium quantification for each individual experiment was used (N=3). The mean and SD of three separate experiments are represented by gray horizontal lines and vertical lines, respectively. One-way ANOVA and Dunnett’s multiple comparisons were performed between each concentration and 1/1mM Ca/P (*p<0.01).

Figure 3 shows an overview of the IP6 analogues tested in solution.

Figure 4 shows the inhibitory properties of (OEG ₂ ) ₂ -IP4 on Ca/P-induced changes in RPTEC in vitro. (A) Heat map and hierarchical clustering of extracted image features. (B) Depicts some of the single signatures describing cellular changes upon increasing concentrations _{of (OEG2)2 -} IP4. The total number of cells, number of dead cells, single cell area, and single cell density (a measure of cell density) are shown. (C) Depicts some of the single features describing changes in CaP deposition and film patterning upon increasing ( _OEG2 ) ₂ -IP4 concentration. Shown are the total area of binary calcein staining, colorimetric quantification of calcium content extracted from the monolayer, maximum intensity of calcein fluorescence, structural similarity index measure (SSIM) and correlation of CellMask channel images. (D) Shows example brightfield, calcein, CellMask and EthD channel images and two magnified regions of interest (ROI) of RPTEC treated with 13 μM (OEG ₂ ) ₂ -IP4. The average scaled values for each individual experiment are plotted (N=3). The positive control represents treatment with 5/7mM Ca/P and the negative control represents medium without added Ca/P (= 1/1mM Ca/P present in normal cell culture medium). The mean and SD of three separate experiments are represented by gray horizontal lines and vertical lines respectively. One-way analysis of variance and Dunnett's multiple comparisons were performed between each concentration and the positive control (*p<0.01).

Figure 5 shows Ca/P-induced transcriptomic changes in renal epithelial cells, including inflammatory pathways, ECM proteins, cell proliferation and tissue homeostasis processes, and is blocked by (OEG2) _{2 -} IP4 in vitro. A) Heat map and hierarchical clustering of relative gene expression levels determined by RNA sequencing. The top 2000 differentially expressed genes with P ≤ 0.01 and log2 fold change ≥ 0.5 between Ca/P relative to medium control are plotted, and their respective expression levels in other treatment groups (red - relative upregulation, blue - —relatively lowered). B) Abnormal analysis of Ca/P relative to upregulated gene transcripts in medium groups. C) Abnormal analysis of Ca/P relative to downregulated gene transcripts in the medium group. Ca/P was plotted against the weighted set coverage of Gene Ontology terms in the culture control group and their respective enrichment scores of differentially expressed genes (all FDR ≤ 0.05). D) Normalized counts of gene transcripts involved in inflammatory responses, ECM composition, cell proliferation, and tissue homeostasis (FPKM - Exons per kilobase model per million mapped reads) are plotted across treatment groups. (mean + SD, N = 3).

Figure 6 shows that ( _OEG2 ) ₂ -IP4 reduces high phosphate-induced renal injury in vivo. C57BL/6 males consumed a regular diet containing 0.35% inorganic phosphate (NP) or a high-phosphate diet containing 2.0% inorganic phosphate (HP). The mice were injected subcutaneously with ( _OEG2 ) ₂ -IP4 (100 mg/kg) or vehicle (distilled water) three times per week and then sacrificed at 20 weeks of age to harvest their blood and kidneys. Relative mRNA levels of (A) Spp1, (B) IL36a, (C) Ngal, (D) MMP3, and (E) Col1A1 in kidney tissue homogenates are shown (mean ± SD, N = 7, except HP vehicle group N =6, two-way ANOVA with Sidak's multiple comparisons between (OEG ₂ ) ₂ -IP4 versus vehicle control in each dietary group, ns—not significant, *p<0.05). (F) Collagen volume fraction after kidney Picro-Sirius red staining (mean ± SD, N = 6, t test between (OEG ₂ ) ₂ -IP4 and vehicle, **p < 0.01).

Figure 7 shows the inhibition of CaP precipitation by OEG ₂ -IP5, OEG ₁₁ -IP5, (OEG ₂ ) ₂ -IP4, (OEG ₁₁ ) ₂ -IP4 and OEG ₈ -(IP5) ₂ . (A)OEG ₂ -IP5, (B)OEG ₁₁ -IP5, (C)(OEG ₂ ) ₂ -IP4, (D)(OEG ₁₁ ) ₂ -IP4 and (E)OEG ₈ -(IP5) ₂ pairs added The effect of CaP precipitation in RTF with 9mM disodium phosphate and 8mM calcium chloride was assessed by light microscopy and automated image analysis at t = 4h. Quantification of total area covered by CaP deposits/total area of view (N=3, mean+SD, normalized to control).

Figure 8 shows the inhibition of CaP aggregation by OEG ₂ -IP5, OEG ₁₁ -IP5, (OEG ₂ ) ₂ -IP4, (OEG ₁₁ ) ₂ -IP4, and OEG ₈ -(IP5) ₂ . (A)OEG ₂ -IP5, (B)OEG ₁₁ -IP5, (C)(OEG ₂ ) ₂ -IP4, (D)(OEG ₁₁ ) ₂ -IP4 and (E)OEG ₈ -(IP5) ₂ pairs added The effect of CaP precipitation in RTF with 9mM disodium phosphate and 8mM calcium chloride was assessed by light microscopy and automated image analysis at t = 4h. Quantification of average size of aggregates/field normalized (N=3, mean+SD, normalized control).

Figure 9 shows the in vitro reduction of CaP adhesion and prevention of cell damage by IP6 analogues. Confluent RPTEC/TERT 1 cell monolayers were treated with medium supplemented with 7mM disodium phosphate, 5mM calcium chloride and compounds and cultured for 24 hours. The number of CaP deposits and the extent of cell damage were detected by calcein and EthD staining, respectively. Quantify fluorescence images using Matlab. Quantitative CaP deposition coverage on RPTEC monolayers treated with (A) OEG ₂ -IP5, (B) OEG ₁₁ -IP5, (C) (OEG ₂ ) ₂ -IP4, and (D) OEG ₈ -(IP5) ₂ area. (N=3, mean+SD, one-way ANOVA with Dunnett's multiple comparisons, *p<0.05, **p<0.01, ***p<0.001).

Figure 10 shows example images of RPTEC treated with 5/7mM Ca/P and increasing concentrations of ( _OEG2 ) ₂ -IP4. Brightfield, Calcein, CellMask ^™ and EthD channel images and two magnified regions of interest (ROI) are shown (columns 1–4). Cell segmentation is shown overlaid with the Hoechst channel image (blue) (column 4). The positive control represents treatment with 5/7mM Ca/P and the negative control represents medium without added Ca/P (= 1/1mM Ca/P present in normal cell culture medium).

Table 1 shows an overview of the efficacy of the screened compounds in inhibiting CaP precipitation in RTF. Show the minimum concentration of compound required to achieve complete inhibition of CaP precipitation (Figure 3 of chemical structure) (total area covered by crystals <10% of total area control); and the minimum concentration required to prevent aggregation of CaP crystals in the CaP screening assay (CaP aggregate size <50% of aggregate size control), summary table (N=3).

Table 2 shows the measured serum and phosphaturia and calcium levels (mean±SD) in mice of different treatment groups.

Example

Materials and methods

Material

IP6 analogues were custom-synthesized by Chimete Srl (Tortona, Italy). Mass and 1H-NMR spectra were obtained from the supplier to confirm the structure, and the compounds were used as supplied. Phytate dodecasodium salt was purchased from Biosynth AG (Thal, Switzerland). IP5 and IS6 hexapotassium salts were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). IC6 was purchased from Fluorochem (Hadfield, UK). Calcium colorimetric assay kit (MAK022), Bis-Tris, sodium oxalate (NaOx), EthD, Hoechst33342, magnesium chloride hexahydrate, disodium hydrogen phosphate, and calcein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride, anhydrous sodium sulfate and calcium chloride ( _CaCl2 ) dihydrate were obtained from Merck (Kenilworth, NJ, USA). Calcium oxalate monohydrate (CaOx) was purchased from abcr (Karlsruhe, Germany). 8-well glass bottom slides (80 827) were purchased from ibidi (Martinsried, Germany). CellMask Deep Red Plasma stain, standard cell culture plates, and reagents were purchased from ThermoFisher Scientific (Rochester, NY, USA) and TPP (Trasadingen, Switzerland). RPTEC/TERT 1 cells, ProxUp basal medium, and supplements were obtained from Evercyte (Vienna, Austria). The RNeasy kit was purchased from Qiagen (Hilden, Germany), and the TrueSeq RNA kit was purchased from Illumina (San Diego, California, USA). RNAiso Plus was obtained from Takara Biotech (Kusatsu, Japan). ReverTra Ace qPCR RT Master Mix with gDNA Remover and SYBR GreenPCR Master Mix were purchased from Toyobo (Osaka, Japan).

Screening in solution

According to literature reports, artificial renal tubular fluid with final components of 0.05mM oxalate, 0.005mM sulfate, 408mM sodium ion, 424mM chloride ion, 0.26mM potassium ion, 4mM magnesium ion and 0.2mM citrate was prepared in double-distilled water ( RTF) (Fasano, J.M. et al., Kidney Int. 59, 169–178. DOI: 10.1046/j.1523–1755.2001.00477.x, 2001). Filter the solution using a 0.45 μm syringe filter. Add 20 mM Bis-Tris buffer to the reported protocol to ensure pH stability throughout the experiment and set the pH to 7.2. Store artificial RTF at room temperature for up to four months. Stock solutions of 0.25 M phosphate and 1 M calcium were prepared in double-distilled water and stored separately at -20°C.

Prepare dilutions of phosphate to a final concentration of 20x, calcium to a final concentration of 20x, and compound dilutions to a final concentration of 10x in RTF. An assay mixture consisting of 80% RTF, 10% compound diluent, 5% phosphate diluent and 5% calcium diluent was prepared in an Eppendorf tube as follows. Mix RTF (320 μL) with 20 μL of phosphate diluent (final concentration 9mM), 40 μL compound diluent, and 20 μL calcium diluent (final concentration 8mM). After adding each component, the mixture was vortexed and 380 μL of the mixture was immediately added to an 8-well glass bottom slide and incubated at room temperature for 4 hours. CaP precipitation was evaluated using a Leica DM 6000B microscope (Leica Microsystems, Wetzlar, Germany) in bright field mode. For quantification, image 3 wells/condition and 3–4 images/well with a 40X objective. The total area covered by CaP deposits and the average size of CaP aggregates expressed as percent of the field of view were determined.

cell culture

Culture RPTEC/TERT 1 _human proximal tubule cells (RPTEC) in T75 tissue culture flasks using ProxUp basal medium mixed with ProxUp supplement at 37 °C and 5% CO according to the manufacturer's recommendations. Use cells until passage 30 and detect mycoplasma infection regularly. For experiments, RPTEC were cultured in 24-well plates at ^a seeding density of 150,000 cells/cm. Cell viability assessment and cell counting before seeding were performed using an automated cell counter (BioRad TC20, Hercules, CA, USA).

imaging assay

At t = 48 hours after inoculation, RPTEC were treated with ProxUp basal medium supplemented with a first different concentration of phosphate (final concentration 1–7mM) and a second different concentration of calcium (final concentration 1–7mM). Phosphoric acid and calcium are added directly to each well.

To compare selected IP6 analogues, prepare ProxUp basal medium with the selected inhibitor and add to each well. Then, CaP precipitation was induced by direct addition of first phosphate and second calcium. Use final concentrations of 7mM phosphate and 5mM calcium. Cells were cultured at 37 °C and 5% _CO for 24 h. The added medium was removed and the cells were washed twice with PBS before staining.

To evaluate CaP-induced CaOx crystallization, after 24 h of culture, the Ca/P-supplemented medium was removed and the cells were washed twice with PBS. Subsequently, medium containing 1.2mM oxalate and compound was added to each well. After 4 h of incubation at 37 °C and 5% _CO2 , the treatments were removed and the cells were washed once with PBS before staining.

For staining, ProxUp basal medium was mixed with Calcein (500 nM final concentration), EthD (6 μM final concentration), CellMask stain (5 μg/μL), and Hoechst 33342. Add the staining mixture to the RPTEC and _incubate the cells for 30 min at 37 °C and 5% CO in the dark. The staining solution was removed, cells were washed once with PBS and ProxUp basal medium was added. Cells were imaged immediately after staining. Images were acquired by epifluorescence microscopy using a Leica CTR6000 microscope at 37 °C. For quantification, prepare 3 wells/condition and take 3 images/well. For preliminary adhesion experiments, images were taken with a 10x objective, and for imaging assays, images were taken with a 20X objective.

Calcium colorimetric quantification

For colorimetric quantification of calcium content in cell monolayers after Ca/P treatment, a previously reported protocol was used. Briefly, after 24 h Ca/P incubation, cell monolayers were stained and imaged as described above. Then, cells were washed once with PBS and then incubated with 250 μL of 0.1 M HCl overnight at 4°C to remove calcium from the cell monolayer. The HCl solution was collected, centrifuged at 10,000 × g for 4 min at 4°C, and the calcium content was determined using a calcium colorimetric analysis kit (MAK022).

Cell image analysis

Multi-channel images are saved as individual channel images in 8-bit TIFF format. To analyze Hoechst channel images, thresholding was performed using triangle thresholding. The contact nuclei of the binary image are further segmented using a watershed algorithm, where the distance transform of the binary image is taken as input and its local maximum is used as the seed.

For the EthD channel images, a high level of background noise was observed, possibly due to fluorescence bleeding from the CellMask stain. Therefore, it was found that using 99% of all intensity values of each image as a threshold resulted in good detection of foreground pixels. Fixed minimum threshold is set to 60. A watershed segmentation similar to the Hoechst channel is performed to segment touching objects. For calcein channel images, adaptive thresholding by triangle thresholding achieved good detection of CaP deposits, which was verified by visual comparison with brightfield channel images. For compound experiments, an additional upper threshold was set to the minimum threshold of the positive control image for the respective experiment. Thus, overly insensitive thresholds are avoided in images with large amounts of CaP deposition. The total area of CaP deposits was calculated using the sum of the foreground pixels of the binary image. Use the Skimage labeling algorithm to label contact foreground areas.

To segment the image into single cells, the CellMask channel image was first binarized by adaptive thresholding using a block size of 35, followed by median filtering. Perform binary erosion to shrink the contour. Use a version of the watershed algorithm to segment the entire image into individual cells. To do this, the distance transform of the binary erosion image is used as the input image and the seed is set to the local maximum of the Hoechst channel image. Although this protocol allows approximation of single-cell morphology, further improvements in analytical and experimental staining procedures will be necessary to achieve more accurate results.

In addition, texture features of calcein and CellMask channel images are extracted. Calculate the gray level co-occurrence matrix using a set of 5 pixel offsets and an angle of 90°. Here, using larger offsets may improve detection of changes in large-scale features, such as CaP clustering sites. The texture properties of the extracted matrix are contrast, dissimilarity, energy, ASM, homogeneity and correlation. The overlap between calcein and CellMask channel images was measured by calculating the Structural Similarity Index Matrix (SSIM).

The extracted features include single cell or single calcein patch features, as well as entire image features. The complete image features include total cell count based on Hoechst image, total dead cell count based on EthD channel image, texture features of SSIM, calcein and CellMask channel images, maximum, minimum, average and standard deviation of calcein intensity, and total area. and the number of clusters. Single cell, single nucleus, and single calcein block features contain shape, and for calcein and cells only, intensity properties, and are summarized as the median value for each image. Properties include median area, extent, eccentricity, perimeter, compactness, major and minor axis lengths, and maximum, minimum, and average intensity per unit or CaP block. Features are scaled to range from 0 to 1 using MinMaxCasler from the sklearn preprocessing package. For each experiment and three independent experiments used for final analysis, the average of each image feature was calculated over 9 images per condition (3 wells per condition * 3 images/well). Analysis was performed in Python 3 using the Numpy, Pandas, Skimage, sklearn and Seaborn packages.

RNA sequencing

Cells were cultured as described for the imaging assay and cultured with medium control (ProxUp minimal medium with 1/1mM Ca/P), 5/7mM Ca/P, 5/7mM Ca/P with 50 μM ( _OEG2 ) ₂ -IP4 P medium or medium containing 50 μM (OEG ₂ ) ₂ -IP4 for 24 hours. Total RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Three wells were prepared for each sample group, and the total RNA extracted from these 3 wells was combined. The mRNA was purified, and the RNaseq library was prepared using the TrueSeq RNA kit (Illumina). Sequencing was performed on Novaseq 6000 (Illumina). Reads were aligned to the human reference genome GRCh38.p10 using the STAR tool (https://github.com/alexdobin/STAR), and transcripts were quantified using the Kallisto program (42). Ensembl version 91 was used for the definition of gene models. For heatmaps and hierarchical clustering of significantly different genes (p ≤ 0.01, log2 fold change ≥ 0.5), calculate log2 fold change compared to the mean for all samples, log2 fold change >4, set this to 4, use R software draws heat maps. Heat maps were drawn using R software. Webgestalt.org (v2019) (43) was characterized using differentially expressed genes with p ≤ 0.01 and log2 fold change ≥ 0.5. Differentially expressed genes were compared to the Gene Ontology - Functional Database of Biological Processes and using the Human Genome - Protein Coding as the reference group. Weighted set coverage of the top 30 enriched categories is plotted. To compare the expression levels of selected genes, the fragments per kilobase exon model per million mapped reads (FPKM) was used. Three independent experiments were performed. RNA sequencing raw data are available in the EMBL Nucleotide Sequence Database (ENA) under accession number PRJEB38397.

animal research

C57 BL/6 male mice (12 weeks old) were fed a regular diet containing 0.35% inorganic phosphate or a high-phosphate diet containing 2.0% inorganic phosphate. The mice were injected subcutaneously with ( _OEG2 ) ₂ -IP4 (100 mg/kg) or vehicle (distilled water) three times per week and then sacrificed at 20 weeks of age to harvest their blood and kidneys. Some mice were individually transferred to metabolic cages to collect urine for 3 days before sacrifice. Serum FGF23 levels were measured using the intact FGF23 ELISA (Kinos) according to the manufacturer's protocol. Serum and urine phosphate levels were measured using Fuji drI-Chem slides and analyzers (drI-Chem NX500V, Fuji, Tokyo, Japan). Frozen mouse kidneys were homogenized using RNAiso Plus (Takara Biotech, Osaka, Japan). The lysate was extracted with chloroform. Precipitate RNA in the aqueous phase with isopropanol, wash with 75% ethanol, and dissolve in RNase-free water. Reverse transcription of RNA (0.4 g) was performed using ReverTra ace qPCR RT Master Mix and gDNA Remover (FSQ-301, Toyobo, Osaka, Japan) according to the manufacturer's protocol. The quantitative RT-PCR reaction used 20 ng of cDNA incubated with 410 nM of each primer and 6 L of SYBR Green PCR Master Mix (THUNDERBIRD SYBR qPCR Mix QPS-201, Toyobo, Osaka, Japan) in a total volume of 12 L. PCR reactions (95°C for 1 min, followed by 45 cycles of 95°C for 10 sec and 60°C for 40 sec) were performed on a Roche LC480 system (Basel, Switzerland). Relative mRNA levels were calculated by the comparative threshold cycle method using cyclophilin as an internal control. Primer sequences can be found in STable 6. Kidneys not used for RNA extraction were fixed in 10% formalin, processed to make standard paraffin sections, and stained with Picro-Sirius Red to detect collagen as red fibers. Collagen volume fraction (ratio of Sirius red positive area to total area) was quantified using image analysis software (IMAGE PRO 9.32, Medica Cybernetics, Rockville, MD, USA) as previously described (Hirano, Y. Kurosu et al., FEBS Open Bio. 10, 894–903. DOI: 10.1002/2211-5463.1284, 2020). Cortical and cortico-medullary connections were assessed separately. All animal experiments were approved by the Animal Care and Use Committee of Autonomous Medical University.

data analysis

All images were analyzed and plots were prepared using Python 3, except for prescreening in solution experiments. Preliminary cell adhesion data were analyzed using Matlab and graphed using GraphPad Prism (GraphPad, La Jolla, CA, USA). RNA sequencing data were analyzed as described in the section above, and graphs were prepared using R software and GraphPad Prism. Analyze animal data in GraphPadPrism.

result

Example 1: Image-based analysis of calcification process

To enable rapid analysis of molecular effects on multiple renal calcification processes, the inventors developed a cell-based assay capable of monitoring CaP deposition and the cellular changes associated with it. Therefore, the present inventors utilized renal proximal tubule cell (RPTEC) monolayers stained with various dyes to quantify calcification and cell morphological changes (Fig. 1). Monolayer-grown cells were exposed to different ionic conditions found in renal tubules, such as increased calcium and/or phosphate, to trigger CaP crystallization and cell attachment (Figure 1A). CaP deposits were detected by calcein staining (Figure 1B). Calcein has been previously demonstrated as a calcium staining technique for fixed or unfixed cell samples. Fluorescent dyes bind calcium and are imaged using fluorescence microscopy. Furthermore, the present inventors tested CaP-induced induction of CaOx crystallization, which is characteristic of idiopathic kidney stone formation. It was observed that by first inducing CaP deposition and subsequently adding high levels of oxalate, CaOx crystals could be found on CaP deposits. CaOx crystals show strong contrast and a typical twin structure.

Visualize cellular changes by staining membranes with CellMask dye. Hoechst was used as a nuclear dye to aid single-cell segmentation, and ethidium homodimer 1 (EthD) promoted staining of cells with damaged plasma membranes (Figure 1B). Furthermore, textural features of CellMask and Calcein channels were extracted, which showed CaP deposition patterns (i.e., large and high-intensity CaP clusters relatively more diffuse and evenly spread CaP over the cell monolayer) (Figure 1B) .

Example 2: CaP-induced changes in renal epithelial cells

The inventors first studied the effect of increasing calcium and phosphate concentrations on cellular CaP deposition and subsequent cellular changes. The extracted features include single cell shape and fluorescence intensity parameters, texture features of CellMask and calcein images, and CaP deposit shape and intensity features. Hierarchical clustering of experimental conditions and characteristics showed clear dose-dependent trends (Fig. 2A). Low concentrations of calcium and phosphate, such as those in unsupplemented cell culture media (1/1mM Ca/P) and low-supplemented cell culture media (2/2mM Ca/P), do not induce cellular changes; Higher levels resulted in intermittent (2/5mM) and drastic changes (5/7 and 7/7mM) (Figure 2B). When looking at a single parameter, cellular changes related to Ca/P levels become more apparent. Increasing Ca/P results in a loss of tight cell packing, a characteristic of renal epithelium, which is associated with an increase in single cell area and a decrease in single cell compaction (a measure of density). Therefore, these data indicate a shift from the typical rounded shape of epithelial cells to a more irregular shape (Fig. 2B). These effects are accompanied by a reduction in the total number of cells/field. Furthermore, the detection of cells with damaged plasma membranes increased with increasing Ca/P levels (Fig. 2B). These results are consistent with literature reports showing loss of epithelial phenotype and cell damage after stimulation with CaP or CaOx.

The level of CaP deposition was measured first by adaptive thresholding of calcein images and secondly by quantification of the total area covered by each field of view. Using this approach, the inventors found that at higher Ca/P concentrations, CaP coverage of the cell monolayer increased (Figure 2C). These results were confirmed qualitatively by comparison with brightfield images, where CaP is visible as features with a granular structure or, in the case of large sediments, as dark spots (Fig. 2D). Calcein quantification of CaP deposition was determined by using colorimetric calcium quantification, in which calcium is extracted from cell monolayers by acid treatment (Fig. 2C) (Schantl, A.E. et al., Nat. Commun. 11, 721. DOI: 10.1038/s41467-019 -14091-4, 2020). Interestingly, although treatment with 2/5mM Ca/P did not result in a great increase in CaP deposition area, the deposits had high calcein intensity (Fig. 2C,D). Therefore, the inventors hypothesized that, at lower concentrations, CaP would tend to accumulate at sites with enhanced CaP affinity, such as dedifferentiated cells or injured cells with increased surface expression of glycoproteins with calcium-binding properties, rather than in monolayers. Evenly distributed on the top. These findings were supported by high structural similarity index measures (SSIM) of calcein and CellMask channel images, showing that CaP deposition overlapped with areas of membrane staining upon moderate and high Ca/P stimulation (Figure 2C). Overlapping areas show intense membrane staining, which may indicate damage and clumps of detached cell debris. Additionally, the relative texture features of the CellMask channel images reflected the consistency of the images, showing an increase between 1/1 and 2/5mM Ca/P additions and then a decrease again at ≥5/7mM Ca/P (Figure 2C). The highest values observed at moderate Ca/P concentrations may be due to the loss of cell contour when Ca/P is present but large CaP clustering sites are not formed. At high concentrations, CaP sites again caused a decrease in relevant features, which was due to the high staining intensity given by CaP membrane clusters. To further test the inhibitors, a 5/7mM Ca/P concentration was used, which is within the physiological range of the loop of Henle, a demonstrated major site for CaP crystallization.

Example 3: Prescreening of IP6 analogs in solution as renal CaP inhibitors

In a first step, the effect of selected IP6 analogs (Fig. 3) on CaP precipitation and growth was evaluated in vitro using artificial renal tubular fluid (RTF) (Table 1). In previous studies, the present inventors studied the formation of CaP protein particles in serum. However, an important difference with cardiovascular calcification is that renal tubular fluid has very low protein content, whereas blood has high protein content that stabilizes amorphous particles. Two cutoff values were chosen for assessing efficacy. First, complete inhibition was defined as >90% reduction in detected CaP precipitation compared to a control sample without compound. Second, inhibition of CaP aggregation was defined as a >50% reduction in the average size of aggregates compared to the control.

Interestingly, IP6, and to a lesser extent myo-inositol pentaphosphate (IP5), promoted CaP precipitation at 10 and 30 μM, respectively. The formation of IP6-calcium aggregates may accelerate CaP precipitation, as demonstrated by the inventors in phosphate-free medium. Replacing the phosphate group with an oligoethylene glycol (OEG) chain, as in the case of OEG ₂ -IP5, resulted in inhibition of CaP precipitation and aggregation at 30 and 1 μM, respectively (Table 1, Figures 8A and 9A). Comparison of OEG ₂ -IP5 and OEG ₁₁ -IP5 revealed that increasing the length of the OEG chain from 2 to 11 repeat units improved the inhibitory properties of the molecule, reducing its aggregation inhibitory concentration from 1 μM to 300 nM (Table 1, Figure 8B and 9B). However, substitution of phosphate with more OEG (( _OEG2 ) ₂ -IP4 versus _OEG2 -IP5) did not further increase inhibitory activity (Table 1, Figures 8C and 9C). Substitution of phosphate with less charged sulfate and carboxyl groups, such as phytate (IS6) and cyclohexanehexacarboxylic acid (IC6), results in a loss of promoting effect. These compounds showed only weak inhibitory properties against CaP crystallization (Table 1).

The bivalent IP5 molecule, which has been identified as a potent inhibitor of renal CaOx crystallization in previous studies, revealed another interesting trend. The effect of crystallization depends on the length of the linker between IP5 parts. OEG ₄ -(IP5) ₂ , with 4 EG units in the linker, promotes CaP precipitation, while OEG ₈ -(IP5) _{2 ,} with 8 EG repeating units, has an inhibitory effect. Complete inhibition was observed at 30 μM and 50% inhibition of aggregation was obtained at 1 μM (Table 1, Figures 8E, 9E). Taken together, these findings indicate that CaP inhibition by IP6 analogues in completely protein-free media is highly dependent on the charge and stabilizing properties of the molecule.

Example 4: (OEG ₂ ) ₂ -IP4 prevents CaP deposition and cellular changes in vitro

In the next step, the inventors compared the efficacy of OEG ₂ -IP5, OEG ₁₁ -IP5, (OEG ₂ ) ₂ -IP4 and OEG ₈ -(IP5) ₂ in preventing CaP adhesion (Figure 9). The compounds were added to the cell culture medium before adding phosphate and calcium. Here, for all compounds, the efficacy was in a similar range, with complete inhibition of CaP adhesion achieved at 50 μM. Interestingly, (OEG ₂ ) ₂ -IP4 performed best, sharply reducing CaP adhesion to 12.5 μM, coupled with its reported favorable pharmacokinetic profile (Schantl, AE et al., Nat. Commun. 11,721 .DOI:10.1038/s41467-019-14091-4, 2020), prompting the inventors to further characterize the compound.

Treatment of cells with 6–50 μM of (OEG ₂ ) ₂ -IP4 resulted in a dose-dependent reversal of the image signature from the positive control (+5/7mM Ca/P) to the negative control (+1/1mM Ca/P) (Fig. 4A). Compared with the positive control, (OEG ₂ ) ₂ -IP4 dose-dependently reduced the number of dead cells and single cell area, and increased the compactness of single cells and the total number of cells/field of view (Fig. 4B). Therefore, these data indicate that the negative control phenotype was reversed with ( _OEG2 ) ₂ -IP4.

The addition of 6 and 13 μM (OEG ₂ ) ₂ -IP4 to cell culture medium resulted in a decrease in the total CaP area compared to the positive control. However, it still showed higher calcein staining intensity (Fig. 4C). These results show that in the positive control, CaP is evenly distributed on the monolayer, whereas in the case of partial CaP inhibition, large local clusters of high calcium content are formed (Fig. 4C,D, Fig. 10). The inventors hypothesized that, as with the moderate Ca/P concentrations in the concentration range experiments, this reflects a general inhibition of CaP adhesion except in regions of high cellular affinity for CaP. In these areas, a high accumulation of CaP was observed. These effects can be further supported by calcein SSIM and CellMask channel images, such as in experiments with increasing Ca/P concentrations. High SSIM showed that areas of intense membrane staining overlapped with areas of high calcium (Figure 4C). Additionally, the inventors observed overlap of this area with damaged cells, as shown by EthD staining (Fig. 4B). Therefore, these sites may present localized areas of cellular damage, as well as regenerating/proliferating epithelium with surface expression of crystal-binding proteins, which results in high accumulation of CaP. Increasing the concentration of (OEG2) _{2 -} IP4 from 6 μM to 13 μM first increased the correlation measure of CellMask staining to levels above the positive control, which may be attributed to the absence of CaP membrane clusters or CaP membranes before decreasing toward negative control levels. Loss of cell outline in the presence of restricted clusters, where cells display rounded cell outlines.

The inventors then tested the effect of this compound on CaP-induced CaOx crystallization. Although the compound showed limited efficacy on CaOx crystallization in solution in previous studies (Kletzmayr, A. et al., Adv. Sci. 7, 1903337. DOI: 10.1002/advs.201903337, 2020), in CaP-induced In the model, the inventors observed dose-dependent changes. That is, (OEG ₂ ) ₂ -IP4 can first restore the CaOx crystal from the main and most stable form of calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD), which is consistent with the inventor's previous stepwise CaOx inhibition. The report is consistent (Kletzmayr, A. et al., Adv. Sci. 7, 1903337. DOI: 10.1002/advs. 201903337, 2020). Second, by further increasing the concentration to 100 μM, COD was almost completely eliminated. Therefore, (OEG ₂ ) ₂ -IP4 reduces CaP-induced CaOx crystallization, which may be caused by the compound coating and shielding CaP deposits.

Taken together, (OEG ₂ ) ₂ -IP4 prevents CaP adhesion to cell monolayers and associated cellular changes. The compound first limits CaP adhesion and cell damage to localized sites of adhesion formed by CaP membrane clusters before completely preventing CaP deposition and cell damage at higher concentrations.

Example 5: Ca/P-induced transcriptomic changes reflect in vitro vascular calcification and are blocked by ( _OEG2 ) ₂ -IP4

In order to further understand the cellular response to CaP and explain the relevant cell morphological changes observed by imaging, the inventors performed RNA sequencing experiments. Cells were cultured as in the imaging assay and treated with Ca/P-supplemented medium (positive control), Ca/P-supplemented medium premixed with 50 M of ( _OEG2 ) ₂ -IP4, medium alone (negative control), or only ( OEG ₂ ) ₂ -IP4 processing. Hierarchical clustering of differentially expressed genes between positive and negative controls showed drastic changes in gene expression profiles after Ca/P treatment, which was prevented by ( _OEG2 ) ₂ -IP4 addition (Fig. 5A). In positive control vs. negative control samples, 2818 differentially expressed genes were detected with a fold change of 1.5 and p 0.05, similar to Ca/P+(OEG ₂ ) ₂ -IP4 and (OEG ₂ ) ₂ -IP4 vs. Positive control samples (2437 and 2935 differentially expressed genes, respectively). In contrast, only an extremely limited number of differentially expressed genes were detected between Ca/P+( _OEG2 ) ₂ -IP4 relative to the negative control and relative to ( _OEG2 ) ₂ -IP4 (76 and 77 respectively). This confirmed the drastic changes induced by Ca/P treatment and its prevention by (OEG ₂ ) ₂ -IP4.

Overexpression analysis of upregulated genes in positive versus negative control samples revealed enrichment of cell cycle, cell division and related processes (DNA metabolic processes, ribosome biogenesis, chromosome organization), as well as cellular stress response processes (Figure 5B) . Downregulated genes were enriched in structural and developmental processes (Fig. 5C).

The inventors then focused on single gene expression levels, focusing on four groups of genes, namely inflammatory response pathways, extracellular matrix (ECM) proteins, cell cycle and proliferation processes, and genes involved in tissue homeostasis. Ca/P treatment of cells induces inflammatory response pathways, as previously reported for CaOx crystals (Kletzmayr, A. et al., Adv. Sci. 7, 1903337. DOI: 10.1002/advs.201903337, 2020). The upregulated genes include interleukin 6 (IL6) and interleukin 32 (IL32), complement C3 (C3), C-X-C motif chemokine ligands (such as CXCL5), and TNF signaling pathway genes, such as TNFα-induced protein 3 ( TNFAIP3) (Figure 5D). Extracellular matrix and cell surface genes were further studied. Putative calcium crystal-binding proteins, such as the cell surface glycoprotein osteopontin (SPP1) or CD55, are upregulated by Ca/P addition. In contrast, collagen IV family members (COL4A3, COL4A4, COL4A5) were downregulated. Collagen IV is the major protein component of the tubular basement membrane. Therefore, these data suggest strong alterations of the basement membrane that may contribute to the basement membrane calcification observed in kidney stone formers.

Overrepresentation analysis reveals dramatic dysregulation of cell cycle and division processes. The upregulation of the proproliferative gene myc and cyclin D1 (CCND1), which regulates the cell cycle in the G1/S transition, may indicate that renal epithelial cells enter a proliferative state after Ca/P stimulation. This view is further supported by the concurrent upregulation of TP53, a regulator that recognizes and repairs DNA damage at the G1/S regulatory point.

Furthermore, dysregulation of genes involved in developmental processes and tissue homeostasis indicates changes in cell differentiation following Ca/P stimulation. The expression of the epithelial cell marker e-cadherin (CDH1) was reduced after Ca/P treatment, indicating the loss of epithelial phenotype. It has been reported that the wnt signaling pathway promotes osteogenic transdifferentiation of vascular cells and vascular calcification by directly regulating Runx2 gene expression. Ca/P stimulation of renal epithelial cells regulates the expression of several wnt signaling pathway genes, including Wnt family member 7A (WNT7A), sclerostin domain-containing protein 1 (SOSTDC1), and dickkopf WNT signaling pathway inhibitor 1 (DKK1) . In addition, Runx2 expression was up-regulated after Ca/P treatment.

Thus, RNA sequencing revealed dramatic cellular changes following Ca/P stimulation, including a loss of epithelial phenotype toward a more proliferative state and changes in cell differentiation similar to the process of vascular calcification. (OEG ₂ ) ₂ -IP4 largely prevented Ca/P-induced changes, possibly due to reduced cell-crystal interactions.

Example 6: (OEG ₂ ) ₂ -IP4 reduces high phosphate-induced renal injury in vivo

The efficacy of (OEG2) ₂ - _IP4 was further tested in a mouse model of high phosphate-induced renal injury. Based on previously performed characterization of the pharmacokinetics of (OEG2) _{2 -} IP4 in rats, following a subcutaneous injection of 100 mg/kg, plasma concentrations in mice after 30 minutes are expected to be approximately 80 μM. Compared with a normal phosphate diet, a high-phosphate diet induces FGF23 expression, which in turn enhances renal phosphate excretion to maintain serum levels within normal limits. This feedback mechanism has also been shown to contribute to high renal phosphate levels in the early stages of CKD. The phosphate diet induced an increase in urinary phosphate excretion from 1.9 mg/day to 35.6 mg/day, with no significant difference between vehicle and treatment groups (Table 2). Furthermore, no significant differences in serum phosphate, CaP, or urinary calcium were observed between vehicle and treatment groups at the end of the treatment period (Table 2). Mice fed a high-phosphate diet showed elevated markers of kidney injury, such as increased Spp1, IL-36A, and Ngal (Fig. 6A–C). Additionally, elevated expression levels of renal fibrosis markers, such as matrix metallopeptidase 3 (MMP) and collagen 1α1 (Col1a1), were measured in high-phosphate versus normal-phosphate vehicle controls. Concomitant subcutaneous treatment with 100 mg/kg of (OEG ₂ ) ₂ -IP4 3 times/week significantly reduced kidney damage and fibrosis markers compared with the vehicle control group in the high-phosphate diet group (Figure 6A–E). Furthermore, (OEG2) ₂ -IP4 significantly reduced fibrosis, as measured by a decrease in collagen volume fraction after renal Sirius red staining (Fig _. 6F). Thus, the inventors' preliminary results indicate a beneficial effect of ( _OEG2 ) ₂ -IP4 on phosphate-induced renal injury in vivo.

discuss

Renal tubules are exposed to high concentrations of multiple metabolites, sometimes causing their precipitation and cell damage. Calcium precipitation in the form of CaP and CaOx is of particular concern due to associated renal calcification, tissue damage, and potential accelerated development of CKD. Due to the multiple nephrotoxic environmental perturbations, the inventors first aimed to establish simple in vitro image-based analysis tools that could allow rapid testing of a large number of renal perturbations, focusing on calcification conditions, and possible inhibitory molecules.

The proposed image-based measurement platform for calcium analysis allows simple and rapid changes in calcification conditions, i.e., ionic conditions that trigger different types of calcium crystals. The inventors implemented an automated analysis pipeline to quantify single cell changes as well as CaP deposition by fluorescent staining with calcein. The advantages of using image-based analysis methods compared to RNA sequencing of batch cells are the possibility to detect local changes and its adaptability to high-throughput.

The present inventors confirmed that the characteristic profile of renal epithelial cell monolayers gradually changes with increasing Ca/P concentration in the culture medium. A clear shift in the cobblestone-like epithelial phenotype toward an enlarged cell shape was observed. In cell lines, RNA sequencing confirmed the loss of the epithelial marker e-cadherin and a more proliferative state of cells stimulated with Ca/P. These findings are consistent with literature reports indicating that dedifferentiation and cell damage processes occur upon cell-crystal interactions. Altered signaling pathways similar to pathological changes involving vascular calcification suggest possible transdifferentiation of epithelial cells toward an osteoblast-like phenotype. Interestingly, the inventors also observed changes in the CaP deposition pattern. When Ca/P load is reduced, CaP precipitation and/or adhesion is favored at sites of cell damage and high membrane staining. At these sites, CaP accumulates, causing further damage, cell detachment, and the formation of CaP membrane clusters.

Previous studies support the idea that CaP preferentially attaches to specific crystal-binding proteins that can be expressed primarily on dedifferentiated or regenerating renal epithelial cells. RNA sequencing confirmed enhanced expression of crystalline cell surface and ECM proteins such as osteopontin. The enhanced proliferation of Ca/P-stimulated cells may favor uncontrolled multilayer growth and subsequent cell detachment, which may explain the disordered cell membrane staining and contribute to CaP cluster formation.

In addition, collagen IV family members, major components of the renal tubular basement membrane, were downregulated after Ca/P stimulation. Calcification of the basement membrane is thought to be the first step in CaP plaque formation in kidney stone formers, however, to date, the initial calcification process remains unclear. Therefore, collagen IV downregulation may provide the first insights into CaP plaque formation and demonstrate the utility of calcification platforms for modeling pathophysiological processes. Further studies are needed to elucidate whether the initial attachment site is formed by CaP loading or some degree of cellular damage precedes CaP binding and is then amplified by it. The results demonstrate the presence of active cells involved in the renal calcification process, supporting the analysis of multiple molecules that may act at multiple steps of the process.

In a next step, the inventors investigated the efficacy of the IP6 analogue library on the effects of renal CaP precipitation in solution and cell adhesion in vitro. The selected lead compound (OEG ₂ ) ₂ -IP4 dose-dependently restored the cell profile to the negative control profile and inhibited single cell changes and CaP deposition. The protective effect of the compound on high Ca/P-induced cellular changes was confirmed by RNA sequencing. This effect may be the result of both CaP growth and CaP adhesion being inhibited. Importantly, the protective effects of the compounds translated into efficacy in a mouse model of high-phosphate-induced renal injury. Therefore, the inventors believe that ( _OEG2 ) ₂ -IP4 has the potential to prevent CaP-accelerated renal injury by inhibiting CaP precipitation and CaP-cell interactions. Additionally, the compound reduced CaP-induced crystallization of CaOx on cell monolayers in vitro. These results indicate the potential therapeutic benefit of this molecule in CaP-induced nephropathy.

Claims (15)

1. An inositol polyphosphate oligoalkyl ether compound, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a disease associated with the formation of calcium salt crystals, and/or tissue exposure to calcium salt crystals, wherein the disease is selected from the group consisting of:

kidney fibrosis, in particular when associated with calcification of kidney tissue,

nephritis, in particular when associated with calcification of kidney tissue,

-a nephritis, wherein the first and second therapeutic agents,

-a number of times of interstitial nephritis,

-glomerulonephritis, in which the presence of a substance selected from the group consisting of,

phosphate-induced renal fibrosis, in which the presence of a lipid in the kidney,

-a phosphate-induced chronic kidney disease,

chronic kidney disease associated with hyperphosphatemia,

the development of chronic kidney disease,

the toxicity of the phosphate salt,

-a high-phosphate urinary disorder, wherein,

-hyperphosphatemia, and/or

-hyperfgf 23 blood disorder.

2. The inositol polyphosphate polyalkylether compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the inositol polyphosphate polyalkylether compound is represented by formula I

Wherein:

one or two or three X are oligoethylene glycols and the remaining X are OPO3 ² -。

3. The inositol polyphosphate polyalkylether compound of claim 2, or pharmaceutically acceptable salt thereof, wherein two X are oligoethylene glycols and the remaining four X are OPO3 ² -。

4. The inositol polyphosphate polyalkylether compound of claim 2, or pharmaceutically acceptable salt thereof, wherein three X are oligoethylene glycols and the remaining three X are OPO3 ² -。

5. The inositol polyphosphate polyalkylether compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein one X is an oligoethylene glycol and the remaining five X are OPO3 ² -。

6. The inositol polyphosphate polyalkylether compound, or pharmaceutically acceptable salt thereof, according to any one of claims 2 to 5, wherein the oligomeric ethylene glycol is represented by the formula O- (CH 2-O) nCH3, wherein n is selected from integers of 2 to 20, in particular n is 2 to 12.

7. The inositol polyphosphate polyalkylether compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein n is 2.

8. The inositol polyphosphate polyalkylether compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein the compound is described by any one of the following formulas:

9. the inositol polyphosphate polyalkylether compound, or pharmaceutically acceptable salt thereof, according to claim 5, wherein the compound is described by any one of the following formulas:

10. The inositol polyphosphate polyalkylether compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the inositol polyphosphate polyalkylether compound is represented by formula II

Wherein the method comprises the steps of

Each X is OPO ₃ ² -and L is- (O-CH) ₂ –CH ₂ ) _m -O-, wherein m has a value of 5-15, in particular m has a value of 6-12, more in particular m has a value of 7-10, even more in particular m has a value of 8.

11. The inositol polyphosphate polyalkylether compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein the compound is described by any of the following formulas:

12. inositol polyphosphate oligoalkyl ether compound, or pharmaceutically acceptable salt thereof

-according to any of the preceding claims 1 to 7, wherein the inositol moiety is myoinositol

Or (b)

-according to claim 10, wherein both inositol moieties are myo-inositol.

13. The inositol polyphosphate polyalkylether compound, or pharmaceutically acceptable salt thereof, according to any one of the preceding claims 1 to 12, wherein the related disease is a disease related to the formation of calcium phosphate salts or precipitates.

14. The inositol polyphosphate polyalkylether compound, or pharmaceutically acceptable salt thereof, according to any one of the preceding claims 1 to 12, wherein the related disease is a disease related to the formation of calcium oxalate salts or precipitates.

15. The inositol polyphosphate polyalkylether compound, or pharmaceutically acceptable salt thereof, according to any one of the preceding claims 1 to 12, wherein the related disease is a disease related to the formation of mixed calcium oxalate precipitates.