Compositions and methods for the diagnosis, treatment and prevention of renal disease

Abstract

Disclosed herein is a method for detecting the expression of xanthine oxidase in polycystic kidney disease, or for delaying or treating the onset of polycystic kidney disease, or both. The inventors have made the remarkable discovery that abnormal purine metabolism is not associated with the disease, but rather that increased tissue expression of xanthine oxidase / xanthine dehydrogenase (XO / XDH) expression and the production of uric acid and oxygen radicals acting alone or in combination may act as primary mediators of the progression of structural and functional dysfunction. A method involving measuring and evaluating tissue XO / XDH expression in polycystic kidney disease or any cystic tissue is specifically exemplified. Moreover, a method involving administering a composition of agents, including urate lowering agents, to a patient susceptible to developing polycystic kidney disease in a regimen that reduces the expression of XO / XDH and reduces the expression of intracellular uric acid or free oxygen radicals in tissue is specifically exemplified.

Description

[Technical field]

[0001] background Polycystic kidney disease is characterized by a series of anatomical and physiological abnormalities, such as hypertension, endothelial dysfunction, cardiovascular disease, liver disease, abnormalities affecting large, medium and small diameter blood vessels, and other health problems encountered by patients with polycystic kidney disease. Polycystic kidney disease has two main causes that are genetic, classified into two categories: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). Other causes of polycystic kidney disease or cystic disease syndromes can be found in association with cystic fibrosis, renal dysplasia, tuberous sclerosis, von Hippel-Lindau disease, polycystic dysplastic kidney (MCDK), medullary sponge kidney, and acquired renal cystic disease. The origin of polycystic kidney disease is often genetic, the primary cause of the disease, whereas approaches to diagnosing and treating existing disease can be categorized as primary, secondary, and tertiary prevention. Often the progression of the disease is diverse, and some, but not all, identifiable conditions cause or enable polycystic kidney disease to develop, such as 1) pancreatic disease, 2) hormonal abnormalities, 3) drug or chemical induced, 4) tubular abnormalities, 5) genetic syndromes, and 6) others.

[0002] The two main causes of polycystic kidney disease are ADPKD and ARPKD, which are genetic diseases that together contribute to approximately 90% and 5% of all primary polycystic kidney disease cases, respectively. Symptoms that may or may not include hypertension, cardiovascular disease, endothelial dysfunction, hyperuricemia, gout, kidney stones, hematuria, abdominal pain, frequent urinary tract infections, headaches, structural abnormalities of the heart valves, purpura, fatigue, and nausea and vomiting are often associated with polycystic kidney disease.

[0003] Various forms of kidney disease progress with an accelerating loss of filtering capacity.

[0004] overview The embodiments described herein are based on the remarkable discovery that the increased serum concentration of uric acid in polycystic kidney disease is the primary mediator of increased kidney size and decreased kidney function. In addition, the inventors have made the remarkable discovery that increased expression and activity of xanthine oxidase / xanthine dehydrogenase can be found in polycystic kidney disease and can act as a mediator of kidney damage. The discovery that polycystic kidney disease uniquely expresses modulators of damage distinct from the effects of serum uric acid in circulation, and targeting tissue expression of xanthine oxidase or modulators of xanthine oxidase expression provides a novel opportunity to target this newly discovered mechanism of damage. Aspects of the present invention provide a new approach to diagnose polycystic kidney disease and combat the gradual loss of its structural integrity and function.

[0005] In certain embodiments, the present subject matter relates to methods for detecting increased tissue expression of both xanthine oxidase (XO) and xanthine dehydrogenase (XDH) expression in individuals predisposed to polycystic disease.

[0006] In certain embodiments, the present subject matter relates to methods for detecting increased tissue expression of biological signaling molecules, such as modulators of cytokine expression, the inflammatory system, and / or proteome expression, modulated by increased XO / XDH expression in individuals susceptible to progression or accelerated progression of polycystic kidney disease.

[0007] In certain embodiments, methods are provided for detecting, stratifying, identifying and treating individuals whose tissue XO / XDH increases the risk of polycystic kidney disease. For treatment methods, serum or urine samples may be obtained and examined, and serum uric acid, or extracellular vesicles or cell membrane components may be examined to determine overexpression of XO / XDH, and may be monitored with administration of uric acid lowering drugs.

[0008] In another embodiment, compositions and formulations of uric acid lowering agents, and their dosages, routes of delivery or regimens, are provided that provide improved absorption or bioavailability of uric acid lowering agents.As a treatment method, serum or urine samples may be obtained and examined, and serum uric acid, or extracellular vesicles or cell membrane components may be examined to determine overexpression of XO / XDH, and may be monitored with administration of uric acid lowering agents and / or agents that reduce expression of XO / XDH.

[0009] In another embodiment, a method is provided for reducing the expression of xanthine oxidase or xanthine dehydrogenase in tissues, more specifically in the kidney, using inhibitory RNA (iRNA or siRNA).As a treatment method, serum or urine samples may be obtained and examined, and serum uric acid, or extracellular vesicles or cell membrane components may be examined to determine overexpression of XO / XDH, and may be monitored with the administration of uric acid lowering drugs.

[0010] In another embodiment, a method is provided that involves the delivery of iRNA or siRNA to tissues where XO or XDH overexpression occurs, to target the administration of therapy to one or more desired tissues.As a treatment method, serum or urine samples can be obtained and tested, and serum uric acid, or extracellular vesicles or cell membrane components can be tested to determine XO / XDH overexpression, and can be monitored with the administration of uric acid lowering drugs.

[0011] In another embodiment, a method is provided for reducing the risk of developing, delaying the onset of, and / or treating progressive kidney disease, more specifically polycystic kidney disease.

[0012] In another embodiment, methods are provided for reducing the risk of developing, delaying the onset of, and / or treating progressive liver disease, more specifically polycystic liver disease.

[0013] In another embodiment, the present subject matter provides methods for specifically treating polycystic kidney disease to reduce damage associated with tissue-specific expression of xanthine oxidase or XO / XDH.

[0014] Another embodiment relates to a method of specifically treating cystic disease in a tissue in combination with a xanthine oxidase inhibitor and an agent capable of decreasing or modulating expression of XO / XDH in the tissue to reduce damage associated with tissue-specific expression of XO, XDH or XO / XDH expression.

[0015] Also provided are methods for specifically treating cystic disease in tissues to increase expression of upstream modulators, such as expression of sirtuins, more specifically expression of sirtuin-1, as a means of modulating tissue-specific expression of XO / XDH.

[0016] Yet another embodiment relates to a composition comprising gliflozin and a xanthine oxidase inhibitor. Gliflozin is believed to increase the aqueous solubility of the xanthine oxidase inhibitor, and the oral bioavailability of the xanthine oxidase inhibitor. Also, as described herein, gliflozin also has additional functional benefits that make its use in combination with other UALAs particularly beneficial in the context of cystic kidney disease. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 provides a diagram showing purine metabolism and the metabolism of purine-based xanthine oxidase inhibitors. [Diagram 2] 1 provides a diagram showing modulators of xanthine oxidase / xanthine dehydrogenase. [Diagram 3] 1 provides a graph showing that animal models of polycystic kidney disease exhibit high serum allantoin concentrations. [Figure 4]Provides data showing that renal tissue expression of XO is increased in a rodent model of polycystic kidney disease. Figure 4A provides a Western blot showing increased expression of xanthine oxidase. Figure 4B provides data from rat polycystic kidney disease model Han / SPDR (CY / +) or wild type rat kidneys. Light microscopy of kidney tissue sections stained to show the presence of xanthine oxidase enzyme and read data as relative densitometric units (RD). Han / SPDR shows an increased proportion of stained cytoplasm compared to wild type (p<0.05). 4C provides evidence of a similar observation in a mouse polycystic kidney disease model (Pkd1 RC / RC mice) (P<0.05). [Diagram 5] FIG. 1 is a graph showing that XO activity is increased in kidney tissue of a rodent model of polycystic kidney disease compared to wild type. [Figure 6] We provide data showing that uricase inhibition with oxonic acid (OXO) and the corresponding increase in uric acid is associated with an increase in total kidney-to-body weight ratio (2K / BWT) in a rat model of polycystic kidney disease (PCK) and a mouse model of polycystic kidney disease (Pkd1RC / RC). After 70 days of exposure to oxonic acid, both models showed similar results, i.e., increased serum uric acid leading to an increase in 2K / BWT, increased cyst size, and increased serum creatinine (P<0.05; P<0.05; P<0.07 and P<0.05 for rat and mouse creatinine). Increased serum uric acid is associated with structural and functional changes in renal function. Increased creatinine is associated with decreased filtration capacity in renal disease. [Figure 7] We provide data showing that uricase inhibition with oxonic acid and the corresponding increase in uric acid in a rodent model of polycystic kidney disease indicates decreased renal function, as indicated by an increase in serum creatinine. [Figure 8] In a rodent model of polycystic kidney disease (murine RC / RC model), xanthine oxidase inhibition attenuates the progression of renal disease as shown by mean total kidney volume (FIG. 8A) and median kidney size (FIG. 8B). [Figure 9] Figure 1 shows that oxonic acid (OXO) exposure increased kidney size in a rodent model of polycystic kidney disease (mouse RC / RC). OXO and xanthine oxidase inhibition ("OXY+L", oxypurinol and L-arginine) were co-administered, and OXY+L mitigated the effects of oxonic acid and normalized / reduced uric acid-induced kidney enlargement at medium (8 mg / kg / day) or high (24 mg / kg / day) oral doses. [Figure 10] 1 shows an embodiment of a diagnostic device.

[0018] Detailed Description The present invention relates to methods for diagnosing abnormal purine metabolism, methods for treating abnormal purine metabolism and the use of agents that affect abnormal purine metabolism.The present invention relates to methods for diagnosing abnormal purine metabolism in tissue, methods for controlling abnormal purine metabolism in tissue and methods for treating abnormal purine metabolism in tissue.

[0019] The present invention relates to the use of xanthine oxidase inhibitors and / or combinations of agents capable of decreasing tissue expression of the xanthine oxidase enzyme or xanthine oxidase activity in polycystic kidney disease.

[0020] Without being bound to any particular item, the data provided herein suggests that if tissue expression of XO / XOR contributes to intracellular uric acid and oxygen radical production, this may contribute to mitochondrial damage, vascular or structural damage, inflammation or fibrosis. Compositions and methods are provided that reduce such adverse effects.

[0021] Treatment of ADPKD to specifically inhibit intracellular, renal, or other tissue damage through the therapeutic use of xanthine oxidase inhibitors, or free oxygen radical scavengers, or molecules with both properties, is also provided.

[0022] Furthermore, XOIs in combination with anti-inflammatory or anti-fibrotic drugs may be used to protect the kidney from disease progression.

[0023] Additionally, XOIs in combination with "vasypressin receptor antagonists" such as tolvaptan, lixivaptan, or other "-vaptans" slow the functional decline as well as the increase in cyst initiation / cyst growth or total kidney volume, reducing the health effects of both extracellular and intracellular xanthine oxidase.

[0024] Additionally, effectors of xanthine oxidase / xanthine dehydrogenase (XO / XDH) expression or activity or effectors of the XO / XDH ratio, such as SGLT2 inhibitors or sirtuin 1 activators that can directly or indirectly modulate XO / XDH, are administered to subjects exhibiting symptoms of PKD.

[0025] Additionally, silencing of such genes using interference molecules directed against the expression of genes such as xanthine oxidase (XO), thioredoxin interacting protein (TXNIP), and / or nuclear factor erythroid 2-related factor (nrf-2 / heme oxygenase 1 (HO-1)) to reverse the health effects of overexpression is also disclosed.

[0026] In certain embodiments, a method for detecting and treating the progression of kidney disease in a subject is provided, the method comprising obtaining a sample from the subject, detecting a level of expression or activity of xanthine oxidase in the sample, and determining that the subject is in need thereof if the level of expression or activity of xanthine oxidase is equal to or greater than a predetermined level or is elevated relative to a control, and administering to the subject in need thereof a therapeutically effective amount of a xanthine oxidase inhibitor.

[0027] Also provided are methods that include detecting changes to the concentration or activity of xanthine oxidase (XO) or the concentration or activity of xanthine dehydrogenate (XDH), or the concentration or activity ratio of XO / XDH. The methods involve obtaining a sample from a subject exhibiting one or more symptoms of polycystic kidney disease, the sample comprising a blood sample or a urine sample, or an extracellular vesicle sample from a blood sample or a urine sample; detecting the concentration or activity of XO and the concentration or activity of XDH in the sample; and administering UALA, and optionally co-administering an organic base, if the sample comprises a concentration or activity of XO, a concentration or activity of XDH, and / or a concentration or activity ratio of XO / XDH that deviates from that of a healthy subject. In specific examples, the deviation from that of a healthy subject is an XO concentration of greater than 1 mg / L; an XO enzyme activity of greater than 105 (U / L); and / or an XO / XDH that differs from that of a healthy subject by at least 0.1-10%.

[0028] Further method embodiments involve a method for reducing abnormal purine metabolism associated with renal disease in a subject in need thereof. This embodiment involves administering a therapeutically effective amount of one or more urate-lowering agents, wherein the method treats symptoms of cystic disease, and the one or more urate-lowering agents are optionally selected from the group consisting of xanthine oxidase inhibitors and sirtuin-1 activators. In certain embodiments, the administering step includes co-administering oxypurinol and gliflozin.

[0029] In yet another embodiment, a method is provided for reducing a marker of progression of renal disease in a subject. The method involves obtaining a sample from the subject; detecting a marker for renal disease, the marker comprising tissue oxygen radicals, uric acid, cytokines, inflammatory cells, fibrosis, increased number of mitochondria (mitochondriosis), or sirtuin-1 in the sample; and administering a therapeutically effective amount of UALA to the subject in need thereof, optionally co-administering an organic base, if the level of the marker is above baseline. In a specific example, UALA is an interfering molecule that targets the expression of xanthine oxidase or xanthine dehydrogenase, or an eRNA that increases the expression of sirtuin-1. In a more specific embodiment, the administering step comprises co-administering a xanthine oxidase inhibitor and an interfering molecule that targets the expression of xanthine oxidase or xanthine dehydrogenase.

[0030] Also provided is a method comprising detecting the presence of a marker or a ratio of a marker in a biological sample from a subject; and administering to the subject a therapeutically effective amount of at least one urate-lowering agent when the marker is elevated compared to baseline or control, or when the ratio of the marker is disproportionate to baseline or control. The marker can be one or more of xanthine oxidase, xanthine dehydrogenase, sirtuin or sirtuin-1, hypoxia-inducible factor-1 (HIF-1), erythropoietin, PCNA, Wnt / B-catenin, IL-5, IL-6, STAT1, STAT2, mTOR, TNFα, MIF, NLRP3 inflammasome, or cell membrane components, microvesicles, apoptotic bodies, exosomes, or free enzymes or specific parts / fragments of enzymes from blood or urine.

[0031] Another method embodiment relates to a method for monitoring the effectiveness of a treatment for polycystic kidney disease, comprising administering a quantity of a uric acid lowering agent to a subject exhibiting a high xanthine oxidase activity or concentration or a high xanthine oxidase / xanthine dehydrogenase activity or concentration ratio in the subject's urine compared to baseline; and detecting the xanthine oxidase concentration or activity or the xanthine oxidase / xanthine dehydrogenase concentration or activity ratio in the subject's urine, wherein a decrease in the xanthine oxidase activity or concentration or a shift of the xanthine oxidase / xanthine dehydrogenase concentration or activity ratio toward baseline indicates the effectiveness of the treatment.Another method embodiment involves a method for treating polycystic kidney disease, comprising co-administering a therapeutically effective amount of a xanthine oxidative inhibitor and metformin.

[0032] Compositions comprising an amount of oxypurinol and an amount of metformin and / or an SGLT2 inhibitor are also disclosed. In certain embodiments, the composition comprises oxypurinol, metformin and gliflozin.

[0033] In an alternative method embodiment, a method of treating polycystic kidney disease is provided that involves co-administering a therapeutically effective amount of a xanthine oxide inhibitor and tolvaptan and / or lixivaptan.

[0034] In a related embodiment, a composition is provided that includes a therapeutically effective amount of a uricemic agent and a therapeutically effective amount of tolvaptan and / or lixivaptan. In a specific embodiment, UALA is a xanthine oxidase inhibitor.

[0035] Also disclosed is a method of treating cystic disease in a subject in need thereof, the method comprising co-administering therapeutically effective amounts of a uric acid lowering agent and a gliflozin, wherein the gliflozin is co-administered in an amount that increases the aqueous solubility and bioavailability of the uric acid lowering agent.

[0036] In a further embodiment, a method of treating cystic disease is provided that involves co-administering a therapeutically effective amount of a xanthine oxidase inhibitor and a sirtuin-1 activator. In a specific embodiment, the sirtuin-1 activator is an SGLT2 inhibitor.

[0037] In yet another embodiment, a method of treating cystic disease in a subject in need thereof is provided, comprising co-administering a therapeutically effective amount of at least one xanthine oxidase inhibitor and an agent that directly or indirectly reduces the expression of xanthine oxidase. The agent that directly reduces the expression of xanthine oxidase may comprise an interference molecule that targets the expression of xanthine oxidase. The agent that indirectly reduces the expression of xanthine oxidase may comprise a sirtuin-1 activator. In a specific example, the sirtuin-1 activator comprises gliflozin or an eRNA that induces the expression of sirtuin-1, or a combination thereof.

[0038] In related embodiments, compositions are provided that include a therapeutically effective amount of a xanthine oxidase inhibitor and an agent that directly or indirectly reduces expression of xanthine oxidase. The agent that directly reduces expression of xanthine oxidase may include an interfering molecule that targets expression of xanthine oxidase. The agent that indirectly reduces expression of xanthine oxidase may include a sirtuin-1 activator. In certain embodiments, the sirtuin-1 activator includes gliflozin or an eRNA that induces expression of sirtuin-1, or a combination thereof.

[0039] definition Preferred materials and methods are described herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. 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 to which this invention belongs. In describing and claiming the present invention, the following terminology is used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0040] Where a range of values ​​is provided (unless the context clearly dictates otherwise), it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of the range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0041] The articles "a," "an," "the," and other articles are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article, unless specifically stated otherwise. By way of example, "an element" means one element or more than one element. Unless otherwise indicated, "or" includes "and." By way of example, "A, B, or C" means A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C, unless specifically stated otherwise.

[0042] The use of any and all examples provided herein with respect to a particular embodiment, or exemplary language (e.g., "such as"), is intended merely to more clearly describe the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0043] All features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced with alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each disclosed feature is merely one example of a generic series of equivalent or similar features. The examples and embodiments described herein are merely illustrative, and various modifications or variations in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of the present application. Many variations and modifications may be made to the embodiments of the present disclosure without substantially departing from the spirit and principles of the present disclosure. All such modifications and variations are intended to be included within the scope of the present disclosure herein. For example, unless otherwise indicated, the present disclosure is not limited to specific materials, reagents, reactants, manufacturing steps, etc., since these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be carried out in different order, where logically possible.

[0044] As used herein, the term "abnormal purine metabolism" refers to the conversion of purines to metabolites such as xanthine, uric acid (or urate) and / or allantoin at levels above those exhibited in healthy subjects. Abnormal purine metabolism is typically associated with increased expression and / or activity of xanthine oxidase in cells of subjects exhibiting one or more symptoms of disease (e.g., polycystic kidney disease, cystic liver disease or cystic disease, or any disease in which the expression or activity of xanthine oxidase in tissues is increased).

[0045] The terms "administering" or "administration" and their other grammatical forms refer to any route of introducing or delivering a compound or agent to a subject to perform its intended function. Administering or administration can be performed by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, or topical. Administering or administration includes self-administration and administration by another person.

[0046] As used herein, the term "antisense sequence" refers to an oligomeric compound that is at least partially complementary to the target nucleic acid molecule that the oligomeric compound hybridizes to. In certain embodiments, the antisense compound modulates (increases or decreases) the expression of the target nucleic acid. Antisense compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and compounds that are chimeric combinations thereof.

[0047] As used herein, the terms "co-administered," "co-administering," or "co-administration," when used in connection with, for example, administration of an exemplary therapeutic agent with another exemplary therapeutic agent, or administration of a conjunctive agent together with administration of an exemplary therapeutic agent, refer to administration of both the exemplary therapeutic agent and the other exemplary therapeutic agent or conjunctive agent such that both can achieve a physiological effect at the same time. However, the two agents need not be administered together. While in certain embodiments, administration of one agent can precede administration of the other, such co-administration typically results in both agents being present in the subject's body (e.g., in the plasma) at the same time.

[0048] As used herein, a "composition", "pharmaceutical composition" or "therapeutic agent" all include compositions that include at least a uric acid lowering agent. Optionally, a "composition", "pharmaceutical composition" or "therapeutic agent" further includes a pharmaceutically acceptable diluent or carrier, and / or a co-agent. In the case of an interfering molecule, for example, the interfering molecule may be combined with one or more pharmaceutically acceptable diluents, such as, for example, phosphate buffered saline.

[0049] The term "control" when used in the context of describing a comparison to a marker, or a ratio of markers, refers to a representative level of a molecule or a ratio of molecules present in a sample from a healthy subject.

[0050] As used herein, the term "cystic disease" refers to any disease involving tissue in which the occurrence of cysts or cyst growth is increased compared to healthy individuals. Cystic disease refers to a group of conditions that cause cysts (fluid-filled sacs) to form in or around organs. Cyst growth or expansion may be accompanied by increased pressure on healthy tissue surrounding the cyst, resulting in decreased blood circulation, nutrient exchange, or metabolic products that modulate disease progression. Expression of xanthine oxidase / xanthine dehydrogenase (XO / XDH) or the ratio of XO / XDH in cystic tissue may result in direct accumulation of uric acid in the tissue, or accumulation of oxygen radicals in the tissue, which directly or indirectly promotes disease or disease progression in the tissue.

[0051] The term "cystic kidney disease." Cystic kidney disease causes cysts (fluid sacs) to form in or around the kidneys. There are many types of cystic kidney disease. Some are the result of abnormal genes, while others develop in utero or as a result of kidney failure. Both adults and children can have cystic kidney disease. The term cystic kidney disease includes polycystic kidney disease.

[0052] Enhancer RNA, or eRNA, as used herein, refers to a short non-coding RNA molecule that is transcribed from an enhancer locus. Administration of eRNA to the enhancer of a target gene can increase the expression of the target gene. Enhancers are regulatory elements in the genome that cooperate with and control the transcription and cell fate of a target gene in conjunction with a promoter. The promoters of genes such as uricase or sirtuins can be particularly useful for modulating disease progression. The sequence of human sirtuin 1 can be found in accession numbers NM_012238, NM_001142498, and NM_001314049.

[0053] As used herein, "extracellular vesicles" encompass "exosomes" or "microvesicles (MVs)" that are released by nearly all cell types upon fusion of their multivesicular bodies with the cell plasma membrane in some embodiments. The term "extracellular vesicles" can include both exosomes and MVs. Extracellular vesicles are present in many, if not all, eukaryotic body fluids, including blood, urine, and in the medium of cell cultures. Extracellular vesicles, particularly exosomes or MVs, are known for their role in intercellular communication and have demonstrated the ability to unload their contents and contribute to the transformation of normal cells and stem cells to cancerous states. For example, microvesicles can be formed by a variety of processes, including the release of apoptotic bodies, direct budding of microvesicles from the cell's cytoplasmic membrane, and exocytosis from multivesicular bodies. For example, extracellular vesicles are usually formed by their secretion from the endosomal membrane compartment of a cell as a result of fusion of multivesicular bodies with the plasma membrane. Multivesicular bodies (MVBs) are formed by inward budding from the endosomal membrane and subsequent pinching off of the vesicle into the lumen. The internal vesicles present in the MVBs are then released into the extracellular fluid as so-called exosomes or extracellular vesicles.

[0054] The term "healthy subject" refers to a subject who lacks symptoms of disease or a genetic predisposition to disease. In particular embodiments, a healthy subject does not exhibit symptoms of polycystic kidney disease and / or lacks a genetic predisposition to PKD.

[0055] The term "inhibitory oligonucleotide" refers to any oligonucleotide that reduces the production or expression of a protein, such as by interfering with the translation of mRNA into protein in the ribosome, or that is sufficiently complementary to either the gene or mRNA encoding one or more targeted proteins and specifically binds (hybridizes) to the one or more targeted genes or mRNAs, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include isolated or synthetic shRNA or DNA, siRNA or DNA, antisense RNA or DNA, chimeric antisense DNA or RNA, miRNA, and miRNA mimics, among others.

[0056] As used herein, the term "interfering molecule" refers to any molecule that has a direct or indirect effect on gene expression, such as silencing a target gene sequence. Interfering molecules include inhibitory oligonucleotides, RNA interference molecules, and RNA-like interfering molecules. Examples of interfering RNA molecules include antisense sequences, siRNAs, short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), methylated siRNAs or other siRNAs that have been treated to protect siRNAs from degradation by circulating Rnase, and dicer substrate 27-mer duplexes. Examples of "RNA-like" molecules include, but are not limited to, siRNAs, single-stranded siRNAs, microRNAs, and shRNA molecules that contain one or more chemically modified nucleotides, one or more non-nucleotides, one or more deoxyribonucleotides, and one or more non-phosphodiester bonds. Thus, siRNAs, single-stranded siRNAs, shRNAs, miRNAs, and dicer substrate 27-mer duplexes are subsets of "interfering molecules." "Interfering molecules" can also include PMOs. See US Patent Application Publication No. 20200299698, Chinese Patent CN104232644; or Wang et al. Oxid Med Cell Longev, 2022, 2022:4326695, and Origene Technologies (Cat No. TF308350).

[0057] The term "marker" or "biomarker," as used herein, refers to a biological molecule whose presence or relative presence compared to a baseline or control is indicative of a disease or disorder. In the context of this disclosure, examples of markers include xanthine oxidase and / or xanthine dehydrogenase (or a nucleic acid sequence encoding same or a fragment thereof), antigens associated with xanthine oxidase and / or xanthine dehydrogenase, and / or uric acid, and / or genetic markers for PKD.

[0058] The term "metformin" refers to metformin or a pharma- ceutically acceptable salt thereof. See U.S. Patent No. 6,031,004, RE46496, and U.S. Patent No. 10,154,972.

[0059] The term microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs are similar to small interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway, except that miRNAs are derived from a region of an RNA transcript that folds back on itself to form a short hairpin, whereas siRNAs are derived from a longer region of double-stranded RNA. Under a standard naming system, experimentally confirmed miRNAs are assigned names. The prefix "miR" is followed by a dash and a number, the latter of which often indicates the order of the naming. "MIR" refers to the gene that codes for the corresponding miRNA. Different miRNAs that have nearly identical sequences except for one or two nucleotides are annotated with additional lowercase letters. The term miRNA mimic refers to small double-stranded RNA molecules, such as siRNAs, that are designed to mimic endogenous mature miRNA molecules when introduced into a cell.

[0060] The term "organic base" as used herein refers to an organic base that may be (a) a Class 1 base having a pKa1 of about 7 to 13, including, but not limited to, L-arginine, D-arginine, choline, L-lysine, D-lysine, and caffeine. In an alternative embodiment, the organic base is a biguanide (U.S. Pat. No. 9,480,663) or biguanidine, such as metformin, phenformin, buformin, or salts thereof. Metformin is known to have multiple beneficial effects in connection with the compositions and methods described herein. First, it can act as a solubilizer for oxypurinol to increase oral bioavailability. Second, it can act as an alkalizing agent. Third, metformin as used herein can act as a uric acid solubilizer. The unexpected result of metformin has been found where a ratio of oxypurinol to metformin of 1:01 to 1:10 increases the solubility of oxypurinol from 0.2 mg / ml to over 16.8 mg / ml. The gliflozin family of SGLT2 inhibitors are also basic and can act as organic bases according to the teachings herein.

[0061] As used herein, the term "phosphothioate morpholino oligomer," "PMO," or "PMO" refers to molecules that have the same nucleobases (i.e., adenine, cytosine, guanine, uracil, or thymine) as those found naturally in RNA or DNA, but they are linked with a morpholine ring instead of the ribose ring used by RNA. They may also be linked via phosphorodiamidates rather than phosphodiester or phosphorothioate groups. This linkage modification precludes ionization in the normal physiological pH range, so that PMOs within an organism or cell are uncharged molecules. The entire backbone of the PMO is made up of these modified subunits.

[0062] The term "polycystic kidney disease" or "PKD," as used herein, refers to a genetic disorder in which renal tubules become structurally abnormal, resulting in the development and growth of multiple cysts within the kidney. These cysts may begin to develop in utero, during infancy, childhood, or adulthood. Cysts are non-functioning tubules that are filled with fluid that accumulates within them and can range in size from microscopic to giant, crushing or compressing adjacent normal tubules and / or healthy adjacent tissues and blood vessels, eventually rendering them non-functional as well.

[0063] As used herein, the terms "prevent", "prevention" or "preventing" refer to preventing the development of clinical symptoms of a condition, e.g., inhibiting the onset of a disease, in a subject who may be exposed to or predisposed to a condition, but who has not yet experienced or shown symptoms of the condition. The term, as used herein, may further include either 1) a reduction in the frequency or severity of symptoms commonly associated with the disorder; or 2) a delay or avoidance of additional symptoms associated with the condition or disease, or complete prevention of the disease. Those skilled in the art will recognize that when various embodiments are directed to a prophylactic method, a subject in need thereof (i.e., a subject in need of prevention) shall include any subject or patient (preferably a mammal, more preferably a human) who has experienced or shown at least one symptom of the disorder, disease or condition to be prevented. Furthermore, a subject in need thereof may additionally be a subject (preferably a mammal, more preferably a human) who has not shown any symptoms of the disorder, disease or condition to be prevented, but who is considered by a physician, clinician or other medical professional to be at risk of developing the disorder, disease or condition. For example, a subject may be considered to be at risk for developing a disorder, disease or condition (and therefore in need of prevention or prophylactic treatment) as a result of the subject's medical history, including, but not limited to, family history, predisposition, coexisting (comorbid) disorders or conditions, genetic testing, etc.

[0064] As used herein, the term SGLT2 inhibitor refers to a sodium-glucose cotransporter inhibitor. A non-limiting list of SGLT2 inhibitors includes, but is not limited to, "gliflozins", such as empagliflozin, dapagliflozin, canagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin, sergliflozin, sotagliflozin, or tofogliflozin. It has been discovered that gliflozins have multifunctional properties that are particularly beneficial for the treatment of cystic kidney disease. Gliflozins can reduce the expression of xanthine oxidase in response to activating sirtuin-1, and can increase the bioavailability of other urate-lowering agents, such as xanthine oxidase inhibitors, in response to their basicity, and can themselves inhibit xanthine oxidase activity. Thus, gliflozins fall within the definition of urate-lowering agents, xanthine oxidase inhibitors, sirtuin-1 activators, and organic bases. To the extent that gliflozin is described or claimed in combination with or co-administered with one or more of these four agents, it is intended that, unless otherwise stated, reference to gliflozin in that context conveys that gliflozin may serve an additional function relative to that of the agent with which it is combined or co-administered. For example, a composition comprising UALA and gliflozin means that gliflozin may serve as an organic base to increase the bioavailability of UALA and something other than UALA, such as UALA.

[0065] As used herein, "shRNA" (small hairpin RNA) is a short "hairpin turn" RNA sequence that can be used to inhibit or suppress gene expression.

[0066] As used herein, "siRNA" (short interfering RNA), also known as small interfering RNA or silencing RNA, refers to a double-stranded RNA molecule, generally about 15-30 nucleotides in length, that is complementary to the sequence of an mRNA molecule transcribed from a target gene and interferes with the expression of the target gene.

[0067] As used herein, the term "sirtuin-1 activator" refers to an agent that can activate the NAD+-dependent proteolytic deacetylase of the sirtuin family and modulate various functions of sirtuins, such as physiology, metabolism, and stress response. More specifically, an agent that activates sirtuin-1 (SIRT-1) thereby modulates the expression of xanthine oxidase enzyme, xanthine dehydrogenase enzyme, or modulates the ratio of XO / XDH in tissues. Examples of agents that can activate sirtuins include resveratrol, epicatechin, quercetin, SRT2104, SRT1720, 1,4-DHP derivatives, UBCS039, SRT2104, SRT2379, SRT3025, or SGLT-2 inhibitors, including "gliflozins", such as empagliflozin, dapagliflozin, and canagliflozin, and eRNA that induces the expression of sirtuin-1. Other examples of sirtuin-1 activators are described in US Patent Application Publication No. 20070149466.

[0068] As used herein, the term "subject" or "patient" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., who is to be the recipient of a particular treatment.

[0069] As used herein, a "subject in need" is a subject who exhibits one or more symptoms of PKD and / or is genetically predisposed to developing PKD. Symptoms of PKD include increased kidney size, decreased kidney function, increased rate of cyst formation, increased rate of cyst growth, increased high blood pressure, increased abdominal or back pain, increased cardiovascular disease, increased rate of kidney stone formation, increased incidence of gout, increased incidence of renal failure, increased endothelial dysfunction, increased inflammatory conditions, increased rheumatoid arthritis, increased urinary tract infections, increased incidence of heart attack or stroke, increased need for dialysis, increased circulating creatinine or serum uric acid concentrations greater than 6 mg / dL.

[0070] A "therapeutically effective amount" refers to an amount sufficient, when administered in an appropriate dosing regimen, to reduce or alleviate the severity, duration or progression of the disorder being treated (e.g., cancer), prevent the progression of the disorder being treated (e.g., cancer), cause regression of the disorder being treated (e.g., cancer), or enhance or improve the prophylactic or therapeutic effect of another therapy. A complete therapeutic effect does not necessarily occur by administration of one dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered daily in one or more doses per day.

[0071] As used herein, the terms "treat," "treating," "treatment," or "alleviation" refer, by way of example, to therapeutic treatment in which the objective is to halt, slow, or reverse a pathological condition or disorder, and these terms can further include administering a compound to manage the underlying cause of a symptom or condition with the goal of reducing the symptoms or signs of the disease, and to prevent or slow the progression of, halt or potentially reverse the appearance of, or inhibit the underlying mechanisms causing the disease.

[0072] The term "uric acid lowering agent" or UALA refers to a substance known to lower serum uric acid levels in mammals. Typically, UALA can limit serum uric acid levels to at least about 0.2 mg / dl. UALA includes, but is not limited to, xanthine oxidase inhibitors; uricosuric agents, such as benziodarone, benzbromarone, probenecid; uricase derivatives, such as rasburicase and pegylated uricase; gene-based therapy, such as overexpression of uricase or URAT-1 blockade; uricase protein supplements, which can be delivered as a conjugate with polyethylene glycol or another delivery system; interference molecules that target xanthine oxidase or xanthine dehydrogenase, sirtuin-1 activators, such as SGLT-2 inhibitors (e.g., gliflozin), flavonoids, such as resveratrol, or eRNA that upregulates the expression of sirtuin-1; and uric acid channel inhibitors. As noted above, gliflozin has multiple mechanistic functions.

[0073] The term "xanthine oxidase inhibitor" as used herein refers to an agent that reduces the activity or expression of xanthine oxidase. Examples of xanthine oxidase inhibitors include, but are not limited to, allopurinol, oxypurinol, hydroxyakalone, TEI-6720, carprofen, febuxostat, topiroxostat, TMX-049 and y-700; inhibitory oligonucleotides or other interfering molecules that target the expression of xanthine oxidase; or anti-xanthine oxidase antibodies. Xanthine oxidase inhibitors may be further defined by their ability or the ability of prodrug metabolites to inhibit xanthine oxidase or xanthine dehydrogenase, separately or simultaneously, in blood, tissues or fluids or all.

[0074] The compound is preferably formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patch formulations and dry powder inhalers.Typically, the above compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

[0075] In the composition, an effective concentration of one or more compounds or pharma- ceutically acceptable derivatives are mixed with a suitable pharmaceutical carrier or vehicle. The compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acids, bases, solvates, hydrates, or prodrugs prior to formulation as described above. The concentration of the compounds in the composition is effective to deliver an amount that, upon administration, reduces serum uric acid levels by at least 0.5 mg / dl to 5.5 mg / dl or less. In the most preferred embodiment, the effective amount is such that serum uric acid levels are reduced to 5.5 mg / dl or less and 4.0 mg / dl or more. More preferably, the effective amount is such that serum uric acid levels are reduced to 5.2 mg / dl or less and 4.5 mg / dl or more.

[0076] UALA may be administered simultaneously or sequentially (i.e., co-administered) with one or more known antioxidants, including, but not limited to, vitamin C, alpha-lipoic acid, vitamin E, beta-carotene, selenium, zinc, carnosine, green tea, soy and isoflavones, tempol. In another embodiment, the uric acid lowering agent is combined with a vasopressin receptor antagonist, or an agent that can increase the aqueous solubility or bioavailability of an SGLT-2 inhibitor or an inhibitor of xanthine oxidase. Such a combination may be beneficial regardless of uric acid level, but may be particularly helpful when a dosage of UALA is administered that lowers uric acid to less than 5.5 mg / dl. In another embodiment, the uric acid lowering agent is co-administered with metformin. Thus, in another embodiment, a composition is provided that includes both a uric acid lowering agent and metformin.

[0077] Typically, the composition is formulated for single dose administration.To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in the selected vehicle at an effective concentration so that the treated condition is alleviated or relieved.The pharmaceutical carrier or vehicle suitable for administration of the compound provided herein includes any such carrier known to those skilled in the art to be suitable for specific administration mode.

[0078] In-vitro diagnostic devices Figure 10 illustrates diagrammatically an in vitro diagnostic device of the present invention, generally indicated at 10. The in vitro diagnostic device of the present invention includes at least a sample collection chamber 13, an assay module 12 used to detect a biomarker of disease, and a user interface that correlates the measured concentration (level) of the biomarker measured in the assay module. The in vitro diagnostic device may be a portable device, a benchtop device, or a point-of-care device.

[0079] The sample chamber 13 can be any sample collection device known in the art for containing a biological fluid. In one embodiment, the sample collection chamber can contain any one of the biological fluids contemplated herein, such as whole blood, cells, a cell suspension, a cell or tissue lysate, plasma, serum, urine, sweat, or saliva.

[0080] The assay module 12 is preferably made of an assay that can be used to detect proteins in a biological sample, for example, by the use of antibodies in an immunoassay. The assay module 12 may include any assay currently known in the art; however, the assay should be optimized for the detection of a marker used to diagnose a disease (e.g., polycystic kidney disease), severity of injury, or treatment response in a subject. The assay module 12 is in fluid communication with the sample collection chamber 13. In one embodiment, the assay module 12 is configured to perform an immunoassay, which may be any one of a radioimmunoassay, an ELISA (enzyme-linked immunosorbent assay), a "sandwich" immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, a fluorescent immunoassay, a chemiluminescent immunoassay, a phosphorescent immunoassay, or an anodic stripping voltammetric immunoassay. Alternatively, the assay module is configured to perform a nucleic acid hybridization assay. In one embodiment, a colorimetric assay may be used which may include only the assay sample collection chamber 13 and the assay module 12. Although not specifically shown, these components are preferably housed in one assembly 17.

[0081] In one embodiment, the in vitro diagnostic device of the present invention includes a power source 11, an assay module 12, a sample chamber 13, and a data processing module 14. The power source 11 is electrically connected to the assay module and the data processing module 14. The assay module 12 and the data processing module 14 are in electrical communication with each other. As mentioned above, the assay module 12 can include any assay currently known in the art; however, the assay should be optimized for the detection of the biomarkers used herein to detect damage disease or repair in a subject. The assay module 12 is in fluid communication with the sample collection chamber 13. The assay module 12 includes an immunoassay, where the immunoassay can be any one of a radioimmunoassay, an ELISA (enzyme-linked immunosorbent assay), a "sandwich" immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, a fluorescent immunoassay, a chemiluminescent immunoassay, a phosphorescent immunoassay, or an anodic stripping voltammetric immunoassay. A biological sample is placed in the sample chamber 13 and assayed by an assay module 12 that detects for markers. The measured amount of the marker by the assay module 12 is then in electrical communication with a data processing module 14. The data processing 14 module may include any known data processing element known in the art, and may include a chip, a central processing unit (CPU), or a software package that processes information provided by the assay module 12.

[0082] In one embodiment, the data processing module 14 is in electronic communication with a display 15, a memory device 16, or an external device 18 or software package (eg, laboratory information management software (LIMS)).

[0083] In one embodiment, the data processing module 14 is used to process the data into a user-defined usable format. This format includes the measured concentration (level) of one or more markers detected in the sample, which is useful for diagnosing disease (such as polycystic kidney disease), severity of injury, or treatment response in a subject. Information from the data processing module 14 may be displayed on a display 15, stored in a machine-readable format in a memory device, or in electrical communication with an external device 18 for further processing or display. Although not specifically shown, these components are preferably housed in one assembly 17. In one embodiment, the data processing module 14 may be programmed with a comparator algorithm to compare the detected amount of the biomarker transmitted from the assay module 12. The comparator algorithm may compare the measured amount with a user-defined threshold, which may be any limit value useful to the user. In one embodiment, the user-defined threshold is set to the amount of the biomarker measured in a control subject, or a statistically significant average of a control population.

[0084] In one embodiment, an in vitro diagnostic device may include one or more devices, tools, and apparatuses configured to contain or collect a biological sample from an individual. In one embodiment of an in vitro diagnostic device, the tools for collecting the biological sample may include one or more of needles, swabs, scalpels, syringes, scrapers, containers, and other devices and reagents designed to facilitate collection, storage, and transport of the biological sample. In one embodiment, an in vitro diagnostic test may include reagents or solutions for collecting, stabilizing, storing, and processing the biological sample. These reagents include antibodies, aptamers, or combinations thereof, raised against one of the aforementioned biomarkers. In one embodiment, an in vitro diagnostic device disclosed herein may include a microarray device and reagents, as well as additional hardware and software required to assay the sample to detect and visualize the temporally related biomarkers.

[0085] kit In yet another aspect, a kit is disclosed for aiding in the diagnosis of injury, disease or repair, including type, phase, amplitude (severity), and subcellular location, where the kit can be used to detect markers.For example, the kit can be used to detect any one or more of the biomarkers described herein, which markers are differentially present in samples from patients and normal subjects.In another example, the kit can be used to identify compounds that modulate the expression of one or more markers in in vitro or in vivo animal models, and determine the effect of treatment.

[0086] In one embodiment, the kit comprises (a) an antibody, aptamer, or nucleic acid probe that specifically binds to the aforementioned marker; and (b) a detection reagent. Such kits are prepared from the materials described above, and the previous discussion regarding the materials (e.g., antibodies, aptamer detection reagents, immobilization supports, etc.) is fully applicable to this section and will not be repeated.

[0087] In one embodiment of the invention, the kit comprises (a) a composition of detection agents for detecting one or more markers.

[0088] In one embodiment, the invention includes a diagnostic kit for use in screening biological samples for the presence or differential amount of xanthine oxidase and / or xanthine dehydrogenase and / or uric acid and / or sirtuin-1. The diagnostic kit in this embodiment includes a substantially isolated antibody or aptamer specifically immunoreactive with a peptide or polynucleotide antigen, or a nucleic acid probe hybridizing to a polynucleotide biomarker, and a visually detectable label associated with the binding of the polynucleotide or peptide antigen to the antibody or aptamer or nucleic acid probe. In one embodiment, the antibody or aptamer is attached to a solid support. The antibody or aptamer used in the kit of the invention is raised against any one of the biomarkers used herein for time data. In one embodiment, the antibody is a monoclonal or polyclonal antibody or aptamer raised against the rat, rabbit or human form of the biomarker. The detection reagent of the kit includes a second labeled monoclonal or polyclonal antibody or aptamer. Alternatively or additionally, the detection reagent includes a labeled competing antigen.

[0089] In one diagnostic configuration, the test serum is reacted with a solid-phase reagent having a surface-bound antigen obtained by the method of the present invention. After the specific antigen antibody or aptamer binds to the reagent, the reagent reacts with a reporter-labeled anti-human antibody or aptamer, binding the reporter to the reagent in proportion to the amount of anti-antigen antibody or aptamer bound on the solid support. The reagent is washed again to remove unbound labeled antibody or aptamer, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent, or colorimetric substrate.

[0090] The solid surface reagent in the above assay is prepared by known techniques for attaching protein or oligonucleotide materials to solid support materials such as polymer beads, dipsticks, wells (96-well plates) or filter materials. This attachment method generally involves non-specific adsorption of the protein or oligonucleotide to the support, or covalent attachment of the protein or oligonucleotide to chemically reactive groups on the solid support, such as activated carboxyl, hydroxyl, or aldehyde groups, typically via free amino groups. Alternatively, streptavidin-coated plates can be used with biotinylated antigens.

[0091] In another embodiment, the kit may include materials for polymerase chain reaction PCR amplification to facilitate detection of genetic material prior to testing the amount of marker detected for diagnosis of damage, disease or repair, including type, phase, amplitude (severity), subcellular location, pathology and / or treatment effect on the patient.

[0092] In some embodiments, the kits include standard or control information such that a test sample can be compared to the control information standard to determine whether the test amount of a marker detected in the sample is a diagnostic amount consistent with a diagnosis of damage, disease, or repair, including type, phase, amplitude (severity), subcellular location, pathology, and / or effect of treatment on the patient.

[0093] In one embodiment, the kit includes (a) a substrate comprising an adsorbent thereon, the adsorbent being suitable for binding a marker (e.g., xanthine oxidase and / or xanthine dehydrogenase, and / or uric acid), and, optionally, (b) instructions for detecting the marker by contacting a sample with the adsorbent and detecting the marker retained by the adsorbent. In some embodiments, the kit may include an elution agent (as an alternative or in combination with the instructions) or instructions for preparing an elution agent, where the combination of the adsorbent and elution agent enables detection of the marker using gas phase ion spectrometry. Such kits may be prepared from the materials described above, and the previous descriptions of these materials (e.g., probe substrates, adsorbents, wash solutions, etc.) are fully applicable to this section and will not be repeated.

[0094] In certain embodiments, the kit further includes instructions for appropriate operating parameters in the form of a label or insert. For example, the kit may have standard instructions informing the consumer how to wash the probe after the sample has been contacted on the probe. In another example, the kit may have instructions for pre-fractionating the sample to reduce the protein complexity in the sample. In another example, the kit may have instructions for automating fractionation or other steps.

[0095] Biological samples The detection methods provide enhanced diagnostic capabilities by providing the ability to detect and monitor levels of xanthine oxidase and / or xanthine dehydrogenase and / or uric acid present in a biological sample, allowing clinicians to determine the presence, phase and amplitude (severity) of disease. Biological samples operable herein to obtain evidence of increased uric acid concentrations or increased expression of XO include bodily fluids such as blood, plasma, serum, tears, sweat, urine, fecal material, cells, tissues, cell or tissue lysates, whole blood, or other biological samples recognized in the art.

[0096] Baseline levels of a marker (e.g., XO, xanthine dehydrogenase (or a nucleic acid sequence or portion thereof), or uric acid) are levels obtained in a target biological sample of a desired subject species in the absence of known damage, disease, or repair. These levels need not be expressed in hard concentrations, but instead may be known from parallel control experiments and expressed in terms of fluorescence units, density units, etc. Typically, baselines are determined from subjects in which the biomarker is absent or present in negligible amounts in the biological sample. However, some proteins may be underexpressed in damaged, diseased, or repaired patients, or prior to any clinical measurement of damage, disease, or repair. Determining baseline levels of protein biomarkers in a particular species is well within the skill of the art. Typically, elevated levels of xanthine oxidase or uric acid above baseline, or elevated ratios of xanthine oxidase to xanthine dehydrogenase, or otherwise deviating compared to samples from healthy subjects, are indicative of disease and can be compared or quantified over time to determine the severity of the condition or disease, or to determine the effectiveness of treatment.

[0097] A marker associated with tissue containing cysts is a marker obtained in a target biological sample of a desired subject species in the absence of known damage, disease, or repair. The level of a marker that co-localizes with the expression or activity of xanthine oxidase / dehydrogenase is associated with cystic tissue and may be directly or indirectly modulated by xanthine oxidase. The marker may be detectable in the circulatory, lymphatic, or secretory system and / or associated tissue. Examples of baseline markers include, but are not limited to, sirtuins or sirtuin-1, hypoxia inducible factor-1 (HIF-1), erythropoietin, PCNA, Wnt / B-catenin, IL-5, IL-6, STAT1, STAT2, mTOR, TNFa, MIF, NLRP3 inflammasome, or cell membrane components, microvesicles, apoptotic bodies, exosomes, or free enzymes or specific parts / fragments of enzymes from blood or urine.

[0098] In order to provide a correlation between damage, disease, or repair and the measured amount of xanthine oxidase, biological samples are collected from subjects in which these biomarkers need to be measured to assess damage, disease, or repair.

[0099] The detection methods may be implemented in assays or kits for performing the assays. These kits or assays may alternatively be packaged in cartridges for use with the in vitro diagnostic devices of the invention. Such devices utilize these cartridges, kits, or assays in the assay module 12, which may be one of many types of assays.

[0100] Furthermore, markers such as xanthine oxidase, xanthine dehydrogenase or uric acid can be detected in biological samples by a variety of conventional methods. For example, immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, magnetic immunoassay, radioisotope immunoassay, fluorescent immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, fluorescent immunoassay, chemiluminescent immunoassay, phosphorescent immunoassay, anodic stripping voltammetric immunoassay, and others. The in vitro diagnostic device of the present invention can also include any currently available known device that utilizes ion-selective electrode potentiometry, microfluidic technology, fluorescent or chemiluminescent technology, or reflectance technology that optically interprets color changes on protein test strips. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below (but are not intended to be limiting). It should be recognized that currently, none of the existing technologies offer a method for detecting or measuring any of the diseases disclosed herein, nor are there any methods of using such in vitro diagnostic devices to detect any of the disclosed biomarkers and detect their associated damage.

[0101] An exemplary process for detecting the presence or absence, or relative levels above baseline, of markers in a biological sample, alone or in combination, involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or agent, illustratively including an antibody or aptamer, capable of detecting the marker being analyzed, and analyzing binding of the compound or agent to the sample after washing. Samples that specifically bind to the compound or agent express the marker being analyzed.

[0102] In vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioassay, Western blot, Southern blot, Northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assays, cell assays, positron emission tomography, mass spectrometry, combinations thereof, or other techniques known in the art.

[0103] Examples of enzyme (xanthine oxidase) activity assays include, but are not limited to, Cat# 8458 ScienCell Research Laboratories, Cat# MAK078 by Sigma-aldrich, Abcam product number ab102522; cat no. EXOX-100 by BioAssay Systems. Another example of a xanthine oxidase activity assay is provided in Atlante A, Valenti D, Gagliardi S, Passarella S. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells. Brain Res Brain Res Protoc. 2000 Nov;6(1-2):1-5. doi: 10.1016 / s1385-299x(00)00030-1. PMID: 11086257. All of the above are incorporated by reference.

[0104] Further, in vivo techniques for detecting markers include introducing a labeling agent that specifically binds to the marker into a biological sample or test subject. For example, the agent can be labeled with a radioactive marker whose presence and location in the biological sample or test subject can be detected by standard imaging techniques. Assays of bound agents are easily formed, recognizing that the agents bind in a spatially overlapping manner, and detection occurs by distinguishably different detection of each of xanthine oxidase and / or xanthine dehydrogenase and / or uric acid. Color intensity-based quantification of each of the spatially overlapping bound biomarkers is representative of such techniques.

[0105] A preferred agent for detecting a marker in a biological sample is an antibody, an aptamer or a nucleic acid probe sequence capable of binding to the biomarker being analyzed. More preferably, the antibody, aptamer or nucleic acid probe sequence is conjugated with a detectable label. Such an antibody can be polyclonal or monoclonal. Intact antibodies, fragments thereof (e.g., Fab or F(ab')2), or engineered variants thereof (e.g., sFv), or aptamers or bi / trispecific aptamers can also be used. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Antibodies and aptamers against a number of biomarkers of the present invention are available from commercial vendors known to those skilled in the art. Exemplary antibodies operable herein are used to detect biomarkers of the disclosed conditions. In addition, antigens for detecting autoantibodies can also be used to detect late damage of the injuries and disorders mentioned.

[0106] The antibody or aptamer is labeled in some embodiments of the present invention. Those skilled in the art will recognize numerous labels that can be used herein. Labels illustratively include fluorescent labels, biotin, peroxidase, radioactive nucleotides, or other labels known in the art. Alternatively, a detection species of another antibody or aptamer or other compound known in the art is used as a form of detection of the biomarker bound by the antibody or aptamer.

[0107] Antibody-based and aptamer-based assays operable herein include Western blots, immunosorbent assays (e.g., ELISAs and RIAs), and immunoprecipitation assays. As an example, a biological sample or a portion thereof is immobilized on a substrate, such as a membrane made of nitrocellulose or PVDF; or a rigid substrate made of polystyrene or other plastic polymers, such as a microtiter plate, and the substrate is contacted with an antibody or aptamer that specifically binds to the marker under conditions that allow the antibody or aptamer to bind to the biomarker being analyzed. After washing, the presence of the antibody or aptamer on the substrate indicates that the sample contained the marker being evaluated. If the antibody or aptamer is directly conjugated with a detectable label, such as an enzyme, a fluorophore, or a radioisotope, the presence of the label is optionally detected by probing the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody or aptamer that binds to the marker-specific antibody or aptamer is added to the substrate. The presence of the detectable label on the substrate after washing indicates that the sample contained the biomarker.

[0108] Numerous permutations of these basic immunoassays are also workable in the present invention. These include a biomarker-specific antibody or aptamer, as opposed to the sample being immobilized on a substrate, and the substrate is contacted with the biomarker conjugated with a detectable label under conditions that cause the antibody or aptamer to bind to the labeled marker. The substrate is then contacted with the sample under conditions that allow the marker being analyzed to bind to the antibody or aptamer. A reduced amount of detectable label on the substrate after washing indicates that the sample contained the marker.

[0109] Although antibodies or aptamers are preferred for use in the present invention due to their extensive characterization, any other suitable agent (e.g., peptides or small organic molecules) that specifically binds to a biomarker can be used herein in place of an antibody or aptamer in the above immunoassays. Methods for producing aptamers with specific binding specificities are known, as detailed in U.S. Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588; 5,683,867; 5,637,459; and 6,011,020.

[0110] A variety of detectable labels that can be used in diagnostic assays for the expression of biomarkers are known in the art. The agent used in the method for detecting biomarkers is conjugated with a detectable label, for example, an enzyme such as horseradish peroxidase. The agent labeled with horseradish peroxidase can be detected by adding a suitable substrate that produces a color change in the presence of horseradish peroxidase. Several other detectable labels that can be used are known. Common examples of these detectable labels include alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, magnetic particles, biotin, radioisotopes, and other enzymes. It is recognized that a primary / secondary antibody or aptamer system is optionally used to detect one or more biomarkers. A primary antibody or aptamer that specifically recognizes one or more biomarkers is exposed to a biological sample that may contain the biomarker of interest. A secondary antibody or aptamer bearing an appropriate label that recognizes the species or isotype of the primary antibody or aptamer is then contacted with the sample, thereby achieving specific detection of one or more biomarkers in the sample.

[0111] The present invention provides for comparing the amount of one or more markers to normal levels to determine the disease or disorder of the subject. The results of such tests can help a physician determine whether administration of a particular therapy or treatment regimen may be effective and provide for rapid clinical intervention of the injury or disorder to enhance patient recovery.

[0112] It is recognized that other reagents such as assay grade water, buffers, membranes, assay plates, secondary antibodies or aptamers, salts, other ancillary reagents, etc. are available from commercial sources known to those of skill in the art.

[0113] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodological treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 - 3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-lnterscience, New York, 1992 (regularly updated). Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, for example, in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.

[0114] Compositions and Formulations The present invention provides a composition comprising UALA and an organic base that enhances or causes the solubilization of UALA or uric acid or urate in vivo, and optionally a pharma- ceutically acceptable carrier, excipient, vehicle, or diluent. The xanthine oxidase inhibitor and the organic base are preferably selected to ensure maximum solubility, bioavailability, or activity of the xanthine oxidase inhibitor without increasing any side effects. The compositions of the present invention include, in particular, liquid compositions (e.g., solutions, syrups, colloids, or emulsions). Furthermore, the compositions of the present invention contemplate micronized, lyophilized, or dry spray powders composed of XOI combined with one or more organic molecules or organic bases or basic amino acids that enhance equilibrium solubility, dissolution or solubility, or bioavailability in aqueous solution compared to oxypurinol free acid in water. Compositions comprising different combinations of UALA, sirtuin-1 activators, organic bases, and / or co-agents are also contemplated herein.

[0115] The present invention contemplates a pharmaceutical composition comprising a unit dose of at least one UALA (e.g., XOI) and an organic base together with a pharma- ceutically acceptable carrier, excipient, vehicle, or diluent. A "unit dose" refers to a unit, i.e., a single dose, that contains all the components of the composition of the present invention that can be administered to a patient. A "unit dose" can be easily handled and packaged as a physically and chemically stable unit dose of the active agent and the organic base, including the pharmaceutical carrier, excipient, vehicle, or diluent. Alternatively, a dosage form kit containing a xanthine oxidase inhibitor and an organic base, and the remaining components, is provided in separate containers, and the inhibitor, base, and the remaining components are combined before administration. In particular, the dosage form kit contains a xanthine oxidase inhibitor and an organic base in separate containers, and a solution for use is prepared by combining the components with a suitable carrier, e.g., sterile water, before administration. A unit dose of UALA or a xanthine oxidase inhibitor is a dose sufficient to increase circulating or tissue concentrations of UALA, thereby decreasing serum uric acid levels by 1%, 3%, 10%, 30%, 50%, 70%, 90%, 95% or more, and / or inhibiting tissue xanthine oxidase activity by 1%, 3%, 10%, 30%, 50%, 70%, 90%, 95% or more, or both simultaneously. Further, a dose of UALA includes 1, 3, 5, 10, 30, 50, 100, 300, 500, 1000 nanograms, micrograms or milligrams of agent. More specifically, the unit dose comprises UALA and an organic base in a ratio of 1:0.01, 1:0.1, 1:1, 1:2, 1:3, 1:5 or 1:10 to increase the aqueous solubility and / or oral bioavailability of UALA.

[0116] According to another embodiment, a composition is provided that includes an XOI, an organic base and / or choline, and an antioxidant. The antioxidant used for the composition embodiment may include, but is not limited to, alpha lipoic acid, n-acetyl cysteine, and vitamin C.

[0117] Another embodiment relates to a sterile dosage form of a composition comprising UALA and an organic base. In certain embodiments, the composition comprises an XOI in combination with a biguanidine. In certain embodiments, a composition is provided comprising an XOI (e.g., oxypurinol, febuxostat, topoxirostat, or allopurinol) and an organic base. The composition may comprise a dosage unit comprising 1-2000 mg of XOI, or 50-2000 mg of XOI. In even more particular embodiments, the dosage unit comprises a molar or weight-to-weight ratio of oxypurinol to organic base of about 1:1 to about 1:10.

[0118] The formulations can be provided in a lyophilized form suitable for reconstitution and administration in a subject. Formulations are also provided in which the XOI, the organic base, and other components of the composition are provided in non-lyophilized or lyophilized form separately from one another. The components can be reconstituted and / or solubilized in a suitable sterile liquid and combined to produce a pharmaceutical composition suitable for administration to a subject.

[0119] Beneficial effects may also be demonstrated by an increase in serum levels of the active ingredient after administration compared to the active ingredient alone. They may also be demonstrated by a decrease in serum uric acid levels. They may also be demonstrated by an increase in uric acid solubility in animals or, specifically, in humans, or an increase in the bioavailability of nitric oxide or a decrease in oxygen radical production. They may also show a decrease in xanthine oxidase activity.

[0120] The compositions of the present invention may have increased bioavailability (absorbed more rapidly and to a greater extent) which may be demonstrated by increased equilibrium solubility, dissolution rate and solubility compared to the xanthine oxidase inhibitor alone.

[0121] In one aspect, the dissolution rate of the xanthine oxidase inhibitor (i.e., the mass of material dissolved in a given time) can be increased by several times in the composition of the present invention compared to the pure active substance. The solubility of the xanthine oxidase inhibitor contained in the composition of the present invention (i.e., the mass of solvent or the mass of material apparently dissolved in a given volume) can be increased to produce a supersaturated solution. An increase in the final solubility can result, which is maintained for at least several hours, and then the saturation of the solution decreases. In some embodiments, compositions are provided that have a resorption rate increased by 1.5, 2, 3, 4, 5, 10, 15, 20, and 50 times compared to the pure active substance.

[0122] In another embodiment, the present invention provides a composition comprising a xanthine oxidase inhibitor that induces a decrease in intracellular uric acid concentration. The decrease in circulating uric acid levels or intracellular uric acid concentration can correspond to at least about a 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 1, 2, 3, 10, 30 or 100-fold decrease in circulating uric acid levels or intracellular uric acid concentration as measured by in vitro uric acid assay or in vivo measurement of serum uric acid or intracellular or tissue uric acid.

[0123] In certain embodiments of the invention, the organic base is arginine, choline, L-lysine, D-lysine, glucamine, and its N-mono- or di-substituted derivatives including, but not limited to, N-methylglucamine, N,N-dimethylglucamine, N-ethylglucamine, N-methyl,N-ethylglucamine, N,N-diethylglucamine, N-β-hydroxyethylglucamine, N-methyl,N-β-hydroxyethylglucamine, and N,N-di-β-hydroxyethylglucamine, benethamine, benzathine, betaine, deanol, diethylamine, 2-(diethylamino)-ethanol, hydrabamine, 4-(2-hydroxyethyl)-morpholine, 1-(2-hydroxyethyl)-pyrrolidine, tromethamine, diethanolamine (2 Examples of organic bases include, but are not limited to, 2,2',2"-iminobis(ethanol), ethanolamine (2-aminoethanol), 1H-imidazole, piperazine, triethanolamine (2,2',2"-nitrilotris(ethanol), N-methylmorpholine, N-ethylmorpholine, pyridine, dialkylaniline, diisopropylcyclohexylamine, tertiary amines (e.g., triethylamine, trimethylamine), diisopropylethylamine, dicyclohexylamine, N-methyl-D-glutamine, 4-pyrrolidinopyridine, dimethylaminopyridine (DMAP), piperidine, isopropylamine, or caffeine. In other embodiments, the organic base is a biguanide or biguanidine such as metformin. In a further embodiment, the organic base is gliflozin.

[0124] In another embodiment, the organic base is a basic amino acid, particularly lysine and arginine, and the xanthine oxidase inhibitor is allopurinol or oxypurinol.In one embodiment, a liquid composition is provided comprising allopurinol or oxypurinol and L-arginine.

[0125] In a further embodiment, the xanthine oxidase inhibitor and basic amino acid composition may be administered to an animal or human subject as a powder, where the enhanced solubility of the composition is sufficient to produce a liquid dosage form upon contact with water or other aqueous solutions, or with food materials in the digestive tract.

[0126] In a further embodiment, the present invention provides compositions, particularly liquid compositions, comprising a xanthine oxidase inhibitor and choline. Choline is a physiological compound that is used in therapy and does not suffer from disadvantages such as systemic or local toxicity.

[0127] The XOI and the organic base can be in a ratio selected to enhance the solubility of the XOI, enhance the activity of the XOI, or provide a beneficial effect. The ratio of the organic base to the xanthine oxidase inhibitor can range from about 0.01 to 20.0 molar equivalents of organic base to 1.0 molar equivalent of XOI. In some embodiments, the ratio of the organic base to the XOI is 1.0:0.5 molar, particularly 1.0:1.0 molar, and more particularly 1.0:3.0 molar. In certain embodiments, the composition comprises oxypurinol as the XOI and metformin as the organic base, wherein the ratio of oxypurinol to metformin is 1:01 to 1:10 molar.

[0128] The compositions of the present invention may also include a pharma- ceutically acceptable carrier, excipient, vehicle, or diluent. The XOI and organic base may be mixed into a selected pharma- ceutically acceptable carrier, excipient, vehicle, or diluent, and, optionally, other active ingredients, including therapeutic agents, may be added.

[0129] The compositions of the present invention typically contain a suitable pharmaceutical carrier, excipient, vehicle, or diluent that is selected based on the intended administration form and is consistent with conventional pharmaceutical practice.Suitable pharmaceutical carriers, excipients, vehicles, or diluents are described in the standard textbook, Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA 1985).For example, for oral administration in liquid form, the drug components (i.e., XOI and organic base) can be combined with any orally non-toxic pharma-ceutically acceptable inert carrier, such as ethanol, glycerol, water, etc. Suitable binders (e.g., gelatin, starch, corn syrup, natural sugars including glucose; natural and synthetic gums and waxes), lubricants (e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrants (e.g., starch, methylcellulose, agar, bentonite, and xanthan gum), flavorings, targeting agents, coloring agents, and other agents known to those skilled in the art may also be combined in the composition or its components.

[0130] In one embodiment, the compositions of the present invention are formulated to remain active at physiological pH. The compositions may be formulated in the pH range of 4-10, particularly 5-9.

[0131] In one aspect, the present invention relates to an aqueous composition comprising a XOI of the present invention and a solvent system that causes solubilization of the inhibitor to provide an aqueous solution with beneficial properties for incorporation into an oral liquid formulation. The solvent system comprises an organic base, particularly arginine or lysine, more particularly arginine.

[0132] In some embodiments, liquid compositions may be prepared using an XOI, particularly allopurinol or oxypurinol, and arginine, choline, glucamine (n-methylglucamine), or a glucamine salt.

[0133] In one aspect, the invention provides a sterile, pyrogen-free, ready-to-use solution of an XOI, particularly allopurinol or oxypurinol, consisting essentially of an XOI and an organic base dissolved in a physiologically acceptable solvent. In one embodiment, the solution has not been reconstituted from a lyophilizate.

[0134] The present invention also provides an orally applicable composition comprising an XOI and an organic base dissolved in a physiologically acceptable solvent.

[0135] The concentration of oxypurinol in certain liquid compositions of the invention comprising oxypurinol and arginine is about 0.1-100 mg / ml, 0.5-50 mg / ml, 1-25 mg / ml, and 1-10 mg / ml, more preferably 10 mg / ml. The solution may be administered twice daily in a total volume of about 5-100 ml, preferably 30 ml, to achieve a desired dosage of about 200-1000 mg / day, preferably 600 mg / day. Alternatively, if a once-daily dosing regimen is desired, the concentration of oxypurinol in the formulation may be about 6.0-60 mg / ml, preferably 20 mg / ml, administered once daily in about 10-100 ml, preferably 30 ml solution.

[0136] A preferred liquid formulation of the present invention comprises (1) allopurinol, oxypurinol, febuxostat, or a tautomer thereof, in a concentration range of about 0.1 to about 100 mg / ml (w / v), more preferably about 0.5 to about 50 mg / ml (w / v), even more preferably about 1 to about 25 mg / ml (w / v), still more preferably about 1 to 10 mg / ml (w / v), and most preferably about 10 mg / ml; (2) a basic amino acid having a suitable concentration range to provide an appropriate molar ratio of the basic amino acid to XOI or metformin; and (3) a pharma- ceutically acceptable diluent, preferably water, to bring the composition to a specified volume.

[0137] The present invention contemplates lyophilized formulations as described herein. The lyophilized compositions of the present invention can provide products with increased stability, solubility or bioavailability. Lyophilized formulations of XOI include: XOI, an organic base, and a pharma- ceutically acceptable carrier, excipient, or diluent. Storage conditions for lyophilized formulations are typically about 2° C. to about 25° C. XOI with an organic base (e.g., arginine) can be lyophilized at a concentration of about 0.02 mg / ml to about 10 mg / ml of compound in the initial solution. The lyophilization solution preferably includes an organic base (in addition to XOI) and a lyophilization buffer. The preferred pH range for the lyophilization buffer is about 5.5 to about 12.0. The lyophilization buffer can contain sodium citrate, EDTA, and / or sucrose. The lyophilized xanthine oxidase formulation can be reconstituted in sterile water to maintain isotonic conditions of about 290 mOsm. XOI with an organic base can be reconstituted in sterile water, optionally containing a stabilizing amount of an antioxidant. EXAMPLES

[0138] Figure 1 provides a schematic diagram showing purine metabolism and the metabolism of purine-based xanthine oxidase inhibitors. Xanthine oxidase and xanthine dehydrogenase are involved in the enzymatic conversion of hypoxanthine to xanthine and then to uric acid. In most other life forms except humans, apes, and Dalmatians, the uricase enzyme is active and further converts uric acid to allantoin. Aldehyde oxidase and xanthine oxidase enzymes rapidly convert allopurinol to oxypurinol.

[0139] FIG. 2: Provides a diagram showing modulators of xanthine oxidase / xanthine dehydrogenase. Ischemia, oxidative stress, and inflammation are associated with increased xanthine oxidase activity and decreased sirtuin-1 activity. In disease, activity and activation of xanthine oxidase results in increased production of uric acid and oxygen radicals. Intracellular and extracellular inhibition of xanthine oxidase activity may reduce damage to tissues. SGLT2 inhibitors activate sirtuin-1 activity, which in turn reduces xanthine oxidase activity. Inhibition of SGLT2 may indirectly protect cystic tissues that overexpress xanthine oxidase by suppressing xanthine oxidase activity and reducing uric acid and free oxygen radicals in the tissue. Both inhibition of xanthine oxidase and inhibition of SGLT2 may exert their protective effects in cystic tissues. SGLT2 inhibitors increase sirtuin-1 activity, which inactivates xanthine oxidase activity, thereby acting as uricemic agents. SGLT2 inhibitors have also been reported to lower uric acid by reducing renal and intestinal uric acid reabsorption.

[0140] Figure 3: Provides a graph showing that animal models of polycystic kidney disease exhibit high serum allantoin concentrations. Hyperallantoinemia was previously reported in the Han / SPRD rat model of polycystic kidney disease (Edelstein et al.). Hyperallantoinemia occurs early in mouse models of polycystic kidney disease and persists as early as 3-9 months in RC / RC mice. Because allantoin is a breakdown product of uric acid by the uricase enzyme, this finding supports the concept that high chronic exposure to uric acid continues in polycystic kidney disease and may contribute to health effects and progression of decline in renal structure and function.

[0141] Figure 4: Provides data showing that renal tissue expression of XO is increased in rodent models of polycystic kidney disease. Identification and quantification of xanthine oxidase in renal tissue in wild type (WT) rats and advanced PKD rats, CY / +, and RC / RC mice, was performed at late stage polycystic kidney disease. A statistically significant (*;**) increase in xanthine oxidase protein concentration was observed using Western blot. Figure 4A provides a Western blot showing increased expression of xanthine oxidase. Figure 4B provides data from a rat model of polycystic kidney disease, Han / SPDR (CY / +), or wild type rat kidney. Light microscopy of kidney tissue sections stained to show the presence of xanthine oxidase enzyme and read data as relative densitometric units (RD). Han / SPDR shows increased percentage of stained cytoplasm compared to wild type (p<0.05). FIG. 4C provides evidence of a similar observation in a mouse polycystic kidney disease model (Pkd1 RC / RC mice) (P<0.05).

[0142] Figure 5: Graph showing that XO activity is increased in renal tissue of a rodent model of polycystic kidney disease compared to wild type. Quantification of xanthine oxidase activity in renal tissue of wild type (WT) and advanced PKD rats CY / + in late stage polycystic kidney disease was performed. A statistically significant increase in xanthine oxidase activity in Han / SPDR rats was observed.

[0143] Figure 6: Provides data showing that uricase inhibition by oxonic acid (OXO) and corresponding increase in uric acid is accompanied by an increase in total kidney-to-body weight ratio (2K / BWT) in a rat model of polycystic kidney disease (PCK) and a mouse model of polycystic kidney disease (Pkd1RC / RC). A rodent model of polycystic kidney disease was treated with a uricase enzyme inhibitor (oxonic acid (OXO)) to decrease the metabolism of uric acid to allantoin, thereby chronically increasing circulating serum uric acid concentrations. After 8 weeks of treatment with oxonic acid, total kidney volume is significantly increased compared to untreated animals. After 70 days of exposure to oxonic acid, both models showed similar results, i.e., increased serum uric acid resulting in increased 2K / BWT, increased cyst size, and increased serum creatinine (P<0.05; P<0.05; P<0.07 and P<0.05 for rat and mouse creatinine). Increased serum uric acid is associated with structural and functional changes in renal function. Increased creatinine is associated with decreased renal filtering capacity. These results suggest that uric acid modulates cyst development and cyst growth rate, thereby modulating renal structure and disease progression. Figure 6A shows data from the PCK rat model, and Figure 6B shows data from the mouse RC / RC model.

[0144] FIG. 7: Provides data showing that uricase inhibition with oxonic acid and corresponding increase in uric acid in a rodent model of polycystic kidney disease indicates decreased renal function, as indicated by increased serum creatinine.

[0145] Figure 8: In a rodent model of polycystic kidney disease (mouse RC / RC model), inhibition of xanthine oxidase reduces kidney disease progression as shown by mean total kidney volume (Figure 8A) and median cyst size in the kidney (Figure 8B). This suggests that inhibition of uric acid production by xanthine oxidase can reduce kidney size in polycystic kidney disease compared to placebo (vehicle).

[0146] Figure 9: Graph showing that oxonic acid (OXO) exposure increased kidney size in a rodent model of polycystic kidney disease (mouse RC / RC). When OXO and xanthine oxidase inhibition (OXY+L) were administered simultaneously, OXY+L mitigated the effects of oxonic acid and normalized / reduced uric acid-induced kidney enlargement at medium (8 mg / kg / day) or high (24 mg / kg / day) oral doses. This data indicates that the adverse effects of high uric acid can be mitigated by reducing uric acid levels using UALA.

Claims

1. 1. A method for detecting and treating progression of kidney disease in a subject, comprising: obtaining a sample from a subject; and detecting a level of xanthine oxidase expression or activity in the sample, and determining that the subject is in need if the level of xanthine oxidase expression or activity is equal to or greater than a predetermined level or is elevated compared to a control; and administering a therapeutically effective amount of a xanthine oxidase inhibitor to a subject in need thereof. A method comprising:

2. 2. The method of claim 1, wherein the progression of kidney disease is the progression of polycystic kidney disease.

3. 3. The method of claim 2, wherein the progression of polycystic kidney disease is autosomal dominant or autosomal recessive polycystic kidney disease.

4. 10. The method of claim 1, wherein the sample comprises a bodily fluid, optionally blood, plasma, serum, tears, sweat, urine, fecal material, or tissue.

5. The method of claim 1, further comprising detecting a uric acid concentration in the sample or another sample from the subject, wherein a uric acid concentration above a predetermined level or higher than a control further indicates progression of kidney disease.

6. 2. The method of claim 1, wherein the sample is blood, urine, or both, and the detecting step comprises detecting XO or XDH, or both, or an antigen associated with an amino acid chain fragment of XO or XDH.

7. 10. The method of claim 1, wherein the xanthine oxidase inhibitor comprises oxypurinol.

8. 8. The method of claim 7, wherein the composition further comprises an organic base, optionally one or more of L-arginine, D-arginine, choline, L-lysine, D-lysine, caffeine, a biguanide, biguanidine, or gliflozin.

9. 8. The method of claim 7, further comprising co-administering a therapeutically effective amount of an organic base.

10. 1. A method comprising detecting a change in xanthine oxidase (XO) concentration or activity, or xanthine dehydrogenase (XDH) concentration or activity, or XO / XDH concentration or activity ratio, Obtaining a sample from a subject exhibiting one or more symptoms of polycystic kidney disease, wherein the sample comprises a blood sample or a urine sample, or an extracellular vesicle sample from a blood sample or a urine sample; and Detecting the concentration or activity of XO and the concentration or activity of XDH in the sample; and administering UALA and optionally co-administering an organic base if the sample contains an XO concentration or activity, an XDH concentration or activity, and / or an XO / XDH concentration or activity ratio that deviates from that of a healthy subject; A method comprising:

11. Deviations from those of healthy subjects XO concentrations greater than 1 mg / L; XO enzyme activity greater than 105 (U / L); and / or XO / XDH that differs from the XO / XDH of healthy subjects by at least 0.1-10% 11. The method of claim 10, wherein:

12. A method for reducing abnormal purine metabolism associated with renal disease in a subject in need thereof, comprising administering a therapeutically effective amount of one or more uric acid-lowering agents, wherein the method treats a symptom of cystic disease, and the one or more uric acid-lowering agents are optionally selected from the group consisting of xanthine oxidase inhibitors and sirtuin-1 activators.

13. 13. The method of claim 12, wherein the subject in need thereof has one or more cysts present in the kidney or liver.

14. 13. The method of claim 12, wherein the administering step comprises co-administering oxypurinol and gliflozin.

15. 1. A method of reducing a marker of kidney disease progression in a subject, comprising: obtaining a sample from a subject; and detecting markers for kidney disease, the markers including tissue oxygen radicals, uric acid, cytokines, inflammatory cells, fibrosis, increased mitochondriosis, or sirtuin-1 in the sample; and If the level of the marker is above baseline, administering to a subject in need thereof a therapeutically effective amount of UALA, optionally co-administering an organic base. A method comprising:

16. 16. The method of claim 15, wherein the progression of kidney disease is progression of polycystic kidney disease.

17. 17. The method of claim 15 or 16, wherein the UALA is an interference molecule that targets the expression of xanthine oxidase or xanthine dehydrogenase, or an eRNA that increases the expression of sirtuin-1.

18. 18. The method of claim 17, wherein the administering step comprises co-administering a xanthine oxidase inhibitor and an interfering molecule that targets expression of xanthine oxidase or xanthine dehydrogenase.

19. A method comprising detecting the presence of a marker or a ratio of markers in a biological sample from a subject; and administering a therapeutically effective amount of at least one uric acid lowering agent to the subject if the marker is elevated compared to a baseline or control, or if the ratio of the markers is disproportionate to the baseline or control.

20. 20. The method of claim 19, wherein the marker is one or more of xanthine oxidase, xanthine dehydrogenase, sirtuin or sirtuin-1, hypoxia inducible factor-1 (HIF-1), erythropoietin, PCNA, Wnt / B-catenin, IL-5, IL-6, STAT1, STAT2, mTOR, TNFa, MIF, NLRP3 inflammasome, or cell membrane components, microvesicles, apoptotic bodies, exosomes, or free enzymes or specific parts / fragments of enzymes from blood or urine.

21. 21. The method of claim 20, wherein the ratio comprises a xanthine oxidase / xanthine dehydrogenase ratio.

22. 21. The method of claim 20, wherein the uric acid lowering agent is a xanthine oxidase inhibitor or an interfering molecule that targets expression of XO or XDH in the subject's cells.

23. The method of any one of claims 20 to 22, further comprising administering a conjunctive agent.

24. 24. The method of claim 23, wherein the co-agent is a vascipressin receptor antagonist.

25. A method comprising administering a therapeutically effective amount of a uric acid lowering agent to a subject whose urine sample exhibits a higher xanthine oxidase activity or concentration compared to that of a healthy subject, or an XO / XDH concentration or activity ratio that deviates from that of a healthy subject.

26. 26. The method of claim 25, further comprising administering a co-agent.

27. 1. A method for monitoring the effectiveness of a treatment for polycystic kidney disease, comprising: administering an amount of a uric acid lowering agent to a subject exhibiting an elevated xanthine oxidase activity or concentration or an elevated xanthine oxidase / xanthine dehydrogenase activity or concentration ratio in the subject's urine compared to baseline; and detecting a concentration or activity of xanthine oxidase or a concentration or activity ratio of xanthine oxidase / xanthine dehydrogenase in the subject's urine, wherein a decrease in the activity or concentration of xanthine oxidase or a shift in the concentration or activity ratio of xanthine oxidase / xanthine dehydrogenase toward baseline indicates the effectiveness of the treatment. A method comprising:

28. A composition comprising a certain amount of oxypurinol and a certain amount of metformin, and / or an SGLT2 inhibitor.

29. 29. The composition of claim 28, wherein the SGLT2 inhibitor is gliflozin.

30. 29. The composition of claim 28, comprising oxypurinol, metformin and gliflozin.

31. 31. A method of treating polycystic kidney disease, comprising administering a therapeutically effective amount of the composition of any one of claims 28-30.

32. 1. A method of treating polycystic kidney disease comprising co-administering therapeutically effective amounts of a xanthine oxide inhibitor and metformin.

33. A composition comprising a therapeutically effective amount of a uric acid lowering agent and a therapeutically effective amount of tolvaptan and / or lixivaptan.

34. 34. The composition of claim 33, wherein the uric acid lowering agent is a xanthine oxidase inhibitor.

35. 35. The method of claim 34, wherein the xanthine oxidase inhibitor is oxypurinol.

36. 36. A method of treating polycystic kidney disease, comprising administering a therapeutically effective amount of the composition of any one of claims 33 to 35.

37. 1. A method of treating polycystic kidney disease, comprising co-administering a therapeutically effective amount of a xanthine oxide inhibitor and tolvaptan and / or lixivaptan.

38. 1. A method of treating cystic disease in a subject in need thereof, the method comprising co-administering therapeutically effective amounts of a urate-lowering agent and a gliflozin, wherein the gliflozin is co-administered in an amount that increases the aqueous solubility and bioavailability of the urate-lowering agent.

39. A method of treating cystic diseases comprising co-administering therapeutically effective amounts of a xanthine oxidase inhibitor and a sirtuin-1 activator.

40. 40. The method of claim 39, wherein the sirtuin-1 activator is an SGLT2 inhibitor.

41. 41. A method according to claim 39 or 40, which reduces the occurrence or growth of cysts.

42. 40. The method of claim 39, wherein the cystic disease is polycystic kidney disease.

43. 1. A method of treating cystic disease in a subject in need thereof, comprising co-administering a therapeutically effective amount of at least one xanthine oxidase inhibitor and an agent that directly or indirectly decreases the expression of xanthine oxidase.

44. 44. The method of claim 43, wherein the agent that directly reduces the expression of xanthine oxidase comprises an interfering molecule that targets the expression of xanthine oxidase.

45. 44. The method of claim 43, wherein the agent that indirectly decreases expression of xanthine oxidase comprises a sirtuin-1 activator.

46. The method of claim 45, wherein the sirtuin-1 activator comprises gliflozin or an eRNA that induces expression of sirtuin-1, or a combination thereof.

47. A composition comprising a therapeutically effective amount of a xanthine oxidase inhibitor and an agent that directly or indirectly decreases the expression of xanthine oxidase.

48. 48. The composition of claim 47, wherein the agent that directly reduces the expression of xanthine oxidase comprises an interfering molecule that targets the expression of xanthine oxidase.

49. 48. The composition of claim 47, wherein the agent that indirectly decreases the expression of xanthine oxidase comprises a sirtuin-1 activator.

50. The composition of claim 49, wherein the sirtuin-1 activator comprises gliflozin or an eRNA that induces expression of sirtuin-1, or a combination thereof.

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