WO2013142721A1

WO2013142721A1 - Compositions and methods for preventing or treating acute kidney injury using proton pump inhibitors

Info

Publication number: WO2013142721A1
Application number: PCT/US2013/033356
Authority: WO
Inventors: Holger Eltzschig; Almut Grenz
Original assignee: The Regents Of The University Of Colorado, A Body Corporate
Priority date: 2012-03-21
Filing date: 2013-03-21
Publication date: 2013-09-26

Abstract

Embodiments of the present invention generally relate to compositions, methods for preventing or treating acute kidney injury or other kidney conditions in a subject. In certain embodiments, compositions to treat or prevent a kidney condition can include one or more proton pump inhibitors.

Description

COMPOSITIONS AND METHODS FOR PREVENTING OR TREATING ACUTE KIDNEY INJURY USING PROTON PUMP INHIBITORS

PRIORITY

[0001] This PCT application claims priority to U. S. Provisional Application No.

61/613,765 filed March 21, 2012, which is incorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant number

DK097075 (NIDDK) awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0003] Embodiments of the present invention generally relate to compositions and methods for preventing or treating acute kidney injury or other kidney conditions in a subject. Other embodiments concern identifying and treating a subject having or suspected of developing a kidney condition.

BACKGROUND

[0004] Acute kidney injury (AKI), also called acute kidney failure refers to a loss of renal filtration function and an increase in serum creatinine. AKI is a leading cause of morbidity and mortality of hospitalized patients. Early, reliable detection of disease conditions and the identification of novel therapeutic approaches for the treatment of AKI are a highly significant area of biomedical research. Early and reliable detection of AKI is particularly important for trauma, critical care and perioperative patients. In addition to sepsis leading to AKI, another leading cause of AKI is ischemia AKI occurs in about 20-30% of patients after cardiac surgery.

SUMMARY

[0005] Certain embodiments of the present invention provide for methods, compositions and uses of proton pump inhibitors to prevent or to treat an adverse kidney condition. In accordance with these embodiments, certain compositions concern administering proton pump inhibitors to a subject before, during or after surgery to prevent or treat an adverse kidney condition. [0006] Some embodiments concern diagnosing a subject having a kidney condition and treating the subject having the condition with one or more proton pump inhibitors Proton pump inhibitors used in certain embodiments herein can include, but are not limited to, pharmaceutically acceptable compositions of a member of any of the following families and any commercially available composition thereof, for example, pyridyl methylsulfinyl benzimidazoles, omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole, lansoprazole, soraprazan, revaprazan, zegarid and SCH-28080 or others known in the art. Some embodiments provide a pharmaceutically acceptable composition of a member of the proton pump inhibitor family or any combination of the agents contemplated herein. In other embodiments, proton pump inhibitors can be one or more of omeprazole, esomeprazole, and substituted imidazo-l,2a-pyridines (e.g. SCH-28080) and photoaffinity derivative (e.g. Me- DAZIP1). In accordance with these embodiments, a pharmaceutically acceptable composition of one of, omeprazole, pantoprazole, esomeprazole, rabeprazole,

dexlansoprazole, lansoprazole, soraprazan, revaprazan and SCH-28080 or a combination thereof can be used to treat or prevent an adverse kidney condition, including but not limited to, acute kidney injury, kidney failure or the like.

[0007] In some embodiments, proton pump inhibitors can be administered to a subject to reduce kidney injury (e.g. AKI, acute kidney injury) in the subject. In accordance with these embodiments, a proton pump inhibitor can be administered in a therapeutically effective dose to reduce severity of or prevent AKI in a subject for example, in a subject undergoing a surgical procedure (e.g. heart surgery or other surgery), having an organ transplant (e.g. liver, lungs, heart, kidney, having an ischemic event, having a kidney condition or disease that may lead to AKI or experiencing a traumatic event such as an accident or other physical trauma. Causes of AKI can include, but are not limited to, ischemia, reperfusion, trauma such as injury or accident, diabetes or other health condition that causes kidney damage and may result in kidney transplantation.

[0008] In some embodiments, proton pump inhibitors can be administered to a subject to dampen or reduce kidney tissue injury during ischemia and mediate enhanced ischemic tolerance. In other embodiments, a subject may have a pre-existing renal disease or condition that can lead to acute kidney injury in peri-operative period and proton pump inhibitors can be used to treat the subject to reduce severity of or prevent AKI. Renal diseases can include any renal condition that may lead to AKI for example, diabetes. [0009] Any mode for administration to a subject of compositions (e.g. pharmaceutically acceptable compositions) disclosed herein is contemplated. For example, proton pump inhibitors may be administered orally, intranasally, by timed release (e.g. slow release microspheres etc.), by catheter, by suppository, or intravenously, or through any other route known in the art. In some embodiments, proton pump inhibitors are administered intravenously to a subject before, during or after an event or procedure.

[0010] In some embodiments, a pharmaceutically acceptable composition can be used to treat or prevent a kidney condition in a subject through modulating the activity of ATP4A. ATP4A is a subunit of H+-K+-ATPase, a proton pump that uses the energy of ATP hydrolysis to pump hydrogen (and potassium) ions against their concentration gradients. In accordance with these embodiments, proton pump inhibitors can be used to target ATP4A alone or in combination with any other agents used to treat or prevent a kidney condition. In other embodiments, ATP4A activity can also be modulated using anti-sense R A, siRNA, antibodies, antibody fragments, small molecules or aptamers for example alone or in addition to proton pump inhibitors. Antibodies to ATP4A are commercially available and can be obtained by for example from BD Sciences, Labome, Santa Cruz Biotech or other source.

[0011] In some embodiments, proton pump inhibitors can be given to a subject prior to, at the onset of, during or after AKI or a kidney condition or a transplantation event. In accordance with these embodiments, doses of the proton pump inhibitors can be adjusted to achieve desirable effects to treat the kidney condition when administered at different windows of time as deemed appropriate by a health professional.

[0012] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention.

Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

Brief Description of the Drawings

[0013] The following drawings form part of the present specification and are included to further demonstrate certain embodiments. Some embodiments may be better understood by reference to one or more of these drawings alone or in combination with the detailed description of specific embodiments presented.

[0014] Figs. 1A-1C represent exemplary histogram plots illustrating glomerular filtration rate (GFR) in various mouse models (knockout strains) under a control condition or an experimentally induced kidney condition in comparison with corresponding wild type mice. [0015] Figs. 2A-2F represent exemplary histogram plots related to detecting

proinflammatory cytokine levels or other agents under various kidney functions in wild type mice or experimental knockout mice of certain embodiments provided herein. Figs. 2A-2E provide histograms plotting exemplary measurements of serum creatinine (2A) and assessments of renal TNFa (2B), IL6 (2C), MPO levels (2D) and Jablonski Index (2E). Fig. 2F represents exemplary histological staining results of kidney cross sections from the same mice tested in 2A-2E.

[0016] Figs. 3A-3B represent exemplary data illustrating the renal transcript levels (3A) and protein levels (3B) of a target gene of certain embodiments disclosed herein in different mouse strains under various kidney conditions. β-Actin serves as a loading control in Fig. 3B.

[0017] Fig. 4 is a schematic graph illustrating certain regulatory elements and transcription factor binding sites in the promoters of a target gene from mouse and humans disclosed herein in certain embodiments.

[0018] Figs. 5A-5C represent exemplary histogram plots illustrating parameters related to kidney functions in mice under control or a diseased kidney condition in the presence or absence of a proton pump inhibitor. Fig. 5A represents levels of GFR (after a 2h

reperfusion). Fig. 5B illustrates exemplary assessments of Jablonski Index (after a 24h reperfusion). Fig. 5C provides exemplary histological staining images of kidney cross sections of these experiments.

[0019] Fig. 6 represents an exemplary histogram plot illustrating GFR levels in mice under various kidney conditions after increasing doses of a PPI disclosed herein.

[0020] Figs. 7A-7C represent exemplary histogram plots of GFR and Jablonski indices in control and experimental kidney conditions in mice in the presence or absence of a composition provided in certain embodiments herein. Fig. 7A provides GFR detection levels (lh reperfusion). Fig. 7B illustrates exemplary levels of Jablonski Index (24h

reperfusion). Fig. 7C provides exemplary histological staining of kidney cross sections of these mice under the various conditions.

[0021] Figs. 8A-8C represent histogram plots of experiments performed on control and genetic knockout mice related to kidney function. Fig. 8A illustrates GFR (after 2h reperfusion) in absence of treatment. Fig. 8B illustrates Jablonski Index (after 24h reperfusion) in absence of treatment. Fig. 8C provides exemplary histological staining results of kidney cross sections of the control and knockout mouse. [0022] Figs. 9A-9C: Figs. 9A-9B represent exemplary histogram plots related to assessing levels of renal ATP in wild type mice or knockout mice of some embodiments with or without ischemic induced AKI (Fig. 9A) and before and after reperfusion (Fig. 9B). Fig. 9C represents an exemplary histogram plot of control and knockout mice illustrating renal ATP content in the presence or absence of a PPL

[0023] Figs. 10A-10E represent exemplary histogram plots illustrating levels of expression of various agents (10B, 10 C and 10E) in control and knockout mice and demonstrating kidney functions in control and knockout mice, GFR (after lh reperfusion, Fig. 10A; and 24h reperfusion, Fig. 10D).

[0024] Figs. 1 lA-1 ID represent exemplary histogram plots illustrating various parameters of kidney functions in control and knockout mice under conditions of unilateral ischemia (Figs. 1 lA-11C) in the presence or absence of a PPI at various doses (Figs. 11A- 1 IB) 0.5 mg/mouse or 0.35 mg/mouse (Figs.. 1 lC-1 ID) provided in some embodiments herein. GFR (lh reperfusion, Fig. 1 1A; and 24h reperfusion Figs. 1 lB-1 ID) is assessed under each condition.

Definitions:

[0025] As used herein, "a" or "an" may mean one or more than one of an item.

[0026] As used herein the specification, "subject" or "subjects" may include, but are not limited to, mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.

DETAILED DESCRIPTION

[0027] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the details outlined herein, but rather that concentrations, times and other details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.

[0028] In accordance with embodiments of the present invention, there may be employed conventional molecular biology, protein chemistry, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986).

[0029] Certain embodiments of the present invention provide for methods, compositions and uses of proton pump inhibitors to prevent or to treat an adverse kidney condition. In accordance with these embodiments, certain compositions and methods disclosed herein concern treatment or prevention of an acute kidney injury (AKI) in a subject in need thereof. Causes of acute kidney injuries can include, but are not limited to, ischemia/reperfusion, trauma, kidney disease and kidney transplantation.

[0030] In certain embodiments, during renal ischemia, the kidneys can experience profound tissue hypoxia. This can be caused from limited oxygen availability during an ischemic period and no-reflow phenomena during a reperfusion period. Moreover, high oxygen consumption by resident or inflammatory cells can further aggravate renal hypoxia during ischemia and reperfusion. Cellular responses to hypoxia can be regulated by a conserved pathway that involves oxygen sensing prolyl hydroxylases (PHDs) and hypoxia- inducible factors (HIFs). HIFs consist of a constitutively expressed β-subunit (HIF-1 β), and a highly regulated a -subunit (this can either be HIF- 1 a or HIF-2 a). Under normal oxygen conditions, HIF- 1/2 a can be immediately subjected to proteasomal destruction. When oxygen levels fall, HIF- 1/2 a can be stabilized and form active heterodimer with their partner HIF-1 β. The heterodimer translocates into the nucleus where it binds to promoter regions of hypoxia-dependent genes. HIF-binding to a promoter can either result in increased gene expression, or repression of gene expression. The actual oxygen sensing occurs at the level of the PHDs. PHDs use oxygen as a co-factor for HIF-hydroxylation. Therefore, hypoxia results in a functional inhibition of PHDs with subsequent HIF stabilization. Three oxygen sensing PHDs have been implicated in the regulation of HIF stability (PHD 1-3). However, only deletion of PHD 1 can provide a near complete protection from AKI. Pharmacologic HIF inhibition with inhibitor DMAG was associated with abolished protection from ischemic AKI under condition of deletion of PHD 1 gene. Binding sites for HIF in Atp4a promoter have been identified.

[0031] In some embodiments, a pharmaceutically acceptable composition can be used to treat or prevent a kidney condition in a subject through modulating the activity of ATP4A. ATP4A gene codes for the a-subunit of the H+-K+-Atpase, a proton pump that uses the energy of ATP hydrolysis to pump hydrogen (and potassium) ions against their concentration gradients. The H+-K+-Atpase consists of two subunits, a large, transcriptionally regulated 100-kDa a -subunit that contains the catalytic and ion translocating sites and a small a - subunit involved in intracellular processing. ATP4A is an ATP consuming P-type ATPase that is expressed in the gastric parietal cells and in the kidneys (distal and proximal tubules, collecting ducts). ATP4A expressed in the stomach is responsible for the acidification of the gastric fluid. Studies have demonstrated NF-KB-dependent repression of Atp4a during helicobacter pylory infection. Atp4a is one target of certain embodiments for control by compositions disclosed herein in order to reduce the incidence or advancement of AKI or other kidney condition in a subject.

[0032] Some embodiments concern compositions and uses of proton pump inhibitors to treat or prevent an adverse kidney condition in a subject. In certain embodiments, PPIs can be used to inhibit Atp4a activity or expression in a subject, and treat AKI or other kidney condition in the subject. Proton pump inhibitors can include, but are not limited to, pharmaceutically acceptable compositions of a member of any of the following families for inhibitors and any commercially available composition thereof, for example, one class of proton pump inhibitors are those that act by irreversible inhibition of the H+/K+ ATPase. This class of proton pump inhibitors are substituted pyridyl methylsulfinyl benzimidazoles, characterized at thiol-reactive reagents, which covalently inhibit the H,KATPase by forming a disulfide bond with luminally accessible cysteine side chains. These are clinically used proton pump inhibitors. For example, proton pump inhibitors can include omeprazole or a commercially available formulation (e.g. Losec®, Prilosec®, Zegerid®, Ocid®, Lomac®, Omepral®, and Omez®, Zegarid® rapid release form of omeprazole), pantoprazole or a commercially available formulation (e.g. Protonix®, Somac®, Pantoloc®, Pantozol®, Zurcal®, Zentro®, Pan®, Controloc®), esomeprazole or a commercially available formulation (e.g. Nexium®, Esotrex®, Esso®), rabeprazole or a commercially available formulation (e.g Erraz® ,Zechin®, Rabecid®, Nzole-D®, AcipHex®, Pariet®, Rabeloc®. Dorafem®: combination with Domperidon®), dexlansoprazole or a commercially available formulation (e.g. Kapidex®, Dexilant®), lansoprazole or a commercially available formulation (e.g. Prevacid®, Zoton®, Monolitum®, Inhibitol®, Levant®, Lupizole®), soraprazan, and revaprazan or others known in the art. A second class of inhibitors currently under development for clinical use, can include, but are not limited to substituted imidazo- l,2a-pyridines, such as SCH28080, and a photoaffinity derivative, (e.g Me-DAZIPl) that are Kl -competitive, reversible inhibitors and any others known in the art. Some embodiments provide a pharmaceutically acceptable composition of a member of the proton pump inhibitor family or any combination of the agents contemplated herein. In other embodiments, proton pump inhibitors can be one or more of omeprazole, esomeprazole, and SCH-28080. In accordance with these embodiments, a pharmaceutically acceptable composition of one of, omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole, lansoprazole, soraprazan, revaprazan and SCH-28080 or a combination thereof can be used to treat or prevent an adverse kidney condition, including but not limited to, acute kidney injury, kidney failure or the like.

[0033] Esomeprazole is the S-enantiomer of omeprazole, and is clinically used in patients for the treatment of acid reflux, and peptic ulcer disease. It was found that esomeprazole can function as a potent inhibitor of Atp4a, including Atp4a expressed in the kidneys. Pretreatment with esomeprazole (e.g., 0.5 mg/kg BW, 30 min prior to renal ischemia) provided profound kidney protection against AKI. This finding was confirmed by studies in a bilateral mouse model of renal ischemia. In addition, SCH28080, also provide robust kidney protection from ischemic AKI. Furthermore, control of the ATP4A gene may prevent ischemia AKI in a subject.

[0034] In certain embodiments, subjects with pre-existing renal diseases are prone to perioperative AKI. In some embodiments, pre-existing renal diseases can include, but are not limited to, a kidney disease such as diabetes. Proton inhibitors can be administered to a subject with a pre-existing renal disease to prevent or provide protection from AKI.

[0035] In yet other embodiments, kidney samples or other samples can be obtained from a subject and various genes (see for example Tables 1 and 2) can be assessed for transcription or level of protein expression in the subject's sample and presence or propensity to having or developing a kidney condition can be diagnosed in the subject. Then a health professional can assess treatment requirement of the subject. For example, an affected subject can be treated with a proton pump inhibitor to treat an existing or potential adverse kidney condition (e.g. AKI). Once a subject has been treated, additional samples may be obtained from the subject in order to assess whether additional treatments are recommended. Other subjects such as a subject undergoing a transplant could be evaluated for levels or expression of these genes prior to, during or after receiving a transplanted organ in order to assess kidney function in the subject. A transplant patient and/or even the organ being transplanted can be exposed to compositions of PPI disclosed herein in order to treat or preserve kidney function in a subject. One or more PPIs alone or in combination with other transplant agents could be added directly to a transport media of an organ in order to prepare the organ for implantation into a subject.

Transplantation [0036] In yet another embodiment, a subject undergoing transplantation can be treated before, during or after with one or more PPIs or combinations of PPIs with other anti-ATP4a agents and or anti- inflammatory agents (e.g. inhibitors of pro-inflammatory cytokines such as TNF-a, IL-6 etc.). Transplants contemplated herein can include any organ or non-organ transplantation. Organ transplantation can include, but is not limited to, heart, kidney, liver, lung or other transplantation. Other embodiments concern treating a subject undergoing a kidney transplant within 1 week to 48 hours to within hours before and optionally during and after transplantation of the kidneys into the subject. It is also contemplated that the kidneys used for transplantation can be pre-treated with one or more PPIs in an appropriate media (optionally including anti-inflammatory agents and anti-ATP4a agents.

Pharmaceutical Compositions and Administration

[0037] Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By

"biologically compatible form suitable for administration in vivo" is meant a form of the active agent (e.g. pharmaceutical chemical, protein, gene, antibody etc of the embodiments) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. Pharmaceutical compositions that include proton pump inhibitors or a functional variant thereof as an active ingredient may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. Pharmaceutical compositions may include additional cytoprotective or radioprotective agents known by one skilled in the art.

[0038] Dosage form of the pharmaceutical composition can be determined by the mode of administration chosen. Several PPIs are commercially available and have been used to treat other conditions so pharmaceutical considerations for the dose related to safety and efficacy can be readily determined by a health professional. In addition to oral formulations, injectable fluids, inhalational, topical, ophthalmic, peritoneal, and other formulations can be employed. Inhalational preparations can include aerosols, particulates, nasal sprays, inhalers and similar preparations. In general, the goal for particle size for inhalation is about 1 μιη or less in order that the pharmaceutical reach the alveolar region of the lung for absorption. Oral formulations may be liquid (for example, syrups, solutions, or suspensions) or solid (for example, powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art.

[0039] Pharmaceutical compositions disclosed herein can be administered by any route known in the art, including, but not limited to, parenteral administration; for example, intravenous, intramuscular, intraperitoneal, intrasternal, or intra-articular injection or infusion, or by sublingual, oral, topical, intra-nasal, ophthalmic, or transmucosal

administration, or by pulmonary inhalation. When the active compounds are provided as parenteral compositions, for example, for injection or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers. A form of repository or depot slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection, intradermal injection or delivery.

[0040] In certain embodiments, compositions disclosed herein may be administered directly to the site of surgery in a subject in need thereof. In certain examples, the site of surgery can include the region of transplantation (e.g. kidney transplantation).

[0041] In certain embodiments, active compounds (e.g., PPIs, antibodies etc against ATPa4 etc) can be suitably administered by sustained-release systems or slow release formulations. Suitable examples of sustained-release formulations include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, for example, films or microcapsules), suitable hydrophobic materials (for example, as an emulsion in an acceptable oil), suitable microparticles or microbeads, or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release compounds may be administered by intravascular, intravenous, intra arterial, intramuscular, subcutaneous, intra-pericardial, or intra-coronary injection.

Administration can also be oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (as by powders, ointments, gels, drops or transdermal patch), buccal, or as an oral or nasal spray. Pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features that allow a controlled release of the active substance.

[0042] A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises.

[0043] In some embodiments, therapeutic agent(s) can be delivered by way of a pump or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. In another aspect of the disclosure, therapeutic agent(s) are delivered by way of an implanted pump. Implantable drug infusion devices are used to provide subjects with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Essentially, such device may be categorized as either active or passive.

[0044] Depending on the route of administration, the active compound may be coated in a material to protect the compound from the degradation by enzymes, acids and other natural conditions that may inactivate the compound. In a preferred embodiment, the compound may be orally administered. In another preferred embodiment, the compound may be

administered intravenously. In one particular embodiment, the composition may be administered intranasally, such as inhalation.

[0045] Some embodiments disclosed herein concern using a stent or a catheter to deliver one or more agents (e.g. along with compositions disclosed herein) to a subject having or suspected being treated for cancer. Any stent or other delivery method known in the art that can deliver one or more agents directly to tumor site is contemplated. These delivery techniques can be used alone or in combination with other delivery methods.

[0046] A compound (e.g. a peptide, protein or mixture thereof) may be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. The term "pharmaceutically acceptable carrier" as used herein is intended to include diluents such as saline and aqueous buffer solutions. It may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. The active agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0047] Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used.

[0048] Sterile injectable solutions can be prepared by incorporating active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0049] Aqueous compositions can include an effective amount of a therapeutic compound, peptide, epitopic core region, stimulator, inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Compounds and biological materials disclosed herein can be purified by means known in the art. Solutions of the active compounds as free-base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

[0050] A composition including a proton pump inhibitor may be administered prior to, during, or after surgery or other event affecting renal function, or a combination thereof. In some embodiments, one or more doses of the composition can be administered to a subject, such as a human subject, within a week prior to, such as within 5 days, 4 days, 3 days, 48 hours, or from 1-24 hours prior to the event (e.g. surgery). In some embodiments, the composition including proton pump inhibitors can be administered to a subject during an event if applicable (e.g. surgery or transplantation). In some embodiments, one or more doses of the composition including proton pump inhibitor can be administered to a subject within 1 hour to 30 days (or longer) after an event, such as within 14 days, within 7 days, within 5 days, within 4 days, within 3 days, within 48 hours, or within 1-24 hours after an event. In some embodiments, administration can include at least one dose of proton pump inhibitor from about 0.1 to 120 mg/kg within 48 hours prior to the event to within 48 hours after the event or longer. In some embodiments, the administration further includes at least two or more doses from about 0.1 to 120 mg/kg every 24 to 48 hours for a period of from 2 days to 30 days after the event. In some embodiments, the administration includes at least one dose from about 1 to 120 mg/kg prior to an event. In some embodiments, the administration comprises at least one dose of proton pump inhibitor within 24 hours prior to the event. In some embodiments, a subject can be treated in order to achieve a plasma level of about .1 to 10 mg/L.

[0051] In another embodiment, nasal solutions or sprays, aerosols or inhalants may be used to deliver the compound of interest. Additional formulations that are suitable for other modes of administration may include suppositories and pessaries. [0052] In some embodiments, it is understood that certain proton pump inhibitors undergo a shift such as a chiral shift once they are administered to a subject (e.g.

omeprazole). Certain subjects are known to have varying compositions of enzymes to accomplish these shifts or conversions therefore, it is contemplated herein that enzymes or other factors involved in these conversions may be up-regulated or down-regulated in a subject in order to achieve the desired result. Thus, proton pump inhibitor treatment disclosed herein may need to be adjusted for such a subject as determined by a health professional.

[0053] Compositions disclosed herein can be used alone or in combination with other compositions or remedies known in the art for treating or preventing AKI or other kidney condition in a subject.

[0054] Various modifications and additions can be made to the exemplary embodiments discussed without departing from the scope of the present invention. For example, while the embodiments described above refer to particular features, the scope of this invention also includes embodiments having different combinations of features and embodiments that do not include all of the above described features.

[0055] Any method known in the art for isolating and analyzing nucleic acids and/or proteins is contemplated herein. In addition, methods for detecting expression and activity of a DNA, RNA or protein are contemplated herein. For example, assays such as Western blots and PCR assays can be used to analyze such molecules in order to assess the

presence/absence or level of a gene or gene expression in a sample obtained from a subject. It is contemplated herein that a sample from a subject can include a tissue (e.g. kidney), blood or urine sample or other sample associated with kidneys and kidney function.

[0056] Embodiments of the present invention are further illustrated by the following non-limiting examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the instant invention or the scope of the appended claims.

EXAMPLES

Example 1

[0057] In certain exemplary methods, compositions for treating or preventing kidney conditions in a subject are disclosed. Compositions of certain embodiments herein can include one or more proton pump inhibitors or H+-K+-Atpase inhibitors. Exemplary methods using these compositions to treat or prevent acute kidney injury (AKI) or other kidney conditions are provided below.

[0058] Some experiments provided herein were performed to analyze genetic deletion of oxygen sensors in an acceptable mouse model. Animal models were established using murine AKI model systems such as unilateral or bilateral renal artery occlusion to induce AKI in various strains of mice, such as genetically modified mouse strains, that mimic the condition in humans and other animals. These models permitted the discovery of certain target genes that can be used to screen for kidney conditions in a subject and in methods for screening for novel treatment approaches for AKI and other kidney conditions. One skilled in the art could measure the presence/absence or induction/inhibition of one or more target genes in order to assess potency/efficacy of a pharmaceutical agent in prevention or treatment of a kidney condition.

Overview of Renal Ischemia

[0059] During renal ischemia, shifts in the metabolic supply and demand ratio, particularly for oxygen, can result in severe kidney hypoxia. Cellular responses to hypoxia are regulated by enzymes that sense cellular oxygen levels and coordinate transcriptional responses to hypoxia or ischemia. Some of these enzymes are three oxygen sensing prolyl hydroxylases (PHD 1-3). Limited oxygen availability results in inhibition of PHDs with subsequent stabilization of hypoxia-inducible factors (HIFs), a key transcription factor for adaptation to hypoxia. Activation of HIF drives a transcriptionally regulated response that programs cellular metabolism towards hypoxia adaptation. In light of these observations, gene-targeted mice for Phdl, Phd2 or Phd3 were exposed to AKI using a generally acceptable protocol and assessed for renal function, for example, by measuring glomerular filtration rate (GFR), or renal histology. A selective phenotype oiPhdY^1' mice with high protection from ischemic AKI was identified and used in additional studies. In order to gain insight into how Phdl deletion protects the kidneys from ischemia, microarray studies were performed to assess potential genetic links to improved protection in these mice. The genetic screen identified over a 10-fold repression in Atp4a gene expression was observed, when comparing ischemic kidneys from Phdl ^1' mice with controls. Subsequent studies were performed to assess effects of pharmacologically acceptable ATP4A inhibitors (e.g.

omeprazole and isomers and derivatives thereof), which were demonstrated to mimic kidney protection from ischemia observed in Phdl^7" mice. In addition, Atp4a^_/" mice were protected from renal ischemia. Proton pump inhibitors can also be used to prevent or treat ischemic - related AKI as found in many perioperative patients. Acute kidney injury (AKI) in Perioperative Medicine

[0060] Ischemic AKI represents a major threat for surgical patients, with AKI occurring after cardiac surgery in up to 20-30% of patients. Moreover, recent studies indicate that ischemic AKI can set a viscous cycle in motion, leading to intestinal Paneth cell activation and multi-organ failure. Relatively little progress has been made for the treatment of ischemic AKI. Novel therapeutic approaches to prevent or treat AKI are needed to improve outcomes in trauma patients, critical care medicine or during the perioperative period.

[0061] As noted above, one gene of importance regarding protection from AKI appears to be control of ATP4A. The function of ATP4A during ischemic AKI is essentially unknown. The ATP4A gene codes for the a-subunit of the H+-K+-Atpase, a proton pump that uses the energy of ATP hydrolysis to pump hydrogen (and potassium) ions against their concentration gradients. The H+-K+-Atpase consists of two subunits, a large,

transcriptionally regulated 100-kDa a-subunit that contains the catalytic and ion translocating sites and a small α-subunit involved in intracellular processing. ATP4A has been found to be expressed in the stomach and is responsible for the acidification of the gastric fluid, e.g. previous studies showed NF-KB-dependent repression of Atp4a during helicobacter pylory infection. Another organ expressing ATP4A are the kidneys [distal and proximal tubules, collecting ducts]. Pharmacologic and genetic studies in Atp4a^_/" mice demonstrate a functional role for ATP4A in the acidification of the urine.

[0062] Compositions including proton pump inhibitors and the H+-K+-Atpase inhibitors were tested in some experiments for their effects on kidney conditions. For example, two such agents included but are not limited to, omeprazole and SCH-28080, both of which can efficiently inhibit renal ATP4A activity. It was observed that pharmacologic inhibition of ATP4A with omeprazole was associated with protection from ischemic AKI, in conjunction with improved energy balance. Proton pump inhibitors (such as omeprazole) are used clinically for the treatment of acid reflux in surgical patients and have a great safety profile. Thus, these findings are significant for AKI in patients and could be translated into the practice of perioperative medicine.

Example 2

Genetic models to study the relative roles of PHDs

[0063] In some exemplary experiments, animal models for studying ischemic AKI were established (see above). Two exemplary murine model systems for renal ischemia were used: isolated renal artery occlusion and bilateral clamping of whole pedicle with micro-vascular clamps to induce renal ischemia. Following renal ischemia (20 to 30 min), the kidneys were reperfused over 2-24h and glomerular filtration rates (GFR), renal histology, and creatinine clearance was measured as an indication of kidney function and health of the animals. GFR measurements via infusion of FITC-labeled inulin through a central venous catheter placed into the jugular vein were used.

[0064] In addition, functional roles of cellular oxygen sensors during ischemic AKI were pursued in some studies. It was hypothesized that ischemic AKI and renal hypoxia would result in a functional inhibition of PHDs, subsequent HIF activation and kidney protection from ischemia. To test this hypothesis, in one exemplary method, a genetic approach was taken to identify the relative contribution of individual oxygen sensing PHDs. Genetic models used to study PHDs included PhdV^A, Phd2^~/~ and Phd3^~A mice. PhdP^A and Phd3 ^A mice appear healthy and breed normally while Phd2^~A mice die at mid-gestation.

Heterozygote Phd2^+A mice appear healthy, and breed normally. In other exemplary methods, heterozygous Phd2^+A mice were used to study the consequences of partial Phd2 deficiency. Renal tissues were harvested and examined for transcript levels of Phds in these mouse lines. The renal Phd levels correlate with their genotype, while transcript levels of other Phds were unaltered. In some studies, ischemic AKI was established in all three mouse lines, followed by evaluation of relative contributions of PHD 1-3 to kidney protection from ischemia. In one exemplary method, a "head-to-head" comparison of "Phd knockout mice" during ischemic AKI was examined. Briefly, individual Phd knockout mice were exposed to renal ischemia for 30 min. GFR was measured via infusion of FITC-labeled insulin following 2h of reperfusion. Surprisingly, no difference was found between heterozygous Phd2^+A mice, heterozygous Phd3 ^A mice or their littermate controls (data not shown). In contrast, gene- targeted mice for Phdl^~A were unexpectedly protected from ischemic tissue injury (Figs. 1 A- 1C, n=4).

[0065] Based on these findings, more detailed studies in PhdV^A mice exposed to ischemic AKI were performed. Consistent with the original findings of improved GFR following renal ischemia, additional data confirmed a protected phenotype oiPhdl^~A mice. Kidney function as assessed by serum creatinine (Fig. 2A), renal inflammatory parameters, e.g. TNFa (Fig. 2B), IL6 (Fig. 2C), MPO levels (Fig. 2D) and Jablonski Index (Fig. 2E), and renal histology (Fig. 2F) demonstrated attenuated kidney injury in PhdV^A mice (Figs. 2A-2F, n=4).

[0066] In order to study how Phdl deficiency protects the kidneys from ischemic AKI, further analysis of ischemic kidneys from genetic models was conducted. Microarray analysis was performed on wild type and PhdT ^" AKI mice. Briefly, three PhdT ^" mice or littermate controls matched in age, sex and gender were exposed to renal ischemia (30 min), followed by 2h of reperfusion. Kidney samples were analyzed to determine the transcript levels of genome via microarray. A number of genes were identified to be differentially regulated. The top 5 differentially induced or repressed genes are provided in Table 1 (n=4; subset of genes were validated by RT-PCR and Western plot, Figs. 3A-3B and data not shown). It is contemplated that genes disclosed herein can be used to diagnose or to assess a kidney condition in an affected subject.

Table 1 Microarray analysis of AKI mouse models

Table 2 Exemplary Genes** Identified in the Microarray

[0067] Figs. 1A-1C represent exemplary histograms plotting functions of Phd genes in genetic animal models exposed to ischemic kidney injury. Fig. 1A demonstrates glomerular filtration rates (GFR) observed in Phdl full knockout, wild type littermates, and the corresponding mice having ischemia induced AKI. A significant difference of GFR between the knockouts having AKI and the control mice is observed. Fig. IB illustrates GFR observed from heterozygous Phd2^+/~ mice, wild type mice and the corresponding AKI mice. Fig. 1C provides GFR observed from wild type mice and Phd3^~A mice (with or without induced AKI). A role of Phdl in kidney protection from ischemic AKI is supported by these observations.

[0068] Figs. 2A-2E represents exemplary histograms illustrating kidney functions of wild type and Phdl^''^' ischemic AKI mouse models, assessed by various renal inflammatory parameters. Fig. 2F provides exemplary histology staining results illustrating attenuated kidney injury in AKI Phdl ^1' mice compared to the wild type AKI control mice. Wild type and Phdl ^1' mice without AKI were included as additional controls.

Identification of Atp4a as a PHD-1 target gene

[0069] Gene expression of renal Atp4a, observed from microarray analyses, was confirmed by additional studies. As illustrated in Figs. 3A-3B, renal Atp4a transcript (Fig. 3A) and protein levels (Fig. 3B) were repressed following renal ischemia in wild-type mice. The reduction was profoundly enhanced in the absence of Phdl (e.g. Phdl^''^' mice). Renal Atp4a levels following kidney ischemia were hardly detectable in Phdl ^1' mice.

[0070] Fig. 3A represents an exemplary histogram illustrating transcription levels of renal Atp4a. Levels of renal Atp4a transcript significantly decreased in Phdl deficient mice compared to wild type controls, while exposure to ischemic AKI resulted in further reduction in each strain. Fig. 3B provides exemplary Western blots representing renal Apt4a protein levels in WT and Phdl knockout mice with or without induction of ischemic AKI. This data confirmed that the protein levels correlated with transcription of Atp4a transcript.

Example 3

[0071] ATP4A consists of two subunits, a 114-kDa a-subunit (gene locus Atp4a) and a 35-kDa (protein moiety) b-subunit (gene locus Atp4b). The a-subunit contains ATP and cation binding sites and carries out the catalytic and transport functions of the enzyme.

Previous studies implicated Atp4a in the regulation of the acid base status of the kidneys. Renal ATP4A can be blocked pharmacologically by proton pump inhibitors that are clinically used for the treatment of acid reflux.

[0072] Fig. 4 is a schematic diagram illustrating promoters of a mouse and a human Apt4a gene. Certain regulatory elements and transcription factor binding sites of these genes are demonstrated.

Functional role of Atp4a in ischemic kidney injury

[0073] Some studies were performed to investigate whether transcriptional repression of Atp4a might play a functional role in kidney protection from ischemic AKI. Some exemplary experiments were conducted with genetic models exposed to ischemic AKI, such as Phdl^''^' mice. Various compositions were provided to study prevention or treatment of ischemic AKI and other kidney conditions. In some experiments, compositions including proton pump inhibitors were studied. One example of these inhibitors is esomeprazole.

[0074] Esomeprazole is the S-enantiomer of omeprazole, and is clinically used in patients for the treatment of acid reflux, and peptic ulcer disease. Esomeprazole functions as a potent inhibitor of Atp4a, including Atp4a expressed in the kidneys. In certain

experiments, Esomeprazole was given to mice 30 min prior to renal ischemia exposure to prevent induction of ischemic AKI (Figs. 5A-5C, n=4). In other experiments, Esomeprazole was administered to affected AKI mice to assess whether the agent could alleviate the condition. Optimal doses of Esomeprazole for preventing or treating ischemic AKI were determined. Administration of 0.5 mg kg BW (by weight) of Esomeprazole to mice demonstrated significant increase in GFR compared to doses of 0.1, 0.25 and 0.75 mg/kg BW after 2h of reperfusion (Fig. 6, n=4). In one exemplary experiment, an optimal dose of 0.5 mg/kg BW was given to mice 30 min prior to ischemic exposure (Figs. 5A-5C). Mice that did not receive Esomeprazole or mice that were not exposed to ischemic AKI were included in the experiment. Analysis including GFR (2h reperfusion) (Fig. 5A) and Jablonski Index (Fig. 5B) were performed on samples from all mice. Significant increase of GFR in

Esomeprazole treated ischemic AKI mice compared to mice of the same disease without treatment was observed. Inflammation was reduced in the treated AKI mice as verified by evaluation of inflammatory parameter Jablonski Index. Subsequent histological studies performed on these mouse samples further confirmed profound kidney protection from AKI with the regiment (see for example, Fig. 5C; n=4). Studies in a bilateral model of renal ischemia also confirmed the findings (data not shown).

[0075] Figs. 5A-5C represent data demonstrating protection from ischemic AKI after administration of a proton pump inhibitor. Fig. 5 A provides an exemplary histogram plotting GFR observed in mice treated under various conditions. Fig. 5B represents an histogram plot illustrating reduced inflammation reflected by change of Jablonski Index in treated AKI mice compared to untreated control mice. Fig. 5C demonstrates exemplary histological staining results of kidney sections from the same mice as in Figs. 5A-5B. As presented in Figs. 5A- 5B, a group of mice did not have ischemic induced AKI (-1) and were untreated (white bar); a second group of mice did not have ischemic induced AKI (-1) but were treated with Esomeprazole (black bar); a third group of mice had ischemic induced AKI (+1) but were not treated (white bar); and a fourth group of mice had ischemic induced AKI (+1) and were treated with Esomeprazole (black bar). [0076] Fig. 6 represents an exemplary histogram demonstrating protection against ischemic AKI in mice by administration of various doses of Esoeprazole (ranging from 0.1- 0.75 mg/kg BW).

[0077] In other experiments, compositions including pharmacologic inhibitors of H+- K+-Atpase were examined. Other H+-K+-Atpase inhibitors were used in these studies. One agent used is the proton-pump inhibitor, SCH28080. Similar to the above studies, robust kidney protection was observed from ischemic AKI (100μg/mouse i. v.; Figs. 7A-7; n=4). These functional experiments indicated the likelihood that the previously observed kidney protection from ischemic AK in Phdl^^' mice is mediated, at least in part through repression oiAtp4a. Inhibition of Apt4A with pharmacological inhibitors such as proton pump inhibitors and H+-K+-Atpase inhibitors, provided protection against ischemic AKI was also observed using acceptable animal models.

[0078] Figs. 7A-7C represent exemplary experimental results illustrating protection from ischemic AKI achieved by administration of SCH28080, a H+-K+-Atpase inhibitor. Fig. 7A provides an exemplary histogram plotting GFR observed in mice under various conditions. Fig. 7B represents an exemplary graph illustrating reduced inflammation represented by change of Jablonski Index in treated AKI mice compared to the untreated. Mice without ischemic AKI demonstrated much lower Jablonski Index compared to mice having ischemia induced AKI. Fig. 7C represent exemplary histological staining results of kidney sections from the same mouse models used for experiments represented by Figs. 7A-7B. As presented in Figs. 7A-7B, a group of mice did not have ischemic induced AKI (-1) or were untreated (white bar); a second group of mice did not have ischemic induced AKI (-1) but were treated with SCH28080 (black bar); a third group of mice had ischemic induced AKI (+1) but were not treated (white bar); and a fourth group of mice had ischemic induced AKI (+1) and were treated with SCH28080 (black bar).

Example 5

Studies of ischemic AKI in Atp4a-/- mice

[0079] In order to further investigate treatment for AKI and other kidney conditions, in some studies, a mouse strain with targeted deletion of Atp4a was generated. Based on findings of treatment of proton pump inhibitors (e.g. omeprazole) and H+-K+-Atpase inhibitors (such as, SCH28080), it was hypothesized that gene-targeted mice for Atp4a would be protected during ischemic AKI. Atp4a ^/~ mice thrive, are indistinguishable from their wild- type and heterozygous littermates in both behavior and outward appearance, and are fertile. In some experiments, Atp4a ^/~ mice having ischemia induced AKI were created and renal function was subsequently assessed (Figs. 8A-8C; n=4). These studies were consistent with previous findings in Phdl-/- mice, and the pharmacologic studies with inhibitors. Deficiency of Atp4a caused very robust kidney protection during ischemic AKI. While previous studies had shown that the baseline acid base status οΐΑΐρ4α ^/~ mice is normal, the findings provided herein are the first to identify a functional role of Atp4a in renal physiology, and particularly suggest potential treatment and a preventive method for kidney ischemia.

[0080] Based on the observation that the kidneys become profoundly hypoxic during ischemic AKI, a functional role of individual oxygen sensing enzymes in renal protection from ischemia was established. Studies demonstrating a selective protection in Phdl^7" mice lead to the surprising finding of a functional role of ATP4A repression in kidney protection. Furthermore, pharmacologic studies using proton pump inhibitors and H+-K+-Atpase inhibitors on mice with genetic deletion of the Atp4a gene provide support for potential methods of prevention and treatment for renal ischemia in humans. These findings also provide novel approaches to prevent or treat acute kidney injury in subject undergoing or that have undergone a surgical procedure, a heart attack patient, trauma or during critical illness.

[0081] Figs. 8A-8C represent exemplary histogram plots illustrating effects of various agents on ischemic AKI conferred by deletion of Atp4a gene. Fig. 8A represents GFR in +Atp4a or Atp4a deficient mice with or without induced ischemic AKI (2h reperfusion). Development of ischemic AKI is accompanied by significant reduction of GFR in both strains, but deletion of Atp4a caused a significant increase of GFR in AKI mice. Fig. 8B represents an exemplary graph illustrating reduced inflammation reflected by level of Jablonski Index in Atp4a knockout AKI mice compared to the corresponding wt control mice. Fig. 8C demonstrates exemplary histological staining results of kidney sections from the same mice as in Figs. 8A-8B. As presented in Figs. 8A-8B, a group of wt mice (white bar) that did not have ischemia induced AKI (-1); a second group were Apt4a knockout mice (black bar) that did not have ischemia induced AKI (-1); a third group of mice were wt (white bar) having ischemia induced AKI (+1); and a fourth group were Apt4a knockout mice (black bar) having ischemia induced AKI (+1).

ATP conversation during ischemic AKI

[0082] In other exemplary methods, treatment of renal ischemia and AKI was further studied. For example, some studies included analyzing renal ATP levels following renal ischemia, and ATP recovery in Phdl^''^' mice, or following treatment with proton pump inhibitors. A luminometric assay may be used to assess renal ATP levels. In certain experiments, ATP recovery was determined following treatment with Omeprazole. As presented in Figs. 9A-9C, baseline ATP levels were comparable between wt and Phdl deficient mice, and in normal mice with or without treatment of Esomeprazole. These observations were consistent with previous studies. However, an accelerated ATP recovery was observed in Phdl^''^' mice (Fig. 9A-9B; n=4) and an attenuated ATP depletion was observed with omeprazole treatment (0.5 mg/kg BW) (Fig. 9C; n=4). To further confirm these findings, ATP recovery was examined following ischemic AKI in mice treated with SCH28080 (e.g. 100μg/mouse i.v.), as done in Phdl ^1' mice. In addition, ATP depletion was studied following different time periods of renal ischemia \xv Atp4a ^~ mice. In addition, Atp4 ^/~ mice were exposed to 30 min of renal ischemia and ATP recovery assessed with different reperfusion times (e.g. 10, 20 and 30 min).

[0083] Figs. 9A-9B represent exemplary histograms illustrating changes of ATP content during ischemia (Fig. 9A), and at various time periods during reperfusion (Fig. 9B) in wild type and Phdl knockout mice. Phdl knockouts demonstrated an attenuated loss of energy rich phosphates (ATP) during renal ischemia compared to the control. Fig. 9C is a histogram plot illustrating renal ATP content before and after ischemia exposure in wild-type mice, and before and after treatment of a pharmacologic ATP4A inhibitor, such as Esomeprazole. Significant reduction in loss of ATP during renal ischemia was observed in treated versus untreated mice.

Other Studies of AKI in Atp4a-/- mice

[0084] Some other studies were performed on Atp4a deficient mice with induced AKI. Renal functions of these mice were assessed. For example, glomerular filtration rate (GFR) was observed and found to be significantly higher in Atp4a-/- mice following 30 min ischemia and 1 hour reperfusion (Fig. 10A). Inflammatory cytokines, such as TNF-a (Fig. 10 B) and IL-6 (Fig. IOC) were reduced in these mice compared to wt mice. In another set of experiments, reperfusion time was increased to 24 hours. Interestingly, GFR in Atp4a-/- mice was observed and found to be similar to the very low GFR in wild-type mice (Fig. 10D). Infiltration with neutrophils was significantly reduced in Atp4a-/- mice (Fig. 10E). Therefore, pro-inflammatory indicators are reduced in the knockout mice and this can be used to develop agents that target Atp4a to reduce inflammatory responses in a subject in need thereof.

[0085] Figs. 10A-10E represent exemplary histogram plots of data that illustrate reduced detrimental effects of AKI mice with the knockout of Atp4a: increased GFR (lh reperfusion, Fig. 10A); reduced TNFa (Fig. 10B) and IL-6 (Fig. IOC); GFR (24h reperfusion, Fig. 10D) and decreased neutrophil infiltration reflected by MPO (Fig. 10E). Wild type mice treated under the normal and ischemic induced AKI conditions were included as controls. Knockout mice under the normal conditions were also provided as controls.

Studies with pharmacologic inhibitors

[0086] In some other experiments, a unilateral ischemia animal model was used. In yet other experiments, a bilateral model of renal ischemia was examined. In some experiments, mice were pretreated with esomeprazole at various doses, such as 0.5 mg/mouse

(pretreatment 30 min prior to renal ischemia; Fig. 1 1 A). To study long term outcome, 24 hours reperfusion was also performed in some experiments before GFR analysis, from which a robust improvement of the GFR was found in mice treated with esomeprazole compared to non treated mice (Fig. 1 IB). In addition, a lower dose of esomeprazole (0.035mg/mouse), which is equivalent to 80 mg in humans was tested. Esomeprazole at the lower dose was administered by IP injection 90 min before ischemia exposure. It was observed that kidney function was much better in mice treated with esomeprazole (Fig. 11C). Studies in a bilateral model of renal ischemia confirmed the above findings (Fig. 1 ID).

[0087] Figs. 1 lA-1 ID represent exemplary histogram plots illustrating protection from ischemia by administration of Esomeprazole at an optimal dose and a lower dose in different models. Figs. 11 A- 1 IB represent histogram plots demonstrating affect on GFR (Fig. 11A, lh reperfusion; Fig. 1 IB, 24 h reperfusion) in the presence of Esomeprazole at 0.5mg/mouse prior to ischemia compared to untreated mice in a unilateral ischemia model. Fig. 1 1 C represent an exemplary histogram plot change of GFR (24 h reperfusion) as a result of Esomeprazole at 0.035mg/mouse in comparison with untreated mice in the unilateral ischemia model. Fig. 1 ID is a histogram plot illustrating change of GFR (24 h reperfusion) in the presence of Esomeprazole at 0.035mg/mouse compared to untreated mice in the bilateral ischemia model.

Study medication

[0088] In one exemplary method, subject in one study received either esomeprazole as an infusion (Nexium™, 80mg) or matching placebo as an infusion (e.g. physiological saline, a control). Esomeprazole is a commonly used agent for the treatment of dyspepsia. But until the instant application, it was not used for prevention or treatment of ischemia/reperfusion injury following renal transplantation or other event to reduce or prevent onset or progression of AKI. Pharmacokinetic

[0089] Proton pump inhibitors (PPIs) are the standard medication used in the treatment of dyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease (GORD/GERD), gastrinoma, gastritis/esophagitis and Zollinger-Ellison syndrome. PPIs are a popular and prevalent medication based on their proven efficacy and safety. Esomeprazole is the [S]- enantiomer of omeprazoel, a prototype of proton pump inhibitors.

[0090] All proton pump inhibitors are prodrugs that require activation in an acidic environment by converting to an active sulfenamide form. This sulfonamide reacts with a cysteine residue on the H+/K+-ATPase and inhibits the activity of the proton pump irreversibly as previously observed. PPIs produce a profound suppression of acid secretion that persists longer than their presence in the plasma (nearly complete suppression of acid secretion for up to 24 hours). Due to irreversible inhibition, resumption of H+/K+-ATPase action requires de novo synthesis of the pump. Comparisons of effects of this agent suggest esomeprazole inhibits acid secretion more effectively than other proton pump inhibitors at similar therapeutic doses.

[0091] Peak concentration after a standard oral dose is 0.5 to 2mg/ml where protein binding of all PPIs is 95% or greater. It is known that PPIs easily pass cell membranes. All PPI's undergo hepatic metabolization and elimination in urine (80%) and feces (20%) with a half life time of about 1.5 h or less.

[0092] PPIs are metabolized by two cytochrome P450 isoenzymes (see below). These PPI metabolites are excreted via the kidney. Subject with chronic kidney disease generally do not require adjustment of the standard dose. However, subjects with liver failure should be treated with lower doses.

Adverse effects of PPIs

[0093] PPIs are generally very well tolerated at standard doses. Even a fivefold increase in the AUC of PPIs appears to cause only negligible side effects. The risk of minor adverse effects (e.g. headache, nausea, disturbed bowl function, abdominal pain, rash) from PPIs is low (1-3%). Serious adverse effects (e.g. interstitial nephritis, hepatitis and visual disturbances) are rare as well and occurred after chronic PPI treatment. E.g. the mean duration of PPI therapy before developing interstitial nephritis was 11 weeks in one retrospective study. Another study demonstrated a treatment duration of about 10 days to 18 months in 15 patients who developed interstitial nephritis (previously presented). A systematic review article reported 64 cases with interstitial nephritis between 1970 and 2006 with a mean PPI treatment duration of 13 weeks. Pharmacogenomics

[0094] Proton pump inhibitors, PPIs, (e.g. omeprazole, esomeprazole, pantoprazole) are metabolized by cytochrome P450 isoenzyme 2C19 (CYP2C19). CYP2C19 is responsible for the major metabolism of PPIs, while CYP3A4 functions as an ancillary metabolic pathway when the main pathway is saturated. There are genetic differences that affect the activity of this enzyme and they are classified into three groups: homozygous extensive metabolizer (homEM), heterozygous extensive metabolizer (hetEM), and poor metabolizer (PM). Plasma PPI's are the lowest in homEM group and the highest in the PM group.

[0095] It has been demonstrated, that acid secretion in individuals with a poor metabolizer status of CYP2C19 who are undergoing an omeprazole therapy is therefore assumed to be more strongly inhibited than for those with the extensive metabolizer status. However, the standard recommended doses of PPIs take into account these differences, and most patients reach a sufficient degree of acid inhibition regardless of the variability in metabolism of this drug.

Drug interactions

[0096] To date only omeprazole has been found to interact with other drugs metabolized by CYP2C19. Clinically significant interactions generally do not occur but, awareness should be high in patients on warfarin, phenytoin, diazepam or carbamazepine medication. However, drug interaction is unlikely to happen after a single day treatment as presented herein.

Immunosuppressive drugs are metabolized via the CYP450 pathway as well. Chronic treatment with PPIs has been shown to increase tacrolimus blood levels by inhibiting its metabolism via CYP3A4/5.

Dosing

[0097] Standard doses of esomeprazole e.g. for the treatment of GERD is about 20- 40mg/day for up to 8 weeks depending on the disease severity. In the case of Zollinger- Ellison Syndrome, the doses can be safely increased up to lOOmg per day. Therefore, it is contemplated herein that a dose range to treat or prevent AKI in a subject can be about 10 to 150 mgs per dose or per day or other regimen.

Material and Methods

Mice generation and breeding

[0098] All animal protocols were in accordance with the United States Guidelines IACUC. All mice were housed in a 12-h-light-dark cycle and were used gender-, age- and weight-matched between 12 and 16 weeks. In transcript and pharmacological studies C57BL/6J mice obtained from Jackson Laboratories were used. Mice deficient in Atp4a on the C57BL/6J strain were generated, validated, and characterized.

Murine model for renal ischemia

[0099] Mice underwent right nephrectomy followed by left renal artery ischemia (30 minutes of ischemia) using a hanging weight system, as previously described. Briefly, a right nephrectomy is performed, and then the left kidney is carefully isolated and the left adrenal gland dissected away. The kidney is placed in a lucite cup and a 7-0 nylon suture is threaded under the renal artery. Weights are attached at both ends of the suture and ischemia is performed for indicated time points. Ischemia is confirmed by color change of the kidney from red to pale white. At the end of the designated ischemic time period, the weights are re- supported and reperfusion ensues. At the end of surgery, mice received 0.3 ml normal saline and were allowed to recover for 2 hours under a heating lamp for metabolic cage

investigations. Inulin clearance was performed one hour following reperfusion.

Assessment of Renal Function via Metabolic Cage Investigation

[00100] Mice were placed in metabolic cages (Techiplast) for urine collection 2 hours following renal ischemia for 24 hours. Thereafter plasma and urine creatinine were measured by HPLC as described below. From these data creatinine clearance was calculated. Kidneys were harvested immediately after 24 hours of reperfusion and were stored at -80°C until further analysis.

Inulin Clearance

[00101] Inulin clearance was measured 2 hours after renal ischemia (30 minutes) as described previously. Briefly mice were anesthetized using 50mg/kg IP pentobarbital.

Animals were then placed on a temperature-controlled operating table to keep rectal temperature at 37°C. The right jugular vein was cannulated for continuous infusion. Blood samples were taken via retroorbital vein plexus puncture. A catheter was placed in the urinary bladder for timed urine collection after removal of the right kidney. After surgery, mice received a bolus of 0.45% sodium chloride solution in an amount equal to 20% body weight. Continuous infusion was maintained at a rate of 800μl/h/25g body weight and FITC- labeled inulin (0.75g/100ml, Sigma) was added to the infusion for evaluation of whole kidney glomerular filtration rate (GFR) as described before. After stabilization of the animals for 20 minutes, 20 minute timed urine collections were performed. Blood was obtained in the middle of every urine collection period for measurement of FITC-inulin. Concentration of inulin in plasma and urine were performed by measurement of wavelength using a spectophotometer (Biotek Synergy 2) and GFR was calculated by standard formulas.

Renal Histology

[00102] Kidneys were excised and harvested 24 hours following 30 minutes of renal ischemia. Renal tissues were fixed in 4.5% buffered formalin, dehydrated, and embedded in paraffin. Sections (3μιη) were stained with hematoxylin and eosin (H&E). Examination and scoring of three representative sections of each kidney (n = 4-6 for each condition) were carried out blinded.

Transcriptional Analysis

[00103] Total RNA was isolated from mouse kidneys or human proximal tubule cells (HK-2) using the Trizol Reagent according to the manufacturer^'s instructions (Invitrogen). Therefore frozen tissue or HK-2 cell suspensions were homogenized in Trizol Reagent and chloroform. After spinning at 12,000xg for 15 minutes, the aqueous phase was removed and the RNA was precipitated with isopropanol. RNA was pelleted, washed with ethanol, treated with DNAse, and the concentration was quantified. The PCR reactions contained ΙμΜ sense and ΙμΜ antisense oligonucleotidase with SYBR Green (Bio-Rad). Each target sequence was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, 72°C for lmin. Quantification of transcript levels was measured by real-time reverse transcription (RT)-PCR (iCycler, Bio-Rad Laboratories Inc.).

Statistical Analysis

[00104] Renal injury score data are given as median and range, all other data are presented as mean ± SD. Renal injury was analyzed with the Kruskal-Wallis test, with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test. For all other outcomes, data were compared by two-factor ANOVA with Bonferroni's post test, or by Student's t test where appropriate. Data are expressed as mean ± SD. P<0.05 was considered statistically significant. For all statistical analysis GraphPad Prism 5.0 software for Windows XP was used.

All of the COMPOSITIONS and/or METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variation may be applied to the COMPOSITIONS and/or METHODS described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

What is claimed is:

1. A method for preventing an adverse kidney condition comprising: administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising one or more proton pump inhibitors, wherein the administration prevent onset of the adverse kidney conditions.

2. The method of claim 1, wherein the adverse kidney condition is acute kidney injury (AKI).

3. The method of claim 1 , wherein the proton pump inhibitors comprise substituted pyridyl methylsulfinyl benzimidazoles, omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole, lansoprazole, soraprazan, revaprazan, substituted imidazo-l,2a-pyridines, SCH-28080 or a combination thereof.

4. The method of claim 1, wherein the proton pump inhibitors comprise omeprazole, esomeprazole, and SCH-28080 or a combination thereof.

5. A method of preventing progression of an adverse kidney condition in a subject with a pre-existing renal condition comprising administering to the subject a

therapeutically effective amount of a pharmaceutical composition comprising one or more proton pump inhibitors, wherein the administration prevents progression of the kidney condition in the subject.

6. A method of treating acute kidney injury (AKI) in a subject comprising

administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more proton pump inhibitors, wherein the administration to the subject treats acute kidney injury in the subject.

7. The method of claim 6, wherein the proton pump inhibitors comprise omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole, lansoprazole, soraprazan, revaprazan and SCH-28080 or a combination thereof.

8. The method of claim 6, wherein the proton pump inhibitors comprise omeprazole, esomeprazole, and SCH-28080 or a combination thereof.

9. The method of claim 6, wherein the proton pump inhibitors are administered to the subject before, during or after an event.

10. A composition for preventing acute kidney injury (AKI) in a subject comprising, an agent or molecule that modulates the expression level or activity of ATP4a and one or more proton pump inhibitors.

1 1. The composition of claim 10, wherein the proton pump inhibitors comprise

omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole,

lansoprazole, soraprazan, revaprazan and SCH-28080 or a combination thereof.

12. The composition of claim 10, wherein the agent or molecule comprises anti-sense RNA, siRNA, antibodies, antibody fragments, small molecules or aptamers that bind to and inhibit ATP4a activity.

13. The composition of claim 10, wherein the proton pump inhibitor is omeprazole, esomeprazole, SCH-28080 or a combination thereof.

14. A method for treating a subject having a kidney condition comprising: administering a proton pump inhibitor to the subject when a sample from the subject has at least one of increased levels of one or more genes comprising ATP4A, Egln2, LGalS4, QPCTL, and NAPA, and reduced levels of one or more genes comprising WDFYl, BLVRB, C19, FXYDl and NNT compared to a control subject no having a kidney condition.

15. The composition of claim 10, wherein the proton pump inhibitors comprise

omeprazole, pantoprazole, esomeprazole, rabeprazole, dexlansoprazole,

lansoprazole, soraprazan, revaprazan and SCH-28080 or a combination thereof.

16. A method for diagnosing a kidney condition in a subject, comprising

a. obtaining one or more samples from the subject;

b. analyzing the sample for increased levels of one or more genes comprising ATP4A, Egln2, LGalS4, QPCTL, and NAPA, and for reduced levels of one or more genes comprising WDFYl, BLVRB, CI 9, FXYDl and NNT compared to a control sample; and c. diagnosing onset or presence of a kidney condition in the subject based on the genetic analysis of the sample.

17. The method of claim 16, further comprising administering to the subject, a proton pump inhibitor when one or more genes is increased or decreased compared to the control.

18. The method of claim 17, further comprising administering an anti-inflammatory agent to the subject.

19. The method of claim 17, further comprising administering an agent to reduce

expression or activity of one or more genes comprising ATP4A, Egln2, LGalS4, QPCTL, and NAPA in the subject.

20. The method of claim 17, further comprising administering an agent to increase

expression or activity of one or more of genes comprising WDFY1, BLVRB, CI 9, FXYD1 and NNT in the subject.