WO2018064112A1 - Treatment of acute kidney injury - Google Patents

Treatment of acute kidney injury Download PDF

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Publication number
WO2018064112A1
WO2018064112A1 PCT/US2017/053615 US2017053615W WO2018064112A1 WO 2018064112 A1 WO2018064112 A1 WO 2018064112A1 US 2017053615 W US2017053615 W US 2017053615W WO 2018064112 A1 WO2018064112 A1 WO 2018064112A1
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amino
arginase
dihydroxyboranyl
propyl
methyl
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French (fr)
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Alaa S. Awad
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Penn State Research Foundation
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Penn State Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • Acute kidney injury is a serious complication affecting >5-7% of hospitalized patients and is associated with a high mortality, morbidity, and increased healthcare costs (Bellomo et al., 2012, Lancet, 380(9843):756-766, Liangos et al., 2006, Clin J Am Soc Nephrol, 1(1):43-51, Star et al., 1998, Kidney Int, 54(6): 1817-1831, Bonventre et al., 2003, J Am Soc Nephrol, 14(8):2199-2210).
  • IRI is the major cause of AKI in native kidneys and in kidney allografts (Bonventre et al., 2003, J Am Soc Nephrol, 14(8):2199-2210, Rabb et al., 2002, Kidney Int, 61(6): 1935-1946, Rabb H et al., 2006, Nat Clin Pract Nephrol, 2(3): 124-125).
  • Ischemia and/or reperfusion initiate changes in renal blood flow, hypoxic cell death, ATP depletion, vascular endothelial cells, tubular epithelial cells and leukocytes that result in the loss of immune system homeostasis in the kidney (Thurman et al., 2006, The Journal of clinical investigation, 116(2):357-368, Li et al., 2007, J Immunol, 178(9):5899-5911, Kelly et al., 1996, The Journal of clinical investigation, 97(4): 1056-1063, Day et al., 2005, American journal of physiology, 288(4):F722-F731, Day et al., 2006, J Immunol, 176(5):3108-3114).
  • compositions and methods for treating AKI there is a need in the art for compositions and methods for treating AKI.
  • the present invention addresses this unmet need in the art.
  • Figure 1 depicts experimental results demonstrating increased Arginase 2 expression and activity in renal IRI. Results are means ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01 vs. zero hrs.
  • Figure 2 depicts experimental results demonstrating Arginase 2 deficiency or arginase inhibition improves kidney function after IRI. Results are means ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01 vs. sham, #p ⁇ 0.05; ##p ⁇ 0.01 vs. vehicle-treated IRI. .
  • Figure 3 depicts experimental results demonstrating arginase inhibition reduces kidney damage after IRI. Results are means ⁇ SEM. *p ⁇ 0.01; **p ⁇ 0.001 vs. sham, #p ⁇ 0.01; ##p ⁇ 0.001 vs. vehicle-treated IRI.
  • Figure 4 depicts experimental results demonstrating arginase inhibition reduces kidney inflammatory and apoptotic cells after IRI. Results are means ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.001 vs. sham, #p ⁇ 0.001 vs. vehicle-treated IRI.
  • Figure 5 depicts experimental results demonstrating arginase inhibition reduces kidney inflammatory cytokines after IRI. Results are means ⁇ SEM. *p ⁇ 0.001; vs. sham, #p ⁇ 0.05 vs. vehicle-treated IRI.
  • Figure 6 depicts experimental results demonstrating arginase inhibition increases kidney NO after IRI. Results are means ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 vs. sham, #p ⁇ 0.05 vs. vehicle-treated IRI.
  • Figure 7 depicts experimental results demonstrating Arginase 2 deficiency or arginase inhibition improves kidney oxidative stress after IRI. Results are means ⁇ SEM. *p ⁇ 0.05 vs. sham, #p ⁇ 0.05 vs. vehicle-treated IRI.
  • Figure 8 depicts experimental results demonstrating Arginase inhibition increases kidney PGC- ⁇ after IRI. Results are means ⁇ SEM. *p ⁇ 0.05 vs. sham, #p ⁇ 0.05 vs. vehicle-treated IRI.
  • the present invention provides a composition for treating acute kidney injury.
  • the composition comprises an arginase inhibitor.
  • the arginase inhibitor inhibits Arginase 1, Arginase 2, or a combination thereof.
  • the arginase inhibitor is one or more selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an
  • oligonucleotide a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule and any combination thereof.
  • the arginase inhibitor is a small molecule.
  • the small molecule is selected from the group consisting of BEC,
  • the arginase inhibitor comprises an oligonucleotide specific for Arginase.
  • the oligonucleotide is specific for Arginase 1 or Arginase 2.
  • the arginase inhibitor comprises an anti-Arginase antibody.
  • the anti-Arginase antibody is an anti-Arginase 1 antibody or an anti-Arginase 2 antibody.
  • the acute kidney injury comprises ischemia reperfusion injury.
  • the present invention provides a method for treating acute kidney injury in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one arginase inhibitor.
  • the at least one arginase inhibitor is selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an
  • the arginase inhibitor inhibits Arginase 1, Arginase 2, or a combination thereof.
  • the arginase inhibitor is a small molecule.
  • the small molecule is selected from the group consisting of BEC,
  • the arginase inhibitor comprises an oligonucleotide specific for Arginase.
  • the oligonucleotide is specific for Arginase 1 or Arginase 2.
  • the arginase inhibitor comprises an anti-Arginase antibody.
  • the anti-Arginase antibody is an anti-Arginase 1 antibody or an anti-Arginase 2 antibody.
  • the composition is administered by nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal, epidural injection, oral or any combination thereof.
  • acute kidney injury comprises ischemia reperfusion injury.
  • the subject a mammal. In one embodiment, the subject is a human.
  • the composition is administered before kidney allograft. In one embodiment, the composition is administered after renal IRI. In one embodiment, the method further comprises administering a second therapeutic agent.
  • the second therapeutic is selected from the group consisting of calcium, glucose, sodium polystyrene sulfonate, Kayexalate, Kionex or any combination thereof. In one embodiment, the second therapeutic agent is acute dialysis.
  • the present invention relates to compositions and methods for inhibiting arginase.
  • the invention is based, in part, on the unexpected finding that inhibition of arginase provided treatment and prevention to AKI.
  • the composition of the invention comprises an inhibitor of arginase.
  • the inhibitor of arginase inhibits the expression, activity, or both of arginase.
  • the method of the present invention comprises treating and preventing AKI. In one embodiment, the method comprises administering to a subject an effective amount of a composition comprising an inhibitor of arginase.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • AKI acute kidney injury
  • SCr serum creatinine
  • GFR glomerular filtration rate
  • ischemia reperfusion injury refers to renal injury due to one or more identified occurrences of renal ischemia or ischemia and reperfusion and/or toxemia and/or hypoperfusion or hemorrhage leads to hypoperfusion.
  • the term ischemic kidney injury may be used interchangeably herein. Ischemia reperfusion injury can be identified by clinicians, for example by recognizing ischemic conditions or by reference to certain intrinsic renal causes of acute kidney injury.
  • an "arginase inhibitor” refers to any agent capable of inhibiting the function, expression, activity or combinations thereof, of Arginase 2, Arginase 1, or both Arginase 1 and Arginase 2 and/or any agent capable of increasing levels of arginase's substrate, arginine and/or citrulline.
  • the terms "Arginase,” “Arginase 1 " and “Arginase 2,” are inclusive of all species, including human.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of a disease or disorder, for the purpose of diminishing or eliminating those signs or symptoms.
  • treating a disease or disorder means reducing the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a patient.
  • Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • a "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
  • a "coding region" of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
  • the coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
  • “Complementary” as used herein to refer to a nucleic acid refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%), or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • DNA as used herein is defined as deoxyribonucleic acid.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein,
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • fusion polypeptide refers to a chimeric protein containing a protein of interest (e.g., luciferase) joined to a heterologous sequence (e.g., a non- luciferase amino acid or protein).
  • a protein of interest e.g., luciferase
  • a heterologous sequence e.g., a non- luciferase amino acid or protein
  • homology refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in its normal context in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated when used in relation to a nucleic acid, as in “isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state they exist in nature.
  • isolated nucleic acid e.g., DNA and RNA
  • a given DNA sequence e.g., a gene
  • RNA sequences e.g., a specific mRNA sequence encoding a specific protein
  • isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • the isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand (i.e., the
  • oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
  • oligonucleotide specific for refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
  • oligonucleotide As used herein, the terms “oligonucleotide,” “siRNA,” and “antisense oligonucleotide” are used interchangeably throughout the specification and include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate,
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • isolated when used in relation to a polypeptide, as in "isolated protein” or “isolated polypeptide” refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated polypeptide is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state they exist in nature.
  • nucleic acid is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases
  • nucleic acid typically refers to large polynucleotides.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • Antisense refers particularly to the nucleic acid sequence of the non- coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • expression cassette is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter/regulatory sequences necessary for transcription and, optionally, translation of the coding sequence.
  • operably linked refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of sequences encoding amino acids in such a manner that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in an inducible manner.
  • an “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds to the promoter is present.
  • a “constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • nucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • polypeptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
  • polypeptides derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • peptidomimetic is a compound containing non- peptidic structural elements that is capable of mimicking the biological action of a parent peptide.
  • a peptidomimetic may or may not comprise peptide bonds.
  • RNA as used herein is defined as ribonucleic acid.
  • Recombinant polynucleotide refers to a polynucleotide having sequences that are not naturally joined together.
  • An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • recombinant polypeptide as used herein is defined as a polypeptide produced by using recombinant DNA methods.
  • conjugated refers to covalent attachment of one molecule to a second molecule.
  • transdominant negative mutant gene refers to a gene encoding a polypeptide or protein product that prevents other copies of the same gene or gene product, which have not been mutated (i.e., which have the wild-type sequence) from functioning properly (e.g., by inhibiting wild type protein function).
  • the product of a transdominant negative mutant gene is referred to herein as "dominant negative” or "DN” (e.g., a dominant negative protein, or a DN protein).
  • inhibitor means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
  • inflammatory means relating to inflammation.
  • inflammation refers to the process by which vascular tissues responds to harmful stimuli, such as pathogens, damaged cells, or irritants. "Inflammation includes, but is not limited to secretion of and response to inflammatory factors, e.g., inflammatory cytokines.
  • Variant is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear
  • vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • subject refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • patient, subject or individual is a human.
  • treatment is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder contemplated herein, a sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a disease or disorder contemplated herein, at least one sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein.
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention is based on novel therapeutic modalities using
  • Arginase inhibitors in the treatment of acute kidney injury provides methods and compositions for treating or preventing AKI.
  • the present invention provides a composition for treating and preventing AKI in a subject.
  • the present invention provides a composition for treating AKI, the composition comprising an inhibitor of arginase.
  • the arginase is Arginase 1 or Arginase 2.
  • the inhibitor of Arginase is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule. .
  • the inhibitor of Arginase 1 is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule.
  • the inhibitor of Arginase 2 is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule.
  • the arginase inhibitor can include any agent capable of inhibiting the function, expression, activity or combinations thereof Arginase.
  • Arginase is Arginase 1, Arginase 2, or both Arginase 1 and Arginase 2.
  • the arginase inhibitor can also inhibit Arginase 1, Arginase 2 or both.
  • the invention provides a method for treating and preventing AKI in a subject.
  • the method comprises administering to a subject an effective amount of an arginase inhibitor, an analog thereof, or a combination thereof.
  • the method comprises administering to a subject an effective amount of an Arginase inhibitor, wherein the inhibitor is Arginase 1 or Arginase 2.
  • the method further comprises administering a second therapeutic agent.
  • the second therapeutic agent is an inhibitor of AKI.
  • the subject is a mammal. In another embodiment, the mammal is a human. Small molecule inhibitors
  • the inhibitor is a small molecule.
  • a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.
  • a small molecule inhibitor of the invention comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.
  • Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries.
  • the method may use a variety of techniques well- known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.
  • an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles.
  • the shape and rigidity of the core determines the orientation of the building blocks in shape space.
  • the libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries") or synthesized with less structural bias using flexible cores.
  • the small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted and it is understood that the invention embraces all salts and solvates of the inhibitors depicted here, as well as the non-salt and non-solvate form of the inhibitors, as is well understood by the skilled artisan.
  • the salts of the inhibitors of the invention are pharmaceutically acceptable salts.
  • tautomeric forms may be present for any of the inhibitors described herein, each and every tautomeric form is intended to be included in the present invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2- pyridone tautomer is also intended.
  • the invention also includes any or all of the stereochemical forms, including any enantiomeric or diasteriomeric forms of the inhibitors described.
  • the recitation of the structure or name herein is intended to embrace all possible stereoisomers of inhibitors depicted. All forms of the inhibitors are also embraced by the invention, such as crystalline or non-crystalline forms of the inhibitors.
  • Compositions comprising an inhibitor of the invention are also intended, such as a composition of substantially pure inhibitor, including a specific stereochemical form thereof, or a composition comprising mixtures of inhibitors of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture.
  • the small molecule inhibitor of the invention comprises an analog or derivative of an inhibitor described herein.
  • the small molecules described herein are candidates for derivatization.
  • the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development.
  • new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.
  • small molecule inhibitors described herein are derivatized/analoged as is well known in the art of combinatorial and medicinal chemistry.
  • the analogs or derivatives can be prepared by adding and/or substituting functional groups at various locations.
  • the small molecules described herein can be converted into derivatives/analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs.
  • the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms.
  • the ring groups can be changed so as to have a different number of atoms in the ring and/or to include hetero atoms.
  • aromatics can be converted to cyclic rings, and vice versa.
  • the rings may be from 5-7 atoms, and may be homocycles or heterocycles.
  • an analog is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions.
  • an analog can be a structure having a structure similar to that of the small molecule inhibitors described herein or can be based on a scaffold of a small molecule inhibitor described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically.
  • An analog or derivative of any of a small molecule inhibitor in accordance with the present invention can be used to treat AKI or an AKI -related disease or disorder.
  • the small molecule inhibitors described herein can independently be derivatized/analoged by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative/analog can be used.
  • the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic having one or more hetero atoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo-substituted aliphatics, and the like.
  • any ring group on a compound can be derivatized to increase and/or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms.
  • the arginase inhibitor is selected from 2(S)amino- 6- boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L-cysteine (BEC), ⁇ - ⁇ -L-arginine (NOHA), ⁇ -hydroxynor-L-arginine (nor-NOHA), a- difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl- Lysine, L-Lysine, N5-(benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)- L-thiocitrulline tert-butyl ester, N5-[N-(benzyloxycarbonyl)-N'- (methoxycarbonylmethoxy)amidino]-N2-(tertbutoxycarbonyl)-L-or
  • the arginase inhibitor is S-(2-boronoethyl)-L-cysteine (BEC). In one embodiment, the arginase inhibitor is l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32. In one embodiment, arginase inhibitors include those described in Ivanenkov et al. (2014), the contents of which are incorporated herein by reference. Nucleic acid inhibitors
  • the invention includes an isolated nucleic acid.
  • the inhibitor is an siRNA, miRNA, or antisense molecule, which inhibits Arginase.
  • the siRNA, miRNA, or antisense molecule inhibits Arginase 1.
  • the siRNA, miRNA, or antisense molecule inhibits Arginase 2.
  • the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the nucleic acid.
  • the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012,
  • Arginase can be inhibited by way of inactivating and/or sequestering Arginase.
  • Arginase 1 can be inhibited by way of inactivating and/or sequestering Arginase 1.
  • inhibiting the activity of Arginase 1 can be accomplished by using a transdominant negative mutant.
  • Arginase 2 can be inhibited by way of inactivating and/or sequestering Arginase 2.
  • inhibiting the activity of Arginase 2 can be accomplished by using a transdominant negative mutant.
  • siRNA is used to decrease the level of Arginase protein. In one embodiment, siRNA is used to decrease the level of Arginase 1 protein. In one embodiment, siRNA is used to decrease the level of Arginase 2 protein.
  • RNA interference is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer.
  • siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process.
  • RISC RNA-induced silencing complex
  • Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA.
  • the bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R.
  • RNA Interference RNA Interference
  • RNAi RNA Interference
  • C/T C/T content at the termini
  • Tm nucleotide content of the 3' overhang
  • the present invention also includes methods of decreasing levels of Arginase 1 or Arginase 2 using RNAi technology.
  • the invention includes a vector comprising an siRNA or antisense polynucleotide.
  • the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide, wherein the target polypeptide is
  • the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor.
  • shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target.
  • the encoded shRNA is expressed by a cell, and is then processed into siRNA.
  • the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.
  • siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein.
  • at least one module in each promoter functions to position the start site for RNA synthesis.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.
  • the invention relates to a vector, comprising the nucleotide sequence of the invention or the construct of the invention.
  • the vector of the invention is an expression vector.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector.
  • Prokaryote- and/or eukaryote- vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.)
  • the vector in which the nucleic acid sequence is introduced can be a plasmid which is or is not integrated in the genome of a host cell when it is introduced in the cell.
  • Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.
  • the vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012).
  • the vector is a vector useful for transforming animal cells.
  • the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of invention, described elsewhere herein.
  • a promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012).
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • the recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells.
  • Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol
  • the selectable markers may be introduced on a separate vector from the nucleic acid of interest.
  • the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett.
  • Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.
  • an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit Arginase protein expression.
  • the antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Arginase 1.
  • an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit Arginase 2 protein expression.
  • the antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Arginase 2.
  • Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).
  • Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule thereby inhibiting the translation of genes.
  • antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5, 190,931.
  • antisense molecules of the invention may be made synthetically and then provided to the cell.
  • Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell.
  • Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).
  • a ribozyme is used to inhibit
  • Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence encoding Arginase.
  • Ribozymes targeting Arginase may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.
  • Ribozymes targeting Arginase 1 or Arginase 2 may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.
  • the inhibitor of Arginase may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene.
  • the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA.
  • the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
  • the inhibitor of Arginase 1 may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase 1, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene.
  • the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA.
  • the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
  • the inhibitor of Arginase 2 may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase 2, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene.
  • the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA.
  • the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
  • the arginase inhibitor is an oligonucleotide specific for Arginase. In one embodiment, the arginase inhibitor is an oligonucleotide specific for Arginase 1. In one embodiment, the invention provides a method for treating and preventing AKI, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase 2. Polypeptide inhibitors
  • the invention includes an isolated peptide inhibitor that inhibits Arginase.
  • the peptide inhibitor of the invention inhibits Arginase directly by binding to Arginase thereby preventing the normal functional activity of Arginase.
  • the peptide inhibitor of the invention inhibits Arginase 1 by competing with endogenous Arginase 1.
  • the peptide inhibitor of the invention inhibits the activity of Arginase 1 by acting as a transdominant negative mutant.
  • the peptide inhibitor of the invention inhibits Arginase 2 by competing with endogenous Arginase 2. In yet another embodiment, the peptide inhibitor of the invention inhibits the activity of Arginase 2 by acting as a transdominant negative mutant.
  • the variants of the polypeptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • the fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the invention also contemplates an inhibitor of Arginase comprising an antibody, or antibody fragment, specific for Arginase. That is, the antibody to Arginase can provide a beneficial effect.
  • the invention also contemplates an inhibitor of Arginase 1 comprising an antibody, or antibody fragment, specific for Arginase 1. That is, the antibody to Arginase 1 can provide a beneficial effect.
  • the arginase inhibitor comprises an antibody, or antibody fragment, specific for Arginase 1.
  • the arginase inhibitor comprises an antibody, or antibody fragment, specific for Arginase 2.
  • the antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab) 2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras may be prepared using methods known to those skilled in the art.
  • Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest.
  • the polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired.
  • Suitable carriers that may be chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin.
  • the coupled polypeptide may then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • the composition of the present invention comprises a combination of an Arginase inhibitor and second therapeutic agent.
  • the second therapeutic agents include, but are not limited to calcium, glucose or sodium polystyrene sulfonate, Kayexalate, Kionex.
  • the second therapeutic agent may also be acute dialysis.
  • a composition comprising a combination of inhibitors described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual inhibitor. In other embodiments, a composition comprising a combination of inhibitors described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual inhibitor.
  • a composition comprising a combination of inhibitors comprises individual inhibitors in any suitable ratio.
  • the composition comprises a 1 : 1 ratio of two individual inhibitors.
  • the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • the present invention also provides methods of treating or preventing AKI in a subject.
  • the AKI is treated or prevented by inhibiting arginases.
  • the invention provides a method for treating AKI, wherein Arginase is inhibited.
  • the invention provides a method for treating AKI, wherein Arginase 1 is inhibited.
  • the invention provides a method for treating AKI wherein Arginase 2 is inhibited.
  • the invention provides a method for treating AKI, by inhibiting Arginase, wherein the arginase inhibitor is S-(2- boronoethyl)-L-cysteine (BEC).
  • the invention is not limited to treatment of AKI that is already established.
  • the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of AKI do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing AKI, in that a composition, as discussed previously elsewhere herein, can be
  • the method of preventing AKI comprises administering a
  • the method of preventing AKI comprises administering a composition of the invention a kidney ex vivo prior to kidney allograft. In one embodiment, the method of preventing AKI comprises administering a composition of the invention to a patient and to a kidney ex vivo prior to kidney allograft.
  • the prevention of AKI encompasses administering to a subject a composition as a preventative measure against the development of, or progression of AKI.
  • methods of modulating the level or activity of a gene, or gene product encompass a wide plethora of techniques for modulating not only the level and activity of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both.
  • the invention provides a method for treating and preventing AKI, by inhibiting Arginase, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase.
  • the invention provides a method for treating and preventing AKI, by inhibiting Arginase, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase 1. In one embodiment, the invention provides a method for treating and preventing AKI, wherein the Arginase inhibitor is an oligonucleotide that specifically bind Arginase 2
  • the invention encompasses administration of an inhibitor of Arginase.
  • an inhibitor of Arginase To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate modulator composition to a subject.
  • the present invention is not limited to any particular method of administration or treatment regimen.
  • the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase. In one embodiment, the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase 1. In one embodiment, the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase 2.
  • inhibitors of the invention can be administered singly or in any combination. Further, the inhibitors of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other.
  • inhibitors of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other.
  • compositions of the invention can be used to prevent or treat AKI, and that an inhibitor composition can be used alone or in any combination with another modulator to effect a therapeutic result.
  • any of the inhibitor compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with AKI.
  • the invention provides a method for treating AKI, by administering an Arginase inhibitor, wherein the arginase inhibitor is Arginase 1, Arginase 2, S-(2-boronoethyl)-L-cysteine (BEC) or any combination thereof.
  • the methods of modulating arginase described herein can also treat one or more diseases or disorders associated with abnormal arginase expression or levels in a patient as compared to healthy normal controls.
  • Diseases or disorders associated with abnormal arginase expression or levels in a patient as compared to healthy normal controls can include renal diseases or disorders, renal injury, diabetic nephropathy, angina, congestive heart failure, cancer, azotemia, albuminuria, nephritis, renal failure or cardiovascular diseases.
  • the modulation of arginase can be used to treat one or more diseases or disorders associated with abnormal arginase expression or levels in a patient.
  • the invention provides a method for treating and preventing AKI, wherein Arginase modulated.
  • the invention provides a method for treating and preventing AKI, wherein Arginase 1, Arginase 2 or any combination thereof is modulated.
  • the invention includes a method comprising administering a combination of inhibitors described herein.
  • the method has an additive effect, wherein the overall effect of the administering a combination of inhibitors is approximately equal to the sum of the effects of
  • the method has a synergistic effect, wherein the overall effect of administering a combination of inhibitors is greater than the sum of the effects of administering each individual inhibitor.
  • the method comprises administering a combination of inhibitors in any suitable ratio.
  • the method comprises administering two individual inhibitors at a 1 : 1 ratio.
  • the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • the invention provides compositions for treating AKI in a subject.
  • the invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention.
  • a pharmaceutical composition may consist of at least one modulator composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one modulator composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • the compound or conjugate of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • compositions useful for practicing the methods of the invention may be administered to deliver a dose of between
  • the pharmaceutically acceptable salt 1 ng/kg/day and 100 mg/kg/day.
  • the pharmaceutically acceptable salt 1 ng/kg/day and 100 mg/kg/day.
  • compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • Methods of introduction include but are not limited to nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the pharmaceutical compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of
  • Such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory
  • a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • compositions are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such
  • compositions are generally suitable for administration to animals of all sorts.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • the pharmaceutically acceptable carrier is not DMSO alone.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents;
  • sweetening agents such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • additional ingredients that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack
  • the composition of the invention may comprise a preservative from about 0.005% to 2.0%) by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • a particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
  • the composition preferably includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound.
  • Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent is present in an amount of from 0.01%) to 0.5% by weight by total weight of the composition.
  • Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01%> to 0.20% and more preferably in the range of 0.02% to 0.10%) by weight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin, and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water, and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present invention may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be
  • an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the compound in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease in a subject.
  • compositions of the invention are administered to the subject in dosages that range from one to five times per day or more.
  • compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
  • the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.
  • Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.
  • the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.
  • the term "container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical
  • composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.
  • the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.
  • Example 1 The Problem of Acute Kidney Injury
  • mice Male C57B1/6J wild type (WT) and Arginase T 1' mice (6-8 weeks old) were subjected to bilateral renal ischemia for 28 minutes followed by reperfusion for 24 hours, which produces a large increase in plasma creatinine and BUN as indicative of kidney injury in (WT) mice ( Figure 1).
  • BEC (17 mg/kg) or vehicle was administered once intraperitoneally 18 hours before ischemia as described previously (Gao et al., 2014, J Clin Invest, 124(11):4989-5001, Awad et al., 2006, Am J Physiol Renal Physiol, 290(6):F1516-F1524, Gao et al., 2013, Am J Physiol Renal Physiol, 304(5):F515-F521). Renal function, histology, kidney inflammatory cell recruitments, oxidative stress, and NO was then determined.
  • Arginase 2 mRNA expression ( Figure 1) increased by 6 hours, and peaked at 48 hours, while Arginase 2 protein expression (Figure 1) demonstrated sustained increases after 24 hours after renal IRI, paralleling the increase in kidney arginase activity ( Figure 1).
  • the transient increase in plasma arginase activity is due to release of Arginase 2 from damaged renal cells during IR.
  • an increase in renal Arginase 1 mRNA expression ( Figure 1) at 24-48 h was observed, the Arginase 1 protein ( Figure 1) was undetectable by Western blot post IRI. This data confirmed previous results of Arginase 2 but not Arginase 1 expression in the kidney (Morris et al., 2011, Diabetes, 60(11):3015- 3022).
  • WT and Arginase 2 ⁇ ' ⁇ mice were subjected to bilateral renal ischemia for 28 minutes followed by reperfusion for 24 hours (Figure 2). WT mice displayed a significant increase in plasma creatinine and BUN after renal IRI. In contrast, the increased in plasma creatinine and BUN were significantly reduced in Arginase 2 '1' mice.
  • Periodic acid-Schiff (PAS) staining of kidney sections showed severe histologic kidney damage in vehicle-treated compared to sham mice after IRI. Inhibition of arginases after IRI exhibited significantly reduced histologic kidney damage, as shown by less cast formation, preservation of brush border membranes, less sloughing of epithelial compared to vehicle-treated IRI mice. Similarly, kidney injury marker-1 (KIM-1) mRNA expression is elevated in vehicle-treated IRI mice; an effect significantly reduced using BEC treatment.
  • KIM-1 kidney injury marker-1
  • Cleaved caspase-3 an early marker of cells undergoing apoptosis, staining of kidney sections ( Figure 4) showed no difference in the number of apoptotic cells between vehicle-treated or BEC-treated sham mice, and apoptotic cells were only observed rarely. In contrast, vehicle-treated mice showed increased number of apoptotic cells compared to BEC-treated mice 24 h after renal IRI.

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Abstract

The present invention provides compositions and methods comprising novel arginase inhibitors to treat and prevent Acute Kidney Injury.

Description

TITLE OF THE INVENTION
Treatment of Acute Kidney Injury
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
This invention was made with government support under R01 DK 094930 awarded by the National Institute of Health. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is entitled to priority to U.S. Provisional application No. 62/400,351, filed September 27, 2016, which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
Novel therapeutic interventions for preventing or attenuating renal tissue injury following ischemia reperfusion injury (TRI) remain a focus of significant interest. Acute kidney injury (AKI) is a serious complication affecting >5-7% of hospitalized patients and is associated with a high mortality, morbidity, and increased healthcare costs (Bellomo et al., 2012, Lancet, 380(9843):756-766, Liangos et al., 2006, Clin J Am Soc Nephrol, 1(1):43-51, Star et al., 1998, Kidney Int, 54(6): 1817-1831, Bonventre et al., 2003, J Am Soc Nephrol, 14(8):2199-2210). Apart from nephrotoxins, IRI is the major cause of AKI in native kidneys and in kidney allografts (Bonventre et al., 2003, J Am Soc Nephrol, 14(8):2199-2210, Rabb et al., 2002, Kidney Int, 61(6): 1935-1946, Rabb H et al., 2006, Nat Clin Pract Nephrol, 2(3): 124-125). Ischemia and/or reperfusion initiate changes in renal blood flow, hypoxic cell death, ATP depletion, vascular endothelial cells, tubular epithelial cells and leukocytes that result in the loss of immune system homeostasis in the kidney (Thurman et al., 2006, The Journal of clinical investigation, 116(2):357-368, Li et al., 2007, J Immunol, 178(9):5899-5911, Kelly et al., 1996, The Journal of clinical investigation, 97(4): 1056-1063, Day et al., 2005, American journal of physiology, 288(4):F722-F731, Day et al., 2006, J Immunol, 176(5):3108-3114). One of the early events in renal IRI is activation of the endothelium leading to an increase in vascular permeability (Sutton et al., 2003, American journal of physiology, 285(2):F191- F198) which promotes extravasation of leukocytes into the kidney. Currently, there are no definitive therapeutic or protective approaches available for AKI.
Thus, there is a need in the art for compositions and methods for treating AKI. The present invention addresses this unmet need in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Figure 1 depicts experimental results demonstrating increased Arginase 2 expression and activity in renal IRI. Results are means ± SEM. *p<0.05; **p<0.01 vs. zero hrs.
Figure 2 depicts experimental results demonstrating Arginase 2 deficiency or arginase inhibition improves kidney function after IRI. Results are means ± SEM. *p<0.05; **p<0.01 vs. sham, #p<0.05; ##p<0.01 vs. vehicle-treated IRI. .
Figure 3 depicts experimental results demonstrating arginase inhibition reduces kidney damage after IRI. Results are means ± SEM. *p<0.01; **p<0.001 vs. sham, #p<0.01; ##p<0.001 vs. vehicle-treated IRI.
Figure 4 depicts experimental results demonstrating arginase inhibition reduces kidney inflammatory and apoptotic cells after IRI. Results are means ± SEM. *p<0.05; **p<0.001 vs. sham, #p<0.001 vs. vehicle-treated IRI.
Figure 5 depicts experimental results demonstrating arginase inhibition reduces kidney inflammatory cytokines after IRI. Results are means ± SEM. *p<0.001; vs. sham, #p<0.05 vs. vehicle-treated IRI. Figure 6 depicts experimental results demonstrating arginase inhibition increases kidney NO after IRI. Results are means ± SEM. *p<0.05, **p<0.01 vs. sham, #p<0.05 vs. vehicle-treated IRI.
Figure 7 depicts experimental results demonstrating Arginase 2 deficiency or arginase inhibition improves kidney oxidative stress after IRI. Results are means ± SEM. *p<0.05 vs. sham, #p<0.05 vs. vehicle-treated IRI.
Figure 8 depicts experimental results demonstrating Arginase inhibition increases kidney PGC-Ια after IRI. Results are means ± SEM. *p<0.05 vs. sham, #p<0.05 vs. vehicle-treated IRI.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a composition for treating acute kidney injury. In one embodiment, the composition comprises an arginase inhibitor. In one embodiment, the arginase inhibitor inhibits Arginase 1, Arginase 2, or a combination thereof.
In one embodiment, the arginase inhibitor is one or more selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an
oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule and any combination thereof.
In one embodiment, the arginase inhibitor is a small molecule. In one embodiment, the small molecule is selected from the group consisting of BEC,
2(S)amino- 6-boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L- cysteine (BEC), ΝΩ-ΟΗ-L-arginine (NOHA), ΝΩ-hydroxynor-L-arginine (nor-NOHA), a-difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl- Lysine, L-Lysine, N5-(benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)-L-thiocitrulline tert- butyl ester, N5-[N-(benzyloxycarbonyl)-N' -(methoxycarbonylmethoxy)amidino]-N2- (tertbutoxycarbonyl)-L-ornithine tert-butyl ester, 6-(dihydroxyboranyl)-2-(3- phenoxypropyl)norleucine hydrochloride, (6-(dihydroxyboranyl)-2-(hydroxymethyl)-L- norleucine, 2-amino-6-(dihydroxyboryl)-2-[3-(4-piperidinyl) propyl ]hexanoic acid, 2-[4- (dihydroxyboranyl) butyl] lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-L-yl)ethyl]- lnorleucine dihydrochloride, (2S,3S)-3-amino-2-[3-(dihydroxyboranyl) propyl]tetrahydrofuran-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminocyclopentyl)-4-[3- (dihydroxyboranyl) propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[3-(4- carboxyphenyl) propyl]-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminoethyl)-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l,3'-bipyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l-[2-(pyrrolidin-l- yl)ethyl]pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[2-(benzylamino)ethyl]-4-[3- (dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[(7-chloro- l,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboranyl) propyl] pyrrolidine- 3-carboxylic acid, (2-[3-[4-(3,4-dichlorobenzyl)piperazin-l-yl]propyl]-6- (dihydroxyboranyl)norleucine, 6-(dihydroxyboranyl)-2-(3-[4-[4-(methylsulfonyl)benzyl] piperazin-l-yl]propyl) norleucine, (2S)-2-amino-6-(dihydroxyboryl)-2-[cis-3-[[[4'- (trifluoromethyl)biphenyl-3-yl]methyl]amino]cyclobutyl] hexanoic acid, (2S)-2-amino-6- (dihydroxyboryl)-2-[cis-3-[[(4-fluoro-l-naphthyl)methyl]amino]cyclobutyl]hexanoic acid, (2S)-2-amino-2-[cis-3-[[(3',4'-dichlorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, (2S)-2-amino-2-[cis-3-[[(4'-chlorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[[4'-(trifluoromethyl)biphenyl-4-yl]methyl]amino]cyclobutyl] hexanoic acid), (2 S)-2-amino-2- [ci s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid), (2S)-2-amino-2-[cis-3- [[(2,2'-difluoro-5'-methylbiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32 or any combination thereof.
In one embodiment, the arginase inhibitor comprises an oligonucleotide specific for Arginase. In one embodiment, the oligonucleotide is specific for Arginase 1 or Arginase 2.
In one embodiment, the arginase inhibitor comprises an anti-Arginase antibody. In one embodiment, the anti-Arginase antibody is an anti-Arginase 1 antibody or an anti-Arginase 2 antibody.
In one embodiment, the acute kidney injury comprises ischemia reperfusion injury. The present invention provides a method for treating acute kidney injury in a subject. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one arginase inhibitor. In one embodiment, the at least one arginase inhibitor is selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an
oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule and any combination thereof. In one embodiment, the arginase inhibitor inhibits Arginase 1, Arginase 2, or a combination thereof.
In one embodiment, the arginase inhibitor is a small molecule. In one embodiment, the small molecule is selected from the group consisting of BEC,
2(S)amino- 6-boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L- cysteine (BEC), ΝΩ-ΟΗ-L-arginine (NOHA), ΝΩ-hydroxynor-L-arginine (nor-NOHA), a-difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl- Lysine, L-Lysine, N5-(benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)-L-thiocitrulline tert- butyl ester, N5-[N-(benzyloxycarbonyl)-N' -(methoxycarbonylmethoxy)amidino]-N2- (tertbutoxycarbonyl)-L-ornithine tert-butyl ester, 6-(dihydroxyboranyl)-2-(3- phenoxypropyl)norleucine hydrochloride, (6-(dihydroxyboranyl)-2-(hydroxymethyl)-L- norleucine, 2-amino-6-(dihydroxyboryl)-2-[3-(4-piperidinyl) propyl jhexanoic acid, 2-[4- (dihydroxyboranyl) butyl] lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-L-yl)ethylj- lnorleucine dihydrochloride, (2S,3S)-3-amino-2-[3-(dihydroxyboranyl)
propyl]tetrahydrofuran-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminocyclopentyl)-4-[3- (dihydroxyboranyl) propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[3-(4- carboxyphenyl) propyl]-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminoethyl)-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l,3'-bipyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l-[2-(pyrrolidin-l- yl)ethyl]pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[2-(benzylamino)ethyl]-4-[3- (dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[(7-chloro- l,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboranyl) propyl] pyrrolidine- 3-carboxylic acid, (2-[3-[4-(3,4-dichlorobenzyl)piperazin-l-yl]propyl]-6- (dihydroxyboranyl)norleucine, 6-(dihydroxyboranyl)-2-(3-[4-[4-(methylsulfonyl)benzyl] piperazin-l-yl]propyl) norleucine, (2S)-2-amino-6-(dihydroxyboryl)-2-[cis-3-[[[4'- (trifluoromethyl)biphenyl-3-yl]methyl]amino]cyclobutyl] hexanoic acid, (2S)-2-amino-6- (dihydroxyboryl)-2-[cis-3-[[(4-fluoro-l-naphthyl)methyl]amino]cyclobutyl]hexanoic acid, (2S)-2-amino-2-[cis-3-[[(3',4'-dichlorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, (2S)-2-amino-2-[cis-3-[[(4'-chlorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[[4'-(trifluoromethyl)biphenyl-4-yl]methyl]amino]cyclobutyl] hexanoic acid), (2 S)-2-amino-2- [ci s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid), (2S)-2-amino-2-[cis-3- [[(2,2'-difluoro-5'-methylbiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32 or any combination thereof.
In one embodiment, the arginase inhibitor comprises an oligonucleotide specific for Arginase. In one embodiment, the oligonucleotide is specific for Arginase 1 or Arginase 2.
In one embodiment, the arginase inhibitor comprises an anti-Arginase antibody. In one embodiment, the anti-Arginase antibody is an anti-Arginase 1 antibody or an anti-Arginase 2 antibody.
In one embodiment, the composition is administered by nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal, epidural injection, oral or any combination thereof.
In one embodiment, acute kidney injury comprises ischemia reperfusion injury.
In one embodiment, the subject a mammal. In one embodiment, the subject is a human.
In one embodiment, the composition is administered before kidney allograft. In one embodiment, the composition is administered after renal IRI. In one embodiment, the method further comprises administering a second therapeutic agent. In one embodiment, the second therapeutic is selected from the group consisting of calcium, glucose, sodium polystyrene sulfonate, Kayexalate, Kionex or any combination thereof. In one embodiment, the second therapeutic agent is acute dialysis.
DETAILED DESCRIPTION
The present invention relates to compositions and methods for inhibiting arginase. In one aspect, the invention is based, in part, on the unexpected finding that inhibition of arginase provided treatment and prevention to AKI.
In one embodiment, the composition of the invention comprises an inhibitor of arginase. For example, in one embodiment, the inhibitor of arginase inhibits the expression, activity, or both of arginase.
In one embodiment, the method of the present invention comprises treating and preventing AKI. In one embodiment, the method comprises administering to a subject an effective amount of a composition comprising an inhibitor of arginase.
Definitions
Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well-known and commonly employed in the art.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%), ±5%), ±1%), or ±0.1%) from the specified value, as such variations are appropriate to perform the disclosed methods.
The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
As used herein, "acute kidney injury" refers to the abrupt loss of kidney function, resulting in the retention of urea and other nitrogenous waste products and in the dysregulation of extracellular volume and electrolytes. The loss of kidney function that defines AKI is most easily detected by measurement of the serum creatinine (SCr), which is used to estimate the glomerular filtration rate (GFR). AKI is defined as any of the following: Increase in SCr by >=0.3 mg/dl within 48 hours; or increase in SCr to >=1.5 times baseline, which is known or presumed to have occurred within the prior 7 days; or urine volume <0.5 ml/kg/h for 6 hours.
As used herein, "ischemia reperfusion injury" as used herein refers to renal injury due to one or more identified occurrences of renal ischemia or ischemia and reperfusion and/or toxemia and/or hypoperfusion or hemorrhage leads to hypoperfusion. The term ischemic kidney injury may be used interchangeably herein. Ischemia reperfusion injury can be identified by clinicians, for example by recognizing ischemic conditions or by reference to certain intrinsic renal causes of acute kidney injury. As used herein, an "arginase inhibitor" refers to any agent capable of inhibiting the function, expression, activity or combinations thereof, of Arginase 2, Arginase 1, or both Arginase 1 and Arginase 2 and/or any agent capable of increasing levels of arginase's substrate, arginine and/or citrulline. The terms "Arginase," "Arginase 1 " and "Arginase 2," are inclusive of all species, including human.
A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of a disease or disorder, for the purpose of diminishing or eliminating those signs or symptoms.
As used herein, "treating a disease or disorder" means reducing the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a patient.
"Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.
By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.
A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
A "coding region" of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
"Complementary" as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%), or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term "DNA" as used herein is defined as deoxyribonucleic acid. The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein,
polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
The term "fusion polypeptide" refers to a chimeric protein containing a protein of interest (e.g., luciferase) joined to a heterologous sequence (e.g., a non- luciferase amino acid or protein).
The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710
University Avenue. Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. The term "isolated" when used in relation to a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences (e.g., a specific mRNA sequence encoding a specific protein), are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand (i.e., the
oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
As used herein, the term "oligonucleotide specific for" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
As used herein, the terms "oligonucleotide," "siRNA," and "antisense oligonucleotide" are used interchangeably throughout the specification and include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate,
methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. The term "isolated" when used in relation to a polypeptide, as in "isolated protein" or "isolated polypeptide" refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated polypeptide is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state they exist in nature.
By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, phosphorothioate,
methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases
(adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid" typically refers to large polynucleotides.
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.
The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."
"Antisense" refers particularly to the nucleic acid sequence of the non- coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
By "expression cassette" is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter/regulatory sequences necessary for transcription and, optionally, translation of the coding sequence.
The term "operably linked" as used herein refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of sequences encoding amino acids in such a manner that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in an inducible manner.
An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds to the promoter is present.
A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.
As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
As used herein, a "peptidomimetic" is a compound containing non- peptidic structural elements that is capable of mimicking the biological action of a parent peptide. A peptidomimetic may or may not comprise peptide bonds.
The term "RNA" as used herein is defined as ribonucleic acid.
"Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.
As used herein, "conjugated" refers to covalent attachment of one molecule to a second molecule.
As used herein, the term "transdominant negative mutant gene" refers to a gene encoding a polypeptide or protein product that prevents other copies of the same gene or gene product, which have not been mutated (i.e., which have the wild-type sequence) from functioning properly (e.g., by inhibiting wild type protein function). The product of a transdominant negative mutant gene is referred to herein as "dominant negative" or "DN" (e.g., a dominant negative protein, or a DN protein).
The phrase "inhibit," as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
As used herein, the term "inflammatory" means relating to inflammation. The term "inflammation" refers to the process by which vascular tissues responds to harmful stimuli, such as pathogens, damaged cells, or irritants. "Inflammation includes, but is not limited to secretion of and response to inflammatory factors, e.g., inflammatory cytokines.
"Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear
polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to
communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
"Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human. As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder contemplated herein, a sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a disease or disorder contemplated herein, at least one sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
Description
The present invention is based on novel therapeutic modalities using
Arginase inhibitors in the treatment of acute kidney injury (AKI). Accordingly, the present invention provides methods and compositions for treating or preventing AKI.
In one embodiment, the present invention provides a composition for treating and preventing AKI in a subject. In one aspect, the present invention provides a composition for treating AKI, the composition comprising an inhibitor of arginase. In one embodiment, the arginase is Arginase 1 or Arginase 2. In one embodiment, the inhibitor of Arginase is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule. . In one embodiment, the inhibitor of Arginase 1 is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule. In one embodiment, the inhibitor of Arginase 2 is selected from a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule.
The arginase inhibitor can include any agent capable of inhibiting the function, expression, activity or combinations thereof Arginase. In one embodiment Arginase is Arginase 1, Arginase 2, or both Arginase 1 and Arginase 2. In one embodiment, the arginase inhibitor can also inhibit Arginase 1, Arginase 2 or both.
In one embodiment, the invention provides a method for treating and preventing AKI in a subject. In one embodiment the method comprises administering to a subject an effective amount of an arginase inhibitor, an analog thereof, or a combination thereof. In one embodiment the method comprises administering to a subject an effective amount of an Arginase inhibitor, wherein the inhibitor is Arginase 1 or Arginase 2.
In one embodiment, the method further comprises administering a second therapeutic agent. In certain embodiments, the second therapeutic agent is an inhibitor of AKI.
In one embodiment, the subject is a mammal. In another embodiment, the mammal is a human. Small molecule inhibitors
In various embodiments, the inhibitor is a small molecule. When the inhibitor is a small molecule, a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule inhibitor of the invention comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.
Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well- known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.
In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure ("focused libraries") or synthesized with less structural bias using flexible cores.
The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted and it is understood that the invention embraces all salts and solvates of the inhibitors depicted here, as well as the non-salt and non-solvate form of the inhibitors, as is well understood by the skilled artisan. In some embodiments, the salts of the inhibitors of the invention are pharmaceutically acceptable salts.
Where tautomeric forms may be present for any of the inhibitors described herein, each and every tautomeric form is intended to be included in the present invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2- pyridone tautomer is also intended.
The invention also includes any or all of the stereochemical forms, including any enantiomeric or diasteriomeric forms of the inhibitors described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of inhibitors depicted. All forms of the inhibitors are also embraced by the invention, such as crystalline or non-crystalline forms of the inhibitors. Compositions comprising an inhibitor of the invention are also intended, such as a composition of substantially pure inhibitor, including a specific stereochemical form thereof, or a composition comprising mixtures of inhibitors of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture.
In one embodiment, the small molecule inhibitor of the invention comprises an analog or derivative of an inhibitor described herein.
In one embodiment, the small molecules described herein are candidates for derivatization. As such, in certain instances, the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development. Thus, in certain instances, during optimization new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.
In some instances, small molecule inhibitors described herein are derivatized/analoged as is well known in the art of combinatorial and medicinal chemistry. The analogs or derivatives can be prepared by adding and/or substituting functional groups at various locations. As such, the small molecules described herein can be converted into derivatives/analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Also, the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms. Also, the ring groups can be changed so as to have a different number of atoms in the ring and/or to include hetero atoms. Moreover, aromatics can be converted to cyclic rings, and vice versa. For example, the rings may be from 5-7 atoms, and may be homocycles or heterocycles.
As used herein, the term "analog," "analogue," or "derivative" is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule inhibitors described herein or can be based on a scaffold of a small molecule inhibitor described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative of any of a small molecule inhibitor in accordance with the present invention can be used to treat AKI or an AKI -related disease or disorder.
In one embodiment, the small molecule inhibitors described herein can independently be derivatized/analoged by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative/analog can be used. For example, the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic having one or more hetero atoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo-substituted aliphatics, and the like. Additionally, any ring group on a compound can be derivatized to increase and/or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms.
In one embodiment, the arginase inhibitor is selected from 2(S)amino- 6- boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L-cysteine (BEC), ΝΩ-ΟΗ-L-arginine (NOHA), ΝΩ-hydroxynor-L-arginine (nor-NOHA), a- difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl- Lysine, L-Lysine, N5-(benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)- L-thiocitrulline tert-butyl ester, N5-[N-(benzyloxycarbonyl)-N'- (methoxycarbonylmethoxy)amidino]-N2-(tertbutoxycarbonyl)-L-ornithine tert-butyl ester, 6-(dihydroxyboranyl)-2-(3-phenoxypropyl)norleucine hydrochloride, (6- (dihydroxyboranyl)-2-(hydroxymethyl)-L-norleucine, 2-amino-6- (dihydroxyboryl)-2-[3-(4-piperidinyl) propyl]hexanoic acid, 2-[4-(dihydroxyboranyl) butyl] lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-L-yl)ethyl]-lnorleucine
dihydrochloride, (2S,3S)-3-amino-2-[3-(dihydroxyboranyl) propyl]
tetrahydrofuran-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminocyclopentyl)-4-[3- (dihydroxyboranyl) propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[3-(4- carboxyphenyl) propyl]-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-(2-aminoethyl)-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l,3'-bipyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-4-[3-(dihydroxyboranyl)propyl]-l-[2- (pyrrolidin-l-yl)ethyl]pyrrolidine-3 -carboxylic acid, (3R,4S)-3-amino-l- [2-(benzylamino)ethyl]-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3 -carboxylic acid, (3R,4S)-3-amino-l-[(7-chloro-l,2,3,4-tetrahydroisoquinolin-3-yl)
methyl]-4-[3-(dihydroxyboranyl) propyl] pyrrolidine-3 -carboxylic acid, (2-[3-[4-(3,4- dichlorobenzyl)piperazin-l-yl]propyl]-6-(dihydroxyboranyl)norleucine, 6- (dihydroxyboranyl)-2-(3-[4-[4-(methylsulfonyl)benzyl] piperazin-l-yl]propyl) norleucine, (2 S)-2-amino-6-(dihy droxybory l)-2- [ci s-3 - [ [[4 ' - (trifluoromethyl)biphenyl-3-yl]methyl]amino]cyclobutyl] hexanoic acid, (2S)-2-amino-6- (dihydroxyboryl)-2-[cis-3-[[(4-fluoro-l-naphthyl)methyl]amino]cyclobutyl]hexanoic acid, (2S)-2-amino-2-[cis-3-[[(3',4'-dichlorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, (2S)-2-amino-2-[cis-3-[[(4'-chlorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[[4'-(trifluoromethyl)biphenyl-4-yl]methyl]amino]cyclobutyl] hexanoic acid), (2 S)-2-amino-2- [ci s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid), (2S)-2-amino-2-[cis-3- [[(2,2'-difluoro-5'-methylbiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid or combinations thereof. In one embodiment, the arginase inhibitor is S-(2-boronoethyl)-L-cysteine (BEC). In one embodiment, the arginase inhibitor is l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32. In one embodiment, arginase inhibitors include those described in Ivanenkov et al. (2014), the contents of which are incorporated herein by reference. Nucleic acid inhibitors
In other related aspects, the invention includes an isolated nucleic acid. In some instances, the inhibitor is an siRNA, miRNA, or antisense molecule, which inhibits Arginase. In one embodiment, the siRNA, miRNA, or antisense molecule inhibits Arginase 1. In one embodiment, the siRNA, miRNA, or antisense molecule inhibits Arginase 2. In one embodiment, the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.
In one aspect of the invention, Arginase can be inhibited by way of inactivating and/or sequestering Arginase. In another aspect of the invention, Arginase 1 can be inhibited by way of inactivating and/or sequestering Arginase 1. As such, inhibiting the activity of Arginase 1 can be accomplished by using a transdominant negative mutant. In another aspect of the invention, Arginase 2 can be inhibited by way of inactivating and/or sequestering Arginase 2. As such, inhibiting the activity of Arginase 2 can be accomplished by using a transdominant negative mutant.
In one embodiment, siRNA is used to decrease the level of Arginase protein. In one embodiment, siRNA is used to decrease the level of Arginase 1 protein. In one embodiment, siRNA is used to decrease the level of Arginase 2 protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of Arginase 1 or Arginase 2 using RNAi technology.
In another aspect, the invention includes a vector comprising an siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide, wherein the target polypeptide is
Arginase. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al. (2012), and in Ausubel et al. (1997), and elsewhere herein.
In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.
The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.
In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.
Therefore, in another aspect, the invention relates to a vector, comprising the nucleotide sequence of the invention or the construct of the invention. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and/or eukaryote- vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.
Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.)
By way of illustration, the vector in which the nucleic acid sequence is introduced can be a plasmid which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.
The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells. In one embodiment, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of invention, described elsewhere herein.
A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent
5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol
acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.
Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191- 2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene
Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).
Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.
In one embodiment of the invention, an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit Arginase protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Arginase 1. In one embodiment of the invention, an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit Arginase 2 protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Arginase 2.
Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule thereby inhibiting the translation of genes.
The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem.
172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5, 190,931.
Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).
In one embodiment of the invention, a ribozyme is used to inhibit
Arginase protein expression. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence encoding Arginase. Ribozymes targeting Arginase may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them. Ribozymes targeting Arginase 1 or Arginase 2 may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them. In one embodiment, the inhibitor of Arginase may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
In one embodiment, the inhibitor of Arginase 1 may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase 1, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
In one embodiment, the inhibitor of Arginase 2 may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding Arginase 2, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
In one embodiment, the arginase inhibitor is an oligonucleotide specific for Arginase. In one embodiment, the arginase inhibitor is an oligonucleotide specific for Arginase 1. In one embodiment, the invention provides a method for treating and preventing AKI, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase 2. Polypeptide inhibitors
In other related aspects, the invention includes an isolated peptide inhibitor that inhibits Arginase. For example, in one embodiment, the peptide inhibitor of the invention inhibits Arginase directly by binding to Arginase thereby preventing the normal functional activity of Arginase. In another embodiment, the peptide inhibitor of the invention inhibits Arginase 1 by competing with endogenous Arginase 1. In yet another embodiment, the peptide inhibitor of the invention inhibits the activity of Arginase 1 by acting as a transdominant negative mutant.
In another embodiment, the peptide inhibitor of the invention inhibits Arginase 2 by competing with endogenous Arginase 2. In yet another embodiment, the peptide inhibitor of the invention inhibits the activity of Arginase 2 by acting as a transdominant negative mutant.
The variants of the polypeptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
Antibody inhibitors
The invention also contemplates an inhibitor of Arginase comprising an antibody, or antibody fragment, specific for Arginase. That is, the antibody to Arginase can provide a beneficial effect. The invention also contemplates an inhibitor of Arginase 1 comprising an antibody, or antibody fragment, specific for Arginase 1. That is, the antibody to Arginase 1 can provide a beneficial effect. In one embodiment, the arginase inhibitor comprises an antibody, or antibody fragment, specific for Arginase 1. In one embodiment, the arginase inhibitor comprises an antibody, or antibody fragment, specific for Arginase 2.
The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.
Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled polypeptide may then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit). Combinations of Arginase inhibitors
In one embodiment, the composition of the present invention comprises a combination of an Arginase inhibitor and second therapeutic agent. For example, in one embodiment the second therapeutic agents include, but are not limited to calcium, glucose or sodium polystyrene sulfonate, Kayexalate, Kionex. In one embodiment, the second therapeutic agent may also be acute dialysis.
In certain embodiments, a composition comprising a combination of inhibitors described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual inhibitor. In other embodiments, a composition comprising a combination of inhibitors described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual inhibitor.
A composition comprising a combination of inhibitors comprises individual inhibitors in any suitable ratio. For example, in one embodiment, the composition comprises a 1 : 1 ratio of two individual inhibitors. However, the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed. Therapeutic Methods
The present invention also provides methods of treating or preventing AKI in a subject. In one embodiment, the AKI is treated or prevented by inhibiting arginases. In one embodiment, the invention provides a method for treating AKI, wherein Arginase is inhibited. In one embodiment, the invention provides a method for treating AKI, wherein Arginase 1 is inhibited. In one embodiment, the invention provides a method for treating AKI wherein Arginase 2 is inhibited. In one embodiment, the invention provides a method for treating AKI, by inhibiting Arginase, wherein the arginase inhibitor is S-(2- boronoethyl)-L-cysteine (BEC).
It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of AKI that is already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of AKI do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing AKI, in that a composition, as discussed previously elsewhere herein, can be
administered to a subject prior to the onset of AKI, thereby preventing AKI. For example, in one embodiment, the method of preventing AKI comprises administering a
composition of the invention to a patient prior to kidney allograft. In one embodiment, the method of preventing AKI comprises administering a composition of the invention a kidney ex vivo prior to kidney allograft. In one embodiment, the method of preventing AKI comprises administering a composition of the invention to a patient and to a kidney ex vivo prior to kidney allograft.
One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of AKI, encompasses administering to a subject a composition as a preventative measure against the development of, or progression of AKI. As more fully discussed elsewhere herein, methods of modulating the level or activity of a gene, or gene product, encompass a wide plethora of techniques for modulating not only the level and activity of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both. In one embodiment, the invention provides a method for treating and preventing AKI, by inhibiting Arginase, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase. In one embodiment, the invention provides a method for treating and preventing AKI, by inhibiting Arginase, wherein the arginase inhibitor is an oligonucleotide that specifically binds Arginase 1. In one embodiment, the invention provides a method for treating and preventing AKI, wherein the Arginase inhibitor is an oligonucleotide that specifically bind Arginase 2
The invention encompasses administration of an inhibitor of Arginase. To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate modulator composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.
In one embodiment, the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase. In one embodiment, the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase 1. In one embodiment, the method comprises administering to the subject in need an effective amount of a composition that reduces or inhibits the expression or activity of Arginase 2.
One of skill in the art will appreciate that the inhibitors of the invention can be administered singly or in any combination. Further, the inhibitors of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the inhibitor
compositions of the invention can be used to prevent or treat AKI, and that an inhibitor composition can be used alone or in any combination with another modulator to effect a therapeutic result. In various embodiments, any of the inhibitor compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with AKI. In one embodiment, the invention provides a method for treating AKI, by administering an Arginase inhibitor, wherein the arginase inhibitor is Arginase 1, Arginase 2, S-(2-boronoethyl)-L-cysteine (BEC) or any combination thereof.
The methods of modulating arginase described herein can also treat one or more diseases or disorders associated with abnormal arginase expression or levels in a patient as compared to healthy normal controls. Diseases or disorders associated with abnormal arginase expression or levels in a patient as compared to healthy normal controls can include renal diseases or disorders, renal injury, diabetic nephropathy, angina, congestive heart failure, cancer, azotemia, albuminuria, nephritis, renal failure or cardiovascular diseases.
In one embodiment, the modulation of arginase can be used to treat one or more diseases or disorders associated with abnormal arginase expression or levels in a patient. In one embodiment, the invention provides a method for treating and preventing AKI, wherein Arginase modulated. In one embodiment, the invention provides a method for treating and preventing AKI, wherein Arginase 1, Arginase 2 or any combination thereof is modulated.
In one embodiment, the invention includes a method comprising administering a combination of inhibitors described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of inhibitors is approximately equal to the sum of the effects of
administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of inhibitors is greater than the sum of the effects of administering each individual inhibitor.
The method comprises administering a combination of inhibitors in any suitable ratio. For example, in one embodiment, the method comprises administering two individual inhibitors at a 1 : 1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
Pharmaceutical Compositions and Formulations
In one aspect, the invention provides compositions for treating AKI in a subject. The invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention. Such a pharmaceutical composition may consist of at least one modulator composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one modulator composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compound or conjugate of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In an embodiment, the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between
1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical
compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Methods of introduction include but are not limited to nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of
pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other
pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, PA), which is incorporated herein by reference.
The composition of the invention may comprise a preservative from about 0.005% to 2.0%) by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
The composition preferably includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01%) to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01%> to 0.20% and more preferably in the range of 0.02% to 0.10%) by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent.
Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the therapeutic situation. A non- limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease in a subject.
In one embodiment, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.
Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.
In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., a drug used for treating the same or another disease as that treated by the compositions of the invention) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.
The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical
composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.
EXPERIMENTAL EXAMPLES
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
Example 1 : The Problem of Acute Kidney Injury
The results presented herein demonstrate that Arginase inhibitors are useful for the prevention and treatment of AKI. The results for identifying the mechanisms involved in the development and progression of AKI
Male C57B1/6J wild type (WT) and Arginase T1' mice (6-8 weeks old) were subjected to bilateral renal ischemia for 28 minutes followed by reperfusion for 24 hours, which produces a large increase in plasma creatinine and BUN as indicative of kidney injury in (WT) mice (Figure 1). BEC (17 mg/kg) or vehicle was administered once intraperitoneally 18 hours before ischemia as described previously (Gao et al., 2014, J Clin Invest, 124(11):4989-5001, Awad et al., 2006, Am J Physiol Renal Physiol, 290(6):F1516-F1524, Gao et al., 2013, Am J Physiol Renal Physiol, 304(5):F515-F521). Renal function, histology, kidney inflammatory cell recruitments, oxidative stress, and NO was then determined.
Increased Arginase 2 expression and activity in renal IRI
Arginase 2 mRNA expression (Figure 1) increased by 6 hours, and peaked at 48 hours, while Arginase 2 protein expression (Figure 1) demonstrated sustained increases after 24 hours after renal IRI, paralleling the increase in kidney arginase activity (Figure 1). The transient increase in plasma arginase activity is due to release of Arginase 2 from damaged renal cells during IR. Although an increase in renal Arginase 1 mRNA expression (Figure 1) at 24-48 h was observed, the Arginase 1 protein (Figure 1) was undetectable by Western blot post IRI. This data confirmed previous results of Arginase 2 but not Arginase 1 expression in the kidney (Morris et al., 2011, Diabetes, 60(11):3015- 3022).
Deficiency of Arginase 2 reduces plasma creatinine and BUN after renal IRI
WT and Arginase 2~'~ mice were subjected to bilateral renal ischemia for 28 minutes followed by reperfusion for 24 hours (Figure 2). WT mice displayed a significant increase in plasma creatinine and BUN after renal IRI. In contrast, the increased in plasma creatinine and BUN were significantly reduced in Arginase 2'1' mice.
Inhibition of arginases reduced plasma creatinine and BUN after renal IRI
Vehicle-treated mice had a significant increase in plasma creatinine and BUN compared to sham at 24 hours after IRI (Figure 2), plasma creatinine and BUN were significantly reduced in IRI mice treated with BEC.
Inhibition of arginases decreases renal histological changes after renal IRI
Periodic acid-Schiff (PAS) staining of kidney sections (Figure 3) showed severe histologic kidney damage in vehicle-treated compared to sham mice after IRI. Inhibition of arginases after IRI exhibited significantly reduced histologic kidney damage, as shown by less cast formation, preservation of brush border membranes, less sloughing of epithelial compared to vehicle-treated IRI mice. Similarly, kidney injury marker-1 (KIM-1) mRNA expression is elevated in vehicle-treated IRI mice; an effect significantly reduced using BEC treatment.
Inhibition of arginases decreases renal apoptosis after renal IRI
Cleaved caspase-3, an early marker of cells undergoing apoptosis, staining of kidney sections (Figure 4) showed no difference in the number of apoptotic cells between vehicle-treated or BEC-treated sham mice, and apoptotic cells were only observed rarely. In contrast, vehicle-treated mice showed increased number of apoptotic cells compared to BEC-treated mice 24 h after renal IRI.
Inhibition of arginases reduces kidney inflammatory cell recruitment after renal IRI
Vehicle-treated IRI mice showed significant increases in kidney neutrophils, macrophages, and T cells (Figure 4) by immunostaining compared with sham controls. In contrast, BEC-treated IRI mice had significantly reduced kidney neutrophils, macrophages, and T cells compared to vehicle-treated IRI mice at 24 h following ischemia. Inhibition of arginases reduces kidney inflammatory cytokines after renal IRI
Vehicle-treated IRI mice showed significant increases in kidney IL-Ιβ, KC-GRO, and TNF-a (Figure 5) compared with sham controls. In contrast, BEC-treated IRI mice had significantly reduced kidney IL-Ιβ, KC-GRO, and TNF-a compared to vehicle-treated IRI mice at 24 hours following ischemia.
Inhibition of arginases increased kidney NO after renal IRI.
The renal IRI reduced total kidney NOx, and p-eNOS/eNOS expression, BEC treatment significantly restored total kidney NOx and p-eNOS/eNOS expression after IRI (Figure 6). Deficiency of Arginase 2 or Inhibition of arginases reduced kidney oxidative stress after renal IRI.
Next, the assessment of kidney TBARS and GSH as indicators for oxidative stress was performed. Arginase 2+/+ or vehicle-treated mice significantly increased kidney TBARS and reduced kidney GSH after IRI. In contrast, both Arginase 2~'~ and BEC-treated mice completely prevented oxidative stress after IRI (Figure 7).
Inhibition of arginases increased kidney PGC-Ια after renal IRI.
The renal IRI reduced kidney PGC-Ια expression, BEC treatment significantly restored kidney PGC-Ια expression after IRI (Figure 8).
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. A composition for treating acute kidney injury comprising an arginases inhibitor.
2. The composition of claim 1, wherein the arginase inhibitor inhibits an Arginase selected from Arginase 1 and Arginase 2.
3. The composition of claim 1, wherein the arginase inhibitor is at least one selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule and any combination thereof.
4. The composition of claim 3, wherein the small molecule chemical is at least one selected from the group consisting of BEC, 2(S)amino- 6-boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L-cysteine (BEC), ΝΩ-ΟΗ-L-arginine (NOHA), ΝΩ-hydroxynor-L-arginine (nor-NOHA), a-difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl-Lysine, L-Lysine, N5- (benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)-L-thiocitrulline tert-butyl ester, N5-[N- (benzyloxycarbonyl)-N'-(methoxycarbonylmethoxy)amidino]-N2-(tertbutoxycarbonyl)- L-ornithine tert-butyl ester, 6-(dihydroxyboranyl)-2-(3-phenoxypropyl)norleucine hydrochloride, (6-(dihydroxyboranyl)-2-(hydroxymethyl)-L-norleucine, 2-amino-6- (dihydroxyboryl)-2-[3-(4-piperidinyl) propyl]hexanoic acid, 2-[4-(dihydroxyboranyl) butyl] lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-L-yl)ethyl]-lnorleucine
dihydrochloride, (2S,3 S)-3-amino-2-[3-(dihydroxyboranyl) propyl]tetrahydrofuran-3- carboxylic acid, (3R,4S)-3-amino-l-(2-aminocyclopentyl)-4-[3-(dihydroxyboranyl) propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[3-(4-carboxyphenyl) propyl]- 4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-(2- aminoethyl)-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3- amino-4-[3-(dihydroxyboranyl)propyl]-l,3'-bipyrrolidine-3-carboxylic acid, (3R,4S)-3- amino-4-[3-(dihydroxyboranyl)propyl]-l-[2-(pyrrolidin-l-yl)ethyl]pyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-l-[2-(benzylamino)ethyl]-4-[3-
(dihydroxyboranyl)propyl] pyrrolidine-3 -carboxylic acid, (3R,4S)-3-amino-l-[(7-chloro- l,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboranyl) propyl] pyrrolidine- 3-carboxylic acid, (2-[3-[4-(3,4-dichlorobenzyl)piperazin-l-yl]propyl]-6- (dihydroxyboranyl)norleucine, 6-(dihydroxyboranyl)-2-(3-[4-[4-(methylsulfonyl)benzyl] piperazin-l-yl]propyl) norleucine, (2S)-2-amino-6-(dihydroxyboryl)-2-[cis-3-[[[4'- (trifluoromethyl)biphenyl-3-yl]methyl]amino]cyclobutyl] hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[(4-fluoro-l-naphthyl)methyl]amino]cyclobutyl]hexanoic acid, (2S)-2-amino-2-[cis-3-[[(3',4'-dichlorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, (2S)-2-amino-2-[cis-3-[[(4'-chlorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[[4'-(trifluoromethyl)biphenyl-4-yl]methyl]amino]cyclobutyl] hexanoic acid), (2 S)-2-amino-2- [ci s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid), (2S)-2-amino-2-[cis-3- [[(2,2'-difluoro-5'-methylbiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32 or any combination thereof.
5. The composition of claim 1, wherein the arginase inhibitor comprises an oligonucleotide specific for Arginase.
6. The composition of claim 1, wherein the arginase inhibitor comprises an oligonucleotide specific for Arginase 1, Arginase 2 or a combination thereof.
7. The composition of claim 1, wherein the arginase inhibitors comprises an antibody specific for Arginase.
8. The composition of claim 1, wherein the arginase inhibitor comprises an antibody specific for Arginase 1, Arginase 2 or a combination thereof.
9. The composition of claim 1, wherein the acute kidney injury comprises ischemia reperfusion injury.
10. A method of treating acute kidney injury in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one arginase inhibitor.
11. The method of claim 10, wherein the arginase inhibitor is selected from the group consisting of a protein, a peptide, a peptidomemetic, an antibody, an oligonucleotide, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule and any combination thereof.
12. The method of claim 11, wherein the small molecule chemical is at least one selected from the group consisting of BEC, 2(S)amino- 6-boronohexanoic acid (ABH) or ABH analogues, S-(2-boronoethyl)-L-cysteine (BEC), ΝΩ-ΟΗ-L-arginine (NOHA), ΝΩ-hydroxynor-L-arginine (nor-NOHA), a-difluoromethylomithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl-Lysine, L-Lysine, N5- (benzyloxycarbonyl)-N2-(tertbutoxycarbonyl)-L-thiocitrulline tert-butyl ester, N5-[N- (benzyloxycarbonyl)-N'-(methoxycarbonylmethoxy)amidino]-N2-(tertbutoxycarbonyl)- L-ornithine tert-butyl ester, 6-(dihydroxyboranyl)-2-(3-phenoxypropyl)norleucine hydrochloride, (6-(dihydroxyboranyl)-2-(hydroxymethyl)-L-norleucine, 2-amino-6- (dihydroxyboryl)-2-[3-(4-piperidinyl) propyljhexanoic acid, 2-[4-(dihydroxyboranyl) butyl] lysine, 6-(dihydroxyboranyl)-2-[2-(piperidin-L-yl)ethyl]-lnorleucine
dihydrochloride, (2S,3 S)-3-amino-2-[3-(dihydroxyboranyl) propyl]tetrahydrofuran-3- carboxylic acid, (3R,4S)-3-amino-l-(2-aminocyclopentyl)-4-[3-(dihydroxyboranyl) propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[3-(4-carboxyphenyl) propyl]- 4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-(2- aminoethyl)-4-[3-(dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3- amino-4-[3-(dihydroxyboranyl)propyl]-l,3'-bipyrrolidine-3-carboxylic acid, (3R,4S)-3- amino-4-[3-(dihydroxyboranyl)propyl]-l-[2-(pyrrolidin-l-yl)ethyl]pyrrolidine-3- carboxylic acid, (3R,4S)-3-amino-l-[2-(benzylamino)ethyl]-4-[3- (dihydroxyboranyl)propyl] pyrrolidine-3-carboxylic acid, (3R,4S)-3-amino-l-[(7-chloro- l,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboranyl) propyl] pyrrolidine- 3-carboxylic acid, (2-[3-[4-(3,4-dichlorobenzyl)piperazin-l-yl]propyl]-6- (dihydroxyboranyl)norleucine, 6-(dihydroxyboranyl)-2-(3-[4-[4-(methylsulfonyl)benzyl] piperazin-l-yl]propyl) norleucine, (2S)-2-amino-6-(dihydroxyboryl)-2-[cis-3-[[[4'- (trifluoromethyl)biphenyl-3-yl]methyl]amino]cyclobutyl] hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[(4-fluoro-l-naphthyl)methyl]amino]cyclobutyl]hexanoic acid, (2S)-2-amino-2-[cis-3-[[(3',4'-dichlorobiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, (2S)-2-amino-2-[cis-3-[[(4'-chlorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid, (2S)-2-amino-6- (dihydroxyboiyl)-2-[cis-3-[[[4'-(trifluoromethyl)biphenyl-4-yl]methyl]amino]cyclobutyl] hexanoic acid), (2 S)-2-amino-2- [ci s-3 - [ [(4 ' -chloro-3 -fluorobiphenyl-4- yl)methyl]amino]cyclobutyl]-6-(dihydroxyboryl)hexanoic acid), (2S)-2-amino-2-[cis-3- [[(2,2'-difluoro-5'-methylbiphenyl-4-yl)methyl]amino]cyclobutyl]-6- (dihydroxyboryl)hexanoic acid, l,2,4]triazolo[l,5-a]pyrimidine derivatives 4-32 or any combination thereof.
13. The method of claim 10, wherein the composition is administered by nasal, rectal, intravaginal, parenteral, topical, pulmonary, buccal, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal, epidural injection, oral or any combination thereof.
14. The method of claim 11, wherein the arginase inhibitor inhibits Arginase 1, Arginase 2, or a combination thereof.
15. The method of claim 11, wherein the arginase inhibitor comprises an oligonucleotide specific for Arginase.
16. The method of claim 11, wherein the arginase inhibitor comprises an oligonucleotide specific for Arginase 1, Arginase 2 or a combination thereof.
17. The method of claim 11, wherein the arginase inhibitors comprises an antibody specific for Arginase.
18. The method of claim 11, wherein the arginase inhibitor comprises an antibody specific for Arginase 1, Arginase 2 or a combination thereof.
19. The method of claim 10, wherein the acute kidney injury comprises ischemia reperfusion injury.
20. The method of claim 10, wherein the subject is a human.
21. The method of claim 10, wherein the subject is mammal.
22. The method of claim 10, wherein the composition is administered before kidney allograft.
23. The method of claim 10, wherein the composition is administered after renal IRI.
24. The method of claim 10, wherein the method further comprises administering a second therapeutic agent.
25. The method of claim 24, wherein the second therapeutic agent is at least one selected from the group consisting of calcium, glucose, sodium polystyrene sulfonate, Kayexalate, Kionex or any combination thereof.
26. The method of claim 23, wherein the second therapeutic acute dialysis.
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