RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No.

63/405,014, filed September 9, 2022, the entire disclosure of which is incorporated herein by this reference.

TECHNICAL FIELD

[0002] The presently-disclosed subject matter generally relates to dimeric polypeptide antagonists of the Na/K-ATPase-Src receptor complex. In particular, certain embodiments of the presently-disclosed subject matter relate to dimeric polypeptide antagonists of the Na/K-ATPase- Src receptor complex and the use of such protein dimers in compositions and methods for treating a Src-associated disorder and/or modulating the activity of Src in a cell.

BACKGROUND

[0003] The Na/K-ATPase enzyme is ubiquitously expressed in all tested eukaryotic cells and helps maintain the trans-membrane ion gradient by pumping Na ⁺ out and K ⁺ into cells. The Na/K-ATPase interacts directly with Src via at least two binding motifs: one being between the CD2 of the al subunit and Src SH2 domain; and the other involving the third cytosolic domain (CD3) of the al subunit and Src kinase domain. The formation of this Na/K-ATPase and Src complex serves as a receptor for ouabain (and other cardiotonic steroids (CTS)) and increased oxidative stress to provoke protein kinase cascades. Specifically, binding of ouabain (and other CTS) to Na/K-ATPase will disrupt the latter interaction, and then result in assembly and activation of different pathways including ERK cascades, PLC/PKC pathway and ROS production. Moreover, this interaction between the Na/K-ATPase and Src keeps Src in an inactive state that can then become an active state through CTS or oxidative stress stimulation. Thus, the Na/K-ATPase functions as an endogenous negative Src regulator. See also International Patent Application Nos. WO 2008/054792 and WO 2010/071767, which are both incorporated herein by reference.

[0004] Src family kinases are 52-62-kDa membrane-associated nonreceptor tyrosine kinases and they participate in several tyrosine phosphorylation-related signaling pathways in response to various extracellular ligands. Src, for example, contains at least three protein interaction domains. The SH3 domain binds to polyproline motifs and the SH2 domain interacts with the phosphorylated tyrosine residues. The kinase domain reacts with the nucleotide and phosphorylates the substrate. The binding of protein ligands to the SH3 or SH2 domain can activate Src. Proteins that bind with kinase domain of Src have also been reported as being capable of regulating Src activity.

[0005] It is further appreciated that the Na+/K+-ATPase interacts with Src and Src family kinases to form a functional receptor. The binding of ouabain to this receptor activates Src, which in turn phosphorylates various effectors, resulting in the assembly and activation of different pathways including the Ras/Raf/ERKl/2 and phospholipase C/protein kinase C cascades as well as increases in intracellular Ca ²⁺ and cellular ROS production. The activation of these signaling pathways eventually leads to changes in cardiac and renal functions, stimulation of cell proliferation and tissue fibrosis, protection of tissue against ischemia/reperfusion injury, inhibition of cancer cell growth, and more. While many known Src and Src family kinase inhibitors are developed as ATP analogs that compete for ATP binding to these kinases, such Src inhibitors lack pathway specificity. In this regard, it has previously been observed that blocking NKA/Src activation by a polypeptide designated “pNaKtide” (SEQ ID NO: 5) effectively abolishes the formation of a reactive oxygen species (ROS) amplification loop, resulted in an inhibition of pathological ROS signaling. pNaKtide also reduced ROS stress and signaling in obesity and was effective in blocking and reversing left ventricular hypertrophy and fibrosis in an animal model of uremic cardiomyopathy.

[0006] Accordingly, an improved pNaKtide polypeptide that is capable of antagonizing the Na/K-ATPase-Src receptor complex and that is useful for treating a Src-associated disorder would be both highly desirable and beneficial.

SUMMARY

[0007] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0008] This summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features. [0009] The presently-disclosed subject matter includes dimeric polypeptide antagonists of the Na/K-ATPase-Src receptor complex. In some embodiments, a non-naturally occurring protein dimer is provided that comprises two monomers of a peptide having a sequence of SEQ ID NO: 1 or a functional fragment or functional variant thereof. In some embodiments, each monomer further comprises a cell penetrating peptide operably linked to each peptide of SEQ ID NO: 1. The cell penetrating peptide is, in certain embodiment, is selected from the group consisting of a HIV-TAT peptide, a penetratin peptide, a polyarginine peptide, a pep-1 peptide, and a transportin peptide. In some embodiments, the cell-penetrating peptide is a HIV-TAT peptide and, in some of those embodiments, each peptide monomer comprises the sequence of SEQ ID NO: 5.

[0010] Pharmaceutical compositions are further provided and comprise a non-naturally occurring protein dimer formed of two peptide monomers having a sequence of SEQ ID NO: 1. A pharmaceutically-acceptable vehicle, carrier, or excipient is, in certain embodiments, further included in the pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as, in some embodiments, a chemotherapeutic agent, a toxin, an immunological response modifier, an enzyme, and a radioisotope.

[0011] In further embodiments of the presently-disclosed subject matter are methods for treating a Src-associated disease. In some embodiments, a method of treating a Src-associated disease is provided that comprises administering to a subject in need thereof an effective amount a non-naturally occurring protein dimer described herein and which is formed of two monomers of a peptide having a sequence of SEQ ID NO: 1 or a functional fragment or functional variant thereof. In some embodiments, the Src-associated disease is selected from cancer, vascular disease, cardiovascular disease, tissue fibrosis, osteoporosis, chronic kidney disease, retinopathy, anemia, obesity, and/or pulmonary hypertension. In some particular embodiments, the Src- associated disease is cancer or the Src-associated disease is cardiovascular disease.

|0012] Still further provided, in some embodiments of the presently-disclosed subject matter are methods for reducing Src activity in a cell. In some embodiments, a method for reducing Src activity in a cell is provided that comprises contacting a cell with an effective amount of a protein dimer described herein (i.e., a protein dimer formed of two monomers of a peptide having a sequence of SEQ ID NO: 1 or a functional fragment or functional variant thereof). In some embodiments, the cell contacted by the protein dimer and in which Src activity is reduced is selected from the group consisting of a monocyte, a heart cell, a liver cell, a vascular cell, a breast cell, a prostate cell, a kidney cell, a muscle cell, a brain cell, bone cell, and a tumor cell.

|0013] Further features and advantages of the present invention will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is an image showing the formation of a pNaKtide dimer with H2O2 treatment, where treatment with dithiothreitol (DTT) is shown reversing the formation of the dimer (Lane l=pH 7.0, Lane 2=pH 7.8 (stored in -20 in IxPBS for 4 days), Lane 3=pH 8.0, H2O2 (25mM, Ihr, room temperature), Lane 4=pH 8.0, H2O2 (25mM, Ihr, room temperature) + DTT lOOmM, Lane 5=pH 8.0, H2O2, then pH 7.0 by 3 kD filter (0 hour), Lane 6=pH 8.0, H2O2, then pH 7.0 by 3 kD filter (17 hour); all samples with sample loading buffer, 85 deg heat for 2 min). [0015] FIG. 2 is an image showing that the Cys/Ala mutant variant of pNaKtide cannot form the pNaKtide Dimer (Lane l=pH 7.0, Lane 2=pH 8.0, H2O2 (25mM, Ihr, room temperature), Lane 3=pH 7.0, Lane 4 = pH 8.0, H2O2 (25mM, Ihr, room temperature), Lane 5=pH 7.0 + DTT, Lane 6= pH 8.0, H2O2 (25mM, Ihr, room temperature) + DTT lOOmM; Lane 1&2: sample loading buffer, w/o DTT, w/o heating; Lane 3&4: sample loading buffer, w/o DTT, 85 deg heat for 2 min; Lane 5&6: sample loading buffer, w/DTT (lOOmM), 85 deg heat for 2min.)

[0016] FIG. 3 is a graph showing that as compared with a pNaKtide monomer, the pNaKtide dimer is a more potent inhibitor of c-Src.

[0017] FIG. 4 is a graph showing that as compared with a pNaKtide monomer, the pNaKtide dimer is a more potent inhibitor of c-Src activity, and further showing that the Cys/Ala mutant variant of a pNaKtide (C428A) monomer is a much less potent inhibitor of c-Src activity.

10018] FIG. 5 is a graph showing quantitative data of adipogenesis measured as the relative absorbance of Oil Red O stain.

[0019] FIG. 6 is a graph showing quantitative analysis of the pro-inflammatory cytokine, MCP-1.

[0020] FIG. 7 is a graph showing pNaKtide or pNaKtide dimer administration improves the murine sepsis score (MSS) of C57BL6 mice with cecal ligation and puncture (CLP)-induced experimental sepsis.

[0021] FIG. 8 includes graphs showing pNaKtide or pNaKtide dimer administration improves blood pressure in C57BL6 mice with CLP-induced experimental sepsis.

[0022] FIG. 9 is a schematic diagram showing the formation of a dimer comprised of two

NaKtide monomers in accordance with the presently-disclosed subject matter. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0023] The following is a brief description of the Sequence Listing that is hereby incorporated by reference in its entirety.

[0024] SEQ ID NO: 1 is an amino acid sequence encoding an embodiment of a polypeptide in accordance with the presently-disclosed subject matter (NaKtide, SATWLALSRIAGLCNRAVFQ);

[0025] SEQ ID NO: 2 is an amino acid sequence encoding a TAT cell penetrating peptide (GRKKRRQRRRPPQ);

[0026] SEQ ID NO: 3 is an amino acid sequence encoding a penetratin (AP) cell penetrating peptide (RQIKIWFQNRRMKWK K);

[0027] SEQ ID NO: 4 is an amino acid sequence encoding the N-terminal poly-lysine domain of the al subunit of Na/K-ATPase (AIN, KKGKKGKK); and

[0028] SEQ ID NO: 5 is an amino acid sequence encoding an embodiment of another polypeptide in accordance with the presently-disclosed subject matter (pNaKtide, GRKKRRQRRRPPQSATWLALSRIAGLCNRAVFQ).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

|0029] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0030] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0031 J Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0032] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0033] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0034] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).

[0035] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0036] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® , UNIPROT, and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the fding date of this Application.

[0037] The present application can “comprise” (open ended), “consist of’ (closed ended), or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0038] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0039] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

|0040] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0041] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0042] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0043] The presently-disclosed subject matter is based, at least in part, on the discovery that a protein dimer formed from two monomers of a polypeptide antagonist of the Na/K-ATPase/Src receptor complex (referred to herein as the NaKtide or pNaKtide (SEQ ID NOS: 1 and 5, respectively) can be efficiently prepared and exhibits significantly greater ability to inhibit c-Src activity as compared to the individual monomers alone. Studies further revealed that the protein dimers of the presently-disclosed subject matter had a greater ability to block the Na/K-ATPase signaling mediated oxidant amplification loop in both in vitro and in vivo conditions as compared to the monomer alone.

[0044] In some embodiments, the presently-disclosed subject matter thus includes dimeric polypeptide antagonists of the Na/K-ATPase-Src receptor complex. In particular, certain embodiments of the presently-disclosed subject matter include dimeric polypeptide antagonists of the Na/K-ATPase-Src receptor complex and the use of such protein dimers in compositions and methods for treating a Src-associated disorder and for reducing or otherwise modulating Src activity in a cell.

|0045] The term ‘"protein dimer” and grammatical variations thereof is used herein to refer a macromolecular complex formed by two peptide monomers, or single proteins, and which can be covalently or non-covalently bound to one another, as described in further detail below, to create a larger macromolecule or protein. The terms “polypeptide,” “protein,” and “peptide,” which are used interchangeably herein, of course refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product. Thus, exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

[0046] The terms “polypeptide fragment” or “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.

[0047] A fragment can also be a “functional fragment,” in which case the fragment retains some or all of the activity of the reference polypeptide as described herein. For example, in some embodiments, a functional fragment of the amino acid sequence of SEQ ID NO: 1 retains some or all of the ability of the reference polypeptide to bind to the SH2 domain of Src kinase.

[0048] The terms “modified amino acid,” “modified polypeptide,” and “variant” refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., one or more amino acid substitutions. A variant of a reference polypeptide also refers to a variant of a fragment of the reference polypeptide, for example, a fragment wherein one or more amino acid substitutions have been made relative to the reference polypeptide. A variant can also be a “functional variant,” in which the variant retains some or all of the activity of the reference protein as described herein. For example, a functional variant of the amino acid sequence of SEQ ID NO: 1 retains some or all of the ability of the reference polypeptide to inhibit Src activity.

|0049] The term functional variant includes a functional variant of a functional fragment of a reference polypeptide. The term functional variant further includes conservatively substituted variants. The term “conservatively substituted variant” refers to a peptide comprising an amino acid residue sequence that differs from a reference peptide by one or more conservative amino acid substitutions, and maintains some or all of the activity of the reference peptide as described herein. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine, or methionine for another; the substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. The phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

[0050] The terms “isolated” or “non-naturally occurring” when used herein in the context of a nucleic acid or a polypeptide, is a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated and/or non-naturally occurring nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell. In some embodiments, when referring to a protein dimer of the presently-disclosed subject matter, the terms “isolated” and non-naturally occurring” can be used interchangeably.

|0051] In some embodiments, a non-naturally occurring protein dimer is provided that is comprised of two monomers of a NaKtide peptide with each monomer having the sequence of SEQ ID NO: 1. In some embodiments, the present dimeric polypeptides include polypeptide monomers that share at least 75% homology with the NaKtide polypeptide of SEQ ID NO: 1. In some embodiments, the polypeptide monomers share at least 85% homology with the NaKtide polypeptide of SEQ ID NO: 1. In some embodiments, the polypeptide monomers share at least 90% homology with the NaKtide polypeptide of SEQ ID NO: 1. In some embodiments, the polypeptide monomers share at least 95% homology with the NaKtide polypeptide of SEQ ID NO: 1

[0052] “Percent identity, “ or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul. Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al.

[0053] In some embodiments of the presently-disclosed subject matter, an isolated and non- naturally occurring polypeptide dimer is provided that further comprises a cell penetrating peptide operatively linked to each polypeptide monomer of SEQ ID NO: 1, such that the peptide of SEQ ID NO: 1 can be provided as part of a fusion peptide that is capable of entering a cell and binding to the Src kinase. The term “fusion protein” is intended to describe at least two polypeptides, typically from different sources, which are operatively linked. With regard to the polypeptides, the term “operatively linked” is intended to mean that the two polypeptides are connected in a manner such that each polypeptide can serve its intended function. Typically, the two polypeptides are covalently attached through peptide bonds and can be produced by standard recombinant or chemical synthesis techniques. For example, using recombinant techniques, a DNA molecule encoding a first polypeptide can be directly ligated to another DNA molecule encoding the second polypeptide, and the resultant hybrid DNA molecule can be expressed in a host cell to produce the fusion protein. The DNA molecules are generally ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e., the DNA molecules are ligated to each other in-frame).

[0054] The term “cell penetrating peptide” (CPP) is used herein to generally refer to short peptides that can or that assist in facilitating the transport of molecular cargo across plasma membranes found in a cell. In some instances, the molecular cargo includes another polypeptide, such as the polypeptides described herein. Of course, the cell penetrating peptides can be conjugated to the molecular cargo (e.g., polypeptide) via any number of means, including covalent bonds and/or non-covalent bonds. In a number of instances, however, such cell penetrating peptides will often include a relatively high concentration of positively-charged amino acids, such as lysine and arginine, and will have a sequence that contains an alternating pattern of charged (polar) and non-charged amino acids.

[0055] In some embodiments of the presently-disclosed subject matter, an exemplary leader sequence or cell-penetrating peptide can include the trans-activating transcriptional activator (TAT) cell penetrating peptide, which is represented by the sequence of SEQ ID NO: 2. Addition of a TAT sequence to NaKtide generates pNaKtide. Another exemplary leader sequence includes penetratin (AP), which is represented by the sequence of SEQ ID NO: 3. Yet another exemplary leader sequence includes an amino acid sequence encoding the N-terminal poly-lysine domain of the al subunit of Na/K-ATPase (AIN), which is represented by the sequence of SEQ ID NO: 4 Those of ordinary skill will appreciate though that other leader sequences, including other cell penetrating peptides, can also be used in conjunction with the presently-disclosed polypeptides. In some embodiments, a polypeptide including a leader sequence, such as a cell penetrating peptide, attached to the NaKtide sequence of SEQ ID NO: 1 is referred to herein as a pNaKtide (e.g., SEQ ID NO: 5;

GRKKRRQRRRPPQSATWLALSRIAGLCNR.AVFQ, which includes the TAT cell penetrating peptide of SEQ ID NO: 2 fused to the NaKtide sequence of SEQ ID NO: 1). For additional information and guidance regarding the NaKtide and pNaKtide, see, e.g., U.S. Patent Nos. 8,283,441; 8,691,947; 8,835, 171; 8,906,891; 8,981,051; 9,072,751; 9,114, 126; 9,492,463; 9,226,952; and 9,663,561, each of which are incorporated herein by reference in their entirety.

[0056] In some embodiments of the presently-disclosed protein dimers, each polypeptide monomer described herein for use in constructing the dimer comprises a peptide monomer of SEQ ID NO: 5. Such peptide dimers can be constructed for instance by exposing the monomers to hydrogen peroxide treatment to thereby cause the formation of disulfide bonds between the two monomers. In particular, and without wishing to be bound by any particular theory or mechanism, it is believed that the cysteine residues present in the NaKtide monomers interact with one another via the sulfhydryl group (-SH) present in the two monomers in the presence of H2O2, which, in turn, leads to the formation of a disulfate bond (-S-S-) and the dimer. Indeed, as described in further detail below, experimental results revealed that a monomer variant Cys428/Ala mutant cannot form the dimer (see, e.g., FIG. 2), which indicated the role of the -SH group in the formation of the dimer. This finding was further confirmed by the observation that while an increase in H2O2 introduced by glucose oxidase decreased the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, pretreatment with a pNaKtide monomer was able to reverse or increase the GSH/GSSG ratio.

|0057] In addition to the non-naturally occurring proteins themselves, the presently-disclosed subject matter further includes and makes use of pharmaceutical compositions comprising the polypeptide dimers described herein as well as a pharmaceutically-acceptable vehicle, carrier, or excipient. Indeed, when referring to certain embodiments herein, the terms “polypeptide” and/or “composition” can be used interchangeably herein to refer to a pharmaceutical composition that includes the polypeptide dimers.

[0058] The term “pharmaceutically-acceptable carrier” as used herein refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

[0059] Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of polypeptide to biodegradable polymer and the nature of the particular biodegradable polymer employed, the rate of polypeptide release can be controlled. Depot injectable formulations can also be prepared by entrapping the polypeptide in liposomes or microemulsions, which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0060] Suitable formulations can further include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0061] The compositions can also take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the polypeptides can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0062J The formulations can be presented in unit-dose or multi -dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.

[0063] For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fdlers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods known in the art.

[0064] Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration, the compositions can take the form of tablets or lozenges formulated in a conventional manner. [0065] The compositions can also be formulated as a preparation for implantation or injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). The compounds can also be formulated in rectal compositions, creams or lotions, or transdermal patches.

[0066] In some embodiments, the compositions described herein, in addition to including a peptide dimer of the presently-disclosed subject matter, can also include at least one additional therapeutic agent. As used herein, the term “therapeutic agent” is used to refer to an agent that is capable of “treating” a disease, as defined herein below. In some embodiments, the therapeutic agent can comprise an agent that is useful to a treat a disease selected from the group consisting of: cancer; vascular disease, cardiovascular disease; heart disease; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; ischemia/reperfusion injury; osteoporosis; chronic kidney disease; retinopathy; pulmonary hypertension; and other Src-related diseases. In some embodiments, the additional agent is selected from the group consisting of: chemotherapeutic drug; a toxin; an immunological response modifier; an enzyme; and a radioisotope.

[0067] In some embodiments of the presently-disclosed subject matter, the therapeutic agent that is combined with a peptide of the presently-disclosed subject matter is a chemotherapeutic agent. Examples of chemotherapeutic agents that can be used in accordance with the presently- disclosed subject matter include, but are not limited to, platinum coordination compounds such as cisplatin, carboplatin or oxalyplatin; taxane compounds, such as paclitaxel or docetaxel; topoisomerase I inhibitors such as camptothecin compounds for example irinotecan or topotecan; topoisomerase II inhibitors such as anti-tumor podophyllotoxin derivatives for example etoposide or teniposide; anti-tumor vinca alkaloids for example vinblastine, vincristine or vinorelbine; anti-tumor nucleoside derivatives for example 5 -fluorouracil, gemcitabine or capecitabine; alkylating agents, such as nitrogen mustard or nitrosourea for example cyclophosphamide, chlorambucil, carmustine or lomustine; anti-tumor anthracycline derivatives for example daunorubicin, doxorubicin, idarubicin or mitoxantrone; HER2 antibodies for example trastuzumab; estrogen receptor antagonists or selective estrogen receptor modulators for example tamoxifen, toremifene, droloxifene, faslodex or raloxifene; aromatase inhibitors, such as exemestane, anastrozole, letrazole and vorozole; differentiating agents such as retinoids, vitamin D and retinoic acid metabolism blocking agents (RAMBA) for example accutane; DNA methyl transferase inhibitors for example azacytidine; kinase inhibitors for example flavoperidol, imatinib mesylate or gefitinib; farnesyltransferase inhibitors; HD AC inhibitors; other inhibitors of the ubiquitin-proteasome pathway for example VELCADE® (Millennium Pharmaceuticals, Cambridge, MA); or YONDELIS® (Johnson & Johnson, New Brunswick, NJ).

[0068] Further provided, in some embodiments of the presently-disclosed subject matter, are methods of treating a Src-associated disease or, in other words, diseases caused or whose symptoms are caused, at least in part, by the actions of Src. In some embodiments, a method of treating a Src-associated disease is provided that comprises administering an effective amount of a composition described herein (e.g., a protein dimer comprised of two peptide monomers each having the sequence of SEQ ID NO: 1) to a subject in need of such treatment. As used herein, the terms “treatment” or “treating” relate to any treatment of a condition of interest (e.g., a cancer), including but not limited to prophylactic treatment and therapeutic treatment. As such, the terms “treatment” or “treating” include, but are not limited to: reducing the prevalence of a condition of interest or the development of a condition of interest; inhibiting the progression of a condition of interest; arresting or reducing the further development of a condition of interest; reducing the severity of a condition of interest; ameliorating or relieving symptoms associated with a condition of interest; and causing a regression of a condition of interest or one or more of the symptoms associated with a condition of interest in a subject.

[0069] As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.

[0070] In some embodiments, the Src-associated disease is selected from the group consisting of cancer, vascular disease, cardiovascular disease, tissue fibrosis, osteoporosis, chronic kidney disease, retinopathy, anemia, obesity, and pulmonary hypertension. In some embodiments, the Src-associated disease is cardiovascular disease, and the cardiovascular disease is selected from the group consisting of heart disease, cardiac hypertrophy, congestive heart failure, and ischemia-reperfusion injury.

[0071J In some embodiments, the Src-associated disease is cancer. In some embodiments, treating a cancer can include, but is not limited to, killing cancer cells, inhibiting the development of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the available blood supply to a tumor or cancer cells, promoting an immune response against a tumor or cancer cells, reducing or inhibiting the initiation or progression of a cancer, or increasing the lifespan of a subject with a cancer.

[0072] As used herein, the term “cancer” refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas, melanoma, and sarcomas. By "leukemia" is meant broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, l ' l myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0073] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signetring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0074] The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0075] The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,

Harding-Passey melanomajuvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.

[0076] Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer. In some embodiments, the cancer is selected from the group consisting of prostate cancer, breast cancer, and neuroblastoma.

|0077] For administration of a therapeutic composition as disclosed herein, conventional methods of extrapolating human dosage based on doses administered to a murine animal model can be carried out using the conversion factor for converting the mouse dosage to human dosage: Dose Human per kg = Dose Mouse per kg / 12 (Freireich, et al., (1966) Cancer Chemother Rep. 50: 219-244). Doses can also be given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions. Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as described by Freireich, et al. (Freireich et al., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the dose by the appropriate kg factor. In an adult human, 100 mg/kg is equivalent to 100 mg/kg x 37 kg/sq m=3700 mg/m2.

15 [0078] Suitable methods for administering a therapeutic composition in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and/or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, dermally (e.g., topical application), intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection/bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Patent No. 6,180,082). In some embodiments of the therapeutic methods described herein, the therapeutic compositions are administered orally, intravenously, intranasally, or intraperitoneally to thereby treat a disease or disorder.

|0079] Regardless of the route of administration, the compositions of the presently-disclosed subject matter typically not only include an effective amount of a therapeutic agent, but are typically administered in amount effective to achieve the desired response. As such, the term “effective amount” is used herein to refer to an amount of the therapeutic composition (e.g., a protein dimer comprised of two peptide monomers of SEQ ID NO: 1 and a pharmaceutically vehicle, carrier, or excipient) sufficient to produce a measurable biological response (e.g., an increase in Src inhibition). Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and/or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of doselimiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.

[0080] For additional guidance regarding formulation and dose, see U.S. Patent Nos. 5,326,902; 5,234,933; PCT International Publication No. WO 93/25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, New Jersey; Goodman et al., (1996) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pennsylvania; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland/ Philadelphia; Duch et al., (1998) Toxicol. Lett. 100-101 :255-263.

[0081] By virtue of the antagonistic activity of the protein dimers described herein, in some embodiments, methods for reducing Src activity in a cell can also be provided that comprise the step of contacting a cell with an effective amount of a protein dimer described herein (e g., a protein dimer comprised of two peptide monomers of SEQ ID NO: 1). In some embodiments, the cell is selected from the group consisting of a monocyte, a heart cell, a liver cell, a vascular cell, a breast cell, a prostate cell, a kidney cell, a muscle cell, a brain cell, and a tumor cell. [0082] Various methods known to those skilled in the art can be used to assess Src activity in a cell. Furthermore, although certain embodiments of the methods disclosed herein only call for a qualitative assessment (e.g., the presence or absence of Src activity), other embodiments of the methods call for a quantitative assessment (e.g., an amount of Src activity in a subject or a cell or tissue of the subject). Such quantitative assessments can be made, for example, using any number of methods, as will be understood by those skilled in the art.

[0083] With respect to the administration of or the contacting of a cell with the compositions described above to reduce an amount of Src activity, the terms “reduce,” “reducing,” or “reduction” when used herein in reference to Src activity are used to refer to any decrease or suppression in the amount or rate of Src activity. Of course, it is understood that the degree of reduction need not be absolute (i.e., the degree of inhibition need not be a complete prevention of Src activity) and that intermediate levels of a reduction in Src activity are contemplated by the presently-disclosed subject matter. As such, in some embodiments, the reduction in Src activity can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0084] The skilled artisan will also understand that measuring a reduction in the amount of a certain feature (e.g., Src activity) or an improvement in a certain feature (e.g., tumor size) in a subject is a statistical analysis. For example, an increase in an amount of Src inhibition in a subject can be compared to control level of Src activity, and an Src inhibition of less than or equal to the control level can be indicative of a reduction in the amount of Src activity, as evidenced by a level of statistical significance. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Preferred confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0085] The practice of the presently-disclosed subject matter can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984; Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987; Handbook Of Experimental Immunology, Volumes LIV, D. M. Weir and C. C. Blackwell, eds., 1986.

[0086] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.

EXAMPLES

[0087] Experiments were undertaken to construct and assess the function of a pNaKtide dimer’s ability to control the Na/K-ATPase signaling mediated oxidant amplification loop. As described in further detail below, a dimer of pNaKtide (with about 95% purity) was prepared using a pNaKtide monomer (SEQ ID NO: 5). Compared with the pNaKtide monomer, the prepared dimer showed a significantly higher power to inhibit c-Src activity. Studies also showed that the dimer was more powerful in blocking the Na/K-ATPase signaling mediated oxidant amplification loop in both in vitro and in vivo conditions.

[0088] It was appreciated that the Na+/K+-ATPase signaling-mediated oxidant amplification loop contributes to uremic cardiomyopathy and anemia induced by 5/6 ^th partial nephrectomy (PNx) in mice, which can be prevented by administration of pNaKtide or lentiviral delivery of NaKtide (i.e., the pNaKtide without the HIV TAT leader sequence). The Na/K-ATPase functions not only as a physiological ion transporter, but it also functions as a signaling transducer leading to generation of reactive oxygen species (ROS) and oxidative modification of proteins. The (p)NaKtide, however, which was created from the N domain of the Na/K-ATPase al subunit, can inhibit the Na/K-ATPase signaling-mediated feed forward amplification loop in the generation of ROS, resulting in prevention of chronic kidney disease (CKD)-mediated anemia and attenuation of inflammation and cardiac/renal fibrosis.

[0089] Example 1 - Preparation of pNaKtide Dimer.

[0090] To prepare a pNaKtide dimer, a pNaKtide monomer (SEQ ID NO: 5) was first dissolved in lx PBS (pH 7.0) at concentration of 10 mg/mL as a stock solution. A reaction solution was then prepared by adding the pNaKtide stock solution to lx PBS (pH 8.0) (1 :9, vol/vol) to make Img/mL solution. H2O2 (final concentration 25 mM) was then added to the reaction solution and was allowed to react at room temperature for 60 minutes. The reaction solution was then washed three times with lx PBS (pH 7.0) by passing the solution through a 3 kD filter. The protein concentration was then subsequently measured and evaluated through the use of electrophoresis with GelCode Blue stain (Thermo Fisher Scientific, Waltham, MA, USA), where a 16% Tris-SDS gel and SeeBlue Prestained standard (Thermo Fisher Scientific) was used with 0.5-2 pg of the peptide and where the samples were run and stained with GelCode Blue according to the standard protocol from the manufacturer. Images of the gels were then obtained with FluorChem M imager (Bio-Techne, Minneapolis, MN) followed by quantification calculated with AlphaView software (Protein Simple Corporation, San Jose, CA, USA). As seen in FIG. 1, the pNaKtide dimer was successfully formed through the use of H2O2 and its formation was reversed using dithiothreitol (DTT). In contrast to what was observed in FIG. 1, the use of a pNaKtide mutant including Cys/Ala mutation at position 14 or the Cys428 (ATI A1 HUMAN, Uniprot Entry number P05023-1) of the NaKtide monomer revealed that such a variant NaKtide was unable to form a dimer as shown in FIG. 2 and FIG. 9. In this regard, it was belived that there was a Cys428 to Cys428 interaction through the sulhydryl group (-SH) in the presence of H2O2, leading to the formation of dimer.

[0091J Example 2 - pNaKtide Dimer is More Potent than the pNaKtide Monomer in Inhibiting c-Src Activity.

[0092] Experiments were further undertaken to assess the ability of the pNaKtide dimer to inhibit c-Src. Briefly, ADP-Glo™ Src kinase activity assay kits were obtained from Promega

(Madison, WI, USA) with the assay being performed according to the manufacturer’s instructions. pNaKtide monomer, dimer, and the Cys/Ala mutant variant of pNaKtide (C428A) were tested in parallel for the purpose of comparison. pNaKtide dimer was washed with the buffer that was used for monomer and C428A, to remove possible effect of H2O2. FIG. 3 shows that the pNaKtide dimer was a much more potent inhibitor of c-Src activity than the monomer, while FIG. 4 shows that a Cys/Ala mutant variant of pNaKtide (C428A) was not a potent inhibitor of c-Src activity.

[0093] Example 3 - Effect of various doses of pNaKtide and Dimer on lipid accumulation and inflammatory cytokine level in 3T3L1 cells.

[0094] To assess the effect of various doses of the pNaKtide and the pNaKtide Dimer on lipid accumulation and inflammatory cytokine levels, frozen mouse pre-adipocytes (3T3L-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). After thawing, 3T3L-1 cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA, USA). The cells were plated at a density of 0.05 x 10 ⁶ cells per well in a 24 well plate. The cultures were maintained at 37 °C in a 5% CO2 incubator. Upon attaining 60-70 % confluence, the medium was replaced with adipogenic medium and the cells were cultured for an additional seven days. The cells were treated everyday with varying concentrations of pNaKtide ( 0. 1 pM, 0.5 pM and 1 pM) and Dimer (0.06 pM, 0.25 pM, 0.5 pM).

|0095] Oil Red O (0.21%) in isopropanol (100%) was used for staining of cells differentiated for 6 days in the presence of the adipogenic medium. Briefly, the cells were washed and fixed in 10% formaldehyde for 15 min. The fixed droplets were then stained with Oil Red O solution for 20 min at room temperature, followed by rinsing with PBS. The Oil Red O was eluted by the addition of 100% isopropanol for 10 min in a shaker. Lipid accumulation was measured as the relative absorbance of Oil Red O staining (OD = 490 nm). As shown in FIG. 5, the lipid accumulation was significantly decreased in cells treated with pNaKtide (0.5 pM and 1 pM) and the pNaKtide Dimer (0.25 pM, 0.5 pM) in a dose dependent manner, when compared to control cells (FIG. 1). However, no marked difference was observed in cells treated with the lowest concentrations of pNaKtide (0.1 pM) and Dimer (0.06 pM,) (Values are represented as mean ± SEM (n=6-16). **p<0.01 vs. Control, *p<0.05 vs. Control.)

[0096] For cytokine level estimation, the level of monocyte chemoattractant protein-1 (MCP-1) was determined in conditioned medium using an Enzyme Linked Immunosorbent Assay (ELISA) kit according to the manufacturer’s protocol (Abeam, Cambridge, MA). As shown in FIG. 6, the level of MCP-1 was significantly decreased in cells treated with pNaKtide (0.5 pM) and Dimer (0.06 pM and 0.25 pM). A superior effect was observed with the use of the pNaKtide dimer at a concentration of 0.25 pM (FIG. 2). Though, no significant change was observed in the lowest concentration of pNaKtide (0.1 pM) treated cells. (Values are represented as mean ± SEM (n=6). **p<0.01 vs. Control, *p<0.05 vs. Control.)

[0097] To further assess the effect of the pNaKtide dimer on inflammation, a mouse model of cecal ligation and puncture (CLP)-induced experimental sepsis was utilized followed by assessment using the murine sepsis score (MSS). As detailed in Table 1 below, the murine sepsis score (MSS) developed by Shrum et al., 2014 (PMID: 24725742), is a well-established methodology which involves assessment of seven clinical variables in mice undergoing CLP surgery, and is a reliable way to demonstrate disease progression and mortality in mice undergoing CLP. As part of the experiment, all mice were assessed for MSS at baseline (before Sham or CLP), at 4 hours, 8 hours, and 24 hours after Sham or CLP. Systemic administration of pNaKtide or the pNaKtide Dimer demonstrated improved MSS at 8 hours as well as 24 hours following CLP surgery, as compared to CLP mice without pNaKtide or Dimer as shown in FIG.

7. Statistical significance is shown as: *p<0.05, **p<0.01 vs Sham; #p<0.05, ##p<0.01 vs CLP.

|0098] Further, the systolic and diastolic blood pressure was measured by tail cuff method using CODA 8-Channel Blood Pressure system (Kent Scientific, Boston, MA, USA). Blood pressure was assessed in each experimental group at baseline (before Sham or CLP surgery), at 8 hours, and 24 hours post Sham or CLP surgery. The systolic and diastolic blood pressure was significantly higher in mice injected with dimer before the surgery at baseline. As shown in FIG.

8, the results showed significantly reduced systolic and diastolic blood pressure in CLP mice 8 hours post-surgery, as compared to Sham. pNaKtide and dimer administration showed significant improvement in blood pressure of CLP mice at 8 hours, as compared to CLP alone. Consequently, assessment of blood pressure 24 hours post-surgery also showed significant reduction in systolic and diastolic blood pressure in CLP mice, as compared to Sham. pNaKtide and pNaKtide Dimer administration significantly improved systolic blood pressure in CLP mice at 24 hours, as compared to CLP alone. Each bar represents result as means ± SEM. Statistical significance is shown as: *p<0.05, **p<0.01 vs Sham; #p<0.05, ##p<0.01 vs CLP.

Table 1. Murine Sepsis Score (MSS) to assess the severity of disease in an experimental model of fecal-induced peritonitis.

Variable Score and Description

Appearance:

0 - Coal is smooth.

1 - Patches of hair piloerected.

2 - Majority of back is piloerected. 3 - Piloerection may or may not be present, mouse appears “puffy”.

4 - Piloerection may or may not be present, mouse appears emaciated.

Level of consciousness:

0 - Mouse is active.

1 - Mouse is active but avoids standing upright.

2 - Mouse activity is noticeably slowed. The mouse is still ambulant.

3 - Activity is impaired. Mouse only moves when provoked, movements have a tremor.

4 - Activity severely impaired. Mouse remains stationary when provoked, with possible tremor.

Activity:

0 - Normal amount of activity. Mouse is any of eating, drinking, climbing, running, fighting.

1 - Slightly suppressed activity. Mouse is moving around bottom of cage.

2 - Suppressed activity. Mouse is stationary with occasional investigative movements.

3 - No activity. Mouse is stationary.

4 - No activity. Mouse experiencing tremors, particularly in the hind legs.

Response to Stimulus:

0 - Mouse responds immediately to auditory stimulus or touch.

1 - Slow or no response to auditory stimulus; strong response to touch (moves to escape).

2 - No response to auditory stimulus; moderate response to touch (moves a few steps).

3 - No response to auditory stimulus, mild response to touch (no locomotion).

4 - No response to auditory stimulus. Little or no response to touch. Cannot right itself if pushed over.

Eyes:

0 - Open.

1 - Eyes not fully open, possibly with secretions.

2 - Eyes at least half closed, possibly with secretions. 3 - Eyes half closed or more, possibly with secretions.

4 - Eyes closed or milky.

Respiration rate:

0 - Normal, rapid mouse respiration.

1 - Slightly decreased respiration (rate not quantifiable by eye).

2 - Moderately reduced respiration (rate at the upper range of quantifying by eye).

3 - Severely reduced respiration (rate easily countable by eye, 0.5 s between breaths).

4 - Extremely reduced respiration (>1 s between breaths).

Respiration quality:

0 - Normal.

1 - Brief periods of laboured breathing.

2 - Laboured, no gasping.

3 - Laboured with intermittent gasps.

4 - Gasping.

[0099] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00100] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. SEQUENCE LISTING

SEQ ID NO: 1: SATWLALSRIAGLCNRAVFQ (NaKtide, )

SEQ ID NO: 2: GRKKRRQRRRPPQ (TAT cell penetrating peptide)

SEQ ID NO: 3: RQIKIWFQNRRMKWK K (penetratin (AP) cell penetrating peptide)

SEQ ID NO: 4: KKGKKGKK (AIN, N-terminal poly-lysine domain of Na/K-ATPase al)

SEQ ID NO: 5: GRKKRRQRRRPPQSATWLALSRIAGLCNRAVFQ (pNaKtide,)