WO2013158871A1 - Use of erythropoietin and derivatives for treating hypertension - Google Patents

Abstract

Described herein is the finding that acute administration of rhEPO leads to a significant reduction in arterial blood pressure via nitric oxide signaling. It is further disclosed than an EPO derivative, helix B surface peptide (HBSP), significantly reduces blood pressure, kidney damage and myocardial fibrosis in animal models of hypertension. Provided herein are methods of treating hypertension in a subject, or lowering blood pressure in a subject, by administering a therapeutically effective amount of EPO or an EPO derivative. In some cases, the EPO derivative does not stimulate significant erythropoiesis. Exemplary EPO derivatives that do not stimulate erythropoiesis include carbamylated EPO (CEPO) and HSBP.

Description

USE OF ERYTHROPOIETIN AND DERIVATIVES FOR TREATING HYPERTENSION

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims the benefit of U.S. Provisional Application No. 61/636,547, filed April 20, 2012, U.S. Provisional Application No. 61/638,328, filed April 25, 2012, and U.S.

Provisional Application No. 61/656,698, filed June 7, 2012, all of which are herein incorporated by reference in their entirety.

FIELD

This disclosure concerns the use of erythropoietin (EPO) and EPO derivatives for reducing blood pressure and/or treating hypertension.

BACKGROUND

Erythropoietin (EPO) is a natural hormone produced by the kidney and associated with stimulation of red cell production. Recombinant human erythropoietin (rhEPO) is widely used for treatment of anemia related to cancer, surgery and other conditions associated with anemia or blood loss. In the last decade, new properties of EPO to protect cells from ischemic and other types of damage have been discovered. Numerous studies in different experimental models reported tissue- protective and specifically cardioprotective properties of rhEPO.

Nitric oxide (NO) signaling has previously been associated with EPO, and some studies suggest that rhEPO-induced cardioprotection is the result of NO activation. However, despite the involvement of NO, the primarily limiting factor of long-term rhEPO therapy is systemic hypertension resulting from increased blood viscosity and reduction of hypoxic vasodilation in rhEPO-treated anemic patients. NO activation in rhEPO patients is considered to be a factor limiting the hypertensive response through compensatory vasodilation.

SUMMARY

Disclosed herein is the finding that administration of rhEPO or an EPO derivative leads to a significant reduction in both diastolic and systolic blood pressure.

Provided herein is a method of preventing or treating hypertension in a subject, comprising selecting a subject with hypertension or at risk for hypertension and administering to the subject a therapeutically effective amount of EPO or a derivative thereof. Also provided herein is a method of lowering blood pressure in a subject by selecting a subject with elevated blood pressure and administering to the subject a therapeutically effective amount of EPO or a derivative thereof.

In some embodiments, the EPO is rhEPO. In some embodiments EPO, such as rhEPO, is administered acutely.

In some embodiments, the EPO derivative does not significantly stimulate erythropoiesis. In particular non-limiting examples, the EPO derivative comprises carbamylated EPO (CEPO) or a helix B surface peptide (HBSP).

In some embodiments, the EPO or EPO derivative is administered at a dose of about 100 to about 4000 U/kg, such as about 150 to 3000 U/kg. In other embodiments, the EPO or EPO derivative is administered at a dose of about 10 to about 100 μg/kg, such as less than about 60 /k ·

In some embodiments, the EPO or EPO derivative is administered intravenously. The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1C are graphs showing mean arterial blood pressure (FIG. 1A), systolic blood pressure (FIG. IB) and diastolic blood pressure (FIG. 1C) in rats two hours after a single bolus injection of saline or 3000 U/kg rhEPO.

FIG. 2 is a bar graph showing arterial blood pressure in rats two hours after a single bolus injection of saline (control), or 150, 750 or 3000 U/kg rhEPO. Shown are systolic blood pressure (BPs), diastolic blood pressure (BPd) and mean blood pressure (BPm).

FIG. 3 is a pair of graphs showing the results of open chest pressure-volume loop analysis in rats two hours after a single bolus injection of saline (control) or rhEPO (3000 U/kg).

FIG. 4A is a series of images showing MI size in hearts of rats treated with a single bolus injection of rhEPO, L-NAME or rhEPO + L-NAME.

FIG. 4B is graph quantitatively showing MI size in rats treated with a single bolus injection of rhEPO, L-NAME or rhEPO + L-NAME. Control rats were administered saline by bolus injection.

FIG. 5 is a graph showing systolic blood pressure in a study of Dahl salt- sensitive rats. Rats were placed on a high salt (HS) or low salt (LS) diet and either left untreated (nT), treated with HBSP at the same time as initiating the HS diet (HS+HBSP), or treated with HBSP at week 4 (HS- HBSP delayed) of the HS diet.

FIG. 6 is a series of graphs showing blood pressure of spontaneously hypertensive rats (SHR) treated with HBSP. Shown are systolic (left), diastolic (middle), and mean (right) blood pressure of SHR treated with vehicle (SHR) or SHR treated with HBSP (SHR_HBSP) at 0, 2, 4, 6, and 8 weeks following the initiation of treatment. Wistar- Kyoto rats (WKY) served as controls.

FIG. 7A is a graph quantitating collagen in myocardium of spontaneously hypertensive rats (SHR) treated with HBSP to evaluate myocardial fibrosis. Shown is the percentage of collagen in control rats (WKY), SHR treated with vehicle (SHR) and SHR treated with HBSP (SHR_HBSP).

FIG. 7B is a graph quantitating kidney lesions in spontaneously hypertensive rats (SHR) treated with HBSP. Shown is the percentage of glomeruli lesions in control rats (WKY), SHR treated with vehicle (SHR) and SHR treated with HBSP (SHR_HBSP).

FIG. 8A is a graph showing the concentration of blood nitric oxide metabolites in spontaneously hypertensive rats (SHR) treated with HBSP. Shown is the concentration (μΜ) of nitric oxide metabolites in control rats (WKY), SHR treated with vehicle (SHR) and SHR treated with HBSP (SHR_HBSP).

FIG. 8B is a graph showing blood angiotensin II in spontaneously hypertensive rats (SHR) treated with HBSP. Shown is the concentration (ng/ml) of angiotensin II in control rats (WKY), SHR treated with vehicle (SHR) and SHR treated with HBSP (SHR_HBSP).

FIG. 9 is a series of graphs showing blood pressure of Dahl's salt-sensitive rats treated with

HBSP. Shown are systolic (left), diastolic (middle), and mean (right) blood pressure of Dahl's rats consuming a high salt diet and treated with either vehicle (HS) or HBSP (HS_HBSP). Dahl's rats consuming a low salt diet and treated with vehicle (LS) served as controls.

FIG. 10A is a graph quantitating collagen in myocardium of Dahl's salt- sensitive rats treated with HBSP to evaluate myocardial fibrosis. Shown is the percentage of collagen in Dahl's salt-sensitive rats fed a low-salt diet (LS), Dahl's salt-sensitive rats fed a high-salt diet treated with vehicle (HS) and Dahl's salt- sensitive rats treated with HBSP (HS_HBSP).

FIG. 10B is a graph quantitating kidney lesions in Dahl's salt-sensitive rats treated with HBSP. Shown is the percentage of glomeruli lesions in Dahl's salt- sensitive rats fed a low-salt diet (LS), Dahl's salt- sensitive rats fed a high-salt diet treated with vehicle (HS) and Dahl's salt- sensitive rats treated with HBSP (HS_HBSP).

FIG. 11A is a graph showing the concentration of blood nitric oxide metabolites in Dahl's salt-sensitive rats treated with HBSP. Shown is the concentration (μΜ) of nitric oxide metabolites in Dahl's salt- sensitive rats fed a low-salt diet (LS), Dahl's salt-sensitive rats fed a high-salt diet treated with vehicle (HS) and Dahl's salt-sensitive rats treated with HBSP (HS + HBSP).

FIG. 11B is a graph showing blood angiotensin II in Dahl's salt-sensitive rats treated with HBSP. Shown is the concentration (ng/ml) of angiotensin II in Dahl's salt-sensitive rats fed a low- salt diet (LS), Dahl's salt-sensitive rats fed a high-salt diet treated with vehicle (HS) and Dahl's salt-sensitive rats treated with HBSP (HS + HBSP).

FIG. 12 is a series of graphs showing blood pressure of renal mass reduction (RMR) model rats treated with HBSP. Shown are systolic (left), diastolic (middle), and mean (right) blood pressure of RMR rats treated with either vehicle (RMR) or HBSP (RMR_HBSP). Sham-operated mice treated with vehicle (Sham) served as controls.

FIG. 13A is a graph quantitating collagen in myocardium of RMR rats treated with HBSP to evaluate myocardial fibrosis. Shown is the percentage of collagen in control rats (Sham), RMR rats treated with vehicle (RMR) and RMR rats treated with HBSP (RMR HBSP).

FIG. 13B is a graph quantitating kidney lesions in nephrectomized rats treated with HBSP. Shown is the percentage of glomeruli lesions in control rats (Sham), nephrectomized rats treated with vehicle (NPHR) and nephrectomized rats treated with HBSP (NPHR+HBSP).

FIG. 14A is a graph showing the concentration of blood nitric oxide metabolites in RMR rats treated with HBSP. Shown is the concentration (μΜ) of nitric oxide metabolites in control rats (Sham), RMR rats treated with vehicle (RMR) and RMR rats treated with HBSP (RMR+HBSP).

FIG. 14B is a graph showing blood angiotensin II in RMR rats treated with HBSP. Shown is the concentration (ng/ml) of angiotensin II in control rats (Sham), RMR rats treated with vehicle (RMR) and RMR rats treated with HBSP (RMR+HBSP).

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file, created on April 11, 2013, 2.24 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of HBSP.

SEQ ID NO: 2 is the amino acid sequence of mature human EPO (corresponding to residues 28-193 of GenBank^® Accession No. NP_000790). DETAILED DESCRIPTION

I. Abbreviations

AAR area at risk

ANOVA analysis of variance

BP blood pressure

CEPO carbamylated erythropoietin

EPO erythropoietin

HBSP helix B surface peptide

HS high salt

i.p. intraperitoneal

i.v. intravenous

L-NAME A^-nitro-L-arginine methyl ester

LS low salt

LV left ventricle

MI myocardial infarction

mmHg millimeters of mercury

NO nitric oxide

rhEPO recombinant human erythropoietin

RMR renal mass reduction

rrEPO recombinant rat erythropoietin

SHR spontaneously hypertensive rat

II. Terms and Methods

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Administration: The introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. In some embodiments herein, administration is acute administration, such as administration of no more than five doses, such as one, two, three, four or five doses. In other embodiments, administration is chronic administration, such as at least 2, at least 5, or at least 10 or more doses, such as daily, weekly or monthly administrations. Blood pressure: The pressure exerted by circulating blood upon the walls of blood vessels. Blood pressure generally refers to the arterial pressure of the systemic circulation. During each heartbeat, blood pressure varies between maximum (systolic) and minimum (diastolic) pressure. Blood pressure is typically measured in millimeters of mercury (mmHg). As used herein "elevated blood pressure" generally refers to a systolic blood pressure greater than 120 mmHg and/or a diastolic blood pressure greater than 80 mmHg.

Carbamylated erythropoietin (CEPO): EPO that is at least partially to fully

carbamylated. Carbamylation is a posttranslational modification of proteins resulting from the non- enzymatic reaction between isocyanic acid and primary amino groups on the N-terminal amino acids of proteins as well as the side chains of lysine or arginine residues. In some embodiments, the CEPO has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% free primary amines. Methods of making CEPO have been previously described (see, for example, U.S. Patent Application Publication No. 2008/0305990, which is herein incorporated by reference).

Erythropoiesis: The production of red blood cells. As used herein, an EPO derivative that does not stimulate "statistically significant erythropoiesis" refers to an EPO derivative that does not stimulate an amount of red blood cell production that is statistically significant compared to a control or standard, such as a known non-erythropoietic peptide.

Erythropoietin (EPO): A glycoprotein hormone that controls erythropoiesis. EPO is composed of four alpha helical bundles. The protein is found in the plasma and induces red cell production by promoting erythroid differentiation and initiating hemoglobin synthesis. EPO also has neuroprotective activity against a variety of potential brain injuries and antiapoptotic functions in several tissue types, including but not limited to myocardium. Nucleotide and amino acid sequences for EPO are publically available, such as in the GenBank^® database. For example, human mRNA and amino acid sequences are deposited under GenBank^® Accession No.

NM_000799 and NP_000790, respectively. In one non-limiting embodiment, the EPO is the mature human EPO with the amino acid sequence corresponding to residues 28- 193 of GenBank^® Accession No. NP_000790. An "EPO derivative" is any compound that is derived from EPO but includes at least one modification relative to native EPO. For example, EPO derivatives can include chemically modified EPO (e.g. , carbamylated EPO), EPO fragments (e.g. , HBSP), sequence variants or homologs. In some embodiments of the present disclosure, the EPO derivative retains tissue-protective properties associated with EPO but does not stimulate statistically significant erythropoiesis. Helix B surface peptide (HBSP): An 11 amino acid peptide (QEQLERALNSS; SEQ ID NO: 1) derived from the aqueous face of helix B of EPO. HBSP exhibits tissue-protective activities comparable to EPO, but does not stimulate erythropoiesis (Ueba et al. , Proc Natl Acad Sci USA 107(32): 14357- 14362, 2010). N-terminal glutamine (Q) residues can undergo a spontaneous, irreversible cyclization (particularly at room temperature under acidic conditions) into pyroglutamate (Yu et al., J Pharm Biomed Anal 42:455-463, 2006). Thus, in some embodiments herein, HBSP is pyroglutamate helix B surface peptide (i.e. the N-terminal glutamate is cyclized) (see, for example, Ahmet et al., Mol Med 17(3-4): 194-200, 2011; and Brines et al., Proc Natl Acad Sci USA 105(31): 10925- 10930, 2008).

Hypertension: A condition characterized by elevated arterial blood pressure. Blood pressure measurements are typically reported as a ratio of systolic pressure (arterial pressure during contraction of the heart muscle) to diastolic pressure (residual arterial pressure during relaxation of the heart muscle), reported in units of mmHg. A normal (desirable) diastolic blood pressure in humans is generally between about 60-79 mmHg and a normal (desirable) systolic blood pressure is generally between 90-119 mmHg. In some embodiments herein, a diastolic blood pressure greater than 80 mmHg, such as about 81 mmHg, 85 mmHg, 90 mmHg or 100 mmHg or greater, and/or a systolic blood pressure greater than 120 mmHg, such as about 125 mmHg, 130 mmHg, 140 mmHg or 160 mmHg or greater, is diagnostic of hypertension.

A number of factors have been implicated in the development of hypertension. These include heredity and a number of environmental factors such as salt intake, obesity, occupation, family size, and crowding. Additional factors which may modify the course of hypertension include age, race, sex, stress, diet, smoking, serum cholesterol, and glucose intolerance. The effects of hypertension are numerous, with the most severe being premature death, commonly caused by heart disease related to hypertension. Hypertension imposes an increased work load on the heart; related effects on the heart include angina pectoris, increased myocardial mass or hypertrophy (enlarged heart), and late in the disease, evidence of ischemia or infarction.

Intravenous: Administration into a vein, such as by injection or infusion.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.

The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown in the following table.

Original Residue Conservative Substitutions

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

His Asn; Gin

He Leu, Val

Leu He; Val

Lys Arg; Gin; Glu

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

Preventing, treating or ameliorating a disease: "Preventing" a disease or condition refers to inhibiting the full development of the disease or condition. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease or condition.

Recombinant: A recombinant nucleic acid or polypeptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;

Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; and Altschul et al., Nature Genet. 6: 119-129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol.

215:403-410, 1990.) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Variants and/or fragments of EPO generally comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98% or at least about 99% sequence identity with an EPO sequence. When less than the entire sequence is being compared for sequence identity, fragments will typically possess at least 80% sequence identity over the length of the fragment, and can possess sequence identities of at least 85%, 90%, 95% or 99%. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of compound, such as EPO or an EPO derivative, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to treat or ameliorate elevated blood pressure, or to measurably decrease blood pressure over a period of time, or to measurably inhibit an increase in blood pressure, in a subject. An effective amount of EPO or an EPO derivative may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound.

Unless otherwise explained, 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 disclosure

belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

All publications, patent applications, patents, and other references mentioned herein are

incorporated by reference in their entirety. All GenBank^® Accession numbers are herein

incorporated by reference as they appear in the database on April 25, 2012. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. III. Overview of Several Embodiments

It is disclosed herein that a single bolus administration of rhEPO to rats leads to a significant reduction in both diastolic and systolic blood pressure when administered at doses ranging from 150-3000 U/kg. The reduced blood pressure effect was maintained for more than two hours. The decrease in blood pressure was blocked by an inhibitor of nitric oxide (NO), an important cellular signaling molecule involved in vasodilation. It is further disclosed herein that administration of HBSP, an EPO derivative, to hypertensive rats prevents elevated blood pressure and can also significantly lower elevated blood pressure. It is further disclosed that treatment with HBSP reduces myocardial fibrosis and kidney lesions in animal models of hypertension. These results demonstrate that EPO and EPO derivatives are capable of lowering blood pressure and/or preventing and treating hypertension.

Provided herein is a method of treating or preventing hypertension in a subject by selecting a subject with hypertension or at risk for hypertension, and administering to the subject a therapeutically effective amount of EPO or an EPO derivative.

Also provided is a method of lowering blood pressure in a subject by selecting a subject with elevated blood pressure, and administering to the subject a therapeutically effective amount of EPO or an EPO derivative.

In some embodiments, the subject has a diastolic blood pressure greater than 80 mmHg and/or a systolic blood pressure greater than 120 mmHg. In some examples, the subject has a diastolic blood pressure of at least 90 mmHg, or at least 100 mmHg. In some examples, the subject has a systolic blood pressure of at least 140 mmHg, or at least 160 mmHg.

In some embodiments of the disclosed methods, the EPO is recombinant human EPO (rhEPO). In some examples, rhEPO comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2. In particular examples, rhEPO comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In some embodiments in which EPO (such as rhEPO) is administered to the subject, administration is acute administration. In some examples, acute administration includes no more than five doses, such as one, two, three, four or five doses.

In some embodiments, the subject is administered an EPO derivative. The EPO derivative can be any modified form of EPO, such as chemically modified EPO, or an EPO fragment, variant of homolog, so long as the derivative retains the ability to lower blood pressure. In some examples, the EPO derivative does not stimulate statistically significant erythropoiesis. In particular non- limiting examples, the EPO derivative comprises carbamylated EPO (CEPO) or helix B surface peptide (HBSP), such as pyroglutamate helix B surface peptide.

In some examples, the amino acid sequence of the HBSP is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of the HBSP comprises or consists of SEQ ID NO: 1. In some examples, HBSP comprises the amino acid sequence of SEQ ID NO: 1 with 1 or 2 conservative amino acid substitutions, or a deletion of 1 or 2 amino acids, so long as the HBSP retains the capacity to reduce blood pressure.

The CEPO can be partially or fully carbamylated. In some examples, the CEPO has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% free primary amines.

In some embodiments, the CEPO is human recombinant EPO (rhEPO). In some examples, the rhEPO comprises an amino acid sequence at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2. In particular examples, the rhEPO comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In some embodiments, administration of the EPO derivative comprises chronic

administration. In some examples chronic administration includes as at least 2, at least 5, or at least 10 or more doses, such as daily, weekly or monthly administrations.

The subject can be administered any suitable dose of EPO or EPO derivative to achieve the desired therapeutic endpoint, such as a particular diastolic and/or systolic blood pressure. In some embodiments, the EPO or EPO derivative is administered at a dose of about 100 to about 4000 U/kg, such as about 100, about 150, about 250, about 500, about 750, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500 or about 4000 U/kg.

In particular examples, the EPO or EPO derivative is administered at a dose of about 150 to about 3000 U/kg. In particular non-limiting examples, the EPO or EPO derivative is administered at a dose or 150, 750 or 3000 U/kg.

In some embodiments, the EPO or EPO derivative is administered at a dose of about 1 to about 200 g/kg, such as about 10 to about 100 g/kg, 20 to about 80 g/kg, 30 to about 60 g/kg, or 40 to 50 μg/kg. In particular examples, the EPO or EPO derivative is administered at a dose of less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40 or less than 30 μg/kg.

The EPO or EPO derivative can be administered using any suitable route of administration for lowering blood pressure. In some embodiments, the EPO or EPO derivative is administered intravenously. Hypertension remains a major health problem and is a serious risk factor for stroke and chronic heart failure. Antihypertensive drugs developed on the basis of a nature hormone, such as EPO, have significant advantages in comparison with existing compounds. For example, while most existing pharmaceutical compounds for treating hypertension are based on the blockade of hypertension-associated hormone production or hormone receptors, antihypertensive properties of EPO or EPO derivatives are based on activation of natural vasodilation mechanisms (i.e. via NO signaling). In addition, antihypertensive effects of EPO derivatives are associated with tissue protective properties. IV. EPO and EPO Derivatives

It is known that repeated application of rhEPO is associated with excessive development of red blood cells and increased risk of thrombotic events. However, some derivatives of EPO have been developed that do not significantly stimulate erythropoiesis, but that retain tissue-protective properties. Examples of EPO derivatives that do not promote erythropoiesis but retain tissue protective properties include, for example, carbamylated EPO (CEPO) and helix B surface peptide (HBSP) (Moon et al., Journal of Pharmacology and Experimental Therapeutics 316(3):999-105, 2006; Ahmet et al. , Mol Med 17(3-4): 194-200, 2011 ; each of which is incorporated herein by reference in its entirety). Thus, the present disclosure contemplates the acute use of rhEPO and acute or chronic use of any derivatives or variants of EPO, including derivatives or variants that are not capable of inducing significant erythropoiesis, to reduce blood pressure. In particular non- limiting examples, the EPO derivative is CEPO or HBSP.

A number of EPO variants and derivatives have been described previously, for example those disclosed in U.S. Patent Application Publication Nos. 2009/0221482; 2008/0305990;

2006/0135754; 2003/0104988; 2011/0008363; and 2008/0194475; and European Patent

Application No. 2233504, each of which is herein incorporated by reference in its entirety.

In some embodiments of the present disclosure, the EPO derivatives retain tissue protective properties but do not stimulate erythropoiesis. U.S. Patent Application Publication No.

2009/0221482 describes a number of EPO-derived peptide fragments that exhibit tissue protective properties (such as HBSP). The peptides are derived from the three dimensional structure of the EPO protein, such as from regions of EPO that face away from the ligand binding sites and/or the internal portion of the EPO receptor homodimer. Any of the EPO-derived peptide fragments disclosed in U.S. Patent Application Publication No. 2009/0221482 are contemplated for use in the present disclosure. In other embodiments of the present disclosure, the EPO derivative that retains tissue protective properties but does not significantly induce erythropoiesis is carbamylated EPO (CEPO). U.S. Patent Application Publication No. 2008/0305990 teaches a method of producing CEPO having less than about 10% (or less than about 7.5%) free primary amines on the lysine residues and the N-terminal amino acid. U.S. Patent Application Publication No. 2006/0135754 also teaches a method of producing CEPO, specifically a method of producing EPO that is nearly fully carbamylated with a low level of polymers or aggregates. CEPO produced according to the methods taught in U.S. Patent Application Publication No. 2008/0305990 or 2006/0135754 are contemplated for use in the methods disclosed herein.

U.S. Patent Application Publication No. 2011/0008363 teaches EPO variants expressed in neuronal tissue with cell protective properties, but little to no hematopoietic activity. Any of the EPO variants and homologs disclosed in U.S. Patent Application Publication No. 2011/0008363 can be used with the methods disclosed herein.

U.S. Patent Application Publication No. 2003/0104988 teaches EPO molecules with carbohydrate modifications, such as EPO having no sialic acid moieties, EPO having no N-linked or O-linked carbohydrates, and EPO having reduced carbohydrate content due to treatment with a glycosidase. Any of the EPO molecules and derivatives described in U.S. Patent Application Publication No. 2003/0104988 are contemplated for use in the present methods. The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Administration of rhEPO reduces blood pressure via NO signaling

This example describes the finding that a single bolus injection of rhEPO results in a significant reduction of arterial blood pressure in rats.

Materials, Animals and Experimental Design

Recombinant human erythropoietin, Procrit^® (rhEPO) was purchased from Amgen.

Recombinant rat erythropoietin (rrEPO) was purchased from R&D Systems (Minneapolis, MN). Two to three-month old male Wistar rats were purchased from Charles River. Experiment 1 - Arterial blood pressure during the first 2 hours after EPO administration

Ten rats were anesthetized with isoflurane (2% in oxygen), intubated and artificially ventilated. Body temperature was maintained at 37°C using a heating pad and heat lamp. The left femoral artery and vein were isolated, and a pressure catheter (Milar, IF) was inserted into the femoral artery and advanced proximally to the level of the thoracic aorta. Arterial blood pressure was recorded continuously at lk/sec using a multi-channel recording system (PowerLab). After 10 minutes of baseline recording, 3000 U/kg of rhEPO (n=6) or 1 ml of physiological saline (n=4) were injected as a bolus via the right femoral vein. Blood pressure monitoring was continued for 2 hours after drug administration.

Experiment 2 - Arterial blood pressure after different doses of EPO administration

Under general anesthesia (2% of isoflurane in oxygen) rats were injected via the femoral vein either with 3000 U/kg of rhEPO (n=7), 750 U/kg of rhEPO (n=4), 150 U/kg of rhEPO (n=4), or lml/kg of physiological saline (n=l 1). Rats were allowed to recover from anesthesia. Two hours after injection, rats were re-anesthetized and a IF pressure catheter was inserted via the femoral artery into the thoracic aorta (as described above). Arterial blood pressure was recorded for 5 minutes.

Experiment 3 - Arterial blood pressure and cardiovascular performance 2 hours after EPO administration

Under general anesthesia, 6 groups of rats were subjected to intravenous injections of 3000 U/kg of rhEPO (n=7), 10 μg/kg of rrEPO (the equivalent of 3000 U/kg rhEPO) (n=7), or 1 ml/kg of physiological saline (n=l 1). In 3 groups (n=5 in each), these injections were preceded with 15 mg/kg of a non- selective inhibitor of nitric oxide synthase (L-NAME). Rats were allowed to recover from anesthesia, but 2 hours later were re- anesthetized and hemodynamic and

cardiovascular performances were analyzed via pressure-volume loops analyses (Ahmet et ah, Circulation 110(9): 1083- 1090, 2004). Briefly, after bilateral thoracotomy in the sixth intercostal space, a 2-0 suture was placed around vena cava inferior for volume manipulation and a 1.4F- combined pressure-conductance catheter (Millar Instruments Inc., Houston, TX) was inserted into the left ventricle (LV) through the apex. Prior to collecting cardiac data, the catheter was advanced to the ascending thoracic aorta to record arterial blood pressure for 5 minutes. Then the catheter was repositioned into the LV and measurements of cardiac function were commenced. Pressure- volume loops were recorded and analyzed using PVAN analyzing software (v3.6; Millar Inc.). Traditional load-dependent hemodynamic indices, such as ejection fraction (EF), +dP/dt, -dP/dt, end-diastolic pressure (EDP), and isovolumic relaxation time constant (τ) were measured, and load- independent indices, such as end-systolic elastance (Ees), preload recruitable stroke work (PRSW), and end-diastolic stiffness (Eed) were determined or calculated. Arterial elastance (Ea) was calculated as index of vascular tension. Arterio-ventricular coupling (AV coupling), an index of cardiac work efficiency, was calculated as Ea/Ees.

Experiment 4 - Myocardial infarction size 24 hours after coronary ligation

Under general anesthesia (2% of isoflurane in oxygen), 40 rats were subjected to a permanent ligation of the coronary artery. After opening the left side of the chest, the left anterior descending coronary artery was ligated by 7-0 surgical suture at the level of the left atrial apex to induce myocardial infarction (MI), as previously described (Ahmet et ah, Heart Fail Rev 10:289- 296, 2005). Immediately after coronary ligation (<5 minutes), all animals received a single i.p. injection of either 3000 U/kg of rhEPO at (n=l l), lcc/kg of physiological saline (n=l l), 15 mg/kg of L-NAME (n=8) or both rhEPO and L-NAME (n=10). The chest was surgically closed, the residual air was evacuated through a needle puncture, and rats were weaned from anesthesia.

Twenty-four hours after surgery, rats were anesthetized by isoflurane, chests were opened bilaterally, and 2 ml of 5% Evans blue (Sigma) was injected rapidly into the left ventricle (LV) via the apex, while the aorta was tightly closed by forceps to distinguish the perfused from

underperfused areas. The hearts were removed, rapidly rinsed in phosphate-buffered cold saline, and atriums and great vessels were dissected from the ventricles. The ventricle was cut into 4 pieces along the short axis and kept in 4% triphenyltetrazolium chloride (TTC, Sigma) at 37 C for 30 minutes to distinguish the infarct area (unstained) from the area at risk (AAR, brick red stained). TTC sections were photographed, digitized, and analyzed off-line. The MI size from TTC staining sections was assessed, as described previously (Ahmet et ah, Heart Fail Rev 10:289-296, 2005) using NIH Image software. MI size was expressed as a percent of either AAR, or LV.

Statistical evaluation

Data is presented as means + SEM. Results of experiments were analyzed using one way analyses of variances (ANOVA) with post-hoc comparison via Bonferroni corrections.

Results

In a first experiment, rats (Wistar, male, 3-month old) were administered a single bolus injection of rhEPO (3000 U/kg) or saline. Mean blood pressure, systolic blood pressure and diastolic blood pressure were determined under anesthesia two hours after injection of rhEPO or saline. As shown in FIG. 1, arterial blood pressure began to fall almost immediately after an intravenous bolus injection of 3000 U/kg of rhEPO (FIG. 1A). Both systolic and diastolic pressures continued to fall until leveling off 90 minutes following the EPO injection (FIG. IB and FIG. 1C). The 30-minute averages of both systolic and diastolic blood pressure were consistently and significantly lower in rhEPO treated group compared to control.

The effect of different doses of rhEPO on blood pressure were tested in a second experiment performed using male Wistar rats. Rats were administered a single bolus injection of saline (control), 150 U/kg rhEPO, 750 U/kg rhEPO or 3000 U/kg rhEPO. Arterial blood pressure was measured under anesthesia two hours after injection. As shown in FIG. 2, a significant reduction in arterial blood pressure was observed with all three doses of EPO. These results are also shown quantitatively in Table 1 below.

Table 1. Arterial blood pressure 2 hours after intraperitoneal bolus injection of different doses of rhEPO (Mean ± SEM)

*- p<0.05; - p<0.05 vs. Control, post-hoc comparison

In a third experiment, the acute effect of EPO on hemodynamic and cardiovascular function was evaluated (FIG. 3). Bolus injections of 3000 U/kg of rhEPO or an equal dose of recombinant rat EPO (rrEPO) were administered intravenously immediately after pretreatment either with L- NAME (15 mg/kg) or with physiological saline (vehicle). Two hours after injection, rats were anesthetized and their hemodynamic and cardiovascular functions were assessed via pressure- volume loop analyses. Systolic, diastolic and mean arterial pressures were significantly lower in rats treated with either human or rat recombinant EPO compared to the control group (Table 2, column 2 or column 3 vs. column 1). All parameters reflecting cardiovascular performance were similar in rats treated with either rhEPO or rrEPO. Indices reflecting cardiac systolic function

(ESP, dP/dt+, and PRSW) were significantly lower in EPO treated rats compared with control (in rrEPO treated animals however reduction in PRSW did not reach statistical significance).

Pretreatment with L-NAME (Table 2, columns 4, 5 and 6) completely blocked the effects of EPO on blood pressure and cardiovascular performance. Table 2. Cardiovascular response 2 hours following a bolus injection of erythropoietin

*- p<0.05; One way ANOVA for four groups: H - experiment involving a human EPO (rhEPO), columns 1, 2, 4, 5; R - experiment involving a rat EPO (rrEPO), columns 1, 3, 4, 6. Values marked by underlined and bold font - p<0.05, post-hoc comparison between control and EPO- treated groups with Bonferroni correction for 2 comparisons.

In a fourth experiment, four groups of rats were subjected to a permanent ligation of descending coronary artery immediately followed by i.v. injection of one of the following:

physiological saline, rhEPO (3000 U/kg), saline+L-NAME (15 mg/kg), or rhEPO+L-NAME.

Twenty four hours following coronary ligation hearts were harvested and myocardial infarct size (MI) was measured histologically. FIG. 4A shows images of MI size in hearts from one rat from each treatment group. MI size was quantitated and the results are shown in FIG. 4B. In the untreated (control) group 24 hours after coronary ligation, the area of myocardium at risk (AAR) covered approximately 50% of the LV. AAR was similar among all groups. The resulting MI in untreated animals occupied 50% of AAR or 25% of LV. Administration of rhEPO immediately after coronary ligation resulted in 60% smaller MI than in control rats. Treatment with L-NAME without subsequent EPO administration resulted in the MI size similar to that of untreated animals. Pretreatment with L-NAME did not affect the reduction of MI size produced by rhEPO treatment. Discussion

Despite widespread agreement that EPO treatment is associated with activation of NO signaling, the studies disclosed herein are the first to demonstrate that an acute hemodynamic response to a bolus injection of rhEPO is a significant and immediate reduction of arterial blood pressure. The very short latent period of reaction suggests that the acute hemodynamic response likely resulted from triggering the fastest vasodilating signaling pathway - nitric oxide. This suggestion was confirmed by pretreatment with L-NAME, which completely blocked the acute hemodynamic response. Moreover, the hypotensive effect of rhEPO was similar in doses as high as 3000 U/kg (used is animal experiments on cardioprotection) and in doses as low as 150 U/kg (normal therapeutic dose for treatment of anemia). The present study further demonstrated that the effect of decreased blood pressure was not due to a spurious property of recombinant human EPO when administered to a rat as administration of recombinant rat EPO had the same effect.

The study disclosed herein also demonstrated a significant reduction of systolic heart function 2 hours after rhEPO administration via comprehensive pressure- volume loop analyses. The reduction of ESP, dP/dt max, and especially PRSW is consistent with a reduced arterial pressure, and indicates lower cardiac work and, therefore, suggests lower oxygen requirement of the working myocardium.

In summary, the present disclosure describes the discovery of an acute, NO-mediated, hemodynamic effect of a bolus injection of rhEPO, resulting in a reduction in blood pressure and cardiac work. However, neither activation of NO signaling, nor NO-associated hemodynamic effects mediate the cardioprotective properties of EPO.

Example 2: Administration of helix B surface peptide (HBSP) prevents and/or reduces elevated blood pressure

This example describes the finding that administration of HBSP (ARA290), an EPO derivative, prevents an elevation in blood pressure, and reduces previously elevated blood pressure in a hypertensive rat model.

HBSP is an 1 lmer peptide that is synthesized as the linear sequence of helix B of EPO (QEQLERALNSS; SEQ ID NO: 1). The N-terminal glutamine of HBSP can spontaneously undergo cyclization into pyroglutamate (Bines et al, Proc Natl Acad Sci USA 105(31): 10925- 10930, 2008).

The Dahl rat is a model of hypertension that is sensitive to dietary salt. Dahl rats placed on a high salt diet develop hypertension, which can eventually lead to heart failure and death. Dahl rats maintained on a low salt diet have normal blood pressure.

In the current study, Dahl rats were either placed on a high salt or low salt diet and either untreated or treated with HBSP (ARA290, Araim Pharmaceuticals) at a dose of 985 μg/kg/day (based on the starting weight of the rats). In treated rats, HBSP was delivered continuously through an implanted osmotic pump. Administration of HBSP began either at the same time as the initiation of the high salt diet, or began four weeks after the rats were placed on a high salt diet. As shown in FIG. 5, administration of HBSP at the same time as the high salt diet prevented an increase in systolic blood pressure. When administered at week 4 of the high salt diet, HBSP was capable of reversing the elevation in blood pressure.

These results demonstrate that HBSP, an EPO derivative, is effective for preventing and treating high blood pressure.

Example 3: Helix B surface peptide (HBSP) effectively controls elevation of blood pressure in three different experimental models of hypertension

This example demonstrates that helix B surface peptide (HBSP, ARA290) normalizes elevated blood pressure in three different animal models of hypertension.

Animal Models and Methods

HBSP treatment was evaluated in spontaneously hypertensive rats (SHR), salt sensitive Dahl's rats consuming a high salt diet, and Wistar rats subjected to a resection of 5/6^ώ of the total renal mass (RMR). Osmotic pumps delivering approximately 1 mg/kg to 0.4 mg/kg of HBSP daily (depending on body weight) for 8 weeks were implanted into the abdominal cavity of the test animals. For all three models, control rats were implanted with vehicle charged osmotic pumps. Wistar- Kyoto (WKY) rats served as control animals for the SHR model. Salt-sensitive Dahl's rats fed a low salt diet were used as controls in the Dahl rat model. For the RMR model, sham-operated animals served as controls.

In all animal models, control and treated rats were evaluated for blood pressure, myocardial fibrosis, kidney lesions, blood nitric oxide metabolites and angiotensin II during and/or at the conclusion of the 8-week treatment period (see FIGS. 6-14). Spontaneously hypertensive rat (SHR) model

Systolic, diastolic and mean blood pressure gradually increased in untreated SHR animals. In contrast, treatment with HBSP significantly decreased blood pressure in SHR animals (FIG. 6). Treatment with HBSP also significantly reduced myocardial fibrosis (FIG. 7A) and kidney lesions (FIG. 7B) in the SHR model. These data demonstrate that HBSP treatment completely normalizes elevated blood pressure, kidney damage and myocardial fibrosis in the SHR model. Measurements of NO metabolites and angiotensin II in the blood (FIGS. 8 A and 8B) suggested that blood pressure was controlled through activation of NO signaling. Dahl's salt-sensitive rat model

Systolic, diastolic and mean blood pressure gradually increased in untreated animals over the course of 8 weeks on a high salt diet. However, animals fed a high salt diet and treated with HBSP exhibited normal blood pressure, as did control rats maintained on a low salt diet (FIG. 9). Myocardial fibrosis (FIG. 10A) and kidney lesions (FIG. 10B) were also significantly reduced in HBSP-treated animals on a high salt diet, relative to untreated animals on a high salt diet. These data demonstrate that HBSP prevents high salt diet-induced elevation of blood pressure, kidney damage and myocardial fibrosis in the salt- sensitive Dahl's rat model. The increase of NO metabolites in blood (FIG. 11 A) among HBSP treated rats and the lack of change in the

concentration of angiotensin II (FIG. 1 IB) suggested than blood pressure was controlled through activation of NO signaling.

Renal mass reduction (RMR) model

Systolic, diastolic and mean blood pressure steadily increased in untreated RMR rats over the 8-week study period. However, treatment of RMR rats with HBSP significantly reduced blood pressure (FIG. 12). Treatment of RMR with HBSP also significantly reduced myocardial fibrosis (FIG. 13A); kidney lesions were also slightly reduced relative to untreated animals (FIG. 13B). These data demonstrate that HBSP significantly attenuates elevated blood pressure and normalizes myocardial fibrosis in the RMR model. The increase of the concentration of NO metabolites of HBSP treated rats (FIG. 14A) and the absence of changes in angiotensin II (FIG. 14B) suggested that the predominant mechanism for blood pressure control was the activation of NO signaling.

Summary

Systolic blood pressure (SP) gradually increased to 180-200 mmHg in untreated groups of all three experimental models. HBSP treatment resulted in complete normalization of SP in SHR, an approximately 20% reduction of SP (from 190+5 to 150+2 mmHg) in RMR, and prevented the elevation of SP in Dahl's high salt diet rats when high salt diet and treatment were initiated at the same time. In addition, massive pathological changes in kidneys and fibrosis observed in the myocardium of untreated rats were prevented or attenuated by HBSP treatment.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. A method of treating or preventing hypertension in a subject, or a method of lowering blood pressure in a subject, comprising:

selecting a subject with hypertension or at risk for hypertension, or a subject with elevated blood pressure; and

administering to the subject a therapeutically effective amount of an erythropoietin (EPO) derivative, wherein the EPO derivative does not stimulate statistically significant erythropoiesis, thereby treating or preventing hypertension in the subject.

2. The method of claim 1, wherein the EPO derivative comprises helix B surface peptide (HBSP) or carbamylated EPO (CEPO).

3. The method of claim 2, wherein the amino acid sequence of the HBSP comprises SEQ ID NO: 1.

4. The method of claim 2, wherein the amino acid sequence of HBSP consists of SEQ ID NO: 1.

5. The method of claim 2, wherein the CEPO has less than 80%, less than 60%, less than 40%, or less than 20% free primary amines.

6. The method of claim 5, wherein the CEPO is carbamylated recombinant human EPO (rhEPO).

7. The method of claim 6, wherein the rhEPO comprises an amino acid sequence at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 2.

8. The method of claim 6, wherein the rhEPO comprises the amino acid sequence of SEQ ID NO: 2.

9. The method of claim 6, wherein the amino acid sequence of the rhEPO consists of SEQ ID NO: 2.

10. The method of any one of claims 1-9, wherein the EPO derivative is administered at a dose of about 10 to about 100 g/kg.

11. The method of any one of claims 1-9, wherein the EPO derivative is administered at a dose of less than 60 μg/kg.

12. The method of any one of claims 2-4, wherein the HBSP is administered at a dose of less than 60 g/kg.

13. The method of any one of claims 1- 12, wherein the EPO derivative is administered intravenously.

14. A therapeutically effective amount of an EPO derivative for use in a method of treating or preventing hypertension.

15. A therapeutically effective amount of an EPO derivative for use in a method of lowering blood pressure.