WO2015057583A1 - Treatment of chronic kidney disease with sahps - Google Patents

Treatment of chronic kidney disease with sahps Download PDF

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Publication number
WO2015057583A1
WO2015057583A1 PCT/US2014/060304 US2014060304W WO2015057583A1 WO 2015057583 A1 WO2015057583 A1 WO 2015057583A1 US 2014060304 W US2014060304 W US 2014060304W WO 2015057583 A1 WO2015057583 A1 WO 2015057583A1
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subject
polypeptide
ckd
seq
peptide
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PCT/US2014/060304
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French (fr)
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WO2015057583A8 (en
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Peter Yuen
Ana SOUZA
Robert Star
Alexander BOCHAROV
Alan Remaley
Thomas EGGERMAN
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The United States Of America, As Represented By The Secretary
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This disclosure relates to synthetic amphipathic helical peptides, analogs thereof, and their use, for example for the treatment and inhibition of disease, such as chronic kidney disease, or dyslipidemic or vascular disorders.
  • Kidney disease including chronic and acute disease, causes over 800,000 deaths worldwide each year.
  • Acute kidney disease involves loss of kidney function typically stemming from an acute causative event (e.g. , sepsis, ischemia, trauma, and/or nephrotoxic drugs).
  • chronic kidney disease involves progressive loss of kidney function over a period of months or years.
  • the pathophysiology of kidney disease varies greatly depending on the type of disease. For example, multiple pathogenic processes such as inflammation, hypoxia, pro-fibrotic cell accumulation, extracellular matrix remodeling, and vascular drop-out have been proposed to be involved in CKD progression.
  • angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers
  • ARBs are included in current standard-of-care for CKD, they do not slow progression in about half of patients with CKD; the remaining CKD patients progress to end stage renal disease or die prematurely of other causes regardless of CKD therapy.
  • Recent novel therapeutic advances, including bardoxolone and pirfenidone and dual ACE-ARB therapy to slow down the progression of CKD have largely failed, due in part to the complexity of CKD and the divergent pathophysiologies associated with kidney disease, and/or undesirable side effects.
  • SAHP Synthetic Amphipathic Alpha-Helical Peptide
  • Differentiation 36 (CD36) antagonist are provided.
  • the subject can have CKD or be at risk of CKD.
  • the subject with or at risk of CKD is selected prior to administration of the therapeutically effective amount of the SAHP.
  • a therapeutically effective amount of a polypeptide comprising a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 3 (5A- 37pA), SEQ ID NO: 1 (L37pA), SEQ ID NO: 66 (ELK-B/B2 consensus), SEQ ID NO: 67 (ELK- B), or SEQ ID NO: 68 (ELK-B2) is administered to the subject to inhibit and/or treat CKD in a subject with or at risk of CKD.
  • polypeptides comprising SAHPs and analogs thereof have been identified and are described herein.
  • the SAHP includes multiple amphipathic alpha- helical domains and comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 66, 67, or 68.
  • a disclosed SAHP is a CD36 antagonist.
  • a disclosed SAHP is a selective CD36 antagonist that inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
  • a method of inhibiting and/or treating a dyslipidemic or vascular disorder by administering a therapeutically effective amount of a disclosed SAHP to a subject is provided.
  • the subject can be a subject with or at risk of a dyslipidemic or vascular disorder.
  • the subject is selected prior to administration of the therapeutically effective amount of the SAHP.
  • Dyslipidemic and vascular disorders amenable to treatment with the isolated SAHPs disclosed herein include, but are not limited to, hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,
  • a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, and/or SEQ ID NO: 68 is administered to the subject.
  • FIGs. 1A-1E Effects of CD36KO and 5A peptide therapy on CKD progression.
  • untreated WT mice subjected to 5/6Nx+AngII had increased levels of BUN (a) and serum creatinine (b), glomerulosclerosis (c), interstitial fibrosis (d), and (e) urinary albumin-to-creatinine ratio (ACR).
  • BUN serum creatinine
  • c glomerulosclerosis
  • d interstitial fibrosis
  • ACR urinary albumin-to-creatinine ratio
  • FIGs. 2A-2L Effects of CD36KO and 5A peptide therapy on metabolic and electrolyte abnormalities associated with CKD.
  • WT mice Four weeks after 5/6Nx with Angll infusion, WT mice had hypercalcemia (a), hyperphosphatemia (b), increased calcium x phosphorus product (c), increased levels of alkaline phosphatase (d), hypermagnesemia (e), hypoglycemia (f), and hypercholesterolemia (g). All these metabolic changes were prevented in CD36KO mice and in WT mice treated with 5A. ALT (j) and AST (k) were not different between the groups. WT mice subjected to 5/6Nx with Angll infusion had very high serum FGF-23 (1) levels, which was significantly lower in CD36KO mice.
  • FIGs. 3A-3D Effects of CKD and CK36KO on telemetry blood pressure. Mice were subjected to 5/6Nx, Angiotensin II infusion, and telemetry: WT and CD36KO. Upper graph shows model-based weekly mean values: Systolic (a), diastolic (b), and mean arterial blood (c) pressures (Y-axis) significantly increased over time (X-axis), from baseline to 4 weeks, p ⁇ 0.001, in both groups.
  • Pulse pressure (d) increased over time in the WT group (p ⁇ 0.001), but it was kept almost constant and did not significantly increase in the KO group. Differences in pulse pressure (d) between the 2 groups started to become statistically different (p ⁇ 0.05) after 2 weeks. Lower graph shows estimates by group-specific sinusoid-by-weeks model: Systolic (a), diastolic (b), and mean arterial blood (c) pressures (Y-axis) increased over time, from baseline to 4wks, p ⁇ 0.001 , in both groups.
  • time in weeks is indicated: baseline (B), week 1 (1), week 2 (2), week 3 (3), week 4 (4).
  • X-axis represents time elapsed after 13:00 (1 PM), time when recording of telemetry data was started, for 24h after it.
  • FIGs. 4A-4F Effects of 5 A therapy on renal mRNA expression of cytokines and NLRP3 on the progressive CKD model.
  • IL-6 IL-6
  • CXCL-1 b
  • 5 A therapy There is also a trend toward increased renal mRNA expression of TNF-OC (c) and TGF- ⁇ (d), which were lower in mice that received 5A.
  • TNF-OC c
  • TGF- ⁇ ⁇
  • N 4-6/group, ANOVA with Dunnett's post-hoc test).
  • FIGs. 5A-5C Effects of 5A therapy on kidney fibrosis and renal mRNA expression of cytokines and NLRP3 on the UUO model.
  • UUO Unilateral Uretheral Obstruction
  • untreated WT CD-I mice had substantial interstitial fibrosis, cortex thinning, and macrophage infiltration of the obstructed kidney (a).
  • FIGs. 6A and 6B Localization of CD36 and fluorescent 5A peptide in the kidney,
  • (a) Kidney tissue immunofluorescence staining for CD36 showing CD36 expression in the kidney (light grey) of WT mouse. CD36 is not expressed in the kidney of CD36KO mouse; dark grey DAPI.
  • (b) Kidney tissue immunofluorescence staining for CD36 showing CD36 expression in the kidney (light grey) of WT mouse. CD36 is not expressed in the kidney of CD36KO mouse; dark grey DAPI.
  • Immunohistochemistry shows CD36 expression on proximal tubular cells of WT mice but not in
  • CD36KO mice (200X).
  • FIGs. 7A and 7B 5A-DMPC-BODIPY-CE uptake in human CD36 expressing HeLa cells.
  • CD36 stably transfected HeLa cells were incubated without any ligands, with 5A-POPC-BP-CE for 2 h at 37°C followed by quantification on a Victor3 fluorimeter (Perkin Elmer).
  • Dose-dependent uptake of 5A- DMPC-BP-CE and DMPC-BP-CE to cultured HeLa cell expressing CD36 (triangle) and mock- transfected cells (square) is shown in panels a and b, respectively.
  • DMPC-BP-CE also binds to CD36 by itself, but weakly.
  • FIG. 8 Effects of CKD and 5A peptide treatment on biochemistry profiles.
  • FIG. 10 List of TaqMan Real-Time PCR assays used in Example 1.
  • FIG. 11 Outline of progressive CKD model. After collecting baseline urine sample, C57BL/6 mice were subjected to 2/3 nephrectomy of the left kidney (week -1), by removing both superior and inferior poles of the left kidney. One week later (0), mice were subjected to complete right nephrectomy. After right nephrectomy was complete, mice received one subcutaneous osmotic minipump without Angiotensin II (control) or with Angiotensin II (progressive CKD) - continuous infusion of
  • mice were followed with weekly urine sample collection, until euthanasia 4 weeks after 5/6 nephrectomy was complete.
  • Mice that were subjected to telemetry data analysis were subjected to carotid artery catheter implantation at week -2, one week prior to the start of all following surgeries. For more details, please refer to Methods.
  • FIG. 12 Summary of Unilateral Urethral Obstruction (UUO) model. Twenty-four hours (-1 day) before sham or surgery (UUO), CD-I mice received a subcutaneous osmotic minipump without or with 5A (5 or 15mg/kg/day). All mice were euthanized 10 days after UUO.
  • UUO Unilateral Urethral Obstruction
  • FIGs. 13A and 13B Uptake of scavenger receptor ligand in HeLa cells stably expressing SR-BI, SR-BII, or CD36.
  • SR-BI or SR-BII stably transfected HeLa cells were incubated without any ligands, or with the scavenger receptor ligands Alexa 488-labeled HDL, Alexa 488-labeled LDL or Alexa 488- labeled L37pA for 2 hours at 37°C followed by FACS analysis (FIG. 13A).
  • FIGs. 14A and 14B Effects of SAHP on lipopolysaccharide (LPS)-induced Interleukin-8 (IL-8) secretion in human embryonic kidney (HEK)293 cells.
  • LPS lipopolysaccharide
  • IL-8 Interleukin-8
  • CD36 expressing cells were incubated with 50 ng/ml of LPS in the presence or absence of increasing concentrations of the L37pA (SEQ ID NO: 1), ELK (SEQ ID NO: 46), ELK-B (SEQ ID NO: 67), 5A-37pA (SEQ ID NO: 3), or control L3D-37pA (SEQ ID NO: 1 synthesized with L-amino acids except that D-amino acids were used for alanine, lysine, and aspartic acid) SAHP for 24 hours in serum- free media, and the resulting amount of IL-8 secreted from the cells was assayed (FIG. 14B). IL-8 was measured in conditioned media and presented as pg/mg of cell protein (FIG. 14A) or as a percentage of inhibition in hCD36-HEK293 (FIG. 14B). Results are presented as mean +SEM.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence.txt (-36 kb), which was created on September 22, 2014, which is incorporated by reference herein.
  • SEQ ID Nos: 1 and 3-68 are the amino acid sequences of SAHPs.
  • SEQ ID NO: 2 and 69-70 are the amino acid sequences of polyproline linkers.
  • SEQ ID NO: 71-87 are exemplary amino acid sequences of functional domain peptides.
  • ApoA-I the predominant protein constituent of HDL (Panagotopulos et al, J. Biol. Chem.
  • ApoA-I has been tested and shown to be effective as a therapeutic agent in animal models to ameliorate development and progression of atherosclerosis by accelerating cholesterol efflux from lipid-laden macrophages and removing proinflammatory oxidized phospholipids from arterial cell walls.
  • pharmaceutical grade quality ApoA-I protein is expensive and may not be commercially feasible as a therapeutic agent.
  • SAHP amphipathic helical peptides
  • SAHPs have multiple amphipathic alpha-helical structures that recapitulate the secondary structure of the native ApoA-I protein, which contains at least eight amphipathic alpha helices.
  • the helical amphipathic structure enables their interactions with class B scavenger receptors including CD36 and its homologues, SR-BI and SR-BII.
  • SAHPs such as L37pA and 5A-37pA, target equally both CD36 and SR-BI.
  • Novel SAHPs are disclosed herein, that are designed to target primarily only one scavenger receptor, such as the ELK-B (SEQ ID NO: 67) and ELK-B2 (SEQ ID NO: 68) SAHPs, which selectively target the CD36 scavenger receptor.
  • ELK-B SEQ ID NO: 67
  • ELK-B2 SEQ ID NO: 68
  • SAHPs are useful for the treatment of CKD.
  • CKD progression is believed to be quite different from that of dyslipidemic or vascular disorders.
  • prior therapeutic agents for the treatment of kidney disease chronic or acute disease
  • CKD treatment via SAHP administration requires the CD36 scavenger receptor.
  • Scavenger receptors, including CD36 are known as "cleaners" of the vascular system, due to their extensive uptake of numerous foreign substances and waste materials, as well as cholesterol and lipids. Due to the multi-faceted role of CD36 in human biology, prior studies have concluded that determining CD36 function in any tissue or system requires empirical evidence (see, e.g., Silverstein and Febbraio, Sci. Signal., 72:re3, 2009).
  • CKD comprising administration of therapeutically effective amount of a SAHP, such as a SAHP that is a CD36 receptor antagonist, to a subject in need thereof.
  • a SAHP such as a SAHP that is a CD36 receptor antagonist
  • the disclosed methods can be utilized for the treatment and/or inhibition of CKD, as well as dyslipidemic or vascular disorders.
  • a polypeptide includes single or plural polypeptides and can be considered equivalent to the phrase “at least one polypeptide.”
  • the term “comprises” means “includes.”
  • “comprising an antigen” means “including an antigen” without excluding other elements.
  • the phrase “and/or” means “and” or “or.” It is further to be understood that any and 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 descriptive purposes, unless otherwise indicated.
  • Administration The introduction of a composition into a subject by a chosen route.
  • Administration can be local or systemic.
  • the composition such as a composition including a SAHP
  • the composition is administered by introducing the composition into a vein of the subject.
  • long-term administration such as is accomplished using a continuous release pump or a coated, implanted device (such as a stent).
  • Agent Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for treating or inhibiting CKD or a dyslipidemic or vascular disorder in a subject.
  • Agents include proteins, peptides, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest, such as viruses, such as recombinant viruses.
  • An agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent.
  • the agent is a polypeptide agent (such as a SAHP). The skilled artisan will understand that particular agents may be useful to achieve more than one result.
  • amphipathic An amphipathic molecule contains both hydrophobic (non-polar) and hydrophilic (polar) groups.
  • the hydrophobic group can be an alkyl group, such as a long carbon chain, for example, with the formula: CH3(CH2) n , (where n is generally greater than or equal to about 4 to about 16).
  • Such carbon chains also optionally comprise one or more branches, wherein a hydrogen is replaced with an aliphatic moiety, such as an alkyl group.
  • a hydrophobic group also can comprise an aryl group.
  • the hydrophilic group can be one or more of the following: an ionic molecule, such as an anionic molecule (e.g.
  • a fatty acid, a sulfate or a sulfonate) or a cationic molecule or a cationic molecule, an amphoteric molecule (e.g. , a phospholipid), or a non-ionic molecule (e.g. , a small polymer).
  • an amphoteric molecule e.g. , a phospholipid
  • a non-ionic molecule e.g. , a small polymer
  • amphipathic molecule is an amphipathic peptide.
  • An amphipathic peptide can also be described as a helical peptide that has hydrophilic amino acid residues on one face of the helix and hydrophobic amino acid residues on the opposite face.
  • peptides and polypeptides described herein form amphipathic helices in a physiological environment, such as for instance in the presence of lipid or a lipid interface.
  • Analog, derivative or mimetic An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization, and so forth.
  • Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in
  • a derivative is a biologically active molecule derived from the base structure.
  • a mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule.
  • Biologically active molecules can include chemical structures that mimic the biological activities of a compound.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.
  • Anti-CKD agent An agent that inhibits or treats CKD, such as an agent that inhibits the progression of CKD or treats a symptom of CKD.
  • anti-CKD agents include Renin-Angiotensin-Aldosterone System (RAAS) inhibitors such as ACE inhibitors and ARBs, as well as peroxisome proliferator-activated receptor (PPAR) antagonists, endothelin receptor A antagonists, endothelin receptor B antagonists, anti-ocvP6-integrin antibodies, hepatic growth factors (HGFs), Plasminogen activator inhibitor- 1 (PAI-1) inhibitors, Lysyl oxidase-like-2 (LOXL2) inhibitors, Nrf2 agents ⁇ e.g., bardoxolone); Pirfenidone and other anti-fibrotics such as VEGF inhibitors) (See, e.g.
  • RAAS Renin-Angiotensin-Aldosterone System
  • CD36 Cluster of Differentiation 36: An integral membrane protein that is a member of the class B scavenger receptor family of cell surface proteins. CD36 is also known as FAT, SCARG3, GP88, and glycoprotein IV. Class B scavenger receptors recognize oxidized low density lipoprotein. Additional class B scavenger receptors include scavenger receptor BI (SR-BI) and scavenger receptor BII (SR-BII). CD36 binds many ligands including oxidized low density lipoprotein (LDL) and long chain fatty acids, and is involved, for example, in long chain fatty acid uptake into cells and lipid metabolism. Upon ligand binding, CD36 and the ligand are internalized into the cell where processing of the ligand can occur. A non-limiting example of CD36 protein sequence is provided as GenBank® Accession No.
  • NP_001001547.1 (incorporated by reference herein as present in GenBank on September 24, 2013).
  • CD36 antagonist An agent that inhibits CD36 activity.
  • the CD36 antagonist can be a direct or indirect inhibitor of CD36 activity.
  • Non-limiting examples of "CD36 activity" that can be inhibited by a CD36 antagonist include protein-protein interaction, receptor internalization, ligand binding, ligand transport, and/or signaling activity of CD36.
  • a CD36 antagonist can be an agent that inhibits the internalization activity of CD36, or the signaling activity of CD36 in vitro or in vivo, or a combination thereof.
  • the CD36 activity inhibited by a CD36 antagonist is LPS-induced IL-8 secretion from cells expressing CD36.
  • a “selective CD36 antagonist” is an agent that inhibits at least 2-fold (such as at least 3-fold, at least 4-fold or at least 5-fold) more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
  • Methods of determining CD36, SR-BI, and/or SR-BII activity, as well as methods of determining if an agent is an antagonist of CD36, SR-BI, and/or SR-BII activity are known to the person of ordinary skill in the art, and example methods are described herein (see, for example, Example 2).
  • the SAHPs disclosed herein are CD36 antagonists.
  • Nonlimiting examples of SAHPs that are CD36 antagonists include SAHPs with an amino acid sequence set forth as SEQ ID NO: 1 (L37pA), SEQ ID NO: 3 (5A-37pA), SEQ ID NO: 67 (ELK-B), and SEQ ID NO: 68 (ELJK-B2). Of these, SEQ ID NO: 67 (ELK-B), and SEQ ID NO: 68 (ELJK-B2) are considered to be selective CD36 antagonists.
  • CKD Chronic Kidney Disease
  • GFR glomerular filtration rate
  • cardiovascular disease CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, pericarditis.
  • the severity of CKD can be classified in five stages based on level of kidney function, with stage 1 being the mildest and usually causing few symptoms and stage 5 being a severe illness with poor life expectancy.
  • the stages include:
  • Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.
  • Stage 5 Established kidney failure (GFR ⁇ 15 mL/min/1.73 m 2 , permanent renal replacement therapy (RRT), or end stage renal disease (ESRD). Stage 5 CKD is also known as end stage renal disease (ESRD) or end-stage kidney disease (ESKD).
  • CKD does not include acute kidney disease, the etiology of which typically includes an acute causative event ⁇ e.g. , sepsis, ischemia, trauma, and/or nephrotoxic drugs) which leads to onset of kidney disease in less than three months.
  • an acute causative event ⁇ e.g. , sepsis, ischemia, trauma, and/or nephrotoxic drugs
  • Control A reference standard.
  • the control is a negative control sample obtained from a healthy patient.
  • the control is a positive control, such as a sample obtained from a patient diagnosed with CKD or a dyslipidemic or vascular disorder.
  • control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of CKD or dyslipidemic or vascular disorder patients with known prognosis or outcome, or group of samples that represent baseline or normal values).
  • a difference between a test sample and a control can be an increase or conversely a decrease.
  • the difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference.
  • a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 68%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.
  • a domain of a protein is a part of a protein that shares common structural, physiochemical and functional features; for example hydrophobic, polar, globular, helical domains or properties, for example a DNA binding domain, an ATP binding domain, and the like.
  • Dyslipidemic disorder A disorder associated with any altered amount of any or all of the lipids or lipoproteins in the blood.
  • Dyslipidemic disorders include, for example, hyperlipidemia,
  • hyperlipoproteinemia hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, and cardiovascular disease (i.e. , coronary artery disease, atherosclerosis and restenosis).
  • cardiovascular disease i.e. , coronary artery disease, atherosclerosis and restenosis.
  • Proteins can be expressed and remain intracellular, can become a component of a cell membrane, or be can secreted into the extracellular matrix or medium.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, and stop codons.
  • ATG start codon
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
  • a promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al , Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells such as metallothionein promoter or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used.
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
  • a polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
  • lipid efflux refers to a process whereby lipid, such as cholesterol and phospholipid, is complexed with an acceptor, such as an apolipoprotein or apolipoprotein peptide mimic, and removed from vesicles or cells.
  • acceptor such as an apolipoprotein or apolipoprotein peptide mimic
  • ABCA1-dependent lipid efflux refers to a process whereby apolipoproteins or peptide mimics of apolipoproteins bind to a cell and efflux lipid from the cell by a process that is facilitated by the ABCA1 transporter.
  • Hydrophilic A hydrophilic (or lipophobic) group is electrically polarized and capable of H- bonding, enabling it to dissolve more readily in water than in oil or other "non-polar" solvents.
  • Hydrophobic A hydrophobic (or lipophilic) group is electrically neutral and nonpolar, and thus prefers other neutral and nonpolar solvents or molecular environments. Examples of hydrophobic molecules include alkanes, oils and fats.
  • Inhibiting or treating a disease Inhibiting the full development or progression of a disease or condition, for example, in a subject who is at risk for a disease, such as CKD, atherosclerosis or cardiovascular disease.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
  • a "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • Isolated/purified An "isolated” or “purified” biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids or proteins.
  • an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell.
  • a preparation is purified such that the biological component represents at least 50%, such as at least 68%, at least 90%, at least 95%, or greater of the total biological component content of the preparation.
  • Linker A molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds.
  • Lipid A class of water-insoluble, or partially water insoluble, oily or greasy organic substances, that are extractable from cells and tissues by nonpolar solvents, such as chloroform or ether.
  • Types of lipids include triglycerides (i.e. , natural fats and oils composed of glycerin and fatty acid chains), phospholipids (e.g. , phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and
  • sphingolipids e.g. , sphingomyelin, cerebrosides and gangliosides
  • sterols e.g. , cholesterol
  • Nucleic acid A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide -nucleic acids (PNAs), and the like.
  • PNAs peptide -nucleic acids
  • polynucleotides can be synthesized, for example, using an automated DNA synthesizer.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e. , A, T, G, C), this also includes an RNA sequence (i.e. , A, U, G, C) in which "U" replaces "T.”
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyriniidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • nucleotide sequences the left-hand end of a single-stranded nucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5 ' -direction.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5 '-end of the RNA transcript are referred to as "upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences.”
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a
  • polynucleotide such as a gene, a cDNA, or an mRNA
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • nucleic acid encodes a disclosed PreF antigen.
  • Recombinant nucleic acid refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a "recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, such as a "recombinant polypeptide.”
  • a recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome -binding site, etc.) as well.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Peptide and 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 "peptide” or “polypeptide” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • the term “residue” or "amino acid residue” includes reference to an amino acid that is incorporated into a peptide, polypeptide, or protein.
  • Peptide modifications can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity and conformation as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C 1 -C 1 6 ester, or converted to an amide of formula NR1R2 wherein Ri and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically-acceptable acid addition salt such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy or to a C1-C16 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.
  • 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.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions ⁇ e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • 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.
  • 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.
  • the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired response. It may also be accompanied by medications for its use for treatment purposes.
  • the unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.
  • Sequence identity/similarity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences.
  • 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • sequence comparison For sequence comparison of nucleic acid sequences and amino acids sequences, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, . Mol. Biol. 48:443, 1968, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see for example, Current Protocols in Molecular Biology (Ausubel et al. , eds 1995 supplement)).
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. , J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.
  • BLAST Altschul et al., . Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (World Wide Web address ncbi.nlm.nih.gov).
  • the BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff , Proc. Natl. Acad. Sci. USA 89: 10915, 1989).
  • Another indicia of sequence similarity between two nucleic acids is the ability to hybridize the sequences to each other, or to the same target sequence.
  • the stringency of hybridization conditions are sequence-dependent and are different under different environmental parameters. Thus, hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength
  • stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • “Stringent conditions” encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. “Stringent conditions” may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency” are those under which sequences with more than 6% mismatch will not hybridize. In contrast nucleic acids that hybridize under “low stringency conditions include those with much less sequence identity, or with sequence identity over only short subsequences of the nucleic acid.
  • SAHP Synthetic alpha- helical peptide
  • the alpha-helical domains are typically linked by an amino acid linker (such as a proline residue).
  • SAHPs promote cholesterol efflux from cells by the ABCA1 transporter.
  • the person of ordinary skill in the art is familiar with certain SAHPs (such as the L37pA (SEQ ID NO: 1) and 5A-37pA (SEQ ID NO: 3) peptides).
  • Novel SAHPs are also provided herein, such as the ELK-B (SEQ ID NO: 67) and ELK-B2 (SEQ ID NO: 68) peptides.
  • methods of treating CKD by administering a therapeutically effective amount of a SAHP to a subject in need thereof are provided.
  • Therapeutically effective amount A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a SAHP or peptide analog useful in inhibiting and/or treating CKD or a dyslipidemic or vascular disorder in a subject.
  • a therapeutically effective amount of an agent is an amount sufficient to prevent, inhibit and/or treat CKD or a dyslipidemic or vascular disorder in a subject without causing a substantial cytotoxic effect in the subject.
  • the effective amount of an agent useful for inhibiting and/or treating CKD or a dyslipidemic or vascular disorder in a subject will be dependent on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • Therapeutic peptides are described herein that include or consist of multiple amphipathic alpha- helical domain peptides, termed Synthetic Amphipathic Helical Peptides (SAHPs).
  • SAHPs Synthetic Amphipathic Helical Peptides
  • the peptides are useful, for example, for the treatment of CKD, as wells as dyslipidemic or vascular disorders.
  • SAHPs were known prior to the disclosure provided herein (for example, the L37pA and 5A-37pA peptides). Description of known SAHPs is provided, for example, in PCT. Pub Nos.
  • the SAHP includes multiple amphipathic alpha-helical domains, wherein a first amphipathic alpha-helical domain and a second amphipathic alpha-helical domain exhibit equivalent hydrophobicity (as measured, e.g., by their hydrophobic moments; see Eisenberg et al , Faraday Symp. Chem. Soc. 17: 109-120, 1982; Eisenberg et al. , PNAS 81 : 140-144, 1984; and Eisenberg et al. , . Mol. Biol. 179: 125-142, 1984).
  • the first domain and second domain are covalently linked by a linking peptide, such as a glycine, alanine or proline, or other bridging molecule.
  • the degree of amphipathicity ⁇ i.e. , degree of symmetry of hydrophobicity) in the SAHPs or peptide analogs can be conveniently quantified by calculating the hydrophobic moment ( ⁇ #) of each of the amphipathic alpha-helical domains.
  • Methods for calculating ⁇ # for a particular peptide sequence are well- known in the art, and are described, for example in Eisenberg et al , Faraday Symp. Chem. Soc. 17: 109- 120, 1982; Eisenberg et al. , PNAS 81 : 140-144, 1984; and Eisenberg et al., J. Mol. Biol. 179: 125-142, 1984.
  • the actual ⁇ # obtained for a particular peptide sequence will depend on the total number of amino acid residues composing the peptide.
  • the amphipathicities of peptides of different lengths can be directly compared by way of the mean hydrophobic moment.
  • the mean hydrophobic moment per residue can be obtained by dividing ⁇ # by the number of residues in the peptide.
  • the SAHP includes or consists of the amino acid sequence set forth as DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF (L37pA; SEQ ID NO: 1).
  • the SAHP includes or consists of the amino acid sequence set forth as DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (5A-37pA; SEQ ID NO: 3).
  • the L37pA and 5A-37pA peptides include two amphipathic alpha-helical domains linked by a proline residue, and as disclosed herein, are antagonists of CD36 in vitro. Therefore, in several embodiments, a polypeptide including or consisting of a disclosed SAHP, such as a polypeptide including or consisting of the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3, is a CD36 receptor antagonist. Additional, non-limiting examples of SAHPs with two amphipathic alpha-helical domains are provided in Table 1.
  • Additional SAHPs are provided herein including peptides with multiple amphipathic alpha-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway.
  • the SAHP includes or consists of the consensus amino acid sequence set forth as EKLXiELLX 2 KLLELLKKLLPEKLX 3 ELLX 4 KLLELLKKLL (SEQ ID NO: 66), wherein, Xi is L or K, X 2 is K or E, X3 is L or K, and X4 is K or E.
  • the SAHP includes or consists of the amino acid sequence set forth as
  • the SAHP includes or consists of the amino acid sequence set forth as
  • EKLKELLEKLLELLKKLLPEKLKELLEKLLELLKKLL (ELK-B 2; SEQ ID NO: 68).
  • a polypeptide including or consisting of the amino acid sequence set forth as SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68 is a CD36 receptor antagonist.
  • the disclosed SAHP (such as a peptide including the amino acid sequence set forth as SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68) is a selective CD36 antagonist that inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
  • the SAHP can inhibit CD36 activity to a greater extent than SR-BI and/or SR-BII activity in a comparable assay.
  • the SAHP has an IC5 0 value for CD36 inhibition that is no more than 50% (such as no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the corresponding IC5 0 value for SR-BI or SR-BII inhibition.
  • Methods of determining CD36, SR-BI, and/or SRT-BII activity, and inhibition thereof, are known to the person of ordinary skill in the art, and further described herein (see, e.g. , Example 2).
  • the SAHP is included on a polypeptide having a maximum length, for example no more than 40, 50, 60, 68, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids amino acids in length.
  • the polypeptide including the SAHP can include, consist or consist essentially of the SAHP.
  • the disclosed SAHPs bind to CD36.
  • the dissociation constant for SAHP binding to CD36 is less than about 10 "4 Molar, such as less than about 10 5 Molar, 10 "6 Molar, 10 "7 Molar, or less than 10 "8 Molar. Binding to CD36 can be determined by methods known in the art. The determination that a particular agent specifically binds to a particular polypeptide may readily be made by using or adapting routine procedures.
  • the disclosed SAHPs specifically bind to CD36 as opposed to other scavenger receptors, such as SR-BI or SR-BII receptors.
  • Specific binding to CD36 can be determined by methods known in the art, for example according to methods disclosed in Example 2, below. The determination that a particular agent specifically binds to a particular polypeptide compared to other polypeptides may readily be made by using or adapting routine procedures.
  • a disclosed SAHP is a CD36 receptor antagonist.
  • a SAHP including the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68 is a CD36 receptor antagonist (see example 2, below).
  • Methods of assaying a peptide for effects on CD36 receptor signaling activity are known in the art and described herein (see, e.g. , Example 2, below).
  • the linkage between amino acid residues can be a peptide bond or amide linkage (i.e., -C-C(O)NH-).
  • one or more amide linkages are optionally replaced with a linkage other than amide, for example, a substituted amide.
  • Substituted amides generally include, but are not limited to, groups of the formula -C(0)NR-, where R is (Ci-C6) alkyl, substituted (Ci-C6) alkyl, (C1-C6) alkenyl, substituted (Ci-C6) alkenyl, (Ci-C6) alkynyl, substituted (Ci-C6) alkynyl, (C5-C20) aryl, substituted (C5-C20) aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl, and substituted 6-26 membered alkheteroaryl. Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides.
  • Suitable amide mimetic moieties are described, for example, in Olson et al , J. Med. Chem. 36:3039-3049, 1993.
  • the amino- and carboxy-terminal ends can be modified by conjugation with various functional groups.
  • Neutralization of the terminal charge of synthetic peptide mimics of apolipoproteins has been shown to increase their lipid affinity (Yancey et al. , Biochem. 34:7955-7965, 1995; Venkatachalapathi et al , Protein: Structure, Function and Genetics 15:349-359, 1993).
  • acetylation of the amino terminal end of amphipathic peptides increases the lipid affinity of the peptide (Mishra et al , J. Biol. Chem. 267:7185-7191, 1994).
  • the amino acid proline is used to link the multiple amphipathic oc-helices included on the SAHP.
  • suitable amino acids such as glycine, serine, threonine, and alanine, that functionally separate the multiple amphipathic alpha-helical domains can be used.
  • the linking amino acid will have the ability to impart a ⁇ - turn at the linkage, such as glycine, serine, threonine, and alanine.
  • larger linkers containing two or more amino acids or bifunctional organic compounds, such as H 2 N(CH 2 ) n COOH, where n is an integer from 1 to 12, can also be used.
  • linkers examples include Hunig et al , Chem. Ber. 100:3039-3044, 1974 and Basak et al , Bioconjug. Chem. 5:301-305, 1994).
  • SAHPs useful within the disclosure are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D-amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
  • proline analogs can be made in which the ring size of the proline residue is changed from a 5-membered ring to a 4-, 6-, or 7-membered ring.
  • Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.
  • Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl ⁇ e.g.
  • morpholino oxazolyl, piperazinyl ⁇ e.g. , 1-piperazinyl), piperidyl ⁇ e.g. , 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl ⁇ e.g. , 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl ⁇ e.g.
  • heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
  • Peptides, as well as peptide analogs and mimetics can also be covalently bound to one or more of a variety of nonproteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkenes, as described in U.S. Patent Nos. 4,640,835; 4,496,668; 4,301,144; 4,668,417; 4,791,192; and 4,179,337.
  • amino acid residues in the SAHPs may be substituted with naturally occurring non-encoded amino acids and synthetic amino acids.
  • Certain commonly encountered amino acids which provide useful substitutions include, but are not limited to, ⁇ -alanine and other omega-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionic acid, 4-aminobutyric acid and the like; oc-aminoisobutyric acid; ⁇ -aminohexanoic acid; ⁇ -amino valeric acid; N-methylglycine or sarcosine; ornithine; citrulline; t-butylalanine; t-butylglycine; N- methylisoleucine; phenylglycine; cyclohexylalanine; nor leucine; naphthylalanine; 4-chlorophenylalanine; 2-fluorophenylalanine; 3-fluorophen
  • the amino acids of the SAHPs will be substituted with L-amino acids, the substitutions are not limited to L-amino acids.
  • modified forms of the SAHPs wherein an L-amino acid is replaced with an identical D- amino acid (e.g. , L-Arg ⁇ D-Arg) or with a conservatively-substituted D-amino acid (e.g. , L-Arg ⁇ D- Lys), and vice versa.
  • peptide analogs and mimetics within the scope of the disclosure include glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues (e.g. , lysine or arginine).
  • Acyl groups are selected from the group of alkyl-moieties including C3 to CI 8 normal alkyl, thereby forming alkanoyl aroyl species. Also embraced are versions of a native primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.
  • a detectable moiety can be linked to the SAHP or peptide analogs disclosed herein, creating a peptide/peptide analog-detectable moiety conjugate.
  • Detectable moieties suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • the detectable moieties contemplated for the present disclosure can include, but are not limited to, an immunofluorescent moiety (e.g. , fluorescein, rhodamine, Texas red, and the like), a radioactive moiety (e.g. , 3 H, 32 P, 125 1, 35 S, 18 F, 64 Cu, "Tc), an enzyme moiety (e.g.
  • the detectable moiety can be linked to the SAHP or peptide analog at either the N- and/or C-terminus.
  • a linker can be included between the SAHP or peptide analog and the detectable moiety.
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • an additional functional domain or peptide can be linked to the SAHPs or peptide analogs disclosed herein, creating a peptide/peptide analog-additional functional domain/peptide conjugate.
  • the additional functional domain or peptide can be linked to the SAHP or peptide analog at either the N- and/or C-terminus.
  • Exemplary additional functional domains/peptides are known in the art, see for example, PCT. Pub Nos. WO2006/044596, WO2009/129263, and WO 2011/066511 , each of which is incorporated by reference herein in its entirety.
  • Exemplary additional functional domains/peptides are known in the art, see for example, PCT. Pub Nos. WO2006/044596, WO2009/129263, and WO 2011/066511 , each of which is incorporated by reference herein in its entirety.
  • Exemplary additional functional domains/peptides are known in the art, see for example, PCT. Pub Nos. WO2006/044596,
  • domains/peptides include those shown in Table 2. Table 2. Exemplary additional functional domains.
  • Cell recognition sequences can increase the ability of the SAHPs or peptide analogs containing these sequences to bind to cells, the prerequisite first step in ABCA1 -mediated cholesterol efflux (Remaley et al, Biochem. Biophys. Res. Commun. 280:818-823, 2001).
  • Cell internalization sequences can increase the cellular uptake of the SAHPs or peptide analogs into intracellular compartments, where the initial lipidation of the peptides has been proposed to occur (Neufeld et al, J. Biol. Chem. 279: 15571- 15578, 2004), thus facilitating lipid efflux.
  • Sequences that activate neutral cholesterol hydrolase Kisilevsky et al , J. Lipid Res.
  • lipoprotein lipase activation sequences result in triglyceride levels of no greater than 200 mg/dl.
  • a linker can be included between the SAHP or peptide analog and the additional functional domain or peptide.
  • the additional functional domain or peptide can enhance the ability of the SAHP or peptide analog to efflux lipids from cells in a non-cytotoxic manner, and/or enhance its therapeutic efficacy.
  • the linkers contemplated by the present disclosure can be any bifunctional molecule capable of covalently linking two peptides to one another.
  • suitable linkers are bifunctional molecules in which the functional groups are capable of being covalently attached to the N- and/or C- terminus of a peptide.
  • Functional groups suitable for attachment to the N- or C-terminus of peptides are well known in the art, as are suitable chemistries for effecting such covalent bond formation.
  • the linker may be flexible, rigid or semi-rigid.
  • Suitable linkers include, for example, amino acid residues such as Pro or Gly or peptide segments containing from about 2 to about 5, 10, 15, 20, or even more amino acids, bifunctional organic compounds such as H2N(CH2) n COOH where n is an integer from 1 to 12, and the like. Examples of such linkers, as well as methods of making such linkers and peptides incorporating such linkers, are well-known in the art (see, e.g. , Hunig et al. , Chem. Ber. 100:3039-3044, 1974 and Basak et al. , Bioconjug. Chem. 5:301-305, 1994).
  • Conjugation methods applicable to the present disclosure include, by way of non-limiting example, reductive animation, diazo coupling, thioether bond, disulfide bond, amidation and
  • the amphipathic alpha-helical domains are "activated" prior to conjugation. Activation provides the necessary chemical groups for the conjugation reaction to occur.
  • the activation step includes derivatization with adipic acid dihydrazide.
  • the activation step includes derivatization with the N-hydroxysuccinimide ester of 3-(2-pyridyl dithio)-propionic acid.
  • the activation step includes derivatization with succinimidyl 3-(bromoacetamido) propionate.
  • derivatizing agents include succinimidylformylbenzoate and succinimidyllevulinate .
  • mixtures of two or more such as 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the disclosed peptides are provided.
  • SAHPs or peptide analogs wherein the multiple amphipathic alpha-helical domains are comprised of dimers, trimers, tetramers and even higher order polymers (i.e. , "multimers") comprising the same or different sequences. Such multimers may be in the form of tandem repeats.
  • the amphipathic alpha-helical domains may be directly attached to one another or separated by one or more linkers.
  • the amphipathic alpha-helical domains can be connected in a head- to-tail fashion (i.e. , N-terminus to C-terminus), a head-to-head fashion, (i.e.
  • the multimers are tandem repeats of two, three, four, and up to about ten amphipathic alpha-helical domains, but any number of amphipathic alpha-helical domains can be used. 4. Synthesis and Production of SAHPs
  • the SAHPs or peptide analogs of the disclosure can be prepared using virtually any technique known to one of ordinary skill in the art for the preparation of peptides. Examples of methods for preparing SAHPs are disclosed herein and known in the art (see, e.g., PCT. Pub Nos. WO2006/044596, WO2009/129263, and WO 2011/066511, each of which is incorporated by reference herein in its entirety).
  • the SAHPs can be prepared using step-wise solution or solid phase peptide syntheses, or recombinant DNA techniques, or the equivalents thereof.
  • SAHPs of the disclosure having either the D- or L-configuration can be readily synthesized by automated solid phase procedures well known in the art. Suitable syntheses can be performed by utilizing "T-boc" or "F-moc” procedures. Techniques and procedures for solid phase synthesis are described in Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the SAHPs may be prepared by way of segment condensation, as described, for example, in Liu et al, Tetrahedron Lett. 37:933-936, 1996; Baca et al, J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al, Int. J. Peptide Protein Res. 45:209-216, 1995;
  • SAHPs of the disclosure having either the D- or L-configuration can also be readily purchased from commercial suppliers of synthetic peptides.
  • Such suppliers include, for example, Advanced ChemTech (Louisville, KY), Applied Biosystems (Foster City, CA), Anaspec (San Jose, CA), and Cell Essentials (Boston, MA).
  • the SAHP or the relevant portion can also be synthesized using conventional recombinant genetic engineering techniques.
  • a polynucleotide sequence encoding the SAHP is inserted into an appropriate expression vehicle, that is, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the expression vehicle is then transfected into a suitable target cell which will express the SAHP.
  • the expressed peptide is then isolated by procedures well-established in the art.
  • the polynucleotide can be designed to encode multiple units of the SAHP separated by enzymatic cleavage sites.
  • the resulting polypeptide can be cleaved ⁇ e.g., by treatment with the appropriate enzyme) in order to recover the peptide units.
  • This can increase the yield of peptides driven by a single promoter.
  • a polycistronic polynucleotide can be designed so that a single mRNA is transcribed which encodes multiple peptides, each coding region operatively linked to a cap-independent translation control sequence, for example, an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • the translation of each peptide encoded by the mRNA is directed internally in the transcript, for example, by the IRES.
  • the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual peptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase yield of peptide driven by a single promoter.
  • host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors ⁇ e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors ⁇ e.g.
  • cauliflower mosaic virus or tobacco mosaic virus (TMV)
  • TMV tobacco mosaic virus
  • recombinant plasmid expression vectors ⁇ e.g. , Ti plasmid
  • the expression elements of the expression systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements can be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
  • promoters such as the baculovirus polyhedron promoter can be used.
  • promoters derived from the genome of plant cells e.g.
  • heat shock promoters the promoter for the small subunit of RUBISCO, the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV, the coat protein promoter of TMV) can be used.
  • plant viruses e.g., the 35S RNA promoter of CaMV, the coat protein promoter of TMV
  • promoters derived from the genome of mammalian cells e.g. , metallothionein promoter
  • mammalian viruses e.g. , the adenovirus late promoter, the vaccinia virus 7.5 K promoter
  • the SAHPs or peptide analogs of the disclosure can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like.
  • the actual conditions used to purify a particular SAHPs or peptide analog will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art.
  • any antibody which specifically binds the SAHPs or peptide analog may be used.
  • various host animals including but not limited to, rabbits, mice, rats, and the like, may be immunized by injection with a SAHP or peptide analog and antibodies purified according to standard methods.
  • Polynucleotides encoding the SAHPs disclosed herein are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the SAHPs.
  • the polynucleotides encoding a SAHP include a recombinant DNA which is incorporated into a vector into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single and double forms of DNA.
  • DNA sequences encoding the antigen can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • Polynucleotide sequences encoding SAHPs can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e. , ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing
  • DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human).
  • Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, CI 29 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture.
  • the host cells include HEK293 cells or derivatives thereof, such as GnTI 7" cells (ATCC® No. CRL-3022).
  • Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as, but not limited to, E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCb. method using procedures well known in the art.
  • MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman Ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • a number of viral vectors have been constructed, that can be used to express the disclosed SAHPs, including polyoma, i.e. , SV40 (Madzak et al , 1992, . Gen. Virol , 73: 15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Hopkins et al , 1988, Bio Techniques, 6:616- 629; Gorziglia et al , 1992, /. Virol, 66:4407-4412; Quantin et al, 1992, Proc. Natl Acad. Sci.
  • Baculovirus vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, CA; Protein Sciences Corp., Meriden, Conn.;
  • the peptides or peptide analogs of the disclosure (and mixtures thereof), or a polynucleotide encoding same can be used to treat any disorder in animals, especially mammals (e.g. , humans), for which promoting lipid efflux is beneficial, such as dyslipidemic or vascular disorders.
  • Such conditions include, but are not limited to, hyperlipidemia (e.g. , hypercholesterolemia), cardiovascular disease (e.g., atherosclerosis), restenosis (e.g., atherosclerotic plaques), peripheral vascular disease, acute coronary syndrome, reperfusion myocardial injury, asthma, chronic pulmonary obstructive disorder and the like.
  • the peptides or peptide analogs of the disclosure can also be used during the treatment of thrombotic stroke and during thrombolytic treatment of occluded coronary artery disease.
  • the methods include selecting a subject in need of treatment, such as a subject with or at risk of a dyslipidemic or vascular disorder.
  • the methods include administration of a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP as disclosed herein to a subject with or at risk of a dyslipidemic or vascular disorder.
  • the polypeptide can comprise or consist of a SAHP including the amino acid sequence set forth as SEQ ID NO: 66, wherein, Xi is L or K, X 2 is K or E, X3 is L or K, and X4 is K or E (ELK-B/B2 consensus), such as a SAHP including the amino acid sequence set forth as SEQ ID NO: 67 (ELK-B) or SEQ ID NO: 68 (ELK-B2).
  • the method can further include combination therapy with other lipid lowering compositions or drugs used to treat the foregoing conditions, or with agents (such as peptides) that activate LPL activity.
  • combination therapies include, but are not limited to, simultaneous or sequential administration of the drugs involved.
  • the peptide or peptide analog formulations can be administered with any one or more of the cholesterol lowering therapies currently in use, for example, bile-acid resins, niacin and statins.
  • the SAHP or peptide analog formulations can be administered with a lipoprotein lipase activating agent, such as an apoC-II protein, variant or fragment thereof, to prevent, reduce or inhibit hypertriglyceridemia associated with the administration of any of the disclosed peptides or peptide analogs.
  • a lipoprotein lipase activating agent such as an apoC-II protein, variant or fragment thereof
  • the SAHPs or peptide analogs can be used in conjunction with statins or fibrates to treat hyperlipidemia, hypercholesterolemia and/or cardiovascular disease, such as
  • the SAHPs or peptide analogs of the disclosure can be used in combination with an anti-microbial agent and/or an anti-inflammatory agent.
  • the SAHPs can also be expressed in vivo, by using any of the available gene therapy approaches.
  • the peptides or peptide analogs of the disclosure (and mixtures thereof), or a polynucleotide molecule encoding same can be used to treat or inhibit CKD in animals, especially mammals (e.g. , humans).
  • the methods include selecting a subject in need of treatment, such as a subject with early or late CKD, or at risk of CKD.
  • a subject with CKD is selected based on glomerular filtration rate (GFR), for example the selected subject can have a GFR of less than 60 mL/min/1.73 m 2 for three consecutive months (see also, the National Kidney Foundation's guidelines for diagnosing CKD (Levey et al, Ann Intern. Med., 139:137-147, 2003), incorporated by reference herein in its entirety).
  • GFR glomerular filtration rate
  • CKD is often diagnosed in the course of screening individuals known to be at risk of CKD, such as those with high blood pressure or diabetes, or those with a family history of CKD.
  • CKD may also be identified when it leads to one of its recognized complications, such as cardiovascular disease, anemia or pericarditis.
  • a subject with a particular stage of CKD can also be selected, for example a subject with stage 1,
  • CKD 2, 3, 4or 5 CKD.
  • the severity of CKD can be classified in five stages based on level of kidney function, with stage 1 being the mildest and usually causing few symptoms and stage 5 being a severe illness with poor life expectancy.
  • the stages include:
  • Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.
  • Stage 3 Moderate reduction in GFR (30-59 mL/min/1.73 m 2 ).
  • Stage 4. Severe reduction in GFR (15-29 mL/min/1.73 m 2 ). Preparation for renal replacement therapy.
  • Stage 5 Established kidney failure (GFR ⁇ 15 mL/min/1.73 m 2 , permanent renal replacement therapy (RRT), or end stage renal disease (ESRD). Stage 5 CKD is also known as end stage renal disease (ESRD) or end-stage kidney disease (ESKD).
  • Therapy can be initiated before onset of CKD, or after a subject is diagnosed with CKD.
  • therapy can be initiated when a subject has stage 1, stage 2, stage 3, stage 4, or stage 5 CKD.
  • the methods of preventing and/or treating CKD delay or prevent progression of the CKD in the subject (for example compared to a control subject not receiving the SAHP), for example delay or prevention of progression to stage 5 CKD.
  • the methods include administration of a therapeutically effective amount of a polypeptide comprising or consisting of a disclosed SAHP to a subject with or at risk of CKD.
  • the polypeptide comprises or consists of a SAHP including the amino acid sequence set forth as one of SEQ ID NO: 1 (L37pA) or SEQ ID NO: 3 (5A-37pA).
  • the polypeptide comprises or consists of a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 66, wherein, Xi is L or K, X 2 is K or E, X3 is L or K, and X4 is K or E (ELK-B/B2 consensus), such as a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 67 (ELK-B) or SEQ ID NO: 68 (ELK-B2).
  • the method can further include combination therapy with other anti-CKD agents.
  • combination therapies include, but are not limited to simultaneous or sequential administration of the drugs involved.
  • the peptide or peptide analog formulations can be administered with any one or more of a known anti-CKD agent, such as ACE inhibitors, ARBs, peroxisome proliferator-activated receptor (PPAR) antagonists, endothelin receptor A antagonists, endothelin receptor B antagonists, anti-ocvP6-integrin antibodies, hepatic growth factors (HGFs), Plasminogen activator inhibitor-1 (PAI-1) inhibitors, Lysyl oxidase-like-2 (LOXL2) inhibitors,
  • ACE inhibitors such as ACE inhibitors, ARBs, peroxisome proliferator-activated receptor (PPAR) antagonists, endothelin receptor A antagonists, endothelin receptor B antagonists, anti-ocvP6-integrin antibodies, hepatic growth factors (HGFs), Plas
  • the CKD treated or inhibited by the disclosed methods is not acute kidney disease, obstructive kidney disease, atherosclerotic kidney disease, or polycystic kidney disease.
  • the SAHPs can also be expressed in vivo, by using any of the available gene therapy approaches.
  • a polypeptide comprising or consisting of a SAHP or peptide analogs can be synthesized or isolated from various sources and administered directly to the subject.
  • therapeutic compositions including at least one SAHP or analog thereof are administered to a subject suffering from a dyslipidemic or vascular disorder, such as hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury, in an amount sufficient to inhibit or treat the dyslipidemic or vascular disorder.
  • a dyslipidemic or vascular disorder such as hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute
  • compositions including at least one SAHP or analog thereof are administered to a subject at risk of CKD, or suffering from CKD. Amounts effective for this use will depend upon the severity of the disorder and the general state of the subject's health. A therapeutically effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Treatment of a disease or condition in a subject can include delaying the development of the disease or condition (such as CKD or a dyslipidemic or vascular disorder) in the subject.
  • Treatment of the disease or condition also includes reducing signs or symptoms associated with the disease or condition (such as CKD or a dyslipidemic or vascular disorder).
  • treatment using the methods disclosed herein prolongs the time of survival of the subject.
  • the disease or condition (such as CKD or a dyslipidemic or vascular disorder) does not need to be completely eliminated or cured for the methods to be effective.
  • treatment with one or more of the provided polypeptides can decrease the signs or symptoms of the disease or condition (such as CKD or a dyslipidemic or vascular disorder), or delay the onset or progression of the disease or condition by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 68%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (such as CKD or a dyslipidemic or vascular disorder), as compared a control, such as a population of subjects with the disease or condition (such as CKD or a dyslipidemic or vascular disorder) in the absence of the treatment with the therapeutic polypeptide.
  • CKD or a dyslipidemic or vascular disorder can decrease the signs or symptoms of the disease or condition (such as CKD or a dyslipidemic or vascular disorder), or delay the onset or progression of the disease or condition by a desired amount, for example by at least 10%, at least 20%, at least 50%, at
  • a polypeptide comprising or consisting of a disclosed SAHP or peptide analog can be administered by any means known to one of skill in the art (see, e.g. , Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995), such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection.
  • the polypeptide comprising or consisting of a disclosed SAHP or peptide analog can be provided as an implant, an oily injection, or as a particulate system.
  • the particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle (Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995).
  • the amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes.
  • Administration may begin whenever a subject has developed, or is at risk for developing a disease or condition, such as a dyslipidemic or vascular disorder or CKD.
  • a polypeptide comprising or consisting of a disclosed SAHP is administered that includes one or more of the amino acid sequences set forth in SEQ ID NOs: 1, 3, 66, 67, or 68. 4.
  • administration of the polypeptide comprising or consisting of a disclosed SAHP or mixture of such polypeptides, or the relevant portion can be achieved by an appropriate nucleic acid expression vector (or combination of vectors) which is administered so that it becomes intracellular, for example, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g.
  • nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, for example, by homologous or non-homologous recombination.
  • a DNA expression vector e.g. , the vector pCDNA
  • a strong viral promoter e.g. ,
  • cytomegalovirus to drive the expression.
  • retroviral vectors such as pRETRO-ON, BD Biosciences, Palo Alto, CA
  • pRETRO-ON BD Biosciences, Palo Alto, CA
  • pRETRO-ON BD Biosciences, Palo Alto, CA
  • these plasmids can be allowed to transfect the cells, then administer a course of tetracycline to achieve regulated expression.
  • Plasmid vectors such as pMAM-neo (BD Biosciences, Palo Alto, CA) or pMSG
  • MMTV-LTR promoter which can be regulated with steroids
  • pSVL Invitrogen, Carlsbad, CA
  • pBPV metallothionein-responsive promoter
  • retroviruses examples include adenovirus, AAV (adeno-associated virus), recombinant HSV, poxviruses (vaccinia) and recombinant lentivirus (such as HIV). All these vectors achieve the basic goal of delivering into the target cell the cDNA sequence and control elements needed for transcription.
  • Retroviruses have been considered a preferred vector for gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al, Prog. Med. Genet. 7: 130-142, 1988).
  • a nucleic acid encoding the polypeptide comprising or consisting of a disclosed SAHP can be cloned into a retroviral vector and driven from either its endogenous promoter (where applicable) or from the retroviral LTR (long terminal repeat).
  • Other viral transfection systems may also be utilized for this type of approach, including adenovirus, AAV (McLaughlin et al, J. Virol. 62: 1963-1973, 1988), vaccinia virus (Moss et al, Annu. Rev. Immunol. 5:305-324, 1987), Bovine Papilloma virus (Rasmussen et al, Methods Enzymol. 139:642-654, 1987) or members of the herpesvirus group such as Epstein-Barr virus
  • lipidic and liposome-mediated gene delivery has recently been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, Mol. Biotechnol, 11 : 175-180, 1999; Lee and
  • cationic liposomes have been analyzed for their ability to transfect monocytic leukemia cells, and shown to be a viable alternative to using viral vectors (de Lima et al. , Mol. Membr. Biol, 16: 103-109, 1999).
  • Such cationic liposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al , Cancer
  • nucleic acid molecule or vector including the nucleic acid molecule is administered that encodes one or more of the amino acid sequences set forth in SEQ ID NOs: 1, 3, 66, 67, or 68.
  • the provided polypeptide comprising or consisting of a disclosed SAHP or peptide analogs, constructs, or vectors encoding such peptides, can be combined with a pharmaceutically acceptable carrier ⁇ e.g. , a phospholipid or other type of lipid) or vehicle for administration to human or animal subjects.
  • a pharmaceutically acceptable carrier e.g. , a phospholipid or other type of lipid
  • more than one SAHP or peptide analog can be combined to form a single preparation.
  • the SAHPs or peptide analogs can be conveniently presented in unit dosage form and prepared using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze -dried
  • lyophilized condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use.
  • a sterile liquid carrier for example, water for injections
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.
  • unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, formulations encompassed herein may include other agents commonly used by one of ordinary skill in the art.
  • the pharmaceutical compositions provided herein, including those for use in treating dyslipidemic and vascular disorders or CKD, may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes.
  • polypeptide comprising or consisting of a disclosed SAHP or peptide analogs with suitable features of ABCAl-specificity and low cytotoxicity can be precomplexed with phospholipids or other lipids into either discoidal or spherical shape particles prior to administration to subjects.
  • compositions may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment.
  • This may be achieved by, for example, and not by way of limitation, local or regional infusion or perfusion during surgery, topical application (e.g. , wound dressing), injection, catheter, suppository, or implant (e.g. , implants formed from porous, non-porous, or gelatinous materials, including membranes, such as silastic membranes or fibers), and the like.
  • one or more of the disclosed peptides may be administered either by coating or impregnating an implant such as stent to treat a dyslipidemic or vascular disorder or CKD.
  • an implant such as stent to treat a dyslipidemic or vascular disorder or CKD.
  • these peptides are prepared and purified as described herein.
  • the implant can be partially or completely coated with the peptide.
  • the luminal surface of the implant may be coated with the peptide.
  • the peptide may be attached to the implant by any chemical or mechanical bond or force, including linking agents.
  • the coating may be directly linked (tethered) to the first surface, such as through silane groups.
  • the implant may be impregnated with at least one peptide by methods known to those of skill in the art so that multiple surfaces (such as the outer and inner surfaces) of the implant include the peptide.
  • the implant may be coated or impregnated according to methods known to one of ordinary skill in the art.
  • Exemplary, non-limiting examples, of peptide attachment to an implant are discussed in Smith (Radiology 230: 1-2, 2004), United States Patent No. 6,675,920, United States Patent No. 7,402,329, Wessely (Nat. Rev. Cardiol. 7(4): 194-203, 2010), Puskas et al. (Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1(4): 451-62, 2009), Butt et al. (Future Cardiol. 2009 5(2): 141-57,
  • Bosiers et al. (Vase. Health Risk Manag. 4(3): 553-9, 2008) and Kukreja et al. (Pharmacol. Res. 57(3): 171-80, 2008), each of which is incorporated by reference herein in its entirety.
  • the implant may be coated or impregnated with materials in addition to the disclosed peptides to further enhance their bio-utility.
  • suitable coatings are medicated coatings, drug-eluting coatings, hydrophilic coatings, smoothing coatings.
  • administration can be by direct injection at the site (or former site) of a tissue that is to be treated, such as the heart or the peripheral vasculature.
  • the pharmaceutical compositions are delivered in a vesicle, in particular liposomes (see, e.g. , Langer, Science 249: 1527-1533, 1990; Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).
  • the pharmaceutical compositions can be delivered in a controlled release system.
  • a pump can be used (see, e.g., Langer Science 249: 1527-1533, 1990; Sefton Crit. Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al. , Surgery 88:507-516, 1980; Saudek et al. , N. Engl. J. Med. 321 :574-579, 1989).
  • polymeric materials can be used (see, e.g. , Ranger et al. , Macromol. Sci. Rev. Macromol. Chem. 23:61-64, 1983; Levy et al.
  • therapeutic agent(s) are delivered by way of an implanted pump, described, for example, in U.S. Patent No. 6,436,091 ; U.S. Patent No. 5,939,380; and U.S. Patent No. 5,993,414.
  • Implantable drug infusion devices are used to provide subjects with a constant and long term dosage or infusion of a drug or any other therapeutic agent.
  • Active drug or programmable infusion devices feature a pump or a metering system to deliver the drug into the patient' s system.
  • An example of such an active drug infusion device currently available is the Medtronic SynchroMedTM programmable pump.
  • Such pumps typically include a drug reservoir, a peristaltic pump to pump the drug out from the reservoir, and a catheter port to transport the pumped out drug from the reservoir via the pump to a patient' s anatomy.
  • Such devices also typically include a battery to power the pump, as well as an electronic module to control the flow rate of the pump.
  • the Medtronic SynchroMedTM pump further includes an antenna to permit the remote programming of the pump.
  • Passive drug infusion devices in contrast, do not feature a pump, but rather rely upon a pressurized drug reservoir to deliver the drug. Thus, such devices tend to be both smaller as well as cheaper as compared to active devices.
  • An example of such a device includes the Medtronic IsoMedTM. This device delivers the drug into the patient through the force provided by a pressurized reservoir applied across a flow control unit.
  • the amount of the pharmaceutical compositions that will be effective depends on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each subject's circumstances. An example of such a dosage range is 0.1 to 200 mg/kg body weight in single or divided doses. Another example of a dosage range is 1.0 to 100 mg/kg body weight in single or divided doses.
  • about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, or about 50 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to the subject.
  • from 1-5, 1-10, 1-20, 5-10, 5-20, 10-20, 10-30, 10-50, 20-30, or 20-50 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to the subject.
  • no more than 20 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject, such as no more than 5, 10, or 15 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject. In one preferred embodiment, 5, 10, or 15 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject.
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy.
  • compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g. , in which the loading dose is about two to five times the maintenance dose).
  • the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art.
  • the volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to about 1.0 ml.
  • exemplary delivery methods include, but are not limited to, those provide by Malik et al. , . Curr. Drug Deliv. 4(2): 141-151, 2007 which is hereby incorporated by reference in its entirety).
  • compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use.
  • Sterile solutions can be prepared by incorporating the conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the disclosed antigen and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the disclosed antigen plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Kits are also provided.
  • kits for treating CKD or a dyslipidemic or vascular disorder in a subject will typically include a polypeptide comprising or consisting of a disclosed SAHP as disclosed herein. More than one of the SAHPs can be included in the kit.
  • the kit can include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container typically holds a composition including one or more of the disclosed polypeptide comprising or consisting of a disclosed SAHP.
  • the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a label or package insert indicates that the composition is used for treating the particular condition.
  • the label or package insert typically will further include instructions for use of a disclosed polypeptide comprising or consisting of a disclosed SAHP in a therapeutic method, such as a method of treating or inhibiting CKD or a dyslipidemic or vascular disorder in a subject.
  • the package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
  • kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • This example illustrates use of SAHPs for the treatment of CKD.
  • CKD Chronic kidney disease
  • ESRD end stage renal disease
  • CVD cardiovascular disease
  • Renin- Angiotensin-Aldosterone System (RAAS) inhibitors are standard-of-care in CKD, but a significant fraction of treated individuals still progress to ESRD, or die of CVD.
  • RAAS Renin- Angiotensin-Aldosterone System
  • CD36 is a widely expressed cell surface class B scavenger receptor that recognizes a large variety of ligands, including apoA-1, apoL-1, thrombospondin-1, serum amyloid A (SAA), lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL), free fatty acids, advanced glycated end products, apoptotic cell surfaces, phosphatidyl-serine (PS), and microparticles (Madhavan et al. , Journal of the American Society of Nephrology 22, 2119-2128, 2011 ; Lopez-Dee et al. , Mediators of inflammation 2011 , 296069, 2011 ; Baranova et al.
  • SAA serum amyloid A
  • LPS lipopolysaccharide
  • oxLDL oxidized low-density lipoprotein
  • PS phosphatidyl-serine
  • CD36 is upregulated by oxLDL (Liu et al , Inflammation research, 63, 33-43, 2014) or LPS (Bocharov et al , J Biol Chem, 279, 36072-36082, 2004) in macrophages, and contributes to the formation and accumulation of foam cells during atherosclerosis.
  • ApoA- I the main apolipoprotein constituent of HDL, acts as a ligand for many receptors including CD36 (Baranova et al , J Biol Chem, 280, 8031-8040, 2005).
  • ApoA-I mimetic peptide, 5A also binds to CD36 and acts as a CD36 antagonist (FIGs. 7A and 7B).
  • This example illustrated that targeting CD36 is protective against CKD progression and its associated risk factors for CVD in a progressive CKD model that resembles human disease. This protection is achieved either genetically in CD36 knockout (KO) mice, or pharmacologically, by using the peptide 5A-37pA (5A), which is a CD36 antagonist. It is also demonstrated that, in a kidney fibrosis model using an outbred strain (CD-I), that 5A protects mice from interstitial fibrosis and exerts renal antiinflammatory effects while down-regulating renal inflammasome genes.
  • CD-I outbred strain
  • CD36KO mice and WT mice treated with 5A are protected against CKD progression after 5/6 nephrectomy with Angiotensin II infusion. Because CD36KO mice are on C57BL/6 background and C57BL/6 WT mice are resistant to CKD progression following 5/6 nephrectomy (Nx), the 5/6Nx plus continuous Angiotensin II (Angll) infusion model was used to induce progressive CKD in this strain (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010). C57BL/6 WT mice subjected to 5/6Nx without Angll infusion did not develop CKD and were used as controls.
  • Nx 5/6Nx plus continuous Angiotensin II
  • mice subjected to 5/6Nx+AngII which received continuous infusion of 5A peptide by osmotic minipump (5mg/kg/day) were also significantly protected from CKD progression, and had albuminuria levels similar to KO mice (FIGs. 1A-1E).
  • CD36KO and WT mice treated with 5A are protected from a metabolic profile typical of CKD, including risk factors for CVD.
  • CKD a metabolic profile typical of CKD
  • risk factors for CVD CKD
  • 5/6Nx+AngII developed a metabolic profile typical of patients with CKD, including hypercalcemia, hyperphosphatemia, hypermagnesemia, dyslipidemia, and high serum levels of fibroblast growth factor- 23 (FGF-23) (FIGs. 2A-2L).
  • FGF-23 is a phosphate regulatory hormone that is involved in left ventricular hypertrophy, and is associated with subclinical and clinical cardiac disease, including heart failure and coronary vascular events (Kestenbaum et al , Circulation. Heart failure 7, 409-417, 2014).
  • High serum phosphate and FGF-23 levels are independent risk factors for cardiovascular events, particularly among CKD patients (Scialla et al , Journal of the American Society of Nephrology 25, 349-360, 2014; Scialla et al , Nature reviews. Nephrology 10, 268-278, 2014), and FGF-23 can accelerate phosphate-induced uremic vascular calcification (Moe et al, Kidney Int., 85, 1022-1023, 2014; Jimbo et al, Kidney Int., 85, 1103-1111, 2014). WT mice subjected to 5/6Nx+ Angll also had lower serum levels of glucose at 4 weeks.
  • CD36KO mice and WT mice treated with 5A (WT+5A) subjected to 5/6Nx+AngII had a more normal metabolic profile, similar to the control group, which did not develop progressive CKD.
  • WT mice also had significantly increased levels of acute -phase serum amyloid A (SAA) when compared to control group (5/6Nx alone): mean ⁇ standard deviation 131+ 7.07 (control) vs. 378.4+ 80.1 ug/ml (5/6Nx+AngII), p ⁇ 0.05.
  • SAA is a known ligand to CD36 (Baranova et al , J Biol Chem, 285, 8492-8506, 2010) that is increased in CKD patients (Lavin-Gomez et al, Advances in peritoneal dialysis. Conference on Peritoneal Dialysis 27, 33-37, 2011 ; Weichhart et al. , Journal of the American Society of Nephrology 23, 934-947, 2012). Mice treated with 5A did not have liver toxicity, as assessed by serum transaminases (FIGs. 2J and 2K).
  • Peptide 5A down-regulates renal inflammation- and inflammasome-associated genes.
  • a second set of progressive CKD experiments using the most essential groups was performed to obtain kidney tissue to measure renal mRNA expression of genes associated with inflammation and
  • UUO Unilateral Uretheral Obstruction
  • CD36 can scavenge oxidized-LDL and oxidized-HDL (Calvo et al, Journal of lipid research 39, 777-788, 1998), alleviating lipid/cholesterol imbalance by CD36 inhibition cannot account for the anti-fibrotic effect in the UUO model.
  • CD-I an outbred strain
  • renal vascular resistance increases, which reduces renal blood flow to the obstructed kidney (Eddy et al. , Pediatr Nephrol 27, 1233-1247, 2012), and distribution and metabolism of therapeutic agents become difficult.
  • 5A administration was started 24h before surgery using an osmotic minipump, at two doses: 5 and 15 mg/kg/day.
  • Animals were divided in 4 groups: 1. Sham surgery; 2. UUO; 3. UUO+5A5 (5mg/kg/day); and 4. UUO+5A15 (15mg/kg/day).
  • UUO surgery the contralateral non-obstructed kidney compensates for the obstructed kidney, which complicates meaningful renal function measurements in the model.
  • 5A untreated mice developed a very mild but statistically significant increase in BUN, which was accompanied by hypercalcemia, hyperphosphatemia, and hypermagnesemia; whereas 5A pre- treatment did not show any evidence of liver toxicity (FIG. 9).
  • 5A-treated mice were protected from interstitial fibrosis at the higher dose (15mg/kg/day), and had better preserved kidney structure, as measured by cortical thickness (FIGs. 5A and 5B). 5A treatment prevented F4/80 + macrophage infiltration in the obstructed kidney, even at the lower 5 A dose (FIG. 5A).
  • PCT Proximal Convoluted Tubule
  • CD36KO mice were obtained from the NCI-DCT Laboratory, Bethesda, MD. Sixteen-wk-old male C57BL/6 mice were used for experiments with progressive CKD model. Nine-wk-old male CD-I mice were obtained from Charles River Laboratory,
  • 5A-37pA peptide is a 37 residue amphipathic peptide (DWLKAFYDKVAEKLKEAF-P-DWAKAAYDKAAEKAKEAA, SEQ ID NO: 3) that contains a proline between the two amphipathic helices.
  • the peptide contains a high lipid-affinity helix paired with a low lipid-affinity helix with 5 alanine substitutions.
  • 5A peptide was synthesized by a solid-phase procedure using Fmoc/DIC/HOBt chemistry and purified to > 99% by reverse-phase high-performance liquid chromatography (HPLC) as previously described (Bocharov et al , J Biol Chem, 279, 36072-36082, 2004; Sethi et al , J Biol Chem, 283, 32273-32282, 2008). Purity was assessed by MALDI-TOF-MS (Bruker Ultraflex) (Bocharov et al. Biol Chem, 279, 36072-36082, 2004; Sethi et al , J Biol Chem, 283, 32273-32282, 2008).
  • HeLa cells stably transfected with CD36 were incubated with 10 ⁇ g/ml 5A-DMPC-BP-CE [10/70/1 molar ratio] at 37°C for 2 hours then washed with PBS and detached with Cellstripper® dissociation solution (Cellgro, Herndon, VA). Detached cells were fixed with 4% paraformaldehyde and analyzed by a Victor3 fluorimeter (Perkin Elmer). Mock-transfected HeLa cells were used as controls.
  • mice received a single dose of buprenorphine (0.1 mg/kg) diluted in saline (lml/25g), followed by buprenorphine 0.05mg/kg 18h after the procedure.
  • mice subjected to 5/6Nx without Angll infusion did not develop progressive CKD after 4wk and were considered controls.
  • Kidney Fibrosis Model Unilateral Uretheral Obstruction, UUO. Under isoflurane anesthesia CD-I mice were subjected to right flank incision and right kidney and pelvis were identified. The right ureter was ligated with a double suture below the pelvis and at the end of inferior kidney pole (Shen et al. , Inflammation DOI 10.1007/sl0753-014-9941-y, 2014). Immediately after each surgical intervention, mice received a single dose of buprenorphine (0.1 mg/kg) diluted in saline ( 1 ml/25 g), followed by
  • Angll Val5-AngII 0.75 ⁇ g/kg/min (Sigma-Aldrich, St Louis, MO, USA), diluted in sterile water, or vehicle (sterile water) was continuously infused by subcutaneous osmotic mini- pump (Alzet model 1004, Cupertino, CA). 5 A was continuously administered through a second osmotic mini-pump, at the dose of 5mg/kg/day in the progressive CKD model. Osmotic mini-pumps were inserted at the time of right nephrectomy. In the UUO model, osmotic minipumps with 5 A were started 24h before UUO (5 or 15mg/kg/day).
  • mice were euthanized under isoflurane anesthesia by cardiac puncture, and blood was collected. The remnant kidney was harvested and fixed in 10% formalin. Urine samples were not collected in the UUO model. Serum creatinine was measured by HPLC, and blood urea nitrogen (BUN) by colorimetric assay (QuantiChrom Urea assay kit DIUR-500, Hayward, CA, USA). Urine albumin-to-creatinine -ratio (ACR) was determined from albumin ELISA (Albuwell M; Exocell, Philadelphia, PA, USA) and creatinine by Jaffe method.
  • ACR Urine albumin-to-creatinine -ratio
  • a biochemistry panel was measured using a Siemens Advia 1800 automated chemistry analyzer (Siemens Healthcare Diagnostics, Flanders, NJ). Serum FGF-23 and SAA were measured by ELISA (Immunotopics, Inc., Cat. #60-6300; Abeam, kit abl57723, respectively).
  • Kidney specimens were fixed in 10% formalin, paraffin embedded (FFPE), and stained with Masson's trichrome and periodic acid-Schiff (PAS) reagent (Sigma- Aldrich). Histological changes were assessed semi-quantitatively. The degree of glomerular damage was assessed in 10 randomly selected fields at 400X magnification from the degree of mesangial expansion in PAS-stained tissue and scored as follows: 1, ⁇ 25%; 2, 25-50%; 3, 50-75%; 4, >75%; 5, completely sclerotic glomeruli.
  • Interstitial fibrosis was assessed at 200X magnification on Masson's trichrome- stained sections using 10 randomly selected fields for each animal and scored by the following criteria: 1, area of damage ⁇ 10%; 2, 10-25%; 3, 25-50%; 4, 50-75%; and 5, 75-100%.
  • Immunohistochemistry for CD36 and F4/80 + .
  • Four micron FFPE sections were de- paraffinized, incubated 10 min with an endogenous peroxide blocker (Dako S2003) and then treated for 1 h in an antigen retrieval solution in a steamer (Dako Cat#1700).
  • sections were incubated with a protein blocking solution (CytoQ Background Buster) for 20 min before incubation with the rabbit polyclonal anti-CD36 antibody (Novus, NB400-144) for lh at 1: 1,600.
  • An isotype rabbit IgG Ab was used as a control.
  • Sections were then incubated with a goat anti-rabbit HRP-conjugated Ab for 30 min at 1 :200 followed by DAB Chromogen for 5 min. Sections were next counterstained (Leica Autostainer), coverslipped and digitally captured (Aperio ScanScope). Between each step above, sections were washed with TBST buffer 2X for 5 min. IHC for CD36 was performed on kidney sections from a KO mouse and a WT mouse, both subjected to 5/6Nx+AngII. IHC for F4/80 + cells (Ab from AbD Serotec,
  • mice from each group [2. WT 5/6Nx+AngII (WT); 3. CD36KO 5/6Nx+AngII (KO)] in the progressive CKD model, a telemeter transmitter (model TA11PA-C10, Data
  • WT WT
  • WT WT 5/6Nx+AngII+5A
  • tissue samples preserved in RNAlater (Life Technologies cat#AM7021, Grand Island, NY) were homogenized in TRIzol Reagent using a Precellys 24 homogenizer (Bertin Technologies, France).
  • RNA isolation, reverse transcription and real-time PCR were obtained from Life Technologies.
  • RNA was isolated with the PureLink RNA Mini Kit after DNase treatment.
  • RNA (2 ⁇ g) was reverse-transcribed using a TaqMan Reverse Transcriptase Reagents Kit.
  • Real-time qPCR assays were performed with a StepOne Real-Time PCR System (Applied Biosystems), with 40ng of cDNA per reaction.
  • a list of TaqMan Gene Expression assays used in the study is shown in FIG. 10.
  • the relative levels of gene expression were measured by the comparative CT (AACT) method (Schmittgen et al. , Nature protocols 3, 1101-1108, 2008) with mouse GAPDH used as a reference gene. All gene expression results were analysed using 2 ⁇ € ⁇ formula and presented as normalized fold changes, compared to Control.
  • Fluor-labeled peptide uptake 2-photon microscopy was performed on kidneys of a WT and a CD36KO mouse 3h after IV injection of Alexa Fluor® 488-labeled 5A peptide. 5A labeling with Alexa Fluor® 488 was performed as previously described (Baranova et al. , J Biol Chem, 285, 8492-8506, 2010).
  • Kidney tissue immunofluorescence staining Kidney frozen O.C.T blocks from a WT and a CD36KO mouse were cut into 10-12 ⁇ sections using a Bright cryostat and put on microscope slides.
  • Sections were fixed with 3.7% paraformaldehyde (Electron Microscopy Sciences, PA) for 10 min, washed 3 times, 5 min each, with 0.5% Saponin in PBS. Sections were blocked with 5% Goat Serum-0.05% Saponin-l%BSA-PBS for 1 h then incubated overnight at 4°C with Rabbit polyclonal anti-CD36 antibody (Abeam # ab36977) or Rabbit IgG for nonspecific control, followed by 1 h incubation with secondary antibodies, anti-rabbit-Alexa Fluor 488.
  • tissue were stained with DAPI (Invitrogen) mounted with Vectashield® antifade mounting medium reagent (Vector, cat # H-1400), and subjected to microscopy (Zeiss 510 laser scanning confocal microscope). Images were acquired by using a 488-nm laser line and emission between 505 and 580 nm for Alexa Fluor 488.
  • CD36 antagonistic peptide (5A) may act in the proximal convoluted tubule as injected fluorescent-5A co- localizes with CD36 protein expression in the proximal tubule. 5A exhibits anti-inflammatory effects in the kidney, in part via decreased renal expression of inflammasome gene NLRP3.
  • CD36 has been considered a possible therapeutic target for atherosclerosis (Park et al. , Experimental & molecular medicine 46, e99, 2014)
  • the data provided in this example suggests a direct role for CD36 on CKD progression that is independent of blood pressure and likely involves anti-inflammatory and anti-fibrotic effects.
  • dyslipidemia gradually developed over time in WT mice, and was lessened in CD36KO and 5A-treated mice.
  • CD36 inhibition is working as an oxidized-LDL scavenger receptor or has a local renal mechanism independent of oxidized-LDL.
  • CD36 antagonist peptide 5A was tested in the UUO model using mice fed standard (not high fat) diet. Ten days after sham-surgery or UUO, all CD-I mice (treated or non-treated with 5 A) had similar cholesterol levels. 5A-treated mice subjected to UUO developed less kidney fibrosis than non-treated mice, without altering cholesterol levels; thus, CD36 affects CKD beyond its role as a lipid scavenger receptor.
  • Renin-angiotensin-aldosterone (RAAS) blockers are standard of care for CKD in the clinical practice. RAAS blockers decrease both albuminuria and renal fibrosis in parallel (Leelahavanichkul et al. , Kidney Int., 78, 1136-1153, 2010). Despite the reduction in fibrosis in the 5/6Nx+AngII and UUO models, in the 5/6Nx+AngII model there was only a partial decrease in albumin-to-creatinine -ratio (ACR) in both CD36KO and 5A-treated mice.
  • ACR albumin-to-creatinine -ratio
  • CD36 This partial reduction in albuminuria may be explained by CD36 participating, together with megalin and cubilin (Baines et al., Am J Physiol Renal Physiol 303, F1006- 1014, 2012), in albumin binding and endocytosis in renal proximal tubular cells. Blocking CD36 may reduce the uptake of filtered albumin by the proximal tubule; thus, targeting CD36 prevents CKD progression without dramatic changes in albuminuria. Yang et al. demonstrated that CD36 can mediate albumin-induced cellular fibrosis in cultured proximal tubule cells (Yang et al , J Cell Biochem 101, 735- 744, 2007).
  • inhibiting CD36 might have two competing effects on albuminuria: a) a decrease caused by ameliorating progressive CKD and b) an increase from impaired albumin reuptake.
  • a partial decrease in albuminuria appears to underestimate the benefit of CD36 inhibition on CKD, as CD36 inhibition prevents albumin-induced damage to the proximal tubule.
  • Targeting CD36 may be an adjuvant therapy to RAAS inhibitors, which may be more effective than monotherapy because of non- redundant mechanisms.
  • CKD mice In addition to hypertension and dyslipidemia, CKD mice also developed other risk factors for cardiovascular disease and vascular injury, such as high FGF-23 levels and hyperphosphatemia, which can contribute to endothelial dysfunction and release of microparticles (Agouni et al. , Curr Vase
  • CKD also increases other possible CD36 ligands, including serum amyloid A (SAA) (Baranova et al , J Biol Chem, 285, 8492-8506, 2010), that could contribute to inflammation, oxidative stress, and CKD progression.
  • SAA serum amyloid A
  • Serum amyloid A (SAA) is an acute phase protein that is increased in CKD patients (Weichhart et al , J Am Soc Nephrol 23, 934-947, 2012) and was also increased in the model disclosed herein.
  • Chronic inflammation is thought to be important in the progression of both cardiovascular disease and CKD, and CD36 activation is associated with downstream activation of inflammation (Moore et al., J Biol Chem, 277, 47373-47379, 2002; Febbraio et al., Int J. Biochem Cell Biol 39, 2012-2030, 2007) and inflammasome (Liu et al. , Inflamm Res, 63, 33-43, 2014; Sheedy et al., Nat Immunol 14, 812-820, 2013; Kagan et al , Nat Immunol 14, 112-11 , 2013; Oury et al , Cell Mol Immunol 11, 8-10, 2014).
  • 5A decreases mRNA for renal cytokines in both models, and decreases macrophage infiltration 10 days after UUO. Besides renal effects, CD36 inhibition also can decrease inflammation and fibrosis in other organs. 5A also decreases the expression of genes associated with collagen deposition in the lungs (Yao et al , Chest 140, 1048-1054, 2011). Further, silencing of the CD36 gene results in suppression of silica-induced lung fibrosis in rats (Wang et al , Resp Res 10, 36, 2009).
  • 5A is an apoA-I mimetic peptide that has also been used to attenuate atherosclerosis, acute vascular inflammation, and oxidative stress in experimental animal models (Amar et al. , The Journal of pharmacology and experimental therapeutics 334, 634-641, 2010; Yao et al., Chest 140, 1048-1054, 2011).
  • apoA-I is the major structural protein of HDL that mediates reverse cholesterol transport
  • 5A is thought to actively remove cholesterol from atherosclerotic plaque by enhancing reverse cholesterol transport.
  • 5 A also has anti-inflammatory effects that are independent of HDL levels (Yao et al. , Chest 140, 1048-1054, 2011).
  • CD36 activates the inflammasome (Liu et al , Inflammation research, 63, 33-43, 2014; Sheedy et al, Nat Immunol 14, 812-820, 2013; Kagan et al , Nature immunology 14, 772- 774, 2013; Oury et al. , Cell Mol Immunol 11, 8-10, 2014), which is implicated in renal tissue repair, remodeling, and maladaptive inflammatory responses (Leemans et al , Nature reviews. Nephrology, 2014).
  • NLRP3 and IL- ⁇ renal mRNA expression is increased in both kidney injury models. 5A prevented this increase, suggesting that it may have an effect on down-regulating inflammasome in the kidney. 5A likely exerts some of its actions on the proximal tubule cells.
  • CD36 is a new therapeutic target and 5A is a new potential therapy to slow CKD progression.
  • the blood-pressure-independent benefit of CD36 inhibition suggests it might complement RAAS inhibition through non-overlapping effects, possibly leading to combination therapy.
  • Targeting CD36 should be examined as a possible alternative or second line therapy for CKD, especially when RAAS inhibitors are contra-indicated. New therapies to slow CKD progression would benefit millions of individuals with CKD worldwide.
  • Example 2 CD36 selective SAHP Peptides
  • ELK-B and ELK-B2 are CD36 antagonists.
  • a panel of 20 SAHP were tested in HEK293 cell lines stably transfected with CD36.
  • SR-BI or SR-BII, to identify SAHP with preferential selectivity towards CD36.
  • ELK-B and ELK-B 2 acted selectively inhibited CD36 activity compared to SR-BI and SR-BII activity under similar conditions.
  • This peptide consists of two type A amphipathic a-helices connected by a proline.
  • the hydrophobicity of the second helix was reduced by substitution of hydrophobic amino acids with alanine.
  • Four derivatives of 5A were synthesized to test the impact of the introduction of two amino acids with antioxidant potential, cysteine and histidine, on its properties.
  • the second prototype peptide, ELK contains only three amino acid residues: glutamic acid, leucine, and lysine. It consists of two identical canonical type A amphipathic a-helices with hydrophobic interface turned by 180° and neutral net charge. The helices within this peptide are connected by a proline residue.
  • the original ELK peptide was used to make sixteen derivatives.
  • HEK 293 cells stably transfected to express hSR-BI, hSR-BII and hCD36 were reported in previous publications. The cells were grown to 50% confluency in
  • DMEM additionally containing 10% FBS and antibiotics. After 24 hour incubation in serum free DMEM, cells were challenged with various concentrations of LPS in the presence or absence of various peptides
  • L37pA binds to SR-BI, SR-BII, CD36, and blocks LPS-induced IL8-secretion.
  • SR-BI, SR-BII and CD36 bind various lipoproteins including HDL and its apolipoproteins A-I and -AIL Because specificity of SAHP is not well understood, the uptake of the most known SAHP, L37pA and its inactive analogue L3D-37pA was first evaluated in HeLa cell lines stably transfected with human SR-BI, SR-BII and CD36 and compared with canonical SR-B ligands, HDL and LDL.
  • Alexa 488 HDL, Alexa 488 LDL and Alexa 488 L37pA was significantly increased in both hSR-BI and CD36 expressing HeLa cells when compared to mock-transfected controls (FIG. 13A). No increase was found for L3D-37pA. Furthermore, no increase in Alexa 488-BSA or Alexa 488- lactoferrin uptake was seen in these cells when compared to Mock-transfected HeLa controls. Uptake of L37pA was dose-dependent and demonstrated similar dose-response in SR-BI, SR-BII, and CD36 expressing HeLa. Both mock-HeLa and LDL receptor HeLa minimally bound Alexa 488-L37pA (FIG. 13B).
  • L37pA, 5A, and ELK-based SAHP differentially inhibit LPS-induced IL-8 secretion by SR-
  • ELK- B and ELK-B2 blocked IL-8 secretion induced by LPS in cells expressing CD36, but were less effective in blocking LPS-induced IL-8 secretion in cells expressing SR-BI, or SR-BII (Table 4).
  • the majority of tested peptides had either no effect or affected both receptors, leading to suppression of LPS-induced IL-8 secretion in all cell lines in a dose-dependent manner (FIGs. 14A and 14B).
  • ELK-B and ELK-B2 peptides are the most efficient and relatively selective antagonists of LPS-induced IL-8 secretion in cells expressing the CD36 receptor.
  • Table 4 Effect of ELK based SAHP on LPS-induced IL-6 secretion from hSR-BI, hSR-BI and hCD36 stably transfected HEK293 cells.
  • This example describes an exemplary method for treating and/or inhibiting CKD in a human subject.
  • the method includes administration of a therapeutically effective amount of a polypeptide including or consisting of a SAHP as disclosed herein to a subject with or at risk of chronic kidney disease.
  • CKD can be treated by administering a therapeutically effective amount of a polypeptide including or consisting of a SAHP, such as a SAHP including the amino acid sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 3 to a subject in need thereof.
  • a SAHP such as a SAHP including the amino acid sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 3
  • the method can include screening subjects to determine if they have CKD, or if they are at risk of developing CKD.
  • Subjects having or at risk of CKD can be selected for treatment.
  • subjects having Stage 1, Stage 2, Stage 3, Stage 4, and/or Stage 5 CKD are selected for treatment.
  • Standard methods are used to identify a subject with CKD, or a particular stage of CKD.
  • a subject with an estimated glomerular filtration rate (GFR) of less than 60 niL/min/1.73 m 2 can be selected for treatment. Pre-screening is not required prior to administration of the therapeutic compositions disclosed herein.
  • the subject is treated prior to administration of a therapeutic agent that includes one or more of the disclosed SAHPs or a nucleic acid molecule encoding a SAHP.
  • a therapeutic agent that includes one or more of the disclosed SAHPs or a nucleic acid molecule encoding a SAHP.
  • the subject can be treated with an established protocol for treatment of CKD (such as a ACEi or ARB therapy).
  • a therapeutic effective amount of a polypeptide including a SAHP or a nucleic acid molecule encoding the polypeptide is administered to the subject (such as an adult human with CKD).
  • the methods can include administering a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP having the amino acid sequence set forth as any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.
  • Additional agents, such as an ACEi or ARB can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous.
  • the amount of the composition administered to inhibit and/or treat CKD depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic
  • a therapeutically effective amount of an agent is the amount sufficient to inhibit and/or treat CKD in the subject without causing a substantial cytotoxic effect in the subject.
  • An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves.
  • from 1 mg/kg/day to 10 mg/kg/day or from 1 mg/kg/day to 20 mg/kg/day of the polypeptide including the SAHP is administered to the subject.
  • the therapeutic compositions can be administered in a single dose delivery, via continuous delivery over an extended time period (for example, using a pump), in a repeated administration protocol (for example, by a daily, weekly, or monthly repeated administration protocol).
  • therapeutic agents that include one or more SAHPs or a nucleic acid molecule encoding the polypeptide are administered intravenously to a human.
  • these compositions may be formulated with an inert diluent or with an inert diluent or with an inert diluent or with an inert
  • Administration of the therapeutic compositions can be taken long term (for example over a period of months or years).
  • subjects having CKD can be monitored for reductions in one or more clinical symptoms associated with CKD, such as glomerular filtration rate or disease progression.
  • subjects are analyzed one or more times, starting 7 days following treatment.
  • Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood and urine, can be obtained and alterations in biomarkers indicative of kidney function and/or injury evaluated.

Abstract

Disclosed herein are polypeptides comprising or consisting of a synthetic amphipathic helical peptide or peptide analog, and nucleic acid molecules encoding such polypeptides. Also disclosed herein are methods of using the disclosed polypeptides, analogs, or nucleic acid molecules to treat and/or inhibit chronic kidney disease, as well as to treat and/or inhibit dyslipidemic or vascular disorders, such as methods to treat or prevent cardiovascular disease including atherosclerosis.

Description

TREATMENT OF CHRONIC KIDNEY DISEASE WITH SAHPS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/890,585, filed October 14, 2013, which is incorporated by reference in its entirety.
FIELD
This disclosure relates to synthetic amphipathic helical peptides, analogs thereof, and their use, for example for the treatment and inhibition of disease, such as chronic kidney disease, or dyslipidemic or vascular disorders.
BACKGROUND
Kidney disease, including chronic and acute disease, causes over 800,000 deaths worldwide each year. Acute kidney disease (AKD) involves loss of kidney function typically stemming from an acute causative event (e.g. , sepsis, ischemia, trauma, and/or nephrotoxic drugs). In contrast, chronic kidney disease (CKD) involves progressive loss of kidney function over a period of months or years. The pathophysiology of kidney disease varies greatly depending on the type of disease. For example, multiple pathogenic processes such as inflammation, hypoxia, pro-fibrotic cell accumulation, extracellular matrix remodeling, and vascular drop-out have been proposed to be involved in CKD progression.
Although angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers
(ARBs) are included in current standard-of-care for CKD, they do not slow progression in about half of patients with CKD; the remaining CKD patients progress to end stage renal disease or die prematurely of other causes regardless of CKD therapy. Recent novel therapeutic advances, including bardoxolone and pirfenidone and dual ACE-ARB therapy to slow down the progression of CKD have largely failed, due in part to the complexity of CKD and the divergent pathophysiologies associated with kidney disease, and/or undesirable side effects. Thus, there exists a need for innovative approaches to successfully treat and/or inhibit CKD, including progression of CKD, in a subject.
SUMMARY
Methods of inhibiting and/or treating CKD in a subject by administering a therapeutically effective amount of a Synthetic Amphipathic Alpha-Helical Peptide (SAHP) that is a Cluster of
Differentiation 36 (CD36) antagonist are provided. The subject can have CKD or be at risk of CKD. In some embodiments, the subject with or at risk of CKD is selected prior to administration of the therapeutically effective amount of the SAHP. In some examples, a therapeutically effective amount of a polypeptide comprising a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 3 (5A- 37pA), SEQ ID NO: 1 (L37pA), SEQ ID NO: 66 (ELK-B/B2 consensus), SEQ ID NO: 67 (ELK- B), or SEQ ID NO: 68 (ELK-B2) is administered to the subject to inhibit and/or treat CKD in a subject with or at risk of CKD. In additional embodiments, polypeptides comprising SAHPs and analogs thereof have been identified and are described herein. In one example, the SAHP includes multiple amphipathic alpha- helical domains and comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 66, 67, or 68. In some embodiments, a disclosed SAHP is a CD36 antagonist. In further embodiments, a disclosed SAHP is a selective CD36 antagonist that inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
In further embodiments, a method of inhibiting and/or treating a dyslipidemic or vascular disorder by administering a therapeutically effective amount of a disclosed SAHP to a subject is provided. The subject can be a subject with or at risk of a dyslipidemic or vascular disorder. In some embodiments, the subject is selected prior to administration of the therapeutically effective amount of the SAHP.
Dyslipidemic and vascular disorders amenable to treatment with the isolated SAHPs disclosed herein include, but are not limited to, hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,
hypertriglyceridemia, high-density lipoprotein (HDL) deficiency, ApoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, reperfusion myocardial injury, vasculitis, inflammation, or combinations of two or more thereof. In some examples, a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, and/or SEQ ID NO: 68 is administered to the subject.
The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
FIGURES
FIGs. 1A-1E. Effects of CD36KO and 5A peptide therapy on CKD progression. After 4 weeks, untreated WT mice subjected to 5/6Nx+AngII had increased levels of BUN (a) and serum creatinine (b), glomerulosclerosis (c), interstitial fibrosis (d), and (e) urinary albumin-to-creatinine ratio (ACR). These changes were reduced in KO mice and in WT mice treated with 5 A (N=l 1-19/group, ANOVA with Dunnett's post-hoc test for a-d; N=l 1-19/group for e, mixed-model analysis, see Example 1, methods). Weekly ACR values were analyzed using mixed-effects models (see Example 1, statistical analysis).
FIGs. 2A-2L. Effects of CD36KO and 5A peptide therapy on metabolic and electrolyte abnormalities associated with CKD. Four weeks after 5/6Nx with Angll infusion, WT mice had hypercalcemia (a), hyperphosphatemia (b), increased calcium x phosphorus product (c), increased levels of alkaline phosphatase (d), hypermagnesemia (e), hypoglycemia (f), and hypercholesterolemia (g). All these metabolic changes were prevented in CD36KO mice and in WT mice treated with 5A. ALT (j) and AST (k) were not different between the groups. WT mice subjected to 5/6Nx with Angll infusion had very high serum FGF-23 (1) levels, which was significantly lower in CD36KO mice. (N=8-10/group, ANOVA with Dunnett's post-hoc test for a-k; N=6-13/group, ANOVA with Dunnett's post-hoc test for 1). FIGs. 3A-3D. Effects of CKD and CK36KO on telemetry blood pressure. Mice were subjected to 5/6Nx, Angiotensin II infusion, and telemetry: WT and CD36KO. Upper graph shows model-based weekly mean values: Systolic (a), diastolic (b), and mean arterial blood (c) pressures (Y-axis) significantly increased over time (X-axis), from baseline to 4 weeks, p<0.001, in both groups. There were not statistical differences between WT and CD36KO mice subjected to 5/6Nx with Angll infusion in a, b, or c. Pulse pressure (d) increased over time in the WT group (p<0.001), but it was kept almost constant and did not significantly increase in the KO group. Differences in pulse pressure (d) between the 2 groups started to become statistically different (p<0.05) after 2 weeks. Lower graph shows estimates by group-specific sinusoid-by-weeks model: Systolic (a), diastolic (b), and mean arterial blood (c) pressures (Y-axis) increased over time, from baseline to 4wks, p<0.001 , in both groups. In these figures, time in weeks is indicated: baseline (B), week 1 (1), week 2 (2), week 3 (3), week 4 (4). X-axis represents time elapsed after 13:00 (1 PM), time when recording of telemetry data was started, for 24h after it. Pulse pressure (d) also increases over time in both, but significantly in the WT group. The circadian pattern on blood pressure in mice can be observed. (N=6/group; mixed-model analysis (see Example 1, methods).
FIGs. 4A-4F. Effects of 5 A therapy on renal mRNA expression of cytokines and NLRP3 on the progressive CKD model. Four weeks after 5/6Nx with Angll infusion, there is a significant increase in renal mRNA expression of IL-6 (a) and CXCL-1 (b) in WT mice, which is prevented by 5 A therapy. There is also a trend toward increased renal mRNA expression of TNF-OC (c) and TGF-βΙ (d), which were lower in mice that received 5A. In the remnant kidney, there is a significant increase on mRNA expression of II- 1 β (e) and NLRP3 (f) genes, which was prevented by 5 A treatment. (N=4-6/group, ANOVA with Dunnett's post-hoc test).
FIGs. 5A-5C. Effects of 5A therapy on kidney fibrosis and renal mRNA expression of cytokines and NLRP3 on the UUO model. Ten days after Unilateral Uretheral Obstruction (UUO), untreated WT CD-I mice had substantial interstitial fibrosis, cortex thinning, and macrophage infiltration of the obstructed kidney (a). Mice treated with 5A, at a higher dose, were protected, (b) = Representative pictures of obstructed kidney sections stained with Masson's-trichrome from all groups (400x) demonstrating increased interstitial fibrosis in obstructed kidneys when compared to sham, (c) = Renal mRNA expression of cytokines and inflammasome-associated genes. (N=8/group for a and b; N=4/group for c; ANOVA with Dunnett's post-hoc test).
FIGs. 6A and 6B. Localization of CD36 and fluorescent 5A peptide in the kidney, (a) Kidney tissue immunofluorescence staining for CD36 showing CD36 expression in the kidney (light grey) of WT mouse. CD36 is not expressed in the kidney of CD36KO mouse; dark grey = DAPI. (b)
Immunohistochemistry shows CD36 expression on proximal tubular cells of WT mice but not in
CD36KO mice (200X). (c) Three hours after intravenous injection of Alexa Fluor® 488-labeled 5A peptide, the fluorescent peptide can be seen on the same location where CD36 is mostly expressed in the kidneys (proximal tubule cells) on 2-photon microscopy.
FIGs. 7A and 7B. 5A-DMPC-BODIPY-CE uptake in human CD36 expressing HeLa cells. CD36 stably transfected HeLa cells were incubated without any ligands, with 5A-POPC-BP-CE for 2 h at 37°C followed by quantification on a Victor3 fluorimeter (Perkin Elmer). Dose-dependent uptake of 5A- DMPC-BP-CE and DMPC-BP-CE to cultured HeLa cell expressing CD36 (triangle) and mock- transfected cells (square) is shown in panels a and b, respectively. DMPC-BP-CE also binds to CD36 by itself, but weakly.
FIG. 8. Effects of CKD and 5A peptide treatment on biochemistry profiles. Experiments with:
1. Control; 2. WT mice subjected to 5/6Nx+AngII; 3. WT mice subjected to 5/6Nx+AngII+5A were performed for renal mRNA expression of cytokines and NLRP3. In this set of experiments, which confirmed the results in Fig. 2, mice that did not receive 5A also had loss of kidney function accompanied by an unhealthy metabolic profile. 5A-treated mice were protected. (N=4-6/group, ANOVA with Dunnett's post-hoc test).
FIG. 9. Effect of UUO and 5 A peptide therapy on biochemistry profile. Ten 10 days after UUO, as expected, there was not any substantial loss of kidney function on any group. These mice also did not have substantial metabolic changes. A very small increase in BUN was observed in mice subjected to UUO that did not receive 5 A treatment. (N=8/group, ANOVA with Dunnett's post-hoc test).
FIG. 10. List of TaqMan Real-Time PCR assays used in Example 1.
FIG. 11. Outline of progressive CKD model. After collecting baseline urine sample, C57BL/6 mice were subjected to 2/3 nephrectomy of the left kidney (week -1), by removing both superior and inferior poles of the left kidney. One week later (0), mice were subjected to complete right nephrectomy. After right nephrectomy was complete, mice received one subcutaneous osmotic minipump without Angiotensin II (control) or with Angiotensin II (progressive CKD) - continuous infusion of
0.75μg/kg/day), and one minipump without or with 5A peptide (continuous infusion of 5mg/kg/day). Then, mice were followed with weekly urine sample collection, until euthanasia 4 weeks after 5/6 nephrectomy was complete. Mice that were subjected to telemetry data analysis were subjected to carotid artery catheter implantation at week -2, one week prior to the start of all following surgeries. For more details, please refer to Methods.
FIG. 12. Summary of Unilateral Urethral Obstruction (UUO) model. Twenty-four hours (-1 day) before sham or surgery (UUO), CD-I mice received a subcutaneous osmotic minipump without or with 5A (5 or 15mg/kg/day). All mice were euthanized 10 days after UUO.
FIGs. 13A and 13B. Uptake of scavenger receptor ligand in HeLa cells stably expressing SR-BI, SR-BII, or CD36. SR-BI or SR-BII stably transfected HeLa cells were incubated without any ligands, or with the scavenger receptor ligands Alexa 488-labeled HDL, Alexa 488-labeled LDL or Alexa 488- labeled L37pA for 2 hours at 37°C followed by FACS analysis (FIG. 13A). Dose-dependent binding of Alexa 488- L37pA to cultured HeLa cells expressing SR-BI (CI), SR-BII (C2), CD36, LDL receptor (LDLr), or controls is shown in FIG. 13B. Results are presented as mean ±SEM.
FIGs. 14A and 14B. Effects of SAHP on lipopolysaccharide (LPS)-induced Interleukin-8 (IL-8) secretion in human embryonic kidney (HEK)293 cells. Control HEK293 cells (WT) or HEK293 cells stably expressing CD36 were treated with the indicated doses of LPS (0, 20, 50, 100, 250 and 500 ng/ml) for 24 hours, and the resulting amount of IL-8 secreted from the cells was assayed (FIG. 14A). Additionally, CD36 expressing cells were incubated with 50 ng/ml of LPS in the presence or absence of increasing concentrations of the L37pA (SEQ ID NO: 1), ELK (SEQ ID NO: 46), ELK-B (SEQ ID NO: 67), 5A-37pA (SEQ ID NO: 3), or control L3D-37pA (SEQ ID NO: 1 synthesized with L-amino acids except that D-amino acids were used for alanine, lysine, and aspartic acid) SAHP for 24 hours in serum- free media, and the resulting amount of IL-8 secreted from the cells was assayed (FIG. 14B). IL-8 was measured in conditioned media and presented as pg/mg of cell protein (FIG. 14A) or as a percentage of inhibition in hCD36-HEK293 (FIG. 14B). Results are presented as mean +SEM.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named "Sequence.txt" (-36 kb), which was created on September 22, 2014, which is incorporated by reference herein. In the accompanying Sequence Listing:
SEQ ID NOs: 1 and 3-68 are the amino acid sequences of SAHPs.
SEQ ID NO: 2, and 69-70 are the amino acid sequences of polyproline linkers.
SEQ ID NO: 71-87 are exemplary amino acid sequences of functional domain peptides.
DETAILED DESCRIPTION
I. Introduction
ApoA-I, the predominant protein constituent of HDL (Panagotopulos et al, J. Biol. Chem.
277:39477-39484, 2002), is believed to promote lipid efflux from cells by a detergent-like extraction process (Remaley et al, J. Lipid Res. 44:828-836, 2003). The ABCA1 transporter has been proposed to facilitate this process by creating a lipid microdomain that promotes the binding of ApoA-I to cells and creates a lipid domain that is susceptible for removal by ApoA-I by a detergent-like extraction process. When lipid efflux occurs by ApoA-I and the other natural exchangeable type apolipoproteins, it occurs by a non-cytotoxic process, whereby the integrity of the cell membrane is maintained (Remaley et al, J. Lipid Res. 44:828-836, 2003). Based on these findings, ApoA-I has been tested and shown to be effective as a therapeutic agent in animal models to ameliorate development and progression of atherosclerosis by accelerating cholesterol efflux from lipid-laden macrophages and removing proinflammatory oxidized phospholipids from arterial cell walls. However, pharmaceutical grade quality ApoA-I protein is expensive and may not be commercially feasible as a therapeutic agent.
To address this problem, a number of short ApoA-I mimetic peptides, termed synthetic amphipathic helical peptides (SAHP) that retain the beneficial effects of ApoA-I have been developed. SAHPs have multiple amphipathic alpha-helical structures that recapitulate the secondary structure of the native ApoA-I protein, which contains at least eight amphipathic alpha helices. The helical amphipathic structure enables their interactions with class B scavenger receptors including CD36 and its homologues, SR-BI and SR-BII. Known SAHPs, such as L37pA and 5A-37pA, target equally both CD36 and SR-BI. Novel SAHPs are disclosed herein, that are designed to target primarily only one scavenger receptor, such as the ELK-B (SEQ ID NO: 67) and ELK-B2 (SEQ ID NO: 68) SAHPs, which selectively target the CD36 scavenger receptor.
Also disclosed herein is the surprising finding that SAHPs are useful for the treatment of CKD. Prior to the disclosure provided herein, there was no connection between use of SAHPs (previously disclosed for treatment of dyslipidemic or vascular disorders) and CKD. Mechanistically, CKD progression is believed to be quite different from that of dyslipidemic or vascular disorders. As noted above, prior therapeutic agents for the treatment of kidney disease (chronic or acute disease), have provided limited success, in part, due to the divergent etiology of different types of kidney disease. The difficulties in developing a successful therapeutic regimen for treatment of kidney disease is exemplified by the finding that ACE inhibitors, a component of the current standard of care for treatment of CKD, can actually cause acute kidney disease in patients with otherwise healthy kidney function (Onuigbo, Nephron Clin. Pract., 118:c407-c419, 2011). Further, clinical trials of other anti-CKD agents such as bardoxolone and aliskiren have failed for lack of efficacy (cardiovascular and renal endpoints) or due to side effects, including hyperkalemia (de Zeeuw, New Engl J Med 369:. 2492-2503, 2013; Parving, New Engl J Med 367:2204-2213, 2013) Given the unpredictable nature of kidney disease, and CKD in particular, it is surprising that multiple SAHPs are shown herein to effectively reduce symptoms of CKD in a mouse model.
Using this in vivo model of CKD, it is shown herein that CKD treatment via SAHP administration requires the CD36 scavenger receptor. Scavenger receptors, including CD36, are known as "cleaners" of the vascular system, due to their extensive uptake of numerous foreign substances and waste materials, as well as cholesterol and lipids. Due to the multi-faceted role of CD36 in human biology, prior studies have concluded that determining CD36 function in any tissue or system requires empirical evidence (see, e.g., Silverstein and Febbraio, Sci. Signal., 72:re3, 2009). Based on the findings reported herein, methods of treating CKD comprising administration of therapeutically effective amount of a SAHP, such as a SAHP that is a CD36 receptor antagonist, to a subject in need thereof, are provided. The disclosed methods can be utilized for the treatment and/or inhibition of CKD, as well as dyslipidemic or vascular disorders.
II. Summary of Terms
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 X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley- VCH in 16 volumes, 2008; and other similar references.
As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term "a polypeptide" includes single or plural polypeptides and can be considered equivalent to the phrase "at least one polypeptide." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The phrase "and/or" means "and" or "or." It is further to be understood that any and 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 descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. 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. To facilitate review of the various embodiments, the following explanations of terms are provided:
Administration: The introduction of a composition into a subject by a chosen route.
Administration can be local or systemic. For example, if the chosen route is intravenous, the composition (such as a composition including a SAHP) is administered by introducing the composition into a vein of the subject. The term also encompasses long-term administration, such as is accomplished using a continuous release pump or a coated, implanted device (such as a stent).
Agent: Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for treating or inhibiting CKD or a dyslipidemic or vascular disorder in a subject. Agents include proteins, peptides, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest, such as viruses, such as recombinant viruses. An agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. In some embodiments, the agent is a polypeptide agent (such as a SAHP). The skilled artisan will understand that particular agents may be useful to achieve more than one result.
Amphipathic: An amphipathic molecule contains both hydrophobic (non-polar) and hydrophilic (polar) groups. The hydrophobic group can be an alkyl group, such as a long carbon chain, for example, with the formula: CH3(CH2)n, (where n is generally greater than or equal to about 4 to about 16). Such carbon chains also optionally comprise one or more branches, wherein a hydrogen is replaced with an aliphatic moiety, such as an alkyl group. A hydrophobic group also can comprise an aryl group. The hydrophilic group can be one or more of the following: an ionic molecule, such as an anionic molecule (e.g. , a fatty acid, a sulfate or a sulfonate) or a cationic molecule, an amphoteric molecule (e.g. , a phospholipid), or a non-ionic molecule (e.g. , a small polymer).
One example of an amphipathic molecule is an amphipathic peptide. An amphipathic peptide can also be described as a helical peptide that has hydrophilic amino acid residues on one face of the helix and hydrophobic amino acid residues on the opposite face. Optionally, peptides and polypeptides described herein form amphipathic helices in a physiological environment, such as for instance in the presence of lipid or a lipid interface.
Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization, and so forth. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in
Remington {The Science and Practice of Pharmacology , 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound.
Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.
Anti-CKD agent: An agent that inhibits or treats CKD, such as an agent that inhibits the progression of CKD or treats a symptom of CKD. Non-limiting examples of anti-CKD agents include Renin-Angiotensin-Aldosterone System (RAAS) inhibitors such as ACE inhibitors and ARBs, as well as peroxisome proliferator-activated receptor (PPAR) antagonists, endothelin receptor A antagonists, endothelin receptor B antagonists, anti-ocvP6-integrin antibodies, hepatic growth factors (HGFs), Plasminogen activator inhibitor- 1 (PAI-1) inhibitors, Lysyl oxidase-like-2 (LOXL2) inhibitors, Nrf2 agents {e.g., bardoxolone); Pirfenidone and other anti-fibrotics such as VEGF inhibitors) (See, e.g. , Perico et al, Nat Rev Drug Discov, 7: 936-953, 2008; Gagliardini <?? a/., Contrib Nephrol, 172: 171-184, 2011 ; Hahm et al, Am J Pathol, 168: 110-125, 2007; Dolman et al, Adv Drug Deliv Rev, 62: 1344-1357, 2010; Iekushi et al, J Hypertens, 28: 2454-2461, 2010; Eddy et al, J Am Soc Nephrol, 17: 2999-3012, 2006; Rodriguez et al, J Biol Chem, 285: 20964-20974, 2010; Song et al, Nephrol Dial Transplant, 25: 77-85, 2010; each of which is incorporated by reference herein in its entirety.)
CD36 (Cluster of Differentiation 36): An integral membrane protein that is a member of the class B scavenger receptor family of cell surface proteins. CD36 is also known as FAT, SCARG3, GP88, and glycoprotein IV. Class B scavenger receptors recognize oxidized low density lipoprotein. Additional class B scavenger receptors include scavenger receptor BI (SR-BI) and scavenger receptor BII (SR-BII). CD36 binds many ligands including oxidized low density lipoprotein (LDL) and long chain fatty acids, and is involved, for example, in long chain fatty acid uptake into cells and lipid metabolism. Upon ligand binding, CD36 and the ligand are internalized into the cell where processing of the ligand can occur. A non-limiting example of CD36 protein sequence is provided as GenBank® Accession No.
NP_001001547.1 (incorporated by reference herein as present in GenBank on September 24, 2013).
CD36 antagonist: An agent that inhibits CD36 activity. The CD36 antagonist can be a direct or indirect inhibitor of CD36 activity. Non-limiting examples of "CD36 activity" that can be inhibited by a CD36 antagonist include protein-protein interaction, receptor internalization, ligand binding, ligand transport, and/or signaling activity of CD36. For example, a CD36 antagonist can be an agent that inhibits the internalization activity of CD36, or the signaling activity of CD36 in vitro or in vivo, or a combination thereof. In one example, the CD36 activity inhibited by a CD36 antagonist is LPS-induced IL-8 secretion from cells expressing CD36. A "selective CD36 antagonist" is an agent that inhibits at least 2-fold (such as at least 3-fold, at least 4-fold or at least 5-fold) more CD36 activity than SR-BI and/or SR-BII activity under similar conditions. Methods of determining CD36, SR-BI, and/or SR-BII activity, as well as methods of determining if an agent is an antagonist of CD36, SR-BI, and/or SR-BII activity are known to the person of ordinary skill in the art, and example methods are described herein (see, for example, Example 2).
In several embodiments, the SAHPs disclosed herein are CD36 antagonists. Nonlimiting examples of SAHPs that are CD36 antagonists include SAHPs with an amino acid sequence set forth as SEQ ID NO: 1 (L37pA), SEQ ID NO: 3 (5A-37pA), SEQ ID NO: 67 (ELK-B), and SEQ ID NO: 68 (ELJK-B2). Of these, SEQ ID NO: 67 (ELK-B), and SEQ ID NO: 68 (ELJK-B2) are considered to be selective CD36 antagonists.
Chronic Kidney Disease (CKD): A condition resulting in progressive loss of kidney function over a period of months or years. In several embodiments, a subject with CKD is one having a glomerular filtration rate (GFR) of less than 60 mL/min/1.73 m2 for three consecutive months (see also, the National Kidney Foundation's guidelines for diagnosing CKD (Levey et al, Ann Intern. Med., 139: 137-147, 2003), incorporated by reference herein in its entirety). CKD is often diagnosed in the course of screening individuals known to be at risk of CKD, such as those with high blood pressure or diabetes, or those with a family history of CKD. CKD may also be identified when it leads to one of its recognized
complications, such as cardiovascular disease, anemia or pericarditis.
The severity of CKD can be classified in five stages based on level of kidney function, with stage 1 being the mildest and usually causing few symptoms and stage 5 being a severe illness with poor life expectancy. The stages include:
Stage 1: Slightly diminished function; kidney damage with normal or relatively high GFR (>90 mL/min/1.73 m2). Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.
Stage 2. Mild reduction in GFR (60-89 mL/min/1.73 m2) with kidney damage.
Stage 3. Moderate reduction in GFR (30-59 mL/min/1.73 m2).
Stage 4. Severe reduction in GFR (15-29 mL/min/1.73 m2). Preparation for renal replacement therapy.
Stage 5. Established kidney failure (GFR <15 mL/min/1.73 m2, permanent renal replacement therapy (RRT), or end stage renal disease (ESRD). Stage 5 CKD is also known as end stage renal disease (ESRD) or end-stage kidney disease (ESKD).
Methods of identifying and treating a subject with CKD are known to the person of ordinary skill in the art. As used herein, CKD does not include acute kidney disease, the etiology of which typically includes an acute causative event {e.g. , sepsis, ischemia, trauma, and/or nephrotoxic drugs) which leads to onset of kidney disease in less than three months. (See, e.g. , Levey et al, Ann Intern. Med., 139: 137-147, 2003, Brenner and Rector, Eds., The Kidney, 9th edition. Philadelphia, Elsevier, Saunders. 2001 ; Lameire et al, Lancet, 382: 168-179, 2013; and Jo et al, Clin J Am Soc Nephrol, 2: 356-365, 2007, each of which is incorporated by reference herein in its entirety.) Control: A reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control, such as a sample obtained from a patient diagnosed with CKD or a dyslipidemic or vascular disorder. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of CKD or dyslipidemic or vascular disorder patients with known prognosis or outcome, or group of samples that represent baseline or normal values).
A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 68%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.
Domain: A domain of a protein is a part of a protein that shares common structural, physiochemical and functional features; for example hydrophobic, polar, globular, helical domains or properties, for example a DNA binding domain, an ATP binding domain, and the like.
Dyslipidemic disorder: A disorder associated with any altered amount of any or all of the lipids or lipoproteins in the blood. Dyslipidemic disorders include, for example, hyperlipidemia,
hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, and cardiovascular disease (i.e. , coronary artery disease, atherosclerosis and restenosis).
Expression: Translation of a nucleic acid into a protein. Proteins can be expressed and remain intracellular, can become a component of a cell membrane, or be can secreted into the extracellular matrix or medium.
Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al , Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
Efflux: The process of flowing out. As applied to the results described herein, lipid efflux refers to a process whereby lipid, such as cholesterol and phospholipid, is complexed with an acceptor, such as an apolipoprotein or apolipoprotein peptide mimic, and removed from vesicles or cells. "ABCA1- dependent lipid efflux" (or lipid efflux by an "ABCA1 -dependent pathway") refers to a process whereby apolipoproteins or peptide mimics of apolipoproteins bind to a cell and efflux lipid from the cell by a process that is facilitated by the ABCA1 transporter.
Hydrophilic: A hydrophilic (or lipophobic) group is electrically polarized and capable of H- bonding, enabling it to dissolve more readily in water than in oil or other "non-polar" solvents.
Hydrophobic: A hydrophobic (or lipophilic) group is electrically neutral and nonpolar, and thus prefers other neutral and nonpolar solvents or molecular environments. Examples of hydrophobic molecules include alkanes, oils and fats.
Inhibiting or treating a disease: Inhibiting the full development or progression of a disease or condition, for example, in a subject who is at risk for a disease, such as CKD, atherosclerosis or cardiovascular disease. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
Isolated/purified: An "isolated" or "purified" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids or proteins. The term "isolated" or "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell. Preferably, a preparation is purified such that the biological component represents at least 50%, such as at least 68%, at least 90%, at least 95%, or greater of the total biological component content of the preparation.
Linker: A molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds.
Lipid: A class of water-insoluble, or partially water insoluble, oily or greasy organic substances, that are extractable from cells and tissues by nonpolar solvents, such as chloroform or ether. Types of lipids include triglycerides (i.e. , natural fats and oils composed of glycerin and fatty acid chains), phospholipids (e.g. , phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and
phosphatidylinositol), sphingolipids (e.g. , sphingomyelin, cerebrosides and gangliosides), and sterols (e.g. , cholesterol).
Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide -nucleic acids (PNAs), and the like. Such
polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term
"oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e. , A, T, G, C), this also includes an RNA sequence (i.e. , A, U, G, C) in which "U" replaces "T."
"Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyriniidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5 ' -direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5 '-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences." "cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
"Encoding" refers to the inherent property of specific sequences of nucleotides in a
polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In some examples, a nucleic acid encodes a disclosed PreF antigen.
"Recombinant nucleic acid" refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, such as a "recombinant polypeptide." A recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome -binding site, etc.) as well.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Peptide and 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 "peptide" or "polypeptide" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a peptide, polypeptide, or protein.
Peptide modifications: Peptides, such as the disclosed SAHPs, can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity and conformation as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2 wherein Ri and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino- terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.
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 {e.g., 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.
In particular embodiments suitable for administration to a subject, the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.
Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman & Wunsch, . Mol. Biol. 48:443, 1968; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al. , Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al. , Meth. Mol. Bio. 24:307-31, 1994. Altschul et al. , J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.
For sequence comparison of nucleic acid sequences and amino acids sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art.
Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, . Mol. Biol. 48:443, 1968, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see for example, Current Protocols in Molecular Biology (Ausubel et al. , eds 1995 supplement)). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. , J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.
Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., . Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (World Wide Web address ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff , Proc. Natl. Acad. Sci. USA 89: 10915, 1989).
Another indicia of sequence similarity between two nucleic acids is the ability to hybridize the sequences to each other, or to the same target sequence. The more similar are the sequences of the two nucleic acids, the more stringent the conditions at which they will hybridize. The stringency of hybridization conditions are sequence-dependent and are different under different environmental parameters. Thus, hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength
(especially the Na+ and/or Mg++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found, for example, in Sambrook et al , Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ; Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic Acid Preparation, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Ltd., NY, NY, 1993; and Ausubel et al. Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, Inc., 1999.
"Stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize. In contrast nucleic acids that hybridize under "low stringency conditions include those with much less sequence identity, or with sequence identity over only short subsequences of the nucleic acid.
Synthetic alpha- helical peptide (SAHP): A polypeptide containing at least two alpha-helical domains that mimics apolipoprotein A-I binding to class B scavenger receptor CD36. The alpha-helical domains are typically linked by an amino acid linker (such as a proline residue). SAHPs promote cholesterol efflux from cells by the ABCA1 transporter. The person of ordinary skill in the art is familiar with certain SAHPs (such as the L37pA (SEQ ID NO: 1) and 5A-37pA (SEQ ID NO: 3) peptides).
Description of known SAHPs is provided, for example, in PCT. Pub Nos. WO2006/044596,
WO2009/129263, and WO 2011/066511, each of which is incorporated by reference herein in its entirety. Novel SAHPs are also provided herein, such as the ELK-B (SEQ ID NO: 67) and ELK-B2 (SEQ ID NO: 68) peptides. In several embodiments, methods of treating CKD by administering a therapeutically effective amount of a SAHP to a subject in need thereof are provided.
Therapeutically effective amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a SAHP or peptide analog useful in inhibiting and/or treating CKD or a dyslipidemic or vascular disorder in a subject. Ideally, a therapeutically effective amount of an agent is an amount sufficient to prevent, inhibit and/or treat CKD or a dyslipidemic or vascular disorder in a subject without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for inhibiting and/or treating CKD or a dyslipidemic or vascular disorder in a subject will be dependent on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
III. Description of Several Embodiments
A. Synthetic Amphipathic Helical Peptides ( SAHP)
Therapeutic peptides are described herein that include or consist of multiple amphipathic alpha- helical domain peptides, termed Synthetic Amphipathic Helical Peptides (SAHPs). The peptides are useful, for example, for the treatment of CKD, as wells as dyslipidemic or vascular disorders.
1. Previously described SAHPs
Several SAHPs were known prior to the disclosure provided herein (for example, the L37pA and 5A-37pA peptides). Description of known SAHPs is provided, for example, in PCT. Pub Nos.
WO2006/044596, WO2009/ 129263, and WO 2011/066511 , each of which is incorporated by reference herein in its entirety.
In one embodiment, the SAHP includes multiple amphipathic alpha-helical domains, wherein a first amphipathic alpha-helical domain and a second amphipathic alpha-helical domain exhibit equivalent hydrophobicity (as measured, e.g., by their hydrophobic moments; see Eisenberg et al , Faraday Symp. Chem. Soc. 17: 109-120, 1982; Eisenberg et al. , PNAS 81 : 140-144, 1984; and Eisenberg et al. , . Mol. Biol. 179: 125-142, 1984). In specific, non-limiting examples, the first domain and second domain are covalently linked by a linking peptide, such as a glycine, alanine or proline, or other bridging molecule.
The degree of amphipathicity {i.e. , degree of symmetry of hydrophobicity) in the SAHPs or peptide analogs can be conveniently quantified by calculating the hydrophobic moment (μ#) of each of the amphipathic alpha-helical domains. Methods for calculating μ# for a particular peptide sequence are well- known in the art, and are described, for example in Eisenberg et al , Faraday Symp. Chem. Soc. 17: 109- 120, 1982; Eisenberg et al. , PNAS 81 : 140-144, 1984; and Eisenberg et al., J. Mol. Biol. 179: 125-142, 1984. The actual μ# obtained for a particular peptide sequence will depend on the total number of amino acid residues composing the peptide. The amphipathicities of peptides of different lengths can be directly compared by way of the mean hydrophobic moment. The mean hydrophobic moment per residue can be obtained by dividing μ# by the number of residues in the peptide.
In some embodiments, the SAHP includes or consists of the amino acid sequence set forth as DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF (L37pA; SEQ ID NO: 1).
In additional embodiments, the SAHP includes or consists of the amino acid sequence set forth as DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (5A-37pA; SEQ ID NO: 3). The L37pA and 5A-37pA peptides include two amphipathic alpha-helical domains linked by a proline residue, and as disclosed herein, are antagonists of CD36 in vitro. Therefore, in several embodiments, a polypeptide including or consisting of a disclosed SAHP, such as a polypeptide including or consisting of the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3, is a CD36 receptor antagonist. Additional, non-limiting examples of SAHPs with two amphipathic alpha-helical domains are provided in Table 1.
Table 1. Exemplary previously described SAHPs.
Figure imgf000020_0001
Peptide Sequence SEQ ID NO:
Pep 18 DWLEAFYDKVAKKLKEAFPDWLKAFYDKVAEKLKEAF 25
Pep 19 DWLEAFYDEVAKKLKKAFPDWLKAFYDKVAEKLKEAF 26
Pep20 DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 27
Pep21 DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 28
Pep22 DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 29
Pep23 DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 30
Pep24 LLDNWDSVTSTFSKLREQPDWAKAAYDKAAEKAKEAA 31
Pep25 LESFKVSFLSALEEYTKKPDWAKAAYDKAAEKAKEAA 32
Pep26 DWAKAAYDKAAEKAKEAAPLLDNWDSVTSTFSKLREQ 33
Pep27 DWAKAAYDKAAEKAKEAAPLESFKVSFLSALEEYTKK 34
Pep28 DWLKAFYDKVAEKLKEAFPSDELRQRLAARLEALKEN 35
Pep29 DWLKAFYDKVAEKLKEAFPRAELQEGARQKLHELQEK 36
Pep30 SDELRQRLAARLEALKENPDWLKAFYDKVAEKLKEAF 37
Pep31 RAELQEGARQKLHELQEKPDWLKAFYDKVAEKLKEAF 38
Pep32 LLDNWDSVTSTFSKLREQPSDELRQRLAARLEALKEN 39
Pep33 LESFKVSFLSALEEYTKKPRAELQEGARQKLHELQEK 40
Pep34 SDELRQRLAARLEALKENPLLDNWDSVTSTFSKLREQ 41
Pep35 LLDNWDSVTSTFSKLREQPLESFKVSFLSALEEYTKK 42
Pep36 DWLKAFYDKVAEKLKEAFPDWLRAFYDKVAEKLKEAF 43
Pep37 DWLKAFYDKVAEKLKEAFPDWLRAFYDRVAEKLKEAF 44
Pep38 DWLKAFYDKVAEKLKEAFPDWLRAFYDRVAEKLREAF 45
ELK EKLKELLEKLLEKLKELLPEKLKELLEKLLEKLKELL 46
ELK-C EELKEKLEELKEKLEEKLPEELKEKLEELKEKLEEKL 47
ELK-C1 EELKAKLEELKAKLEEKLPEELKAKLEELKAKLEEKL 48
ELK-C3 EKLKELLEKLKAKLEELLPEKLKELLEKLKAKLEELL 49
ELK-C4 EKLKAKLEELKAKLEELLPEKLKAKLEELKAKLEELL 50
ELK-D EKLKALLEKLLAKLKELLPEKLKALLEKLLAKLKELL 51
ELK-D2 EKLKELLEKLLAKLKELLPEKLKELLEKLLAKLKELL 52
ELK-E EWLKELLEKLLEKLKELLPEWLKELLEKLLEKLKELL 53
ELK-F EKFKELLEKFLEKFKELLPEKFKELLEKFLEKFKELL 54
ELK-F2 EKFKELLEKLLEKLKELLPEKFKELLEKLLEKLKELL 55
ELK-G EELKELLKELLKKLEKLLPEELKELLKELLKKLEKLL 56
ELK-H EELKKLLEELLKKLKELLPEELKKLLEELLKKLKELL 57
ELK-I EKLKELLEKLLEKLKELLAEKLKELLEKLLEKLKELL 58
ELK-J EKLKELLEKLLEKLKELLAAEKLKELLEKLLEKLKELL 59
ELK-K DWLKAFYDKVACKLKEAFPDWAKAAYNKAAEKAKEAA 60 ELK-L DHLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 61
ELK-A2 EKLKAKLEELKAKLEELLPEKAKAALEEAKAKAEELA 62
ELK-AS EKLKAKLEELKAKLEELLPEHAKAALEEAKCKAEELA 63
37pA DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 1
P5A DHLKAFYDKVACKLKEAFPNWAKAAYDKAAEKAKEAA 64
P5A C12/H2 DWLKAFYDKVAEKLKEAFPDHAKAAYDKAACKAKEAA 65
2. Novel SAHPs
Additional SAHPs are provided herein including peptides with multiple amphipathic alpha-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway.
In some embodiments, the SAHP includes or consists of the consensus amino acid sequence set forth as EKLXiELLX2KLLELLKKLLPEKLX3ELLX4KLLELLKKLL (SEQ ID NO: 66), wherein, Xi is L or K, X2 is K or E, X3 is L or K, and X4 is K or E. In one example, the SAHP includes or consists of the amino acid sequence set forth as
EKLLELLKKLLELLKKLLPEKLLELLKKLLELLKKLL (ELK-B; SEQ ID NO: 67). In another example, the SAHP includes or consists of the amino acid sequence set forth as
EKLKELLEKLLELLKKLLPEKLKELLEKLLELLKKLL (ELK-B 2; SEQ ID NO: 68).
As disclosed herein, the ELK-B and ELK-B2 peptides function as antagonists of the CD36 receptor in vitro. Therefore, in several embodiments, a polypeptide including or consisting of the amino acid sequence set forth as SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68 is a CD36 receptor antagonist.
In some embodiments, the disclosed SAHP (such as a peptide including the amino acid sequence set forth as SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68) is a selective CD36 antagonist that inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions. For example, the SAHP can inhibit CD36 activity to a greater extent than SR-BI and/or SR-BII activity in a comparable assay. In some embodiments the SAHP has an IC50 value for CD36 inhibition that is no more than 50% (such as no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the corresponding IC50 value for SR-BI or SR-BII inhibition. Methods of determining CD36, SR-BI, and/or SRT-BII activity, and inhibition thereof, are known to the person of ordinary skill in the art, and further described herein (see, e.g. , Example 2).
3. Additional description of SAHPs
In some embodiments, the SAHP is included on a polypeptide having a maximum length, for example no more than 40, 50, 60, 68, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids amino acids in length. The polypeptide including the SAHP can include, consist or consist essentially of the SAHP.
In several embodiments, the disclosed SAHPs bind to CD36. In several examples, the dissociation constant for SAHP binding to CD36, is less than about 10"4 Molar, such as less than about 105 Molar, 10"6 Molar, 10"7 Molar, or less than 10"8 Molar. Binding to CD36 can be determined by methods known in the art. The determination that a particular agent specifically binds to a particular polypeptide may readily be made by using or adapting routine procedures.
In some embodiments, the disclosed SAHPs specifically bind to CD36 as opposed to other scavenger receptors, such as SR-BI or SR-BII receptors. Specific binding to CD36 can be determined by methods known in the art, for example according to methods disclosed in Example 2, below. The determination that a particular agent specifically binds to a particular polypeptide compared to other polypeptides may readily be made by using or adapting routine procedures.
In additional embodiments, a disclosed SAHP is a CD36 receptor antagonist. For example, as disclosed herein, a SAHP including the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68 is a CD36 receptor antagonist (see example 2, below). Methods of assaying a peptide for effects on CD36 receptor signaling activity are known in the art and described herein (see, e.g. , Example 2, below).
In the SAHPs disclosed herein, the linkage between amino acid residues can be a peptide bond or amide linkage (i.e., -C-C(O)NH-). Alternatively, one or more amide linkages are optionally replaced with a linkage other than amide, for example, a substituted amide. Substituted amides generally include, but are not limited to, groups of the formula -C(0)NR-, where R is (Ci-C6) alkyl, substituted (Ci-C6) alkyl, (C1-C6) alkenyl, substituted (Ci-C6) alkenyl, (Ci-C6) alkynyl, substituted (Ci-C6) alkynyl, (C5-C20) aryl, substituted (C5-C20) aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl, and substituted 6-26 membered alkheteroaryl. Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides.
Suitable amide mimetic moieties are described, for example, in Olson et al , J. Med. Chem. 36:3039-3049, 1993.
Additionally, in representative SAHPs disclosed herein, the amino- and carboxy-terminal ends can be modified by conjugation with various functional groups. Neutralization of the terminal charge of synthetic peptide mimics of apolipoproteins has been shown to increase their lipid affinity (Yancey et al. , Biochem. 34:7955-7965, 1995; Venkatachalapathi et al , Protein: Structure, Function and Genetics 15:349-359, 1993). For example, acetylation of the amino terminal end of amphipathic peptides increases the lipid affinity of the peptide (Mishra et al , J. Biol. Chem. 267:7185-7191, 1994). Other possible end modifications are described, for example, in Brouillette et al , Biochem. Biophys. Acta 1256: 103-129, 1995: Mishra et al. , J. Biol. Chem. 267:7185-7191, 1994; and Mishra et al. , J. Biol. Chem. 268: 1602- 1611, 1995.
Furthermore, in representative SAHPs disclosed herein, the amino acid proline is used to link the multiple amphipathic oc-helices included on the SAHP. However, other suitable amino acids, such as glycine, serine, threonine, and alanine, that functionally separate the multiple amphipathic alpha-helical domains can be used. In some embodiments, the linking amino acid will have the ability to impart a β- turn at the linkage, such as glycine, serine, threonine, and alanine. In addition, larger linkers containing two or more amino acids or bifunctional organic compounds, such as H2N(CH2)nCOOH, where n is an integer from 1 to 12, can also be used. Examples of such linkers, as well as methods of making such linkers and peptides incorporating such linkers, are well-known in the art (see, e.g. , Hunig et al , Chem. Ber. 100:3039-3044, 1974 and Basak et al , Bioconjug. Chem. 5:301-305, 1994).
In one embodiment, SAHPs useful within the disclosure are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D-amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
heterocyclics. For example, proline analogs can be made in which the ring size of the proline residue is changed from a 5-membered ring to a 4-, 6-, or 7-membered ring. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl {e.g. , morpholino), oxazolyl, piperazinyl {e.g. , 1-piperazinyl), piperidyl {e.g. , 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl {e.g. , 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl {e.g. , thiomorpholino), and triazolyl groups. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. Peptides, as well as peptide analogs and mimetics, can also be covalently bound to one or more of a variety of nonproteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkenes, as described in U.S. Patent Nos. 4,640,835; 4,496,668; 4,301,144; 4,668,417; 4,791,192; and 4,179,337.
In addition to the naturally occurring genetically encoded amino acids, amino acid residues in the SAHPs may be substituted with naturally occurring non-encoded amino acids and synthetic amino acids. Certain commonly encountered amino acids which provide useful substitutions include, but are not limited to, β-alanine and other omega-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionic acid, 4-aminobutyric acid and the like; oc-aminoisobutyric acid; ε-aminohexanoic acid; δ-amino valeric acid; N-methylglycine or sarcosine; ornithine; citrulline; t-butylalanine; t-butylglycine; N- methylisoleucine; phenylglycine; cyclohexylalanine; nor leucine; naphthylalanine; 4-chlorophenylalanine; 2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; penicillamine; 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid; β-2-thienylalanine; methionine sulfoxide; homoarginine; N- acetyl lysine; 2,4-diaminobutyric acid; 2,3-diaminobutyric acid; p-aminophenylalanine; N-methyl valine; homocysteine; homophenylalanine; homoserine; hydroxyproline; homoproline; N-methylated amino acids; and peptoids (N-substituted glycines).
While in certain embodiments, the amino acids of the SAHPs will be substituted with L-amino acids, the substitutions are not limited to L-amino acids. Thus, also encompassed by the present disclosure are modified forms of the SAHPs, wherein an L-amino acid is replaced with an identical D- amino acid (e.g. , L-Arg→D-Arg) or with a conservatively-substituted D-amino acid (e.g. , L-Arg→D- Lys), and vice versa.
Other peptide analogs and mimetics within the scope of the disclosure include glycosylation variants, and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues (e.g. , lysine or arginine). Acyl groups are selected from the group of alkyl-moieties including C3 to CI 8 normal alkyl, thereby forming alkanoyl aroyl species. Also embraced are versions of a native primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.
In another embodiment, a detectable moiety can be linked to the SAHP or peptide analogs disclosed herein, creating a peptide/peptide analog-detectable moiety conjugate. Detectable moieties suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The detectable moieties contemplated for the present disclosure can include, but are not limited to, an immunofluorescent moiety (e.g. , fluorescein, rhodamine, Texas red, and the like), a radioactive moiety (e.g. , 3H, 32P, 1251, 35S, 18F, 64Cu, "Tc), an enzyme moiety (e.g. , horseradish peroxidase, alkaline phosphatase), a colorimetric moiety (e.g. , colloidal gold, biotin, colored glass or plastic, and the like), a label detected by magnetic resonance imaging (e.g. , gadolinium, iron oxide particle). The detectable moiety can be linked to the SAHP or peptide analog at either the N- and/or C-terminus. Optionally, a linker can be included between the SAHP or peptide analog and the detectable moiety.
Means of detecting such moieties are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
In another embodiment, an additional functional domain or peptide can be linked to the SAHPs or peptide analogs disclosed herein, creating a peptide/peptide analog-additional functional domain/peptide conjugate. The additional functional domain or peptide can be linked to the SAHP or peptide analog at either the N- and/or C-terminus. Exemplary additional functional domains/peptides are known in the art, see for example, PCT. Pub Nos. WO2006/044596, WO2009/129263, and WO 2011/066511 , each of which is incorporated by reference herein in its entirety. Exemplary additional functional
domains/peptides include those shown in Table 2. Table 2. Exemplary additional functional domains.
Figure imgf000026_0001
Cell recognition sequences can increase the ability of the SAHPs or peptide analogs containing these sequences to bind to cells, the prerequisite first step in ABCA1 -mediated cholesterol efflux (Remaley et al, Biochem. Biophys. Res. Commun. 280:818-823, 2001). Cell internalization sequences, can increase the cellular uptake of the SAHPs or peptide analogs into intracellular compartments, where the initial lipidation of the peptides has been proposed to occur (Neufeld et al, J. Biol. Chem. 279: 15571- 15578, 2004), thus facilitating lipid efflux. Sequences that activate neutral cholesterol hydrolase (Kisilevsky et al , J. Lipid Res. 44:2257-2267, 2003) can increase the amount of intracellular free cholesterol, the form of cholesterol that effluxes from cells. Similarly, the inhibition of ACAT blocks the esterification of cholesterol to cholesteryl ester, thus increasing the pool of free cholesterol for efflux by the SAHPs or peptide analogs (Kisilevsky et al , J. Lipid Res. 44:2257-2267, 2003). Sequences that target the SAHPs or peptide analogs to the liver can facilitate the last step of reverse cholesterol transport, the hepatic uptake and excretion of cholesterol into the bile (Collet et al. , . Lipid Res. 40: 1185-1193, 1999). Part of the beneficial effect of ApoA-I and synthetic peptide mimics is believed to be due to their antiinflammatory and anti-oxidant properties (Van Lenten et al , J. Clin. Invest. 96:2758-2766, 1995).
Sequences containing domains that sequester oxidized lipids (Datta et al , J. Biol. Chem. 279:26509- 26517, 2004), that act as antioxidants (Bielicki et al, Biochem. 41 :2089-2096, 2002), or that chelate heavy metals (Wakabayashi et al. , Biosci. Biotechnol. Biochem. 63:955-957, 1999), which promote lipid oxidation, can complement the lipid efflux properties of the SAHPs or peptide analogs by also preventing lipid oxidation. Lipoprotein lipase activation sequences can prevent, reduce or inhibit
hypertriglyceridemia associated with administration of any of the disclosed SAHPs or peptide analogs. In an example, lipoprotein lipase activation sequences result in triglyceride levels of no greater than 200 mg/dl.
Optionally, a linker can be included between the SAHP or peptide analog and the additional functional domain or peptide. The additional functional domain or peptide can enhance the ability of the SAHP or peptide analog to efflux lipids from cells in a non-cytotoxic manner, and/or enhance its therapeutic efficacy. The linkers contemplated by the present disclosure can be any bifunctional molecule capable of covalently linking two peptides to one another. Thus, suitable linkers are bifunctional molecules in which the functional groups are capable of being covalently attached to the N- and/or C- terminus of a peptide. Functional groups suitable for attachment to the N- or C-terminus of peptides are well known in the art, as are suitable chemistries for effecting such covalent bond formation. The linker may be flexible, rigid or semi-rigid. Suitable linkers include, for example, amino acid residues such as Pro or Gly or peptide segments containing from about 2 to about 5, 10, 15, 20, or even more amino acids, bifunctional organic compounds such as H2N(CH2)nCOOH where n is an integer from 1 to 12, and the like. Examples of such linkers, as well as methods of making such linkers and peptides incorporating such linkers, are well-known in the art (see, e.g. , Hunig et al. , Chem. Ber. 100:3039-3044, 1974 and Basak et al. , Bioconjug. Chem. 5:301-305, 1994).
Conjugation methods applicable to the present disclosure include, by way of non-limiting example, reductive animation, diazo coupling, thioether bond, disulfide bond, amidation and
thiocarbamoyl chemistries. In one embodiment, the amphipathic alpha-helical domains are "activated" prior to conjugation. Activation provides the necessary chemical groups for the conjugation reaction to occur. In one specific, non-limiting example, the activation step includes derivatization with adipic acid dihydrazide. In another specific, non-limiting example, the activation step includes derivatization with the N-hydroxysuccinimide ester of 3-(2-pyridyl dithio)-propionic acid. In yet another specific, non-limiting example, the activation step includes derivatization with succinimidyl 3-(bromoacetamido) propionate. Further, non-limiting examples of derivatizing agents include succinimidylformylbenzoate and succinimidyllevulinate .
In some embodiments, mixtures of two or more, such as 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the disclosed peptides are provided.
Also encompassed by the present disclosure are SAHPs or peptide analogs, wherein the multiple amphipathic alpha-helical domains are comprised of dimers, trimers, tetramers and even higher order polymers (i.e. , "multimers") comprising the same or different sequences. Such multimers may be in the form of tandem repeats. The amphipathic alpha-helical domains may be directly attached to one another or separated by one or more linkers. The amphipathic alpha-helical domains can be connected in a head- to-tail fashion (i.e. , N-terminus to C-terminus), a head-to-head fashion, (i.e. , N-terminus to N-terminus), a tail-to-tail fashion (i.e. , C-terminus to C-terminus), and/or combinations thereof. In one embodiment, the multimers are tandem repeats of two, three, four, and up to about ten amphipathic alpha-helical domains, but any number of amphipathic alpha-helical domains can be used. 4. Synthesis and Production of SAHPs
The SAHPs or peptide analogs of the disclosure can be prepared using virtually any technique known to one of ordinary skill in the art for the preparation of peptides. Examples of methods for preparing SAHPs are disclosed herein and known in the art (see, e.g., PCT. Pub Nos. WO2006/044596, WO2009/129263, and WO 2011/066511, each of which is incorporated by reference herein in its entirety). For example, the SAHPs can be prepared using step-wise solution or solid phase peptide syntheses, or recombinant DNA techniques, or the equivalents thereof.
SAHPs of the disclosure having either the D- or L-configuration can be readily synthesized by automated solid phase procedures well known in the art. Suitable syntheses can be performed by utilizing "T-boc" or "F-moc" procedures. Techniques and procedures for solid phase synthesis are described in Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the SAHPs may be prepared by way of segment condensation, as described, for example, in Liu et al, Tetrahedron Lett. 37:933-936, 1996; Baca et al, J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al, Int. J. Peptide Protein Res. 45:209-216, 1995;
Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tam, . Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91 :6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31 :322-334, 1988). This is particularly the case with glycine containing peptides. Other methods useful for synthesizing the SAHPs of the disclosure are described in Nakagawa et al. , J. Am. Chem. Soc. 107:6887-6892, 1985.
Additional exemplary techniques known to those of ordinary skill in the art of peptide and peptide analog synthesis are taught by Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis,
Springer Verlag, New York, 1994; and by Jones, J., Amino Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002. The Bodanszky and Jones references detail the parameters and techniques for activating and coupling amino acids and amino acid derivatives. Moreover, the references teach how to select, use and remove various useful functional and protecting groups.
SAHPs of the disclosure having either the D- or L-configuration can also be readily purchased from commercial suppliers of synthetic peptides. Such suppliers include, for example, Advanced ChemTech (Louisville, KY), Applied Biosystems (Foster City, CA), Anaspec (San Jose, CA), and Cell Essentials (Boston, MA).
If the SAHP is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the SAHP or the relevant portion can also be synthesized using conventional recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding the SAHP is inserted into an appropriate expression vehicle, that is, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The expression vehicle is then transfected into a suitable target cell which will express the SAHP. Depending on the expression system used, the expressed peptide is then isolated by procedures well-established in the art. Methods for recombinant protein and peptide production are well known in the art (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).
To increase efficiency of production, the polynucleotide can be designed to encode multiple units of the SAHP separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved {e.g., by treatment with the appropriate enzyme) in order to recover the peptide units. This can increase the yield of peptides driven by a single promoter. In one embodiment, a polycistronic polynucleotide can be designed so that a single mRNA is transcribed which encodes multiple peptides, each coding region operatively linked to a cap-independent translation control sequence, for example, an internal ribosome entry site (IRES). When used in appropriate viral expression systems, the translation of each peptide encoded by the mRNA is directed internally in the transcript, for example, by the IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual peptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase yield of peptide driven by a single promoter.
A variety of host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors {e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors {e.g. , cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors {e.g. , Ti plasmid) containing an appropriate coding sequence; or animal cell systems. The expression elements of the expression systems vary in their strength and specificities.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter can be used. When cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. , heat shock promoters, the promoter for the small subunit of RUBISCO, the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV, the coat protein promoter of TMV) can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g. , metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter, the vaccinia virus 7.5 K promoter) can be used.
The SAHPs or peptide analogs of the disclosure can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify a particular SAHPs or peptide analog will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art.
For affinity chromatography purification, any antibody which specifically binds the SAHPs or peptide analog may be used. For the production of antibodies, various host animals, including but not limited to, rabbits, mice, rats, and the like, may be immunized by injection with a SAHP or peptide analog and antibodies purified according to standard methods.
B. Polynucleotides Encoding SAHPs
Polynucleotides encoding the SAHPs disclosed herein are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the SAHPs.
Methods for the manipulation and insertion of the nucleic acids of this disclosure into vectors are well known in the art (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).
The polynucleotides encoding a SAHP include a recombinant DNA which is incorporated into a vector into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.
DNA sequences encoding the antigen can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
Polynucleotide sequences encoding SAHPs can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e. , ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing
DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non- limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, CI 29 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some embodiments, the host cells include HEK293 cells or derivatives thereof, such as GnTI7" cells (ATCC® No. CRL-3022).
Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCb. method using procedures well known in the art. Alternatively, MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman Ed., 1982).
A number of viral vectors have been constructed, that can be used to express the disclosed SAHPs, including polyoma, i.e. , SV40 (Madzak et al , 1992, . Gen. Virol , 73: 15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al , 1988, Bio Techniques, 6:616- 629; Gorziglia et al , 1992, /. Virol, 66:4407-4412; Quantin et al, 1992, Proc. Natl Acad. Sci. USA, 89:2581-2584; Rosenfeld et al , 1992, Cell, 66: 143-155; Wilkinson et al , 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al , 1990, Hum. Gene Ther., 1 :241-256), vaccinia virus (Mackett et al , 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol, 158:91-123; On et al, 1990, Gene, 89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol, 158:66-90; Johnson et al, 1992, /. Virol,
66:29522965; Fink et al, 1992, Hum. Gene Ther. 3: 11-19; Breakfield et al, 1987, Mol. NeurobioL, 1 :337-371 ; Fresse et al, 1990, Biochem. Pharmacol , 40:2189-2199), Sindbis viruses (H. Herweijer et al, 1995, Human Gene Therapy 6: 1161-1166; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11: 18-22; I. Frolov et al , 1996, Proc. Natl. Acad. Sci. USA
93: 11371-11377) and retroviruses of avian (Brandyopadhyay et al, 1984, Mol. Cell Biol, 4:749-754; Petropouplos et al, 1992, . Virol, 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.
Immunol, 158: 1-24; Miller et al , 1985, Mol. Cell Biol, 5:431-437; Sorge et al, 1984, Mol. Cell Biol, 4: 1730-1737; Mann et al , 1985, . Virol, 54:401-407), and human origin (Page et al, 1990, . Virol, 64:5368-5276; Buchschalcher et al, 1992, . Virol, 66:2731-2739). Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, CA; Protein Sciences Corp., Meriden, Conn.;
Stratagene, La Jolla, CA). C. Pharmaceutical Compositions and Therapeutic Methods
1. Methods of treating or inhibiting dyslipidemic or vascular disorders
In some embodiments, the peptides or peptide analogs of the disclosure (and mixtures thereof), or a polynucleotide encoding same, can be used to treat any disorder in animals, especially mammals (e.g. , humans), for which promoting lipid efflux is beneficial, such as dyslipidemic or vascular disorders. Such conditions include, but are not limited to, hyperlipidemia (e.g. , hypercholesterolemia), cardiovascular disease (e.g., atherosclerosis), restenosis (e.g., atherosclerotic plaques), peripheral vascular disease, acute coronary syndrome, reperfusion myocardial injury, asthma, chronic pulmonary obstructive disorder and the like. The peptides or peptide analogs of the disclosure can also be used during the treatment of thrombotic stroke and during thrombolytic treatment of occluded coronary artery disease. In several embodiments, the methods include selecting a subject in need of treatment, such as a subject with or at risk of a dyslipidemic or vascular disorder.
In some embodiments, the methods include administration of a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP as disclosed herein to a subject with or at risk of a dyslipidemic or vascular disorder. In some embodiments, the polypeptide can comprise or consist of a SAHP including the amino acid sequence set forth as SEQ ID NO: 66, wherein, Xi is L or K, X2 is K or E, X3 is L or K, and X4 is K or E (ELK-B/B2 consensus), such as a SAHP including the amino acid sequence set forth as SEQ ID NO: 67 (ELK-B) or SEQ ID NO: 68 (ELK-B2). The method can further include combination therapy with other lipid lowering compositions or drugs used to treat the foregoing conditions, or with agents (such as peptides) that activate LPL activity. Such combination therapies include, but are not limited to, simultaneous or sequential administration of the drugs involved. For example, in the treatment of hypercholesterolemia or atherosclerosis, the peptide or peptide analog formulations can be administered with any one or more of the cholesterol lowering therapies currently in use, for example, bile-acid resins, niacin and statins.
In other embodiments, the SAHP or peptide analog formulations can be administered with a lipoprotein lipase activating agent, such as an apoC-II protein, variant or fragment thereof, to prevent, reduce or inhibit hypertriglyceridemia associated with the administration of any of the disclosed peptides or peptide analogs.
In another embodiment, the SAHPs or peptide analogs can be used in conjunction with statins or fibrates to treat hyperlipidemia, hypercholesterolemia and/or cardiovascular disease, such as
atherosclerosis. In yet another embodiment, the SAHPs or peptide analogs of the disclosure can be used in combination with an anti-microbial agent and/or an anti-inflammatory agent.
In a further embodiment, the SAHPs can also be expressed in vivo, by using any of the available gene therapy approaches.
2. Methods of treating or inhibiting CKD
In some embodiments, the peptides or peptide analogs of the disclosure (and mixtures thereof), or a polynucleotide molecule encoding same, can be used to treat or inhibit CKD in animals, especially mammals (e.g. , humans). In several embodiments, the methods include selecting a subject in need of treatment, such as a subject with early or late CKD, or at risk of CKD.
In several embodiments, a subject with CKD is selected based on glomerular filtration rate (GFR), for example the selected subject can have a GFR of less than 60 mL/min/1.73 m2 for three consecutive months (see also, the National Kidney Foundation's guidelines for diagnosing CKD (Levey et al, Ann Intern. Med., 139:137-147, 2003), incorporated by reference herein in its entirety). CKD is often diagnosed in the course of screening individuals known to be at risk of CKD, such as those with high blood pressure or diabetes, or those with a family history of CKD. CKD may also be identified when it leads to one of its recognized complications, such as cardiovascular disease, anemia or pericarditis.
A subject with a particular stage of CKD can also be selected, for example a subject with stage 1,
2, 3, 4or 5 CKD. The severity of CKD can be classified in five stages based on level of kidney function, with stage 1 being the mildest and usually causing few symptoms and stage 5 being a severe illness with poor life expectancy. The stages include:
Stage 1: Slightly diminished function; kidney damage with normal or relatively high GFR (>90 mL/min/1.73 m2). Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.
Stage 2. Mild reduction in GFR (60-89 mL/min/1.73 m2) with kidney damage.
Stage 3. Moderate reduction in GFR (30-59 mL/min/1.73 m2). Stage 4. Severe reduction in GFR (15-29 mL/min/1.73 m2). Preparation for renal replacement therapy.
Stage 5. Established kidney failure (GFR <15 mL/min/1.73 m2, permanent renal replacement therapy (RRT), or end stage renal disease (ESRD). Stage 5 CKD is also known as end stage renal disease (ESRD) or end-stage kidney disease (ESKD).
Therapy can be initiated before onset of CKD, or after a subject is diagnosed with CKD. For example, therapy can be initiated when a subject has stage 1, stage 2, stage 3, stage 4, or stage 5 CKD. In several embodiments, the methods of preventing and/or treating CKD delay or prevent progression of the CKD in the subject (for example compared to a control subject not receiving the SAHP), for example delay or prevention of progression to stage 5 CKD.
In some embodiments, the methods include administration of a therapeutically effective amount of a polypeptide comprising or consisting of a disclosed SAHP to a subject with or at risk of CKD. In some embodiments, the polypeptide comprises or consists of a SAHP including the amino acid sequence set forth as one of SEQ ID NO: 1 (L37pA) or SEQ ID NO: 3 (5A-37pA). In other embodiments, the polypeptide comprises or consists of a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 66, wherein, Xi is L or K, X2 is K or E, X3 is L or K, and X4 is K or E (ELK-B/B2 consensus), such as a SAHP comprising the amino acid sequence set forth as SEQ ID NO: 67 (ELK-B) or SEQ ID NO: 68 (ELK-B2).
The method can further include combination therapy with other anti-CKD agents. Such combination therapies include, but are not limited to simultaneous or sequential administration of the drugs involved. For example, in the treatment of CKD, the peptide or peptide analog formulations can be administered with any one or more of a known anti-CKD agent, such as ACE inhibitors, ARBs, peroxisome proliferator-activated receptor (PPAR) antagonists, endothelin receptor A antagonists, endothelin receptor B antagonists, anti-ocvP6-integrin antibodies, hepatic growth factors (HGFs), Plasminogen activator inhibitor-1 (PAI-1) inhibitors, Lysyl oxidase-like-2 (LOXL2) inhibitors,
Bardoxolone, or Pirfenidone.
In some embodiments, the CKD treated or inhibited by the disclosed methods is not acute kidney disease, obstructive kidney disease, atherosclerotic kidney disease, or polycystic kidney disease.
In a further embodiment, the SAHPs can also be expressed in vivo, by using any of the available gene therapy approaches.
3. Administration of Peptides or Peptide Analogs
A polypeptide comprising or consisting of a SAHP or peptide analogs can be synthesized or isolated from various sources and administered directly to the subject. In exemplary applications, therapeutic compositions including at least one SAHP or analog thereof are administered to a subject suffering from a dyslipidemic or vascular disorder, such as hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury, in an amount sufficient to inhibit or treat the dyslipidemic or vascular disorder. In additional embodiments, therapeutic compositions including at least one SAHP or analog thereof are administered to a subject at risk of CKD, or suffering from CKD. Amounts effective for this use will depend upon the severity of the disorder and the general state of the subject's health. A therapeutically effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
Treatment of a disease or condition in a subject (such as CKD or a dyslipidemic or vascular disorder) can include delaying the development of the disease or condition (such as CKD or a dyslipidemic or vascular disorder) in the subject. Treatment of the disease or condition (such as CKD or a dyslipidemic or vascular disorder) also includes reducing signs or symptoms associated with the disease or condition (such as CKD or a dyslipidemic or vascular disorder). In some examples, treatment using the methods disclosed herein prolongs the time of survival of the subject. The disease or condition (such as CKD or a dyslipidemic or vascular disorder) does not need to be completely eliminated or cured for the methods to be effective. For example, treatment with one or more of the provided polypeptides can decrease the signs or symptoms of the disease or condition (such as CKD or a dyslipidemic or vascular disorder), or delay the onset or progression of the disease or condition by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 68%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (such as CKD or a dyslipidemic or vascular disorder), as compared a control, such as a population of subjects with the disease or condition (such as CKD or a dyslipidemic or vascular disorder) in the absence of the treatment with the therapeutic polypeptide.
A polypeptide comprising or consisting of a disclosed SAHP or peptide analog can be administered by any means known to one of skill in the art (see, e.g. , Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995), such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection. To extend the time during which the polypeptide comprising or consisting of a disclosed SAHP or peptide analog is available to inhibit or treat a dyslipidemic or vascular disorder, or CKD, the polypeptide comprising or consisting of a disclosed SAHP or peptide analog can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle (Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995). The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes.
Administration may begin whenever a subject has developed, or is at risk for developing a disease or condition, such as a dyslipidemic or vascular disorder or CKD. In one specific, non-limiting example, a polypeptide comprising or consisting of a disclosed SAHP is administered that includes one or more of the amino acid sequences set forth in SEQ ID NOs: 1, 3, 66, 67, or 68. 4. Administration of Nucleic Acid Molecules
In some embodiments where the therapeutic agent is composed entirely of gene-encoded amino acids, or a portion of it is so composed, administration of the polypeptide comprising or consisting of a disclosed SAHP or mixture of such polypeptides, or the relevant portion, can be achieved by an appropriate nucleic acid expression vector (or combination of vectors) which is administered so that it becomes intracellular, for example, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g. , a gene gun; Biolistic, DuPont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g. , Joliot et al , Proc. Natl. Acad. Sci., 88: 1864-1866,1991). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, for example, by homologous or non-homologous recombination.
Use of a DNA expression vector (e.g. , the vector pCDNA) is an example of a method of introducing the foreign cDNA into a cell under the control of a strong viral promoter (e.g. ,
cytomegalovirus) to drive the expression. However, other vectors can be used. Other retroviral vectors (such as pRETRO-ON, BD Biosciences, Palo Alto, CA) also use this promoter but have the advantages of entering cells without any transfection aid, integrating into the genome of target cells only when the target cell is dividing. It is also possible to turn on the expression of a therapeutic nucleic acid by administering tetracycline when these plasmids are used. Hence these plasmids can be allowed to transfect the cells, then administer a course of tetracycline to achieve regulated expression.
Other plasmid vectors, such as pMAM-neo (BD Biosciences, Palo Alto, CA) or pMSG
(Invitrogen, Carlsbad, CA) use the MMTV-LTR promoter (which can be regulated with steroids) or the SV10 late promoter (pSVL, Invitrogen, Carlsbad, CA) or metallothionein-responsive promoter (pBPV, Invitrogen, Carlsbad, CA) and other viral vectors, including retroviruses. Examples of other viral vectors include adenovirus, AAV (adeno-associated virus), recombinant HSV, poxviruses (vaccinia) and recombinant lentivirus (such as HIV). All these vectors achieve the basic goal of delivering into the target cell the cDNA sequence and control elements needed for transcription.
Retroviruses have been considered a preferred vector for gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al, Prog. Med. Genet. 7: 130-142, 1988). A nucleic acid encoding the polypeptide comprising or consisting of a disclosed SAHP can be cloned into a retroviral vector and driven from either its endogenous promoter (where applicable) or from the retroviral LTR (long terminal repeat). Other viral transfection systems may also be utilized for this type of approach, including adenovirus, AAV (McLaughlin et al, J. Virol. 62: 1963-1973, 1988), vaccinia virus (Moss et al, Annu. Rev. Immunol. 5:305-324, 1987), Bovine Papilloma virus (Rasmussen et al, Methods Enzymol. 139:642-654, 1987) or members of the herpesvirus group such as Epstein-Barr virus
(Margolskee et al, Mol. Cell Biol 8:2837-2847, 1988).
In addition to delivery of a nucleic acid encoding the polypeptide comprising or consisting of a disclosed SAHP to cells using viral vectors, it is possible to use non-infectious methods of delivery. For instance, lipidic and liposome-mediated gene delivery has recently been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, Mol. Biotechnol, 11 : 175-180, 1999; Lee and
Huang, Crit. Rev. Ther. Drug Carrier Syst., 14: 173-206, 1997; and Cooper, Semin. Oncol, 23: 172-187,
1996). For instance, cationic liposomes have been analyzed for their ability to transfect monocytic leukemia cells, and shown to be a viable alternative to using viral vectors (de Lima et al. , Mol. Membr. Biol, 16: 103-109, 1999). Such cationic liposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al , Cancer
Gene Ther., 3:250-256, 1996).
In one specific, non-limiting example, a nucleic acid molecule or vector including the nucleic acid molecule is administered that encodes one or more of the amino acid sequences set forth in SEQ ID NOs: 1, 3, 66, 67, or 68.
5. Representative Methods of Administration, Formulations and Dosage
The provided polypeptide comprising or consisting of a disclosed SAHP or peptide analogs, constructs, or vectors encoding such peptides, can be combined with a pharmaceutically acceptable carrier {e.g. , a phospholipid or other type of lipid) or vehicle for administration to human or animal subjects. In some embodiments, more than one SAHP or peptide analog can be combined to form a single preparation. The SAHPs or peptide analogs can be conveniently presented in unit dosage form and prepared using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze -dried
(lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.
In certain embodiments, unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, formulations encompassed herein may include other agents commonly used by one of ordinary skill in the art. The pharmaceutical compositions provided herein, including those for use in treating dyslipidemic and vascular disorders or CKD, may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. In one embodiment, polypeptide comprising or consisting of a disclosed SAHP or peptide analogs with suitable features of ABCAl-specificity and low cytotoxicity can be precomplexed with phospholipids or other lipids into either discoidal or spherical shape particles prior to administration to subjects.
In another embodiment, it may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment. This may be achieved by, for example, and not by way of limitation, local or regional infusion or perfusion during surgery, topical application (e.g. , wound dressing), injection, catheter, suppository, or implant (e.g. , implants formed from porous, non-porous, or gelatinous materials, including membranes, such as silastic membranes or fibers), and the like.
In a specific embodiment, one or more of the disclosed peptides may be administered either by coating or impregnating an implant such as stent to treat a dyslipidemic or vascular disorder or CKD. These peptides are prepared and purified as described herein. In an example, the implant can be partially or completely coated with the peptide. For instance, the luminal surface of the implant may be coated with the peptide. For treatment of dyslipidemic or vascular disorders, such configuration is believed to reduce atherosclerotic plaques in arteries often associated with atherosclerosis while minimizing the amount of coating material and time required to prepare the implant. The peptide may be attached to the implant by any chemical or mechanical bond or force, including linking agents. Alternatively, the coating may be directly linked (tethered) to the first surface, such as through silane groups. In other examples, the implant may be impregnated with at least one peptide by methods known to those of skill in the art so that multiple surfaces (such as the outer and inner surfaces) of the implant include the peptide.
It is contemplated that the implant may be coated or impregnated according to methods known to one of ordinary skill in the art. Exemplary, non-limiting examples, of peptide attachment to an implant are discussed in Smith (Radiology 230: 1-2, 2004), United States Patent No. 6,675,920, United States Patent No. 7,402,329, Wessely (Nat. Rev. Cardiol. 7(4): 194-203, 2010), Puskas et al. (Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1(4): 451-62, 2009), Butt et al. (Future Cardiol. 2009 5(2): 141-57,
2009), Bosiers et al. (Vase. Health Risk Manag. 4(3): 553-9, 2008) and Kukreja et al. (Pharmacol. Res. 57(3): 171-80, 2008), each of which is incorporated by reference herein in its entirety.
In an additional embodiment, the implant may be coated or impregnated with materials in addition to the disclosed peptides to further enhance their bio-utility. Examples of suitable coatings are medicated coatings, drug-eluting coatings, hydrophilic coatings, smoothing coatings.
In one embodiment, administration can be by direct injection at the site (or former site) of a tissue that is to be treated, such as the heart or the peripheral vasculature. In another embodiment, the pharmaceutical compositions are delivered in a vesicle, in particular liposomes (see, e.g. , Langer, Science 249: 1527-1533, 1990; Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).
In yet another embodiment, the pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer Science 249: 1527-1533, 1990; Sefton Crit. Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al. , Surgery 88:507-516, 1980; Saudek et al. , N. Engl. J. Med. 321 :574-579, 1989). In another embodiment, polymeric materials can be used (see, e.g. , Ranger et al. , Macromol. Sci. Rev. Macromol. Chem. 23:61-64, 1983; Levy et al. , Science 228: 190- 192, 1985; During et αί , Αηη. Neurol. 25:351-356, 1989; and Howard et al. , J. Neurosurg. 71: 105-112, 1989). Other controlled release systems, such as those discussed in the review by Langer (Science 249: 1527-1533, 1990), can also be used.
In another aspect of the disclosure, therapeutic agent(s) are delivered by way of an implanted pump, described, for example, in U.S. Patent No. 6,436,091 ; U.S. Patent No. 5,939,380; and U.S. Patent No. 5,993,414. Implantable drug infusion devices are used to provide subjects with a constant and long term dosage or infusion of a drug or any other therapeutic agent.
Active drug or programmable infusion devices feature a pump or a metering system to deliver the drug into the patient' s system. An example of such an active drug infusion device currently available is the Medtronic SynchroMed™ programmable pump. Such pumps typically include a drug reservoir, a peristaltic pump to pump the drug out from the reservoir, and a catheter port to transport the pumped out drug from the reservoir via the pump to a patient' s anatomy. Such devices also typically include a battery to power the pump, as well as an electronic module to control the flow rate of the pump. The Medtronic SynchroMed™ pump further includes an antenna to permit the remote programming of the pump.
Passive drug infusion devices, in contrast, do not feature a pump, but rather rely upon a pressurized drug reservoir to deliver the drug. Thus, such devices tend to be both smaller as well as cheaper as compared to active devices. An example of such a device includes the Medtronic IsoMed™. This device delivers the drug into the patient through the force provided by a pressurized reservoir applied across a flow control unit.
The amount of the pharmaceutical compositions that will be effective depends on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each subject's circumstances. An example of such a dosage range is 0.1 to 200 mg/kg body weight in single or divided doses. Another example of a dosage range is 1.0 to 100 mg/kg body weight in single or divided doses.
In some embodiments, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, or about 50 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to the subject. In further embodiments, from 1-5, 1-10, 1-20, 5-10, 5-20, 10-20, 10-30, 10-50, 20-30, or 20-50 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to the subject. In some embodiments, no more than 20 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject, such as no more than 5, 10, or 15 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject. In one preferred embodiment, 5, 10, or 15 mg/kg/day of the polypeptide comprising or consisting of a disclosed SAHP is administered to a subject.
The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy.
The pharmaceutical compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g. , in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. The volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to about 1.0 ml. Those of ordinary skill in the art will know appropriate volumes for different routes of administration (for example, exemplary delivery methods include, but are not limited to, those provide by Malik et al. , . Curr. Drug Deliv. 4(2): 141-151, 2007 which is hereby incorporated by reference in its entirety).
The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the disclosed antigen and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the disclosed antigen plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
D. Kits
Kits are also provided. For example, kits for treating CKD or a dyslipidemic or vascular disorder in a subject. The kits will typically include a polypeptide comprising or consisting of a disclosed SAHP as disclosed herein. More than one of the SAHPs can be included in the kit.
The kit can include a container and a label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the disclosed polypeptide comprising or consisting of a disclosed SAHP. In several embodiments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular condition.
The label or package insert typically will further include instructions for use of a disclosed polypeptide comprising or consisting of a disclosed SAHP in a therapeutic method, such as a method of treating or inhibiting CKD or a dyslipidemic or vascular disorder in a subject. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
EXAMPLES
The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims are not intended be limited to those features exemplified.
Example 1: SAHPs for the Treatment of Chronic Kidney Disease
This example illustrates use of SAHPs for the treatment of CKD.
Abstract. Few pharmacological agents are available to slow Chronic Kidney Disease (CKD) progression, which affects millions of people worldwide. To test the role of CD36 in CKD, wild type (WT) vs. CD36 knockout (KO) vs. WT mice treated with 5A-37pA (5A), a CD36 antagonist peptide, were compared in a progressive CKD model that resembles human disease. 5A was further tested in a kidney fibrosis model. KO and 5A-treated WT mice are protected from CKD progression, and this protection occurs without changes in blood pressure. In a model of kidney fibrosis, 5A-treated WT mice are protected from macrophage infiltration and interstitial fibrosis. 5A exerts anti-inflammatory effects in the kidney and decreases renal expression of inflammasome genes. In summary, CD36 is a new therapeutic target for CKD, and 5A is a promising protective agent.
Introduction. Chronic kidney disease (CKD), defined as a prolonged (> 3 months) and progressive decline in kidney function, affects 10-15% of the adult general population in Asia, Australia and Europe, and it is estimated to affect about 11.5% of people in the United States (de Jager et al. ,
Nature reviews. Nephrology 10, 208-214, 2014; 2012 Usrds Annual Data Report. Am J Kidney Dis 61, E1-E459, 2013; Levey et al, Annals of internal medicine 150, 604-612, 2009). Due to its high prevalence and associated morbidity and mortality, CKD is a major public health concern, with its costs due to frequent hospitalizations and renal replacement therapies reaching 18% of Medicare expenditures (2012 Usrds Annual Data Report. Am J Kidney Dis 61, E1-E459, 2013); and increasing rates of diabetes and hypertension contribute to its increasing prevalence (Coresh et al , JAMA, 298, 2038-2047, 2007).
If left untreated, patients with CKD progress to end stage renal disease (ESRD) when they need life-sustaining dialysis and/or kidney transplantation or die of cardiovascular disease. Before and after initiation of dialysis or kidney transplant, this population is at higher risk for cardiovascular disease (CVD) and death: one in five patients undergoing dialysis dies each year in the United States. Adjusted all-cause hospitalization and mortality rates are higher among CKD patients than in the general population (2012 USRDS Annual Data Report. Am J Kidney Dis 61, E1-E459, 2013). Left ventricular dysfunction, volume overload, atherosclerosis, and vascular calcification are common among chronic hemodialysis patients, and cardiac deaths account for 39.4% of all deaths, with ischemic heart disease being responsible for 61.5% of these events (Cheung et al , Kidney Int., 65, 2380-2389, 2004). This population also carries increased morbidities, such as hypertension and dyslipidemia.
To date, few pharmacological interventions are available to slow the progression of CKD. Renin- Angiotensin-Aldosterone System (RAAS) inhibitors are standard-of-care in CKD, but a significant fraction of treated individuals still progress to ESRD, or die of CVD. Several recent approaches to slow the progression of CKD have failed (de Zeeuw et al. , New Eng J Med, 369, 2492-2503, 2013; Fried et al. , New Eng J Med, 369, 1892-1903, 2013; Parving et al. , New Eng J Med, 367, 2204-2213, 2012).
Therefore, there is an urgent need to discover novel therapeutics to reduce and/or prevent CKD progression.
CD36 is a widely expressed cell surface class B scavenger receptor that recognizes a large variety of ligands, including apoA-1, apoL-1, thrombospondin-1, serum amyloid A (SAA), lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL), free fatty acids, advanced glycated end products, apoptotic cell surfaces, phosphatidyl-serine (PS), and microparticles (Madhavan et al. , Journal of the American Society of Nephrology 22, 2119-2128, 2011 ; Lopez-Dee et al. , Mediators of inflammation 2011 , 296069, 2011 ; Baranova et al. , J Biol Chem, 285, 8492-8506, 2010; Ghosh et al , J Clin Res, 118, 1934- 1943, 2008; Baranova et al, J Biol Chem, 280, 8031-8040, 2005). CD36 is upregulated by oxLDL (Liu et al , Inflammation research, 63, 33-43, 2014) or LPS (Bocharov et al , J Biol Chem, 279, 36072-36082, 2004) in macrophages, and contributes to the formation and accumulation of foam cells during atherosclerosis. Absence of CD36 was protective against the development of atherosclerosis in ApoE knockout (KO) mice fed high-fat diet (Kuchibhotla et al , Cardiovascular research 78, 185-196, 2008), and mice lacking CD36 in cardiomyocytes exhibit improved cardiac function after myocardial ischemia/reperfusion (Nagendran et al , J Mol Cell Cardiol, 63, 180-188, 2013). High-density lipoprotein (HDL), which promotes the efflux of excess cholesterol from peripheral cells to the liver, and has anti- inflammatory effects, also binds CD36 (Calvo et al , Journal of lipid research 39, 777-788, 1998). ApoA- I, the main apolipoprotein constituent of HDL, acts as a ligand for many receptors including CD36 (Baranova et al , J Biol Chem, 280, 8031-8040, 2005). ApoA-I mimetic peptide, 5A, also binds to CD36 and acts as a CD36 antagonist (FIGs. 7A and 7B). This example illustrated that targeting CD36 is protective against CKD progression and its associated risk factors for CVD in a progressive CKD model that resembles human disease. This protection is achieved either genetically in CD36 knockout (KO) mice, or pharmacologically, by using the peptide 5A-37pA (5A), which is a CD36 antagonist. It is also demonstrated that, in a kidney fibrosis model using an outbred strain (CD-I), that 5A protects mice from interstitial fibrosis and exerts renal antiinflammatory effects while down-regulating renal inflammasome genes.
Results
CD36KO mice and WT mice treated with 5A are protected against CKD progression after 5/6 nephrectomy with Angiotensin II infusion. Because CD36KO mice are on C57BL/6 background and C57BL/6 WT mice are resistant to CKD progression following 5/6 nephrectomy (Nx), the 5/6Nx plus continuous Angiotensin II (Angll) infusion model was used to induce progressive CKD in this strain (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010). C57BL/6 WT mice subjected to 5/6Nx without Angll infusion did not develop CKD and were used as controls. Four weeks after 5/6Nx, WT mice that received continuous infusion of Angll developed substantial decline in kidney function, with significant elevation in BUN, creatinine, and a progressive rise in urinary albumin-to-creatinine -ratio (ACR), accompanied by histological damage (glomerulosclerosis and interstitial fibrosis) - (FIGs. 1A-1E). CD36KO mice subjected to 5/6Nx with Angll infusion were significantly protected from the decline in kidney function at 4 weeks, with BUN, serum creatinine, and histologic injury similar to controls (FIGs. 1A-1E). This group also had significantly reduced levels of albuminuria compared to WT mice. WT mice subjected to 5/6Nx+AngII, which received continuous infusion of 5A peptide by osmotic minipump (5mg/kg/day) were also significantly protected from CKD progression, and had albuminuria levels similar to KO mice (FIGs. 1A-1E).
CD36KO and WT mice treated with 5A are protected from a metabolic profile typical of CKD, including risk factors for CVD. In addition to CKD, at 4 weeks WT mice subjected to
5/6Nx+AngII developed a metabolic profile typical of patients with CKD, including hypercalcemia, hyperphosphatemia, hypermagnesemia, dyslipidemia, and high serum levels of fibroblast growth factor- 23 (FGF-23) (FIGs. 2A-2L). FGF-23 is a phosphate regulatory hormone that is involved in left ventricular hypertrophy, and is associated with subclinical and clinical cardiac disease, including heart failure and coronary vascular events (Kestenbaum et al , Circulation. Heart failure 7, 409-417, 2014). High serum phosphate and FGF-23 levels are independent risk factors for cardiovascular events, particularly among CKD patients (Scialla et al , Journal of the American Society of Nephrology 25, 349-360, 2014; Scialla et al , Nature reviews. Nephrology 10, 268-278, 2014), and FGF-23 can accelerate phosphate-induced uremic vascular calcification (Moe et al, Kidney Int., 85, 1022-1023, 2014; Jimbo et al, Kidney Int., 85, 1103-1111, 2014). WT mice subjected to 5/6Nx+ Angll also had lower serum levels of glucose at 4 weeks. CD36KO mice and WT mice treated with 5A (WT+5A) subjected to 5/6Nx+AngII had a more normal metabolic profile, similar to the control group, which did not develop progressive CKD. In the model (5/6Nx plus Angll) used here, WT mice also had significantly increased levels of acute -phase serum amyloid A (SAA) when compared to control group (5/6Nx alone): mean ± standard deviation 131+ 7.07 (control) vs. 378.4+ 80.1 ug/ml (5/6Nx+AngII), p<0.05. SAA is a known ligand to CD36 (Baranova et al , J Biol Chem, 285, 8492-8506, 2010) that is increased in CKD patients (Lavin-Gomez et al, Advances in peritoneal dialysis. Conference on Peritoneal Dialysis 27, 33-37, 2011 ; Weichhart et al. , Journal of the American Society of Nephrology 23, 934-947, 2012). Mice treated with 5A did not have liver toxicity, as assessed by serum transaminases (FIGs. 2J and 2K).
CD36KO mice are protected from CKD progression independently of blood pressure changes. Blood pressure was monitored weekly in conscious mice by radiotelemetry for 24h intervals from WT and KO 5/6Nx+AngII groups (N=6/group). WT and KO mice subjected to 5/6Nx+AngII had similar baseline (before surgeries) blood pressure values. Systolic, diastolic, and mean blood pressure gradually increased over time in both WT and KO mice without statistical differences between the two groups (FIGs. 3A-3C). WT 5/6Nx+AngII mice also had progressive increases in pulse pressure, while pulse pressure from KO 5/6Nx+AngII mice was almost constant, and statistical differences between these two groups became apparent after 2 weeks (FIG. 3D). Mice in both groups followed a circadian pattern with blood pressure increased during nighttime and decreased during the day (mice are nocturnal animals). Circadian patterns were not different between the two groups (FIGs. 3A-3D).
Peptide 5A down-regulates renal inflammation- and inflammasome-associated genes. A second set of progressive CKD experiments using the most essential groups was performed to obtain kidney tissue to measure renal mRNA expression of genes associated with inflammation and
inflammasome. This was necessary because histology consumed all of the entire amount of kidney tissue left in this model during initial experiments. As before, WT mice subjected to 5/6Nx+AngII developed CKD while 5A-treated WT mice were protected (FIG. 8). Four weeks after 5/6Nx+AngII, renal gene expression of pro-inflammatory genes IL-6 and CXCL-1 were increased 40- and 10-fold above control, respectively (FIGs. 4A and 4B). There was a trend toward increased renal expression of TNF-OC and TGF-βΙ genes (FIGs. 4C and 4D). 5A-treated WT mice subjected to 5/6Nx+AngII had significantly lower expression of IL-6 and CXCL-1 genes in the remnant kidney (FIGs. 4A and 4B). In this progressive CKD model, there was significant up-regulation of renal inflammasome genes IL-Ιβ and NLRP3 that was prevented by 5A treatment (FIGs. 4E and 4F).
5A treatment prevents renal macrophage infiltration and interstitial fibrosis in the
Unilateral Uretheral Obstruction (UUO) model. The deposition of interstitial extracellular matrix (interstitial fibrosis) is a histological hallmark of CKD, and is a strong predictor of renal functional loss and progression risk in patients (Eddy et al. , Pediatr Nephrol 27, 1233-1247, 2012). We tested the effects of targeting the CD36 receptor in a renal fibrosis model [Unilateral Uretheral Obstruction (UUO)] that is not Angll-dependent and does not cause hypertension. While the progressive CKD model (FIG. 2G) develops hypercholesterolemia on a regular diet, mice do not develop dyslipidemia with the UUO model (FIG. 9). Because CD36 can scavenge oxidized-LDL and oxidized-HDL (Calvo et al, Journal of lipid research 39, 777-788, 1998), alleviating lipid/cholesterol imbalance by CD36 inhibition cannot account for the anti-fibrotic effect in the UUO model. For the UUO model, we employed an outbred strain, CD-I, due to its wider genetic variability. After UUO, renal vascular resistance increases, which reduces renal blood flow to the obstructed kidney (Eddy et al. , Pediatr Nephrol 27, 1233-1247, 2012), and distribution and metabolism of therapeutic agents become difficult. Therefore, 5A administration was started 24h before surgery using an osmotic minipump, at two doses: 5 and 15 mg/kg/day. Animals were divided in 4 groups: 1. Sham surgery; 2. UUO; 3. UUO+5A5 (5mg/kg/day); and 4. UUO+5A15 (15mg/kg/day). After UUO surgery, the contralateral non-obstructed kidney compensates for the obstructed kidney, which complicates meaningful renal function measurements in the model. Surprisingly, 10 days after UUO in the CD-I mice, 5A untreated mice developed a very mild but statistically significant increase in BUN, which was accompanied by hypercalcemia, hyperphosphatemia, and hypermagnesemia; whereas 5A pre- treatment did not show any evidence of liver toxicity (FIG. 9). 5A-treated mice were protected from interstitial fibrosis at the higher dose (15mg/kg/day), and had better preserved kidney structure, as measured by cortical thickness (FIGs. 5A and 5B). 5A treatment prevented F4/80+ macrophage infiltration in the obstructed kidney, even at the lower 5 A dose (FIG. 5A). Ten days after UUO, the obstructed kidney had a higher gene expression of pro-inflammatory cytokines, which trended to be prevented by 5 A peptide treatment. 5A-treatment (5A15) significantly prevented increases gene expression of TGF-βΙ and IL-Ιβ (FIG. 5C).
Both 5A peptide and CD36 receptor co-localize to Proximal Convoluted Tubule (PCT) Cells. The CD36 receptor is expressed in PCT (Baines et al. , American journal of physiology. Renal physiology 303, F1006-1014, 2012; Kennedy et al , Hypertension 61, 216-224, 2013) and interstitial macrophages (Kennedy et al. , Hypertension 61, 216-224, 2013; Okamura et al. , American journal of physiology. Renal physiology 293, F575-585, 2007). Using kidney sections from a KO mouse as a negative control, we found by IHC that CD36 is expressed mainly in PCT (FIG. 6A). To identify whether 5A could have a local direct effect in the kidney, we intravenously injected saline or 5A labeled with Alexa Fluor® 488 into WT or KO mice, and kidney sections were imaged with 2-photon microscopy. After fluorescent-5A injection, 5A was localized within the PCT (FIG. 6B), where the expression of CD36 protein is more abundant.
Methods
Animals. The National Institutes of Health (NIH) criteria for laboratory animal care were used in this study. Generation of CD36KO mice and backcrossing into a C57BL/6 background for more than 10 generations were performed and kindly provided by Dr. Kathryn Moore (Moore et al, J Biol Chem, 277, 47373-47379, 2002; Sheedy et al, Nature immunology 14, 812-820, 2013). CD36KO colony was maintained at an NIH animal facility. WT (CD36+/+) C57BL/6 were obtained from the NCI-DCT Laboratory, Bethesda, MD. Sixteen-wk-old male C57BL/6 mice were used for experiments with progressive CKD model. Nine-wk-old male CD-I mice were obtained from Charles River Laboratory,
Wilmington, MA and used for experiments with kidney fibrosis model. All mice had free access to water and regular chow. Synthesis of peptide antagonist. 5A-37pA peptide (5A) is a 37 residue amphipathic peptide (DWLKAFYDKVAEKLKEAF-P-DWAKAAYDKAAEKAKEAA, SEQ ID NO: 3) that contains a proline between the two amphipathic helices. The peptide contains a high lipid-affinity helix paired with a low lipid-affinity helix with 5 alanine substitutions. 5A peptide was synthesized by a solid-phase procedure using Fmoc/DIC/HOBt chemistry and purified to > 99% by reverse-phase high-performance liquid chromatography (HPLC) as previously described (Bocharov et al , J Biol Chem, 279, 36072-36082, 2004; Sethi et al , J Biol Chem, 283, 32273-32282, 2008). Purity was assessed by MALDI-TOF-MS (Bruker Ultraflex) (Bocharov et al. Biol Chem, 279, 36072-36082, 2004; Sethi et al , J Biol Chem, 283, 32273-32282, 2008).
CD36-dependent 5A-DMPC uptake. For 5 A- dimyristoylphosphatidylcholine-BODIPY
Cholesterol ester complex preparation, 5 mg of dimyristoylphosphatidylcholine (DMPC) and lOOug of BODIPY-CholesterolEster (BP-CE) in chloroform was evaporated with N2 in a glass tube. 5 mg of 5A in 1 ml of dtbO was added to dried lipids and sonicated with 10 sec on/off intervals for 20 minutes on ice. Resulting 5A-DMPC-BP-CE complex was used for experiments. As a negative control, DMPC-BP-CE complex without 5 A was used. Cell uptake studies were performed using DMEM containing 2 mg/ml
BSA. HeLa cells stably transfected with CD36 were incubated with 10 μg/ml 5A-DMPC-BP-CE [10/70/1 molar ratio] at 37°C for 2 hours then washed with PBS and detached with Cellstripper® dissociation solution (Cellgro, Herndon, VA). Detached cells were fixed with 4% paraformaldehyde and analyzed by a Victor3 fluorimeter (Perkin Elmer). Mock-transfected HeLa cells were used as controls.
Progressive CKD Model (5/6 Nephrectomy plus Angiotensin II infusion). 5/6 nephrectomy
(5/6Nx) was performed in C57BL/6 mice in two stages under isoflurane anesthesia. First, via left flank incision, the left kidney was decapsulated to avoid ureter and adrenal damage, and the upper and lower poles were resected. Bleeding was controlled with microfibrillar collagen (Avitene, Davol, USA). The upper and lower poles were weighed. After one week, the entire right kidney was decapsulated and removed via right flank incision. Immediately after each surgical intervention, mice received a single dose of buprenorphine (0.1 mg/kg) diluted in saline (lml/25g), followed by buprenorphine 0.05mg/kg 18h after the procedure. Animals with kidney mass resection [as determined by (removed left kidney weight at week -1) / (removed right kidney weight at week 0) between 0.5-0.65] were used for the study; and others, euthanized (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010). As previously described, C57BL/6 mice are resistant to CKD progression after 5/6Nx, and Angiotensin II (Angll) overcomes this strain-specific resistance (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010; Ma et al , Kidney Int., 64, 350-355, 2003). Therefore, mice subjected to 5/6Nx without Angll infusion did not develop progressive CKD after 4wk and were considered controls. Mice were divided into 4 groups (N= 11- 19/group): 1. WT 5/6Nx without Angll (Control); 2. WT 5/6Nx+AngII (WT); 3. CD36KO 5/6Nx+AngII (KO); 4. WT 5/6Nx+AngII+5A (WT+5A); Angll and 5 A were infused through separate osmotic minipumps (see below) starting at the time of right nephrectomy. Mice were followed for 4wk, with weekly urine collection, and euthanized after 4wk. A second set of experiments was performed to obtain kidney tissue for mRNA expression analysis [(N=4-6/group): 1. Control; 2. WT 5/6Nx+AngII (WT); 3. WT 5/6Nx+AngII+5A (5A)].
Kidney Fibrosis Model (Unilateral Uretheral Obstruction, UUO). Under isoflurane anesthesia CD-I mice were subjected to right flank incision and right kidney and pelvis were identified. The right ureter was ligated with a double suture below the pelvis and at the end of inferior kidney pole (Shen et al. , Inflammation DOI 10.1007/sl0753-014-9941-y, 2014). Immediately after each surgical intervention, mice received a single dose of buprenorphine (0.1 mg/kg) diluted in saline ( 1 ml/25 g), followed by
buprenorphine 0.05 mg/kg 18h after the procedure. Mice were divided in 4 groups (N=8/group): 1. Sham;
2. UUO; 3. UUO+5A 5mg/kg/day (5A 5); 4. UUO+5A 15mg/kg/day (5A 15), and euthanized 10 days after sham or UUO. Timeline schemes of both models are summarized in FIG. 11 and FIG. 12.
Drug administration. Angll (Val5-AngII) 0.75μg/kg/min (Sigma-Aldrich, St Louis, MO, USA), diluted in sterile water, or vehicle (sterile water) was continuously infused by subcutaneous osmotic mini- pump (Alzet model 1004, Cupertino, CA). 5 A was continuously administered through a second osmotic mini-pump, at the dose of 5mg/kg/day in the progressive CKD model. Osmotic mini-pumps were inserted at the time of right nephrectomy. In the UUO model, osmotic minipumps with 5 A were started 24h before UUO (5 or 15mg/kg/day).
Blood and urine measurements. Spot urine samples were collected before (baseline), and at 1, 2,
3, and 4wk after complete 5/6Nx. At 4wk, mice were euthanized under isoflurane anesthesia by cardiac puncture, and blood was collected. The remnant kidney was harvested and fixed in 10% formalin. Urine samples were not collected in the UUO model. Serum creatinine was measured by HPLC, and blood urea nitrogen (BUN) by colorimetric assay (QuantiChrom Urea assay kit DIUR-500, Hayward, CA, USA). Urine albumin-to-creatinine -ratio (ACR) was determined from albumin ELISA (Albuwell M; Exocell, Philadelphia, PA, USA) and creatinine by Jaffe method. A biochemistry panel was measured using a Siemens Advia 1800 automated chemistry analyzer (Siemens Healthcare Diagnostics, Flanders, NJ). Serum FGF-23 and SAA were measured by ELISA (Immunotopics, Inc., Cat. #60-6300; Abeam, kit abl57723, respectively).
Morphologic evaluation of the kidney. Kidney specimens were fixed in 10% formalin, paraffin embedded (FFPE), and stained with Masson's trichrome and periodic acid-Schiff (PAS) reagent (Sigma- Aldrich). Histological changes were assessed semi-quantitatively. The degree of glomerular damage was assessed in 10 randomly selected fields at 400X magnification from the degree of mesangial expansion in PAS-stained tissue and scored as follows: 1, <25%; 2, 25-50%; 3, 50-75%; 4, >75%; 5, completely sclerotic glomeruli. Interstitial fibrosis was assessed at 200X magnification on Masson's trichrome- stained sections using 10 randomly selected fields for each animal and scored by the following criteria: 1, area of damage <10%; 2, 10-25%; 3, 25-50%; 4, 50-75%; and 5, 75-100%.
Immunohistochemistry (IHC) for CD36 and F4/80+. Four micron FFPE sections were de- paraffinized, incubated 10 min with an endogenous peroxide blocker (Dako S2003) and then treated for 1 h in an antigen retrieval solution in a steamer (Dako Cat#1700). Next, sections were incubated with a protein blocking solution (CytoQ Background Buster) for 20 min before incubation with the rabbit polyclonal anti-CD36 antibody (Novus, NB400-144) for lh at 1: 1,600. An isotype rabbit IgG Ab was used as a control. Sections were then incubated with a goat anti-rabbit HRP-conjugated Ab for 30 min at 1 :200 followed by DAB Chromogen for 5 min. Sections were next counterstained (Leica Autostainer), coverslipped and digitally captured (Aperio ScanScope). Between each step above, sections were washed with TBST buffer 2X for 5 min. IHC for CD36 was performed on kidney sections from a KO mouse and a WT mouse, both subjected to 5/6Nx+AngII. IHC for F4/80+ cells (Ab from AbD Serotec,
Cat. #MCA497GA) was performed in FFPE kidney sections from all UUO groups (N=4/group) and the number of positive cells counted in 10 random high-power fields (HPF, 400X).
Radiotelemetry study. In 6 mice from each group: [2. WT 5/6Nx+AngII (WT); 3. CD36KO 5/6Nx+AngII (KO)] in the progressive CKD model, a telemeter transmitter (model TA11PA-C10, Data
Sciences International, St Paul, MN) was implanted in a subcutaneous pocket on the left flank, with the tip of the catheter inserted into the aortic arch (via the carotid artery). The animals were allowed to recover for about lweek, before 5/6Nx. Systolic, diastolic, mean arterial pressure, and pulse pressure were measured and recorded for 10 sec every 30 min for 24 h at baseline, and once a week for 4 weeks after complete 5/6Nx (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010; Doi et al, Kidney Int. , 74, 1017-1025, 2008).
mRNA isolation and RT-qPCR. Because there was no remnant kidney tissue remaining after histology, a new experiment was performed for mRNA expression in the progressive CKD model with 3 groups (N=4-6/group): 1. Control; 2. WT 5/6Nx+AngII (WT); 3. WT 5/6Nx+AngII+5A (5A). In the UUO model mRNA was isolated from mice of all groups (N=4/group). For RNA isolation, tissue samples preserved in RNAlater (Life Technologies cat#AM7021, Grand Island, NY) were homogenized in TRIzol Reagent using a Precellys 24 homogenizer (Bertin Technologies, France). All reagents used for RNA isolation, reverse transcription and real-time PCR were obtained from Life Technologies. RNA was isolated with the PureLink RNA Mini Kit after DNase treatment. RNA (2μg) was reverse-transcribed using a TaqMan Reverse Transcriptase Reagents Kit. Real-time qPCR assays were performed with a StepOne Real-Time PCR System (Applied Biosystems), with 40ng of cDNA per reaction. A list of TaqMan Gene Expression assays used in the study is shown in FIG. 10. The relative levels of gene expression were measured by the comparative CT (AACT) method (Schmittgen et al. , Nature protocols 3, 1101-1108, 2008) with mouse GAPDH used as a reference gene. All gene expression results were analysed using 2~ΑΑ€τ formula and presented as normalized fold changes, compared to Control.
Fluor-labeled peptide uptake. 2-photon microscopy was performed on kidneys of a WT and a CD36KO mouse 3h after IV injection of Alexa Fluor® 488-labeled 5A peptide. 5A labeling with Alexa Fluor® 488 was performed as previously described (Baranova et al. , J Biol Chem, 285, 8492-8506, 2010).
Kidney tissue immunofluorescence staining. Kidney frozen O.C.T blocks from a WT and a CD36KO mouse were cut into 10-12 μιη sections using a Bright cryostat and put on microscope slides.
Sections were fixed with 3.7% paraformaldehyde (Electron Microscopy Sciences, PA) for 10 min, washed 3 times, 5 min each, with 0.5% Saponin in PBS. Sections were blocked with 5% Goat Serum-0.05% Saponin-l%BSA-PBS for 1 h then incubated overnight at 4°C with Rabbit polyclonal anti-CD36 antibody (Abeam # ab36977) or Rabbit IgG for nonspecific control, followed by 1 h incubation with secondary antibodies, anti-rabbit-Alexa Fluor 488. After three washes with PBS, tissue were stained with DAPI (Invitrogen) mounted with Vectashield® antifade mounting medium reagent (Vector, cat # H-1400), and subjected to microscopy (Zeiss 510 laser scanning confocal microscope). Images were acquired by using a 488-nm laser line and emission between 505 and 580 nm for Alexa Fluor 488.
Statistical analysis. Differences in parameter values between the groups at fixed time points were examined for statistical significance by ANOVA, with subsequent post-hoc analysis using Dunnett' s adjustment for multiple comparisons to a reference group; nonparametric Kruskal-Wallis tests were examined when ANOVA assumptions were not met. SAA, measured on 2 groups, was analyzed by Student's t-test. Differences between the groups in weekly mean/sinusoid parameter values (ACR and telemetry data) were examined for statistical significance using mixed-effects models that extend repeated measures ANOVA to imbalanced data settings. Analyses were conducted using SAS v9.3 (SAS Institute, Cary, NC) and R v3.0 (r-project.org). Discussion
In this study, both genetic and pharmacologic inhibition of CD36 were demonstrated to reduce CKD progression and associated cardiovascular risk factors without changes in blood pressure (BP). The CD36 antagonistic peptide (5A) may act in the proximal convoluted tubule as injected fluorescent-5A co- localizes with CD36 protein expression in the proximal tubule. 5A exhibits anti-inflammatory effects in the kidney, in part via decreased renal expression of inflammasome gene NLRP3. Although CD36 has been considered a possible therapeutic target for atherosclerosis (Park et al. , Experimental & molecular medicine 46, e99, 2014), the data provided in this example suggests a direct role for CD36 on CKD progression that is independent of blood pressure and likely involves anti-inflammatory and anti-fibrotic effects.
It was previously demonstrated that targeting CD36 during sepsis prevents sepsis-AKI
(Leelahavanichkul et al, J Immunol 188, 2749-2758, 2012). Here it is demonstrated that targeting CD36 also protects against kidney fibrosis, CKD progression, and co-morbidities associated with CKD. WT mice subjected to 5/6Nx with Angll infusion developed progressive CKD with increasing levels of albuminuria and blood pressure over time. At the end of 4 weeks these mice had a substantial decline in kidney function (as measured by BUN and creatinine), and histological damage (including
glomerulosclerosis and interstitial fibrosis). All these changes were prevented in CD36KO mice and in mice that received 5A. Further, in the UUO model, 5A treatment prevented interstitial fibrosis and cortical thinning in the obstructed kidney. Therefore, 5A had similar effects in two different models of chronic kidney damage and fibrosis.
Other groups have also demonstrated the role of scavenger receptors, including CD36, on kidney fibrosis. Okamura et al. demonstrated that in mice fed a high fat diet subjected to UUO, there was a strong tubular expression of scavenger receptors, especially CD36, in the obstructed kidney. They suggested that these receptors may be involved in the inflammatory and fibrogenic processes activated by oxLDL (Okamura et al , American journal of physiology. Renal physiology 293, F575-585, 2007). Further, the same group demonstrated that CD36KO mice fed high fat diet for 8 weeks and then subjected to UUO have significantly less renal fibrosis compared with WT mice (Okamura et al , Journal of the American Society of Nephrology 20, 495-505, 2009). Here it is demonstrated in a progressive model of CKD and certain metabolic factors known to be associated with development of atherosclerosis in humans, that targeting CD36 - either genetically by using a KO mouse line, or pharmacologically, by using the 5A peptide - is protective against CKD progression. Unlike the previous work, mice were fed standard chow rather than a high fat diet. Hence, dyslipidemia gradually developed over time in WT mice, and was lessened in CD36KO and 5A-treated mice. To help differentiate whether CD36 inhibition is working as an oxidized-LDL scavenger receptor or has a local renal mechanism independent of oxidized-LDL, the
CD36 antagonist peptide 5A was tested in the UUO model using mice fed standard (not high fat) diet. Ten days after sham-surgery or UUO, all CD-I mice (treated or non-treated with 5 A) had similar cholesterol levels. 5A-treated mice subjected to UUO developed less kidney fibrosis than non-treated mice, without altering cholesterol levels; thus, CD36 affects CKD beyond its role as a lipid scavenger receptor.
Renin-angiotensin-aldosterone (RAAS) blockers are standard of care for CKD in the clinical practice. RAAS blockers decrease both albuminuria and renal fibrosis in parallel (Leelahavanichkul et al. , Kidney Int., 78, 1136-1153, 2010). Despite the reduction in fibrosis in the 5/6Nx+AngII and UUO models, in the 5/6Nx+AngII model there was only a partial decrease in albumin-to-creatinine -ratio (ACR) in both CD36KO and 5A-treated mice. This partial reduction in albuminuria may be explained by CD36 participating, together with megalin and cubilin (Baines et al., Am J Physiol Renal Physiol 303, F1006- 1014, 2012), in albumin binding and endocytosis in renal proximal tubular cells. Blocking CD36 may reduce the uptake of filtered albumin by the proximal tubule; thus, targeting CD36 prevents CKD progression without dramatic changes in albuminuria. Yang et al. demonstrated that CD36 can mediate albumin-induced cellular fibrosis in cultured proximal tubule cells (Yang et al , J Cell Biochem 101, 735- 744, 2007). Thus, inhibiting CD36 might have two competing effects on albuminuria: a) a decrease caused by ameliorating progressive CKD and b) an increase from impaired albumin reuptake. The net result, a partial decrease in albuminuria, appears to underestimate the benefit of CD36 inhibition on CKD, as CD36 inhibition prevents albumin-induced damage to the proximal tubule. Targeting CD36 may be an adjuvant therapy to RAAS inhibitors, which may be more effective than monotherapy because of non- redundant mechanisms.
It was previously demonstrated that olmesartan (an Angll type-I receptor antagonist) reduces blood pressure, albuminuria, and CKD progression in CD-I mice subjected to 5/6Nx, whereas mice treated with hydralazine, which reduced blood pressure to the same extent, did not improve CKD progression (Leelahavanichkul et al , Kidney Int., 78, 1136-1153, 2010). In the current experiments, blood pressure (BP) and CKD progression are again disassociated. Despite similar degrees of mean arterial, systolic, or diastolic hypertension in both WT and CD36KO mice subjected to 5/6Nx+AngII, KO mice were protected from CKD, but WT were not. However, WT mice, unlike CD36KO, had significant gradual and progressive increases in pulse pressure throughout the study. There is emerging evidence for a role of pulse pressure on CKD progression and complications. In diabetics, pulse pressure is highly associated with proteinuria, independent of systolic, diastolic, or mean blood pressure levels (Yano et al. , Diabetes Care 35, 1310-1315, 2012). Higher pulse pressure was linearly and independently associated with faster kidney function decline among people with eGFR >60 ml/min/1.73m2 in a large cohort study (4,853 adults) with follow-up of 5 years (Peralta et al. , Am J Kidney Dis 59, 41-49, 2012). Widening arterial pulse pressure is often a consequence of arterial stiffness, which is accelerated by CKD and uremia (Townsend et al. , Am J Hypertens 23, 282-289, 2010), and independently associates with mortality (Foley et al, Kidney Int., 62, 1784-1790, 2002). WT mice subjected to 5/6Nx+AngII also had several other risk factors for vascular calcification and dysfunction, which could have contributed to increased arterial stiffness. Thus, CD36 inhibitory effects are independent of systolic and diastolic pressure, but may alter pulse pressure perhaps via an effect on arterial stiffness.
In addition to hypertension and dyslipidemia, CKD mice also developed other risk factors for cardiovascular disease and vascular injury, such as high FGF-23 levels and hyperphosphatemia, which can contribute to endothelial dysfunction and release of microparticles (Agouni et al. , Curr Vase
Pharmacol 12, 483-492, 2014), also ligands for CD36 (Ghosh et al , J Clin Res, 118, 1934-1943, 2008). CKD also increases other possible CD36 ligands, including serum amyloid A (SAA) (Baranova et al , J Biol Chem, 285, 8492-8506, 2010), that could contribute to inflammation, oxidative stress, and CKD progression. Serum amyloid A (SAA) is an acute phase protein that is increased in CKD patients (Weichhart et al , J Am Soc Nephrol 23, 934-947, 2012) and was also increased in the model disclosed herein. Chronic inflammation is thought to be important in the progression of both cardiovascular disease and CKD, and CD36 activation is associated with downstream activation of inflammation (Moore et al., J Biol Chem, 277, 47373-47379, 2002; Febbraio et al., Int J. Biochem Cell Biol 39, 2012-2030, 2007) and inflammasome (Liu et al. , Inflamm Res, 63, 33-43, 2014; Sheedy et al., Nat Immunol 14, 812-820, 2013; Kagan et al , Nat Immunol 14, 112-11 , 2013; Oury et al , Cell Mol Immunol 11, 8-10, 2014). Here, it was found that 5A decreases mRNA for renal cytokines in both models, and decreases macrophage infiltration 10 days after UUO. Besides renal effects, CD36 inhibition also can decrease inflammation and fibrosis in other organs. 5A also decreases the expression of genes associated with collagen deposition in the lungs (Yao et al , Chest 140, 1048-1054, 2011). Further, silencing of the CD36 gene results in suppression of silica-induced lung fibrosis in rats (Wang et al , Resp Res 10, 36, 2009).
5A is an apoA-I mimetic peptide that has also been used to attenuate atherosclerosis, acute vascular inflammation, and oxidative stress in experimental animal models (Amar et al. , The Journal of pharmacology and experimental therapeutics 334, 634-641, 2010; Yao et al., Chest 140, 1048-1054, 2011). As apoA-I is the major structural protein of HDL that mediates reverse cholesterol transport, 5A is thought to actively remove cholesterol from atherosclerotic plaque by enhancing reverse cholesterol transport. However, 5 A also has anti-inflammatory effects that are independent of HDL levels (Yao et al. , Chest 140, 1048-1054, 2011). CD36 activates the inflammasome (Liu et al , Inflammation research, 63, 33-43, 2014; Sheedy et al, Nat Immunol 14, 812-820, 2013; Kagan et al , Nature immunology 14, 772- 774, 2013; Oury et al. , Cell Mol Immunol 11, 8-10, 2014), which is implicated in renal tissue repair, remodeling, and maladaptive inflammatory responses (Leemans et al , Nature reviews. Nephrology, 2014). Here, it was found that NLRP3 and IL-Ιβ renal mRNA expression is increased in both kidney injury models. 5A prevented this increase, suggesting that it may have an effect on down-regulating inflammasome in the kidney. 5A likely exerts some of its actions on the proximal tubule cells.
Intravenously injected fluorescent-5A peptide localized on proximal tubule cells, the site within the kidney in which CD36 protein is mostly expressed, and where CD36 may have an important role on CKD progression.
In summary, this study suggests that CD36 is a new therapeutic target and 5A is a new potential therapy to slow CKD progression. The blood-pressure-independent benefit of CD36 inhibition suggests it might complement RAAS inhibition through non-overlapping effects, possibly leading to combination therapy. Targeting CD36 should be examined as a possible alternative or second line therapy for CKD, especially when RAAS inhibitors are contra-indicated. New therapies to slow CKD progression would benefit millions of individuals with CKD worldwide. Example 2: CD36 selective SAHP Peptides
This example provides description and characterization of two new SAHPs, termed ELK-B and ELK-B2, which are CD36 antagonists. A panel of 20 SAHP were tested in HEK293 cell lines stably transfected with CD36. SR-BI, or SR-BII, to identify SAHP with preferential selectivity towards CD36. Among several SAHP targeting both SRBI/BII and CD36 receptors, ELK-B and ELK-B 2 acted selectively inhibited CD36 activity compared to SR-BI and SR-BII activity under similar conditions.
Peptide design
Twenty-two ApoA-I mimetic peptides were synthesized. Their sequences, are listed in Table 3. Two peptides were used as prototypes to understand how structural modifications affect their function. The first prototype peptide, 5A-37pA (see Table 3), has been previously described (see, e.g. , PCT. Pub
No. WO2006/044596). This peptide consists of two type A amphipathic a-helices connected by a proline. The hydrophobicity of the second helix was reduced by substitution of hydrophobic amino acids with alanine. Four derivatives of 5A were synthesized to test the impact of the introduction of two amino acids with antioxidant potential, cysteine and histidine, on its properties. The second prototype peptide, ELK (see Table 3), contains only three amino acid residues: glutamic acid, leucine, and lysine. It consists of two identical canonical type A amphipathic a-helices with hydrophobic interface turned by 180° and neutral net charge. The helices within this peptide are connected by a proline residue. The original ELK peptide was used to make sixteen derivatives. Several parameters, in particular, (1) net charge, (2) mean hydrophobicity and the size of hydrophobic face, (3) type of helix and configuration of the proline bridge between the two helices, that may affect the peptide interaction with lipoproteins and cellular receptors, as well as (4) peptide structural asymmetry, shown to affect specificity of cholesterol efflux, have been modified. Table 3. Amino acid sequences of SAHPs.
Figure imgf000053_0001
L37pA uptake. All cell uptake studies were performed using DMEM containing 2 mg/ml BSA as described elsewhere. HeLa cells stably transfected with SR-BI, SR-BII and CD36 were incubated with Alexa 488-L37pA (10 μ /πύ, 37°C, 1 hour) then washed with PBS and detached with Cellstripper™ dissociation solution (Cellgro, Herndon, VA). The detached cells were fixed with 4% paraformaldehyde and analyzed by a fluorescence-activated cell sorter (FACS, model A) or using Victor3™ fluorimeter (Perkin Elmer).
Effect of SAHP on IL-8 secretion in vitro. HEK 293 cells stably transfected to express hSR-BI, hSR-BII and hCD36 were reported in previous publications. The cells were grown to 50% confluency in
DMEM additionally containing 10% FBS and antibiotics. After 24 hour incubation in serum free DMEM, cells were challenged with various concentrations of LPS in the presence or absence of various peptides
(10 or 25 μg/ml) for another 24 hours. Collected culture media was analyzed for IL-8 secretion utilizing ELISA. Peptides demonstrating selective inhibition of LPS- induced IL-8 secretion through CD36 versus SR-BI/II pathway were selected.
Statistical analysis. A one-way ANOVA with a Bonferroni multiple -comparison test or a two- way ANOVA with a Bonferroni posttest test GraphPad Prism™, version 5.0a; GraphPad Software, La Jolla, CA) was used; a p value < 0.05 was considered significant.
Results
L37pA binds to SR-BI, SR-BII, CD36, and blocks LPS-induced IL8-secretion. Previous studies demonstrated that SR-BI, SR-BII and CD36 bind various lipoproteins including HDL and its apolipoproteins A-I and -AIL Because specificity of SAHP is not well understood, the uptake of the most known SAHP, L37pA and its inactive analogue L3D-37pA was first evaluated in HeLa cell lines stably transfected with human SR-BI, SR-BII and CD36 and compared with canonical SR-B ligands, HDL and LDL. The uptake of Alexa 488 HDL, Alexa 488 LDL and Alexa 488 L37pA was significantly increased in both hSR-BI and CD36 expressing HeLa cells when compared to mock-transfected controls (FIG. 13A). No increase was found for L3D-37pA. Furthermore, no increase in Alexa 488-BSA or Alexa 488- lactoferrin uptake was seen in these cells when compared to Mock-transfected HeLa controls. Uptake of L37pA was dose-dependent and demonstrated similar dose-response in SR-BI, SR-BII, and CD36 expressing HeLa. Both mock-HeLa and LDL receptor HeLa minimally bound Alexa 488-L37pA (FIG. 13B).
L37pA, 5A, and ELK-based SAHP differentially inhibit LPS-induced IL-8 secretion by SR-
BI, SR-BI and CD36 expressing cells. Since L37pA binds not only to hSR-BI, but to hSR-BII and hCD36 receptors (FIGs. 13A and 13B), a panel of SAHP based on simple design utilizing combination of three amino acids, the hydrophilic positively charged (K), negatively charged (E) and hydrophobic (L), which are referred to as ELK peptides, was tested. The results of these assays are presented in Table 4. This peptide panel was used to identify SAHP selectively targeting CD36 rather than SR-BI or SR-BII. Using HEK293 cell lines stably transfected with human SR-BI, SR-BII, or CD36, it was found that ELK- B and ELK-B2 blocked IL-8 secretion induced by LPS in cells expressing CD36, but were less effective in blocking LPS-induced IL-8 secretion in cells expressing SR-BI, or SR-BII (Table 4). The majority of tested peptides had either no effect or affected both receptors, leading to suppression of LPS-induced IL-8 secretion in all cell lines in a dose-dependent manner (FIGs. 14A and 14B). These data identify ELK-B and ELK-B2 peptides as the most efficient and relatively selective antagonists of LPS-induced IL-8 secretion in cells expressing the CD36 receptor. Table 4. Effect of ELK based SAHP on LPS-induced IL-6 secretion from hSR-BI, hSR-BI and hCD36 stably transfected HEK293 cells.
Figure imgf000055_0001
EXAMPLE 3
Treatment of CKD in a human subject
This example describes an exemplary method for treating and/or inhibiting CKD in a human subject. The method includes administration of a therapeutically effective amount of a polypeptide including or consisting of a SAHP as disclosed herein to a subject with or at risk of chronic kidney disease. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.
Based upon the teaching disclosed herein, CKD can be treated by administering a therapeutically effective amount of a polypeptide including or consisting of a SAHP, such as a SAHP including the amino acid sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 3 to a subject in need thereof.
Screening of subjects. The method can include screening subjects to determine if they have CKD, or if they are at risk of developing CKD. Subjects having or at risk of CKD can be selected for treatment. In one example, subjects having Stage 1, Stage 2, Stage 3, Stage 4, and/or Stage 5 CKD are selected for treatment. Standard methods are used to identify a subject with CKD, or a particular stage of CKD. For example, a subject with an estimated glomerular filtration rate (GFR) of less than 60 niL/min/1.73 m2 can be selected for treatment. Pre-screening is not required prior to administration of the therapeutic compositions disclosed herein.
Pre-treatment of subjects. In particular examples, the subject is treated prior to administration of a therapeutic agent that includes one or more of the disclosed SAHPs or a nucleic acid molecule encoding a SAHP. However, such pre-treatment is not always required, and can be determined by a skilled clinician. For example, the subject can be treated with an established protocol for treatment of CKD (such as a ACEi or ARB therapy).
Administration of therapeutic agents. Following subject selection, a therapeutic effective amount of a polypeptide including a SAHP or a nucleic acid molecule encoding the polypeptide is administered to the subject (such as an adult human with CKD). For example, the methods can include administering a therapeutically effective amount of a polypeptide comprising or consisting of a SAHP having the amino acid sequence set forth as any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68. Additional agents, such as an ACEi or ARB, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous.
The amount of the composition administered to inhibit and/or treat CKD depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic
composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to inhibit and/or treat CKD in the subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. In one example, from 1 mg/kg/day to 10 mg/kg/day or from 1 mg/kg/day to 20 mg/kg/day of the polypeptide including the SAHP is administered to the subject. The therapeutic compositions can be administered in a single dose delivery, via continuous delivery over an extended time period (for example, using a pump), in a repeated administration protocol (for example, by a daily, weekly, or monthly repeated administration protocol). In one example, therapeutic agents that include one or more SAHPs or a nucleic acid molecule encoding the polypeptide are administered intravenously to a human. As such, these compositions may be formulated with an inert diluent or with an
pharmaceutically acceptable carrier.
Administration of the therapeutic compositions can be taken long term (for example over a period of months or years).
Assessment. Following the administration of one or more therapies, subjects having CKD can be monitored for reductions in one or more clinical symptoms associated with CKD, such as glomerular filtration rate or disease progression. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood and urine, can be obtained and alterations in biomarkers indicative of kidney function and/or injury evaluated.
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

It is claimed:
1. A method of treating or inhibiting chronic kidney disease, comprising:
administering a therapeutically effective amount of a polypeptide comprising a synthetic amphipathic helical peptide, or a nucleic acid molecule encoding the polypeptide, to a subject with or at risk of chronic kidney disease;
wherein the synthetic amphipathic helical peptide is a CD36 receptor antagonist, thereby treating or inhibiting chronic kidney disease in the subject.
2. The method of claim 1, further comprising selecting the subject with or at risk of chronic kidney disease prior to administering the polypeptide.
3. The method of claim 1 or claim 2, wherein the synthetic amphipathic helical peptide inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
4. The method of claim 1 or claim 2, wherein the synthetic amphipathic helical peptide comprises the amino acid sequence set forth as one of SEQ ID NO: 3 (5A-37pA), SEQ ID NO: 1 (L37pA), SEQ ID NO: 66 (ELK-B/B2 consensus), SEQ ID NO: 67 (ELK-B), or SEQ ID NO: 68 (ELK- B2).
5. The method of any one of claims 1-3, wherein the SAHP peptide consists of the amino acid sequence set forth as one of SEQ ID NO: 3 (5A-37pA), SEQ ID NO: 1 (L37pA), SEQ ID NO: 66 (ELK-B/B2 consensus), SEQ ID NO: 67 (ELK-B), or SEQ ID NO: 68 (ELK-B 2).
6. The method of any one of claims 1-5, wherein the chronic kidney disease is not acute kidney disease, obstructive kidney disease, atherosclerotic kidney disease, or polycystic kidney disease.
7. The method of any one of the previous claims, wherein no more than 20 mg/kg/day of the polypeptide is administered to the subject.
8. The method of any one of the previous claims, wherein 1 mg/kg/day to 20 mg/kg/day of the polypeptide is administered to the subject.
9. The method of any one of the previous claims, wherein the polypeptide is administered by continuous release pump.
10. The method of any one of the previous claims, further comprising administering an additional anti-CKD agent to the subject.
11. The method of claim 10, wherein the additional anti-CKD agent is a renin-angiotensin- aldosterone blocker.
12. The method of any one of the previous claims, wherein the subject has chronic kidney disease.
13. The method of claim 12, wherein the subject has stage 1 through stage 4 chronic kidney disease.
14. The method of claim 12, wherein the subject has stage 5 chronic kidney disease.
15. The method of claim 12, wherein the subject has stage 1 chronic kidney disease.
16. The method of any one of the previous claims, wherein treating or inhibiting chronic kidney disease in the subject comprises slowing progression of the subject to stage 5 chronic kidney disease compared to a control subject.
17. The method of any one of claims 1-11, wherein the subject is at risk of chronic kidney disease.
18. The method of any one of the previous claims, wherein the subject is a human.
19. An isolated polypeptide, comprising the amino acid sequence set forth as
EKLX1ELLX2KLLELLKKLLPEKLX3ELLX4KLLELLKKLL (SEQ ID NO: 66), wherein, Xi is L or K, X2 is K or E, X3 is L or K, and X4 is K or E.
20. The isolated polypeptide of claim 19, wherein the polypeptide consists of the amino acid sequence set forth as EKLXiELLX2KLLELLKKLLPEKLX3ELLX4KLLELLKKLL (SEQ ID NO: 66), wherein, Xi is L or K, X2 is K or E, X3 is L or K, and X4 is K or E.
21. The isolated polypeptide of claim 19 or claim 20, wherein the polypeptide comprises or consists of the amino acid sequence set forth as
EKLLELLKKLLELLKKLLPEKLLELLKKLLELLKKLL (ELK-B; SEQ ID NO: 67); or EKLKELLEKLLELLKKLLPEKLKELLEKLLELLKKLL (ELK-B 2; SEQ ID NO: 68).
22. The isolated polypeptide of any one of claims 19-21, wherein the polypeptide specifically binds to CD36 receptor.
23. The isolated polypeptide of any one of claims 19-20, wherein the polypeptide is a CD36 receptor antagonist.
24. The isolated polypeptide of any one of claims 19-20, wherein the polypeptide is a selective CD36 antagonist that inhibits at least 2-fold more CD36 activity than SR-BI and/or SR-BII activity under similar conditions.
25. The isolated polypeptide of any one of claims 19-24, further comprising at least one additional peptide domain.
26. The isolated polypeptide of claim 25, wherein the additional peptide domain comprises a heparin binding site, an integrin binding site, a P-selectin site, a TAT HIV sequence, a panning sequence, a penatratin sequence, a SAA C-terminus sequence, a SAA N-terminus sequence, a LDL receptor sequence, a modified 18A sequence, an ApoA-I Milano sequence, a 6x-His sequence, a lactoferrin sequence, or combinations of two or more thereof.
27. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 19-26.
28. The isolated nucleic acid molecule of claim 27, operably linked to a promoter.
29. A vector comprising the isolated nucleic acid molecule of claim 27 or claim 28.
30 A pharmaceutical composition, comprising the isolated polypeptide of any one of claims 19-26, the isolated nucleic acid molecule of claim 27 or claim 28, or the vector of claim 29, and a pharmaceutically acceptable carrier.
31. A method of treating or inhibiting a dyslipidemic or vascular disorder, comprising: administering a therapeutically effective amount of the isolated polypeptide of any one of claims
19-26, the isolated nucleic acid molecule of claim 27 or claim 28, the vector of claim 29, or the composition of claim 30, to a subject with or at risk of the dyslipidemic or vascular disorder, thereby treating the dyslipidemic or vascular disorder.
32. The method of claim 31, further comprising selecting the subject with or at risk of the dyslipidemic or vascular disorder prior to the administering step.
33. The method of claim 31 or claim 32, wherein the dyslipidemic or vascular disorder comprises hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, ApoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, post-perfusion myocardial injury, vasculitis, inflammation, or combinations of two or more thereof.
33. The method of claim 31, wherein the dyslipidemic or vascular disorder is
hypercholesterolemia.
34. The method of any one of claims 31-33, further comprising administering an additional lipid lowering composition.
35. The method of any one of claims 31-34, wherein no more than 20 mg/kg/day of the isolated polypeptide is administered to the subject.
36. The method of any one of claims 31-35, wherein 1 mg/kg/day to 20 mg/kg/day of the isolated polypeptide is administered to the subject.
37. The method of any one of claims 31-36, wherein the isolated polypeptide is administered by continuous release pump.
38. The method of any one of claims 31-36, wherein the polypeptide is administered on a stent or implanted device.
39. The method of any one of claims 31-38, wherein the subject is a human.
40. A kit, comprising a container comprising the isolated polypeptide of any one of claims 19-26, the isolated nucleic acid molecule of claim 27 or claim 28, the vector of claim 29, or the composition of claim 30, and instructions for use.
41. Use of a therapeutically effective amount of a polypeptide comprising a synthetic amphipathic helical peptide that is a CD36 receptor antagonist, or a nucleic acid molecule encoding the polypeptide, to treat or inhibit chronic kidney disease in a subject.
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