WO2011066511A1 - Synthetic apoa-1 mimetic amphipathic peptides and methods of use thereof - Google Patents

Abstract

Disclosed herein are apolipoprotein A-I mimetic peptides and peptide analogs that promote lipid efflux as well as have anti-inflammatory and anti-oxidant properties. Also disclosed herein are methods of using the disclosed peptides to treat or inhibit dyslipidemic or vascular disorders, such as methods to treat or prevent cardiovascular disease including atherosclerosis.

Description

SYNTHETIC APOA-I MIMETIC AMPHIPA THI C PEPTIDES AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/265,291, filed on November 30, 2009, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to apolipoprotein A-I (apoA-I) mimetic peptides and methods of using such peptides to promote cholesterol efflux, including methods of treatment and/or prevention of cardiovascular disease, such as atherosclerosis.

BACKGROUND

Cardiovascular disease (CVD) is the major cause of morbidity and mortality in developed countries and atherosclerosis is the major cause of CVD.

Accumulation of cholesterol in the arterial wall and vascular inflammation are at the center of pathogenesis of atherosclerosis. Treatments controlling delivery of cholesterol and inflammation (statins) reduced incidence of CVD by 30-40%. There is, however, an urgent need for further reduction.

A most promising direction is complementing reduction in levels of the pro- atherogenic lipoproteins with increasing levels of the anti-atherogenic lipoprotein, high density lipoprotein (HDL), "HDL therapy". The success of HDL therapy depends on the method of elevation of HDL. Presently, the most successful approach is direct infusion of exogenous HDL. Infusion of reconstituted HDL

(rHDL) however has considerable limitations due to high cost and requirement for intravenous delivery making it suitable mainly for acute treatment.

An alternative to rHDL is apolipoprotein A-I (apoA-I) mimetic peptides. Research has demonstrated an inverse correlation between the occurrence of atherosclerosis events and levels of HDL and its most abundant protein constituent, apoA-I. ApoA-I has been shown to promote lipid efflux from ABCAl-transfected cells. However, the nature of the interaction between apoA-I and ABCA1 is not fully understood. Several other exchangeable-type apolipoproteins have also been shown to efflux lipid from ABCAl-transfected cells. Although the exchangeable- type apolipoproteins do not share a similar primary amino acid sequence, they all contain amphipathic helices, a structural motif known to facilitate the interaction of proteins with lipids. Studies have shown that intravenous injections of apoA-I or its variant, apoA-I Milano (which has a cysteine substituted at position 173 for the natural arginine), produced significant regression of atherosclerosis. These results make apoA-I, or derivatives thereof, attractive as potential therapeutic compounds in the treatment and prevention of atherosclerosis.

Short synthetic peptide mimics of apolipoproteins have been used as a model for studying physical and biological properties of apolipoproteins. These include, for instance, single helices taken from native apolipoproteins, synthetic amphipathic alpha helices and variants thereof. Short synthetic amphipathic helical peptides have been shown to promote lipid efflux and inhibit atherosclerosis. However, while some of these peptides exhibit beneficial effects in preventing atherosclerosis, they are also potentially cytotoxic. It is believed that the cytotoxicity is caused by the peptide enabling non-specific, ABCA1 -independent lipid efflux from cells.

Therefore, there exists a need for non-cytotoxic synthetic peptide mimics of apolipoproteins that promote specific lipid efflux from cells for use in the treatment and prevention of cardiovascular diseases, such as atherosclerosis.

SUMMARY OF THE DISCLOSURE

ApoA-I mimetic peptides show remarkable capacity to support cholesterol efflux, mimic anti-inflammatory properties of HDL and reduce development of atherosclerosis in animal models. ApoA-I mimetic peptides cost a fraction of the cost of rHDL; they are safe and well tolerated, approaches for oral delivery are being developed. Also, these peptides offer an opportunity to modify their structure to understand mechanisms of atheroprotective action of HDL with a possibility to further improve it. Surprisingly, limited research has been done to understand structure-function relationships of apoA-I mimetic peptides. Prior to the present disclosure, current understanding of the structure-function relationship of the peptides could be summarized as follows: (1) amphipathic a-helix of 18- 22 residues is essential for a peptide to mimic apoA-I and to be atheroprotective; (2) there is no stereospecificity: peptides made of D-amino acids are as effective as those made of L- amino acids; (3) increasing hydrophobicity by including 2 or 4 phenylalanine residues improves the capacity of peptides to associate with lipids and anti- inflammatory capacity of the peptides; and two helixes connected through proline residue work better than a single helix in cholesterol efflux and inflammation assays; introducing asymmetry in two-helix peptide improves its specificity in cholesterol efflux assay and reduces toxicity. Alignment of negative charges on the

hydrophobic face increases cholesterol efflux.

Presently, the inventors undertook a comprehensive analysis of the structure- function relationships of apoA-I mimetic peptides. Specifically, the inventors investigated twenty two bi-helical apoA-I mimetic peptides in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells and inhibition of low density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face and angle of the link between the helices are the factors determining the efficiency and specificity of efflux. The peptide with optimal parameters was more effective and specific towards the efflux than human apoA-I. Charge and size of hydrophobic face were factors affecting anti-inflammatory properties and the presence of cysteine and histidine residues was the main factor determining anti-oxidant properties.

There was no correlation between capacities of the peptides to support individual functions, each function had its own optimal set of features. None of the disclosed peptides was equally effective in all anti- atherogenic functions, suggesting that different anti- atherogenic functions may have different mechanisms and different structural requirements.

As such, isolated peptides and peptide analogs with multiple amphipathic cc- helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway have been identified and are disclosed herein. In various embodiments, the multi-domain peptides include multiple amphipathic cc-helical domains, wherein a first amphipathic cc-helical domain and a second amphipathic cc-helical domain exhibit equivalent or different hydrophobicity, and wherein the peptide or peptide analog promotes lipid efflux from cells by an ABCA1 -dependent pathway. In one example, the first domain and second domain are covalently linked by a linking peptide, such as a glycine, alanine or proline, or other bridging molecule. In specific examples, the isolated peptide has an amino acid sequence as set forth in SEQ ID NOs: 3-22.

In certain embodiments, the isolated peptides and peptide analogs disclosed herein include an additional functional domain or peptide such as 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, a lipoprotein lipase activating domain (such as a fragment or variant of apoC-II) or combinations of two or more thereof.

Also disclosed herein are pharmaceutical compositions that include one or more isolated peptides or peptide analogs with multiple amphipathic cc-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway. Representative peptides with multiple amphipathic cc-helical domains are shown in SEQ ID NOs: 3-22. In some embodiments, a pharmaceutical composition includes two peptides, a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as SEQ ID NO: 21. In some embodiments, a pharmaceutical composition includes a plurality of peptides selected upon the desired physiological effects (e.g. , anti-oxidant, anti-inflammatory and/or cholesterol efflux properties). In some examples, a pharmaceutical composition comprises at least two peptides, wherein the at least two peptides are selected from a peptide with an amino acid sequence set forth as SEQ ID NO: 12, a peptide with an amino acid sequence set forth as SEQ ID NO: 19, a peptide with an amino acid sequence set forth as SEQ ID NO: 18 or a peptide with an amino acid sequence set forth as SEQ ID NO: 5. In one particular example, a pharmaceutical composition comprises the following four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5. Implants are also disclosed that are coated with one or more of the disclosed isolated peptides or peptide analogs. In some examples, an implant is coated with at least two peptides, a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid set forth as SEQ ID NO: 21. In some examples, an implant is coated with four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

In some examples, an implant is positioned in a heart or peripheral vasculature to treat, inhibit or prevent a dyslipidemic or vascular disorder.

Also described herein is a method of treating dyslipidemic and vascular disorders in a subject, including administering to the subject a therapeutically effective amount of the isolated multi-domain peptide or peptide analog or a mixture of various peptides with different features described herein. For instance, one embodiment includes a mixture comprising a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as SEQ ID NO: 21. Dyslipidemic and vascular disorders amenable to treatment with the isolated multi-domain peptides or mixtures of peptides disclosed herein include, but are not limited to, hyperlipidemia,

hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, reperfusion myocardial injury, vasculitis, inflammation, as well as other disorders affected by these pathological processes, such as memory loss and/or neurodegenerative disorders, septic shock, diabetes, infectious diseases (e.g. , HIV) or combinations of two or more thereof. They also can be used as vectors for delivery of diagnostics or drugs. In various embodiments, a peptide (or mixture of peptides) such as a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3-22, is administered at a therapeutically effective concentration either alone or in combination with a therapeutically effective concentration of a lipoprotein lipase activator, to prevent, inhibit or reduce a dyslipidemic or vascular disorder. In some embodiments, a mixture of peptides including a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide amino acid sequence set forth as SEQ ID NO: 21 is administered at a therapeutically effective concentration either alone or in combination with a therapeutically effective concentration of a lipoprotein lipase activator, to prevent, inhibit or reduce a dyslipidemic or vascular disorder. In other embodiments, a mixture of the following four peptides is administered at a therapeutically effective concentration either alone or in combination with a therapeutically effective concentration of a lipoprotein lipase activator, to prevent, inhibit or reduce a dyslipidemic or vascular disorder: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

Methods are provided for treating or inhibiting dyslipidemic and vascular disorders in a subject. In one embodiment, this method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes one or more of the disclosed isolated peptides individually or as a mixture (such as a mixture comprising a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as SEQ ID NO: 21) that promote lipid efflux from cells via an ABCA1 -dependent pathway. In some embodiments, this method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes a plurality of peptides such as four peptides (e.g. , a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5) for treating or inhibiting dyslipidemic and vascular disorders in a subject.

In specific, non-limiting examples, the dyslipidemic and vascular disorders include hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,

hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, and reperfusion myocardial injury. In certain examples of the provided method, the method includes delivering one or more of the disclosed pharmaceutical composition including at least one of the disclosed isolated peptides or peptide analogs via an implant. In a particular example, the implant is coated with at least one peptide with an amino acid sequence set forth by any one of SEQ ID NOs: 3-22. In some examples, the implant is coated with at least two peptides, such as a first peptide with an amino acid sequence set forth by SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth by SEQ ID NO: 21. In some examples, the implant is coated with a plurality of peptides such as a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5).

The foregoing and other features of the disclosure will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1 and IB are scatter plots showing dependence of the capacity of the peptides to support cholesterol efflux on mean hydrophobicity and charge of the peptides (FIG. 1A) and relationship between cholesterol efflux capacity and contribution of ABC transporters (FIG. IB). Data for cholesterol efflux capacity and specificity are taken from Table 2 and data for hydrophobicity and charge are taken from Table 1. Triangles denote positively charged peptides, squares denote negatively charged peptides.

FIGS. 2A and 2B are graphs illustrating the specificity of cholesterol efflux from THP-1 cells. Cellular cholesterol was labeled by incubation with [ H]- cholesterol for 72 hours in a C0₂ incubator. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium, and fixed or not fixed by incubation for 20 minutes with paraformaldehyde (4%). Cells washed and incubated for another 4 hours at 37°C in serum-free medium containing 80 g/ml of the peptides or lipid-free apoA-I. Cholesterol efflux was expressed as the proportion of [ H]cholesterol transferred from cells to medium. Non-specific efflux (i.e., the efflux in the absence of an acceptor) was subtracted. Open bars denote peptides with charge > +2; cross- hatch bars denote peptides with A for P substitution. FIG. 2A shows efflux from the fixed cells whereas FIG. 2B shows the contribution of the specific efflux (efflux from live cells to the total efflux (efflux from live cells - efflux from fixed cells/efflux from live cells xl00 ). Negative values were shown as "0".

FIGS. 3A and 3B illustrate cholesterol efflux from BHK/ABCA1 cells. Cellular cholesterol was labeled by incubation with [ H] -cholesterol for 48 hours in a C0₂ incubator. Cells were then washed and incubated for 18 hours at 37°C in serum- free medium and then for another 18 hours at 37°C in serum-free medium containing 20 μΜοΙ/ml (or approximately 90 g/ml) of the peptides. Cholesterol efflux was expressed as the proportion of [ H]cholesterol transferred from cells to medium. Non-specific efflux (i.e., the efflux in the absence of an acceptor) was subtracted. FIG. 3A illustrates ABCA1 -dependent efflux from BHK cells. Data presented are a difference between the efflux from BHK/ ABC A 1 cells and BHK/mock cells. FIG. 3B demonstrates the correlation between the ABC-dependent efflux from THP-1 and ABCA1 -dependent efflux from BHK cells. Two peptides, ELK-D and

C12/H12, shown as♦, were excluded from the analysis of correlations.

FIG. 4 is a bar graph showing the effect of disclosed peptides on CD1 lb expression in human monocytes. Resting human monocytes were isolated from blood of healthy volunteers by density centrifugation with Lymphoprep followed by Dynal Negative Monocyte Isolation kit. Monocytes were stimulated with Ιμιηοΐ/L PMA in the presence or absence of the peptides or apoA-I final (concentration 40 μg/mL) and incubated with the FITC conjugated antibody to the active epitope of CD1 lb for 15 minutes at 37°C. Cells were then fixed with 4% formaldehyde.

CD1 lb expression was measured by flow cytometry; results were expressed as percentage of the CD1 lb expression compared to cells stimulated with PMA in the presence of a vehicle. Means ±SEM are presented; *p<0.01. Table 3 shows peptide properties that are likely to influence CD1 lb expression.

FIG. 5 is a bar graph illustrating the effect of peptides on expression of VCAM in mouse endothelial cells. SVEC4 cells were seeded into 96 well plates at the final density of 0.25xl0⁶ cells per well. After 24 hours, cells were washed and apoA-I, HDL or apoA-I mimetic peptides were added at the final concentration of 0.75 mg/ml. After 18 hours incubation cells were washed and tissue necrosis factor (TNF) was added in serum-free medium to the final concentration of 10 ng/ml. Cells were incubated for 5 hours and luciferase activity was measured using Bright- Glo Assay. Data were expressed per milligram of cellular protein and related to the luciferase activity in cells incubated with a vehicle instead of the peptides; means ±SEM are presented; *p<0.01. Table 3 shows peptide properties that are likely to influence VCAM expression.

FIG. 6 is a bar graph illustrating the effect of disclosed peptides on LDL oxidation. Freshly isolated LDL (final concentration 100 g/ml) was incubated at 25°C for the indicated periods of time with CuS0₄ (final concentration 15 μΜοΙ/L) in the presence of the peptides or apoA-I (final concentration 100 g/ml) in the cells of a multi-cell spectrophotometer continuously measuring absorption at 234 nm. Rate of oxidation was calculated as maximum absorbance divided to the length of the lag period. *p<0.01 (calculated from comparing the time-dependence curves presented in FIG. 12). Table 3 shows peptide properties that are likely to influence anti-oxidant properties. G - G-helix, Y-Y-helix, AP- substitution of A for P.

FIG. 7 is a graph showing the dose-dependence of cholesterol efflux from THP- 1 cells activated with LXR agonist. Cellular cholesterol was labeled by incubation in serum-containing medium with [ H] -cholesterol. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium in the presence of the LXR agonist TO-901317 (4 μιηοΙ/L). Cells were washed and incubated for another 4 hours at 37°C in serum-free medium containing indicated concentrations of the peptides or lipid-free apoA-I. Cholesterol efflux was expressed as the proportion of

[ H]cholesterol transferred from cells to medium. Non-specific efflux (e.g., the efflux in the absence of an acceptor) was subtracted. Data from different studies were normalized to the individual concentrations of the peptide 5A, which was included in all studies.

FIG. 8 is a graph showing the dose-dependence of cholesterol efflux from THP- 1 cells not activated with LXR agonist. Cellular cholesterol was labeled by incubation in serum-containing medium with [ H] -cholesterol. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium, washed and incubated for another 4 hours at 37°C in serum-free medium containing indicated concentrations of the peptides or lipid-free apoA-I. Cholesterol efflux was expressed as the proportion of [ H]cholesterol transferred from cells to medium. Non-specific efflux (i.e., the efflux in the absence of an acceptor) was subtracted. Data from different studies were normalized to the efflux to the peptide 5 A, which was included in all studies.

FIG. 9 is a graph illustrating the dose-dependence of ABC-dependent cholesterol efflux from THP-1. Data presented in this figure are a difference between values presented in FIG. 7 and FIG. 8, calculated for each data point. When calculations gave negative values they were interpreted as "0" value.

FIG. 10 is a graph illustrating the contribution of ABC-dependent efflux.

Data presented in this figure show efflux from activated and non-activated THP-1 cells (data taken from FIG. 7 and FIG. 8) at peptide concentration 20 μg/ml.

FIG. 11 is a graph illustrating the specificity of cholesterol efflux from THP- 1 cells. Cellular cholesterol was labeled by incubation with [ H] -cholesterol for 48 hours in a C0₂ incubator. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium, and fixed or not fixed by incubation for 20 minutes with paraformaldehyde (4%). Cells washed and incubated for another 4 hours at 37°C in serum-free medium containing 80 g/ml of the peptides or lipid-free apoA-I.

Cholesterol efflux was expressed as the proportion of [ H] cholesterol transferred from cells to medium. Non-specific efflux (i.e., the efflux in the absence of an acceptor) was subtracted.

FIG. 12 includes tracings illustrating oxidation of LDL. Freshly isolated LDL (final concentration 100 g/ml) was incubated at 25°C for the indicated periods of time with CuS0₄ (final concentration 15 μΜοΙ/L) in the presence of the peptides or apoA-I (final concentration 100 μg/ml) in the cells of a multi-cell

spectrophotometer continuously measuring absorption at 234 nm.

FIGS. 13A-13D are bar graphs illustrating the anti-inflammatory, antioxidant and cholesterol efflux properties of peptides with an amino acid sequence set forth by SEQ ID NO: 12 or 21 when used either alone or in combination. SEQUENCE LISTING

The amino acid sequences listed in the sequence listing are shown using standard three letter code for amino acids, as defined in 37 C.F.R. § 1.822. In the sequence listing:

SEQ ID NO: 1 shows the amino acid sequence of 5A peptide.

SEQ ID NO: 2 shows the amino acid sequence of prototypical ELK peptide.

SEQ ID NOs: 3-22 show the amino acid sequences of variant apoA-I peptides disclosed herein.

SEQ ID NOs: 23-26 show the amino acid sequences of several cell recognition sequences.

SEQ ID NOs: 27-30 show the amino acid sequences of several cell internalization sequences.

SEQ ID NO: 31 shows the amino acid sequence of a neutral cholesterol esterase activation sequence.

SEQ ID NO: 32 shows the amino acid sequence of an AC AT inhibition sequence.

SEQ ID NOs: 33 and 34 show the amino acid sequences of a pair of LDL receptor sequences.

SEQ ID NOs: 35-37 show the amino acid sequences of several anti-oxidant sequences.

SEQ ID NOs: 38 and 39 show the amino acid sequences of a pair of metal chelation sequences.

SEQ ID NO: 40 shows the amino acid sequence of an apoC-II domain with apoC-II-like activity (e.g., lipoprotein lipase activating activity).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Abbreviations

AB: antibody

ABCA1: ATP-binding cassette transporter Al

apoA-I: apolipoprotein A-I apoC-II apolipoprotein C-II

DMPC: dimyristoyl phosphatidyl choline

FPLC: fast protein liquid chromatography

HDL: high-density lipoprotein

HPLC: high-pressure liquid chromatography

LDL: low-density lipoprotein

LPL: lipoprotein lipase

RBC: red blood cell

VLDL: very low density lipoprotein

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 VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A.

Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. The materials, methods and examples are illustrative only and not intended to be limiting. In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Alkane: A type of hydrocarbon, in which the molecule has the maximum possible number of hydrogen atoms, and therefore has no double bonds (e.g. , they are saturated). The generic formula for acyclic alkanes, also known as aliphatic hydrocarbons is C_nH2n+₂; the simplest possible alkane is methane (CH₄).

Alkyl group: A branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, w-propyl, isopropyl, w-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having from 1 to 10 carbon atoms.

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 described herein will 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. 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.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (V_L) and "variable heavy chain" (V_R) refer, respectively, to these light and heavy chains.

As used herein, the term "antibody" includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single- chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and compositions of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Apolipoprotein A-I (apoA-I): A major protein component of high density lipoprotein (HDL) complex in plasma. Apolipoprotein A-l can promote cholesterol efflux from tissues to the liver for excretion. It is a cofactor for lecithin

cholesterolacyltransferase which is responsible for the formation of most plasma cholesteryl esters. In addition, apoA-I has many other pleitropic effects, such as anti-inflammatory, anti-thrombotic, and improving insulin sensitivity, which mechanistically are not understood but may contribute to the anti-atherogenic effect of HDL.

In particular examples, an apoA-I protein, fragment or variant thereof is capable of promoting cholesterol efflux. For example, an apoA-I protein, fragment or variant thereof is administered to a subject to promote cholesterol efflux. Unless the context clearly indicates otherwise, the term apoA-I includes any apoA-I gene, cDNA, mRNA, or protein from any organism and is capable of promoting cholesterol efflux.

Nucleic acid and protein sequences for apoA-I are publicly available. For example, GenBank Accession Nos. NM_144772.2 (human) and NM_009692 (mouse) disclose an apoA-I nucleic acid sequence, and GenBank Accession Nos. NP_658985 (human), AAB21444 (bovine) and NP_033822 (mouse) disclose apoA-I protein sequences, all of which are incorporated by reference as provided by

GenBank on November 30, 2009.

In one example, apoA-I includes a full-length wild- type (or native) sequence.

In other examples, apoA-I includes fragments of a wild-type (or native) sequence. In certain examples, apoA-I variants are those provided by SEQ ID NOs: 3-22. In some examples, apoA-I variants have at least 20% sequence identity, for example, at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, or 98% sequence identity to wild type apoA-I (such as set forth in SEQ ID NO: 2).

Apolipoprotein C-II (apoC-II): A 79 amino acid protein, which plays a role in plasma lipid metabolism as an activator of lipoprotein lipase (LPL). This protein includes three amphipathic helices: helix 1, residues 16-38; helix 2, residues 45-58; and helix 3, residues 64-74. The lipase-activating region of apoC-II has previously been localized to the C-terminal domain of the sequence, from about residue 56, whereas the N-terminal domain (residues 1-50) of the sequence is involved in lipid binding.

In particular examples, an apoC-II protein, fragment or variant thereof is capable of activating lipoprotein lipase. For example, an apoC-II protein, fragment or variant thereof is administered with apoA-I or peptide mimics thereof to prevent, reduce or inhibit hypertriglyceridemia. Unless the context clearly indicates otherwise, the term apoC-II includes any apoC-II gene, cDNA, mRNA, or protein from any organism and is capable of activating lipoprotein lipase.

Nucleic acid and protein sequences for apoC-II are publicly available. For example, GenBank Accession No. NM_009695 (human) discloses an apoC-II nucleic acid sequence, and GenBank Accession Nos. AAH05348 (human),

NP_001078821 (rat), NP_001095850 (bovine), and NP_033825 (mouse) disclose apoC-II protein sequences, all of which are incorporated by reference as provided by GenBank on November 30, 2009.

In one example, apoC-II includes a full-length wild-type (or native) sequence. In other examples, apoC-II includes fragments of a wild-type (or native) sequence. In certain examples, apoC-II fragments include at least residues 51 to 79, such as 56 to 79, 59 to 79, or 61 to 79 of apoC-II.

In some examples, apoC-II variants include 19 amino acids with tyrosine at position 3, He at position 6, Asp at position 9, and Gin at position 10. In some examples, apoC-II has at least 20% sequence identity, for example, at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, or 98% sequence identity to human apoC-II residues 59 to 79.

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, lipoprotein lipase activating domain, and the like. In a particular example, a peptide includes a first domain and a second domain (though they can occur in any order) both capable of promoting lipid efflux in which the first domain is an a-helical domain and the second domain is also an a-helical domain. In some examples, the first and second α-helical domains have the same hydrophobicity. In one particular example, the first and second α-helical domains are identical to each other.

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).

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, lipoprotein 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.

Helix: The molecular conformation of a spiral nature, generated by regularly repeating rotations around the backbone bonds of a macromolecule.

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.

Hydrophobic moment (μίί): One measure of the degree of amphipathicity (i.e., the degree of asymmetry of hydrophobicity) in a peptide or other molecule; it is the vectorial sum of all the hydrophobicity indices for a peptide, divided by the number of residues. Thus, hydrophobic moment is the hydrophobicity of a peptide measured for different angles of rotation per amino acid residue. Methods for calculating μ_Η for a particular peptide sequence are well-known in the art, and are described, for example, in Eisenberg et ah, Faraday Symp. Chem. Soc. 17: 109-120, 1982; Eisenberg et al, J. Mol. Biol. 179: 125-142, 1984; and Kyte & Doolittle, J. Mol. Biol., 157: 105-132, 1982. The actual μ_# obtained for a particular peptide will depend on the type and 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 can be obtained by dividing μ_# by the number of residues in the helix.

The relative hydrophobic moment (μΗΓεΙ) of a peptide is its hydrophobic moment relative to that of a perfectly amphipathic peptide. This measurement provides insight into the amphipathicity using different scales. For example, a value of 0.5 indicates that the peptide has about 50% of the maximum possible

amphipathicity. For the CCS scale, the relative hydrophobic moment is calculated by creating a peptide made up only of Phe (Hi = +10) and Arg (Hi = -10), and placing them so as to obtain perfectly segregated hydrophobic/hydrophilic sectors on the Edmundson projection. For the K&D and Eisenberg scales, He and Arg are used. For a-helical peptides (projection angle = 100°), an 18 residue peptide with 9 Arg, 9 Phe/Ile is used. This results in μΗη^χ of 6.3, 2.8 and 0.83 for the CCS,

K&D and Eisenberg scales, respectively. Table 4 provides the hydrophobicity values for the various amino acid residues using the various hydrophobicity scales. Table 4. HYDROPHOBICITY SCALES

Peptide analysis tool programs (including programs available on the internet) can be used to calculate hydrophobic moment of amphipathic sequences. See, for instance, the peptide sequence analysis tool created by Alex Tossi and Luca Sandri available on the World Wide Web (www) at

bbcm.univ.trieste.it/~tossi/HydroCalc/HydroMCalc.html (maintained by the University of Trieste, Italy). This tool is also discussed in Tossi et al. ("New Consensus hydrophobicity scale extended to non-proteinogenic amino acids", PEPTIDES 2002, Proc. of 27^th European Peptide Symposium, Sorrento, 2002), incorporated herein by reference in its entirety. In particular, this tool utilizes a Java applet that calculates the mean hydrophobicity, the mean hydrophobic moment and the relative hydrophobic moment for peptides using a selected scale and a selected projection angle; this program is known as HydroMCalc.

Ordinary skilled artisans will recognize other ways in which hydrophobic moment and other comparative measurements of amphipathicity can be calculated.

Hydrophilic: A hydrophilic (or lipophobic) group is electrically polarized and capable of hydrogen-bonding, enabling it to dissolve more readily in water than in oil or other "non-polar" solvents.

Implant: A support device. For example, an implant is a device that is employed to enhance and support existing passages, channels and conduits such as the lumen of a blood vessel. In an example, an implant is an endovascular support. In a particular example, an implant is a stent. In one example, an implant is effective to maintain a vessel open. In the present disclosure, an implant can be coated with or impregnated with one or more of the disclosed peptides to assist with the treatment of a dyslipidemic or vascular disorder.

Inhibiting or treating a disease: Inhibiting the full development of a disease, disorder or condition, for example, in a subject who is at risk for a disease such as atherosclerosis and 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. As used herein, the term

"ameliorating," with reference to a disease, pathological condition or symptom, 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, a reduction in the number of relapses 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.

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 70%, at least 90%, at least 95%, or greater of the total biological component content of the preparation.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.

Linker: A molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds. In particular examples, a linker comprises glycine, proline or alanine. In one specific example, the linker is a sequence of one or more prolines.

Lipid: A class of water-insoluble, or partially water insoluble, oily or greasy organic substances, which 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).

Lipid affinity: A measurement of the relative binding affinity of an amphipathic a-helix for lipids. Any number of methods well known to one of skill in the art can be used to determine lipid affinity. In one embodiment, the lipid affinity of an amphipathic a-helix is determined by calculating the hydrophobic moment score of the amphipathic a-helix. For example, an amphipathic a-helix with relatively high lipid affinity will have a hydrophobic moment score per residue greater than or equal to about 0.34 on the Eisenberg scale (100 degree alpha helix), while an amphipathic a-helix with relatively low lipid affinity will have a hydrophobic moment score per residue of less than about 0.34 on the Eisenberg scale (Eisenberg et al., Faraday Symp. Chem. Soc. 17:109-120, 1982). In an alternative embodiment, an amphipathic a-helix with relatively high lipid affinity has a hydrophobic moment score per residue of about 0.40 to about 0.60 on the

Eisenberg consensus scale, while a low lipid affinity helix will have a hydrophobic moment score per residue of about 0.20 to about 0.40 on the consensus scale (Eisenberg et al, PNAS 81: 140-144, 1984 and Eisenberg et al, J. Mol. Biol.

179: 125-142, 1984).

In other embodiments, the lipid affinity of an amphipathic a-helix is determined by one or more functional tests. Specific, non-limiting examples of functional tests include: retention time on reverse phase HPLC, surface monolayer exclusion pressure (Palgunachari et al., Arterioscler. Thromb. Vase. Biol. 16:328- 338, 1996), binding affinity to phospholipid vesicles (Palgunachari et al.,

Arterioscler. Thromb. Vase. Biol. 16:328-338, 1996), and DMPC vesicle solubilization (Remaley et al., J. Lipid Res. 44:828-836, 2003).

Further non-limiting examples of alternative methods of calculating the lipid affinity of an amphipathic a-helix include: total hydrophobic moment, total peptide hydrophobicity, total peptide hydrophobicity per residue, hydrophobicity of amino acids on the hydrophobic face, mean relative hydrophobic moment, hydrophobicity per residue of amino acids on the hydrophobic face, and calculated lipid affinity based on predicted peptide penetration into phospholipid bilayers (Palgunachari et al., Arterioscler. Thromb. Vase. Biol. 16:328-338, 1996). Different types of hydrophobicity scales for amino acids also can be used for calculating hydrophobic moments of amphipathic helices, which can result in a different relative ranking of their lipid affinity (Kyte et al. , J. Mol. Biol. 157: 105- 132, 1982).

Lipoprotein lipase (LPL): An enzyme that hydrolyzes lipids in

lipoproteins, such as those present in chylomicrons and very low-density

lipoproteins (VLDLs), into three free fatty acids and one glycerol molecule. A deficiency in lipoprotein lipase activity can lead to hypertriglyceridemia. In an example, LPL is activated {e.g., stimulated or turned-on) by an apoC-II protein, such as a fragment of an apoC-II protein including the third helix of apoC-II. LPL activation can be measured by methods known of those to skill in the art. In an example, LPL activation can be determined in vivo by obtaining a sample from a subject and measuring triglyceride levels in such sample prior to and after treatment with one or more of the disclosed peptides. A peptide that increases triglyceride levels above the baseline value by more than 3 times (x's; e.g., 3x) the coefficient of variation of the assay, which for most triglycerides assays would be approximately 2-5%, is considered a peptide that inhibits LPL. Any peptide that lowers the serum triglyceride compared to the result obtained with the in vivo administration of the peptide by more than 3x the coefficient of variation of triglyceride assay is considered a peptide that activates LPL. A potentially clinically useful LPL- activating peptide is a peptide that, when administered to subjects, results in a triglyceride level of less than 150 mg/dl, such as less than 145 mg/dl, 140 mg/dl, 135 mg/dl, 130 mg/dl, 125 mg/dl or 120 mg/dl.

Non-cytotoxic compound: A compound that does not substantially affect the viability or growth characteristics of a cell at a dosage normally used to treat the cell or a subject. Furthermore, the percentage of cells releasing intracellular contents, such as LDH or hemoglobin, is low {e.g., about 10% or less) in cells treated with a non-cytotoxic compound. Lipid efflux from a cell that occurs by a non-cytotoxic compound results in the removal of lipid from a cell by a process that maintains the overall integrity of the cell membrane and does not lead to significant cell toxicity. Non-polar: A non-polar compound is one that does not have concentrations of positive or negative electric charge. Non-polar compounds, such as, for example, oil, are not well soluble in water.

Peptide: 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

"peptide" is specifically intended to cover naturally occurring peptides, as well as those which are recombinantly or synthetically produced. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a peptide, polypeptide, or protein.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure 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 one or more therapeutic compounds or molecules, such as one or more multi-domain peptides or peptide analogs and additional pharmaceutical agents.

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.

Phospholipid: A phospholipid consists of a water-soluble polar head, linked to two water- insoluble non-polar tails (by a negatively charged phosphate group).

Both tails consist of a fatty acid, each about 14 to about 24 carbon groups long. When placed in an aqueous environment, phospholipids form a bilayer or micelle, where the hydrophobic tails line up against each other. This forms a membrane with hydrophilic heads on both sides. A phospholipid is a lipid that is a primary component of animal cell membranes.

Polar: A polar molecule is one in which the centers of positive and negative charge distribution do not converge. Polar molecules are characterized by a dipole moment, which measures their polarity, and are soluble in other polar compounds and virtually insoluble in nonpolar compounds.

Recombinant nucleic acid: A sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques such as those described in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,

1989. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.

Therapeutically effective amount: A quantity of a specified agent (or combination of agents) sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a multi-domain peptide or peptide analog useful in preventing, ameliorating, and/or treating a dyslipidemic disorder (e.g., atherosclerosis) in a subject. Ideally, a therapeutically effective amount of an agent is an amount sufficient to prevent, ameliorate, and/or treat a dyslipidemic disorder (e.g., atherosclerosis) in a subject without causing a substantial cytotoxic effect (e.g., membrane micro solubilization) in the subject. The effective amount of an agent useful for preventing, ameliorating, and/or treating a dyslipidemic disorder (e.g., atherosclerosis) 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.

Transformed: A "transformed" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. ///. Overview of Several Embodiments

Isolated peptides and peptide analogs with multiple amphipathic a-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway are disclosed herein. In one embodiment, the multi-domain peptides include multiple amphipathic α-helical domains, wherein a first amphipathic α-helical domain and a second amphipathic α-helical domain exhibit equivalent hydrophobicity (as measured, e.g., by their hydrophobic moments; see Eisenberg et ah, Faraday Symp. Chem. Soc. 17: 109-120, 1982; Eisenberg et al, PNAS 81: 140-144, 1984; and Eisenberg et ah, J. Mol. Biol. 179: 125-142, 1984), and wherein the peptide or peptide analog promotes lipid efflux from cells by an ABCA1 -dependent pathway. 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. In specific, non-limiting examples, the isolated peptide has an amino acid sequence as set forth in SEQ ID NOs: 3-22.

Optionally, the isolated peptides and peptide analogs that promote ABCA1- dependent lipid efflux from cells are also substantially non-cytotoxic, for instance, do not significantly increase triglycerides {e.g., triglyceride levels less than 150 mg/dl or do not show a statically significant increase above baseline triglycerides values).

The isolated peptides and peptide analogs disclosed herein can also include an additional functional domain or peptide. Specific, non-limiting examples of the additional functional domains or peptides include 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, a lipoprotein lipase activating domain or combinations of two or more thereof. Pharmaceutical compositions are disclosed that include one or more isolated peptides or peptide analogs with multiple amphipathic a-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway. Representative peptides with multiple amphipathic α-helical domains are shown in SEQ ID NOs: 3- 22. In some embodiments, a pharmaceutical composition includes a plurality of peptides selected upon the desired physiological effects (e.g. , anti-oxidant, antiinflammatory and/or cholesterol efflux properties). In some examples, a

pharmaceutical composition comprises at least two peptides, wherein the at least two peptides are selected from a peptide with an amino acid sequence set forth as SEQ ID NO: 12, a peptide with an amino acid sequence set forth as SEQ ID NO: 19, a peptide with an amino acid sequence set forth as SEQ ID NO: 18 or a peptide with an amino acid sequence set forth as SEQ ID NO: 5. In one particular example, a pharmaceutical composition comprises the following four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

Implants are also disclosed that are coated with one or more of the disclosed isolated peptides or peptide analogs. In some embodiments, an implant is coated with a mixture of two peptides, a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as

SEQ ID NO: 21. In some examples, an implant is coated with a mixture of four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

In some examples, an implant is positioned in a heart or peripheral vasculature to treat, inhibit or prevent a dyslipidemic or vascular disorder.

Methods are provided for treating or inhibiting dyslipidemic and vascular disorders in a subject. In one embodiment, this method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes one or more of the disclosed isolated peptides or peptide analogs with two or more amphipathic a-helical domains that promote lipid efflux from cells via an ABCA1 -dependent pathway. In specific, non-limiting examples, the dyslipidemic and vascular disorders include hyperlipidemia, hyperlipoproteinemia,

hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, and reperfusion myocardial injury.

In certain examples of the provided method, the isolated peptide includes two amphipathic α-helical domains in which the first domain and second domain have equivalent hydrophobicity and lipid efflux promoting activity. In other examples of the provided method, the isolated peptide includes two or more amphipathic α-helical domain capable of promoting lipid efflux all of which include equivalent hydrophobicity.

In other non-limiting examples of the provided method, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes at least one isolated peptide (or peptide analog) with multiple amphipathic α-helical domains and that promotes lipid efflux from cells via an ABCA1 -dependent pathway (such as an isolated peptide that includes two amphipathic α-helical domains and has an amino acid sequence as set forth in any one of SEQ ID NOs: 3-22) and a separate lipid lowering composition, such as a lipoprotein lipase activating agent, including an apoC-II protein, variant or fragment thereof. The peptide that promotes efflux need not be covalently linked to the peptide that has LPL activating activity. In specific examples, the method includes administering the one or more lipid efflux promoting isolated peptides or peptide analogs (such as a mixture of two peptides, a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid set forth as SEQ ID NO: 21; or a plurality of peptides such as four peptides {e.g., a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5)) and the lipoprotein lipase activating peptide or peptide analog within the same delivery vehicle, such as a liposome. In one particular example, the lipid efflux promoting isolated peptide and the lipoprotein lipase activating peptide are administered as free peptides.

In additional non-limiting examples of the provided method, the method includes delivering one or more of the disclosed pharmaceutical composition including at least one of the disclosed isolated peptides or peptide analogs via an implant. In a particular example, the implant is coated with at least one peptide with an amino acid sequence set forth by any one of SEQ ID NOs: 3-22. In a particular example, the implant is coated with a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as SEQ ID NO: 21. In other examples, the implant is coated with a plurality of peptides such as a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the disclosed peptides (e.g., four peptides, wherein the four peptides are a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5).

IV. Multi-Domain Amphipathic Peptides and Mixtures of Peptides

Isolated peptides and peptide analogs including peptides with multiple amphipathic cc-helical domains that promote lipid efflux from cells via an ABCA1- dependent pathway have been identified and are described in, for instance, PCT

Publication No. WO 2006/044596 (PCT/US2005/036933), as well as the corresponding U.S. National Stage application (U.S. Patent Application No.

11/577,259), U.S. Provisional Patent Application No. 61/045,213 filed April 15, 2008 as well as the corresponding International Application PCT/US2009/040560, filed April 14, 2009, which are incorporated herein by reference in their entirety.

Various peptides described therein included a first amphipathic cc-helical domain that exhibited higher lipid affinity relative to a second amphipathic cc-helical domain, in the same peptide.

ApoA-I, the predominant protein constituent of HDL (Panagotopulos et ah,

J. Biol. Chem. 277:39477-39484, 2002), is believed to promote lipid efflux from cells by a variety of mechanisms including a detergent-like extraction process (Remaley et al., J. Lipid Res. 44:828-836, 2003). One of the proposed mechanisms whereby the ABCA1 transporter facilitates this process is 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. Additional possibilities include active loading of apoA-I and even transporting apoA-I inside the cell for loading and retroendocytosis. Detergent-like properties of the peptides contribute to toxicity, while capacity to efflux cholesterol with minimal detergent effect is ideal. ApoA-I, like most of the other natural exchangeable type apolipoproteins, is almost completely dependent upon the presence of ABCA1 for promoting lipid efflux (Remaley et al., Biochem. Biophys. Res. Commun. 280:818-823, 2001).

Furthermore, 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). ApoA-I contains at least 8 large amphipathic helical domains, which have a wide range of lipid affinity (Gillote et al, J. Biol. Chem. 274:2021-2028, 1999).

Synthetic peptides of each helix of apoA-I have been made, and it has been shown that only 2 of the 8 large amphipathic helices of apoA-I, which have relatively high lipid affinity, can by themselves promote lipid efflux from cells in culture (Gillote et al., J. Biol. Chem. 274:2021-2028, 1999 and Palgunachari et al., Arterioscler. Thromb. Vase. Biol. 16:328-338, 1996). Additionally, synthetic peptide mimics of apolipoproteins have been shown to have anti-inflammatory and anti-oxidant properties (Van Lenten et al., Trends Cardiovasc. Med. 11: 155-161, 2001; Navab et al, Cur. Opin. Lipidol. 9:449-456, 1998; Barter et al, Cur. Opin. Lipidol. 13:285-288, 2002).

Previously, synthetic peptide mimics of apolipoproteins have been designed to have high lipid affinity (Remaley et al., J. Lipid Res. 44:828-836, 2003; Segrest et al., J. Lipid Res. 33: 141-166, 1992; Anantharamaiah et al., J. Biol. Chem.

260: 10248-10255, 1985; Garber et al, J. Lipid Res. 42:545-552, 2001; Navab et al., Circulation 105:290-292, 2002; and U.S. Pat. No. 6,156,727), because high lipid affinity has been shown to be a necessary feature for a peptide to mediate lipid efflux by the ABCA1 transporter (Remaley et al., J. Lipid Res. 44:828-836, 2003). It has also been shown, however, that peptide mimics of apoA-I with high lipid affinity can also promote lipid efflux independent of the ABCAl transporter (Remaley et ah, J. Lipid Res. 44:828-836, 2003). Such peptides have been shown to promote lipid efflux from cells not expressing the ABCAl transporter, and from Tangier disease cells that do not contain a functional ABCAl transporter (Remaley et ah, J. Lipid Res. 44:828-836, 2003). Furthermore, synthetic peptide mimics of apoA-I that possess high lipid affinity can also extract lipid by a passive physical process, based on their ability to remove lipid from cells that have been fixed with paraformaldehyde (Remaley et ah, J. Lipid Res. 44:828-836, 2003). Lipid efflux from cells by this ABCAl -independent pathway has been shown to be cytotoxic to cells, based on the cellular release of LDH (Remaley et ah, J. Lipid Res. 44:828-836, 2003).

In addition to the undesirable cytotoxic effect on cells, ABCAl -independent lipid efflux may also reduce the therapeutic benefit of such peptides by reducing their in vivo capacity for removing lipid from cells affected by the atherosclerotic process. For example, even in subjects with dyslipidemic and vascular disorders, most cells do not have excess cellular cholesterol and, therefore, do not express the ABCAl transporter. Cells, such as macrophages, endothelial cells and smooth muscle cells, which are present in atherosclerotic plaques, are all prone to lipid accumulation, and express ABCAl when loaded with excess cholesterol. The expression of ABCAl by these cells has been shown to be exquisitely regulated by the cholesterol content of cells (Langmann et ah, Biochem. Biophys. Res. Commun. 257:29-33, 1999). Induction of the ABCAl transporter by intracellular cholesterol is a protective cellular mechanism against excess intracellular cholesterol and has been shown to be critical in preventing the development of atherosclerosis (Dean and Chimini, J. Lipid Res. 42: 1007-1017, 2001). Peptide mimics of apolipoproteins that are not specific for removing cholesterol by the ABCAl transporter would be less therapeutically effective in removing cholesterol from ABCAl expressing cells because any cholesterol removed by the peptides from the more abundant non- ABCAl expressing cells will reduce the overall total cholesterol binding capacity of these peptides. The selective and non-cyto toxic removal of lipid from only cells that express the ABCAl transporter would, therefore, be a desirable property for therapeutic peptide mimics of apolipoproteins.

The current disclosure provides isolated multi-domain peptides or peptide analogs that specifically efflux lipids from cells by the ABCAl transporter in a non- cytotoxic manner. In one embodiment, such peptides or peptide analogs contain a first amphipathic cc-helical domain and a second amphipathic cc-helical domain which 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., J. Mol. Biol.

179: 125-142, 1984), and wherein the peptide or peptide analog promotes lipid efflux from cells by an ABCAl -dependent pathway. 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. In specific, non-limiting examples, the isolated peptide has an amino acid sequence as set forth in SEQ ID NOs: 3-22. In particular examples, the disclosed peptides are not only specific for removing lipids from cells by the ABCAl transporter, but are also capable of activating lipoprotein lipase.

The degree of amphipathicity (i.e., degree of symmetry of hydrophobicity) in the multi-domain peptides or peptide analogs can be conveniently quantified by calculating the hydrophobic moment (μ_Η) of each of the amphipathic cc-helical domains. Methods for calculating μ_Η 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_Η 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_Η by the number of residues in the peptide.

In the multi-domain peptides 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 (C -C ) alkyl, substituted (CrC₆) alkyl, (CrC₆) alkenyl, substituted (CrC₆) alkenyl, (CrC₆) alkynyl, substituted (CrC₆) 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 multi-domain peptides 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. 269:7185-7191, 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. 269:7185-7191, 1994; and Mishra et al , J. Biol. Chem. 270: 1602-1611, 1995.

Furthermore, in representative multi-domain peptides disclosed herein, the amino acid proline is used to link the multiple amphipathic cc-helices. However, other suitable amino acids, such as glycine, serine, threonine, and alanine, that functionally separate the multiple amphipathic cc-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). Additional aspects of the disclosure include analogs, variants, derivatives, and mimetics based on the amino acid sequence of the multi-domain peptides disclosed herein. Typically, mimetic compounds are synthetic compounds having a three-dimensional structure (of at least part of the mimetic compound) that mimics, for example, the primary, secondary, and/or tertiary structural, and/or

electrochemical characteristics of a selected peptide, structural domain, active site, or binding region (e.g., a homotypic or heterotypic binding site, a catalytic active site or domain, a receptor or ligand binding interface or domain, or a structural motif) thereof. The mimetic compound will often share a desired biological activity with a native protein, as discussed herein (e.g., the ability to interact with lipids). Typically, at least one subject biological activity of the mimetic compound is not substantially reduced in comparison to, and is often the same as or greater than, the activity of the native protein on which the mimetic was modeled.

A variety of techniques well known to one of skill in the art are available for constructing peptide mimetics with the same, similar, increased, or reduced biological activity as the corresponding native peptide. Often these analogs, variants, derivatives and mimetics will exhibit one or more desired activities that are distinct or improved from the corresponding native peptide, for example, improved characteristics of solubility, stability, lipid interaction, susceptibility to hydrolysis or proteolysis (see, e.g., Morgan and Gainor, Ann. Rep. Med. Chem. 24:243-252, 1989) and/or lipoprotein lipase activity. For example, a peptide mimetic with lipoprotein lipase activity may be generated by incorporating apoC-II amino acid residues known to be involved in apoC-II lipoprotein lipase activation into one of the disclosed multi-domain peptides.

In addition, mimetic compounds of the disclosure can have other desired characteristics that enhance their therapeutic application, such as increased cell permeability, greater affinity and/or avidity for a binding partner, and/or prolonged biological half-life. The mimetic compounds of the disclosure can have a backbone that is partially or completely non-peptide, but with side groups identical to the side groups of the amino acid residues that occur in the peptide on which the mimetic compound is modeled. Several types of chemical bonds, for example, ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of pro tease-resistant mimetic compounds.

In one embodiment, multi-domain peptides 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; 4,496,689;

4,301, 144; 4,670,417; 4,791, 192; and 4,179,337.

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 C18 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 multi- domain peptides 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, ^Cu, ⁹⁹Tc), 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 multi-domain peptide or peptide analog at either the N- and/or C- terminus. Optionally, a linker can be included between the multi-domain peptide 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 multi-domain peptides 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 multi-domain peptide or peptide analog at either the N- and/or C-terminus. Optionally, a linker can be included between the multi-domain peptide or peptide analog and the additional functional domain or peptide. The additional functional domain or peptide can enhance the ability of the multi-domain peptide or peptide analog to efflux lipids from cells in a non-cytotoxic manner, and/or enhance its therapeutic efficacy.

Exemplary additional functional domains/peptides include those shown in Table 6.

Table 6. Exemplary additional functional domains.

Cell recognition sequences can increase the ability of the multi-domain peptides or peptide analogs containing these sequences to bind to cells, the prerequisite first step in ABCA1 -mediated cholesterol efflux (Remaley et ah,

Biochem. Biophys. Res. Commun. 280:818-823, 2001). Cell internalization sequences, can increase the cellular uptake of the multi-domain peptides 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. 44:2257-2269, 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 multi- domain peptides or peptide analogs (Kisilevsky et al., J. Lipid Res. 44:2257-2269, 2003). Sequences that target the multi-domain peptides 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., J. 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 anti-inflammatory and anti-oxidant properties (Van Lenten et al., J. Clin. Invest. 96:2758-2767, 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 multi-domain peptides 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 multi- domain peptides or peptide analogs. In an example, lipoprotein lipase activation sequences result in triglyceride levels of no greater than 200 mg/dl.

LPL activation can be determined by methods known to those of skill in the art. In a particular example, an assay mixture of 200 μΐ containing 2 mg of triacylglycerols from a lipid emulsion can be used. The medium can contain 0.1 M NaCl, 0.1 M Tris-Cl, 20 μg of heparin, and 12 mg of bovine serum albumin (pH 8.5). To study the activation, peptides can be dissolved in 5 M urea, 10 mM Tris-Cl (pH 8.2). Known activators of LPL, such as apoC-II, can be used as a positive control. The protein concentration can be determined by a bicinchoninic acid protein assay. Five μΐ of the stock solution of apoCII, or the same volume of dilutions in 5 M urea, 10 mM Tris (pH 8.2), can be added to the incubations. The reactions can be started by addition of LPL and stopped after incubation for 15 min at 25 °C by addition of organic solvents for extraction of the labeled free fatty acids. The lipase activity can be expressed in units/mg of LPL, where 1 unit corresponds to release of 1 μιηοΐ of fatty acid per min. An at least 10% increase in LPL activity, which is more than 3x the coefficient of variation of most LPL assay, in the presence of a test peptide as compared to LPL activity in the absence of the test peptide, identifies the peptide as an LPL activating peptide.

In particular embodiments, the ability of the test peptides to activate LPL in vivo, can be determined by administering one or more of the peptides. Triglycerides levels in a sample taken from a subject receiving the peptide alone can then be compared to those levels present in a sample taken prior to the subject receiving the peptide or compared to a reference sample containing a known amount of triglycerides. Triglyceride levels of less than 150 mg/dl following peptide administration indicate that the peptide is an LPL activator or a peptide that causes a statistically significant decrease in the baseline triglyceride level when tested in vivo. 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 amination, diazo coupling, thioether bond, disulfide bond, amidation and thiocarbamoyl chemistries. In one embodiment, the amphipathic cc-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. In some examples, a mixture includes a first peptide with an amino acid sequence set forth as SEQ ID NO: 12 and a second peptide with an amino acid sequence set forth as SEQ ID NO: 21. In some examples, a mixture comprises at least two peptides, wherein the at least two peptides are selected from a peptide with an amino acid sequence set forth as SEQ ID NO: 12, a peptide with an amino acid sequence set forth as SEQ ID NO: 19, a peptide with an amino acid sequence set forth as SEQ ID NO: 18 or a peptide with an amino acid sequence set forth as SEQ ID NO: 5. In one particular example, a mixture comprises the following four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

In some examples, a mixture or combination of peptides is chosen based upon the desired physiological effect (e.g. , anti-oxidant, anti-inflammatory, and/or cholesterol efflux). For example, if anti-inflammatory effects (such as inhibition of

VCAM expression) are desired, a mixture includes at least two of the disclosed peptides with larger hydrophobic face, positive or neutral charge which optionally contains a Cys residue, including but not limited to a peptide with an amino acid sequence set forth as SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO:

19, or SEQ ID NO: 21. In some examples, if anti-inflammatory effects (such as anti-inflammatory effects in monocytes) are desired, a mixture includes at least two of the disclosed peptides having a pair of type A a-helices, with a hydrophobic face less than 180° and a neutral or negative charge, including but not limited to a peptide with an amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 11. In some examples, if anti-oxidant effects are desired, a mixture includes at least two of the disclosed peptides having a Cys residue and preferably, does not include a type A a -helix, but a type G or type Y, including but not limited to a peptide with an amino acid sequence set forth as SEQ ID NOS: 9-11. In some examples, if cholesterol efflux is desired, a mixture includes at least two peptides with an amino acid sequence set forth as SEQ ID NO: 12, SEQ ID NO: 7, SEQ ID NO: 20 or SEQ ID NO: 9.

The various peptides can be present in any ratio that is dictated by the specific properties, such as cholesterol efflux, anti-inflammatory or anti-oxidant properties, that are desired. In some examples, the various ratios are chosen based upon relative importance of different function to overall protection against atherosclerosis. For example, if the primary desired property is cholesterol efflux, the mixture can be about 50 to about 99 percent, such as about 60 to about 80 percent or about 70 to about 75 percent, of one or more of the disclosed peptides with cholesterol efflux properties and about 50 to about 1 percent, such as about 40 to about 20 percent or about 30 to about 25 percent (weight/weight) of one or more of the disclosed peptides with additional desirable properties such as antiinflammatory or anti-oxidant properties. If the primary property desired is antiinflammatory, the mixture can be about 50 to about 99 percent of one or more of the disclosed peptides with anti-inflammatory properties and about 50 to about 1 percent, such as about 40 to about 20 percent or about 30 to about 25 percent (weight/weight) of one or more of the disclosed peptides with additional desirable properties such as cholesterol efflux or anti-oxidant properties. If the primary property desired is anti-oxidant activity, the mixture can be about 50 to about 99 percent of one or more of the disclosed peptides with anti-oxidant properties to about 50 to about 1 percent, such as about 40 to about 20 percent or about 30 to about 25 percent (weight/weight) of one or more of the disclosed peptides with additional desirable properties such as cholesterol efflux or anti-inflammatory properties. The ratios may vary according to one of skill in the art and the desired effects.

In one example, the ratio of a first peptide, such as a peptide with an amino acid sequence set forth as SEQ ID NO: 12 to a second peptide, such as a peptide with an amino acid sequence set forth as SEQ ID NO: 21 can be between about

0.01:99.99 to about 99.99:0.01, such as about 0.01:99.99, about 0.1:99.9, about 1:99, about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85: 15, about 90: 10, about 95:5, about 99: 1, about 99.9:0.1, or about 99.99:0.01 (weight/weight). In some examples, the ratio of the mixture comprises about a 1: 1: 1: 1 ratio of the following four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5.

V. Synthesis and Purification of the Peptides

The single and multi-domain peptides 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. For example, the peptides can be prepared using step-wise solution or solid phase peptide syntheses, or recombinant DNA

techniques, or the equivalents thereof.

A. Chemical Synthesis

Peptides of the disclosure comprised of amino acids of 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 multi-domain peptides may be prepared by way of segment condensation, as described, for example, in Liu et ah, Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tarn et al., Int. J. Peptide Protein Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tarn, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tarn, 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 multi-domain peptides of the disclosure are described in Nakagawa et ah, J. Am. Chem. Soc.

107:7087-7092, 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.

Peptides of the disclosure comprised of amino acids of 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).

B. Recombinant Synthesis

If the peptide is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the multi-domain peptide or the relevant portion can also be synthesized using conventional recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding the multi-domain peptide 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 multi-domain peptide. 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. (ed.), Molecular Cloning: A Laboratory Manual, 2^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Ch. 17 and Ausubel et al. Short Protocols in Molecular Biology, 4^th ed., John Wiley & Sons, Inc., 1999).

To increase efficiency of production, the polynucleotide can be designed to encode multiple units of the multi-domain peptide 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 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.

C. Purification

The peptides 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 multi-domain peptide 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 multi-domain peptide 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 multi-domain peptide or peptide analog. The multi-domain peptide or peptide analog can be attached to a suitable carrier (e.g., BSA) by means of a side chain functional group or linker attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, and oil emulsions), keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

Booster injections can be given at regular intervals, and antiserum harvested when the antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, e.g., Ouchterlony et ah, Handbook of Experimental Immunology, Wier, D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μΜ). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher {Manual of Clinical Immunology, Ch. 42, 1980).

Monoclonal antibodies to a peptide or peptide analog may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture, for example the classic method of Kohler & Milstein {Nature 256:495-97, 1975), or a derivative method thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein immunogen {e.g., a multi-domain peptide or peptide analog) over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as enzyme-linked immunosorbent assay (ELISA), as originally described by Engvall {Meth. Enzymol., 70:419-39, 1980), or a derivative method thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999. Polyclonal antiserum containing antibodies can be prepared by immunizing suitable animals with a polypeptide comprising at least one multi-domain peptide or peptide analog, which can be unmodified or modified, to enhance immunogenicity.

Antibody fragments may be used in place of whole antibodies and may be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as

"antibody fragments," are well known and include those described in Better &

Horowitz, Methods Enzymol. 178:476-96, 1989; Glockshuber et ah, Biochemistry 29: 1362-67, 1990; and U.S. Patent Nos. 5,648,237 (Expression of Functional Antibody Fragments); 4,946,778 (Single Polypeptide Chain Binding Molecules); and 5,455,030 (Immunotherapy Using Single Chain Polypeptide Binding

Molecules), and references cited therein. Conditions whereby a polypeptide/binding agent complex can form, as well as assays for the detection of the formation of a polypeptide/binding agent complex and quantitation of binding affinities of the binding agent and polypeptide, are standard in the art. Such assays can include, but are not limited to, Western blotting, immunoprecipitation, immunofluorescence, immunocytochemistry, immunohistochemistry, fluorescence activated cell sorting (FACS), fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA, ELISPOT (Coligan et al, Current Protocols in Immunology, Wiley, NY, 1995), agglutination assays, flocculation assays, cell panning, etc., as are well known to one of skill in the art. VI. Pharmaceutical Compositions and Uses Thereof

The peptides or peptide analogs of the disclosure (and mixtures thereof) can be used to treat any disorder in animals, especially mammals (e.g., humans), for which promoting lipid efflux is beneficial. 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 peptides or peptide analogs can be used alone or in 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 multi-domain peptide 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 multi-domain peptides 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 multi-domain peptides or peptide analogs of the disclosure can be used in combination with an anti-microbials agent and/or an antiinflammatory agent. In a further embodiment, the multi-domain peptides can also be expressed in vivo, by using any of the available gene therapy approaches.

A. Administration of Peptides or Peptide Analogs

In some embodiments, multi-domain peptides or peptide analogs can be isolated from various sources and administered directly to the subject. For example, a multi-domain peptide or peptide analog can be expressed in vitro, such as in an E. coli expression system, as is well known in the art, and isolated in amounts useful for therapeutic compositions.

In exemplary applications, therapeutic compositions 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. 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.

A multi-domain peptide 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 multi-domain peptide or peptide analog is available to inhibit or treat a dyslipidemic or vascular disorder, the multi-domain peptide 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).

In one specific, non-limiting example, a multi domain peptide is

administered that includes one or more of the amino acid sequences shown in SEQ ID NOs: 3-22. In another specific, non-limiting example, a multi domain peptide is administered that includes one or more of the amino acid sequences shown in SEQ ID NOs: 3-22, which promotes lipid efflux, in combination with a lipoprotein lipase activating agent, such as an apoC-II protein, fragment or variant thereof; such LPL activating agent need not be covalently linked, or even administered simultaneously with, the peptide that promotes lipid efflux.

B. 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 multi-domain peptide or mixture of peptides, 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. Set, 88: 1864-1868,1991). 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 multi-domain peptide 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 multi-domain peptide 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 Sy St. , 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 ah, 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).

C. Representative Methods of Administration, Formulations and Dosage

The provided multi-domain peptides 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 multi-domain peptide or peptide analog can be combined to form a single preparation. The multi-domain peptides 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 nonaqueous 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 ampoules 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, 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, multi-domain peptides or peptide analogs with suitable features of ABCA1- 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 sialastic membranes or fibers), and the like.

In a specific embodiment, one or more of the disclosed peptides capable of promoting lipid efflux and/or activating lipoprotein lipase (such as a mixture of peptides including a first peptide with an amino acid sequence set forth by SEQ ID NO: 12 and a second peptide with an amino acid sequence set for the by SEQ ID NO: 21 ; or a mixture of peptides including the following four peptides: a peptide with an amino acid sequence set forth as SEQ ID NO: 12; a peptide with an amino acid sequence set forth as SEQ ID NO: 19; a peptide with an amino acid sequence set forth as SEQ ID NO: 18; and a peptide with an amino acid sequence set forth as SEQ ID NO: 5) may be associated either by coating or impregnating an implant such as stent to treat a dyslipidemic or vascular disorder. 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. 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. 6,695,920, 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 al, Ann. 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.

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.

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 ah, J. Curr. Drug Deliv. 4(2): 141-151, 2007 which is hereby incorporated by reference in its entirety). The subject matter of the present disclosure is further illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Material and Methods

This example provides the material and methods utilized to perform the studies disclosed herein.

Peptide synthesis. All peptides were synthesized by a solid-phase procedure, using an Fmoc /DIC /HOBt protocol on a Biosearch 9600 peptide synthesizer and were, purified to greater than 99% purity by reverse-phase HPLC on an Aquapore RP-300 column, as assessed by MALDI-TOF-MS (Bruker UltraFlex). Retention time of peptides was determined after injection of 1 mg of purified peptide on a C- 18 reverse phase HPLC column and elution with a 25-85% gradient of acetonitrile, containing 0.1% TFA. The mean hydrophobic moment was calculated as the vectorial sum of all the hydrophobicity indices, divided by the number of residues using DNASTAR software (Madison, WI).

Peptides were reconstituted with palmitoyloleoyl phosphatidyl choline (POPC) or dimyrisoylphosphatidyl choline (DMPC), at a molar ratio of 1:7.

Peptides and POPC or DMPC were complexed by co-lyophilization after first being dissolved in glacial acetic acid. The resultant lyophilized cakes were reconstituted with 20 mM NaHC0₃, 0.15 M NaCl and heated to 50°C in a water bath for 3 min and then allowed to cool at room temperature for 3 min for a total of three cycles. Lipoproteins. LDL (1.006-1.063 g/ml) and HDL (1.083-1.21 g/ml) were isolated from human plasma by sequential centrifugation. Apolipoprotein A-I was isolated from HDL as described previously (Sviridov et ah, J. Biol. Chem. 271: 33277-33283, 1996). Cholesterol efflux from THP-1 cells. THP-1 cells were maintained in RPMI medium supplemented with 10% FBS. Cells were differentiated into macrophage-like cells by incubation in RPMI supplemented with 10% FBS and 100 nMol/L phorbol 12- myristate 13-acetate (PMA) for 72 hours. Cellular cholesterol was labeled by incubation in serum-containing medium with [lcc,2cc(n)- H] -cholesterol (GE Health- Amersham, final radioactivity 0.5 MBq/ml) for 48 or 72 hours in a C0₂ incubator. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium in the presence or absence of the Liver X Receptor (LXR) agonist TO-901317 (4 μιηοΙ/L). Cells were washed and incubated for another 4 hours at 37°C in serum-free medium containing indicated concentrations of the peptides or lipid-free apoA-I.

Cholesterol efflux was expressed as the proportion of [ H] cholesterol transferred from cells to medium. Non-specific efflux (i.e. the efflux in the absence of an acceptor) was subtracted. Where indicated cells were fixed by incubation for 20 minutes with paraformaldehyde (4%) prior to the efflux studies. All studies were done in quadruplicates; intra-assay variability was < 5%. Efflux to each peptide was assessed in 2-3 independent studies. Inter-assay variability was assessed by including apoA-I and peptide 5A in all studies; this variability was up to 30% therefore the results from different studies were combined after normalization to the efflux to apoA-I and 5A.

Cholesterol efflux from BHK cells. BHK cells stably transfected with human ABCAl under the mifepristone-inducible promoter were handled as described in Oram et al. , J. Biol. Chem. 276, 39898-39902, 2001. Cellular cholesterol was labeled by incubation with [ H] -cholesterol for 48 hours in a C0₂ incubator. Cells were then washed and incubated for 18 hours at 37°C in serum-free medium and then for another 18 hours at 37°C in serum-free medium containing 20 μΜοΙ/ml (or approximately 90 g/ml) of the peptides. The medium was collected, centrifuged for 15 min at 4°C at 10,000 x g and aliquots of supernatant were counted in a β-counter. Cells were harvested and cell- associated radioactivity was counted. Cholesterol efflux was expressed as the proportion of [ H]cholesterol transferred from cells to medium. Non-specific efflux (i.e., the efflux in the absence of an acceptor) was subtracted. All peptides were tested in the one study done in quadruplicates and repeated twice, inter-assay variability was <10 .

Expression of CD lib on human monocytes. Resting human monocytes were isolated from blood of healthy volunteers by density centrifugation with

Lymphoprep followed by Dynal Negative Monocyte Isolation kit as described previously (Woollard et ah, Clin Exp Immunol 130:256-262, 2002). The CD1 lb assay was described previously (Murphy et ah, Arterioscler. Thromb. Vase. Biol. 28: 2071-2077, 2008). In brief, monocytes were stimulated with Ιμιηοΐ/L phorbol 12- myristate 13-acetate (PMA) in the presence or absence of the peptides or apoA-I final (concentration 40 μg/mL) and incubated with the FITC conjugated antibody to the active epitope of CD1 lb for 15 minutes at 37°C. Cells were then fixed with 4% formaldehyde. Samples were controlled for by using the isotype matched negative control (FITC-anti-mouse IgG). CD1 lb expression was measured by flow cytometry using FACS Calibur (Becton Dickinson, Franklin Lakes, New Jersey). Analysis was conducted using the Cell Quest Pro software. Results were expressed as percentage of the CD1 lb expression compared to cells stimulated with PMA in the presence of a vehicle. Due to considerable inter-assay variability (mainly due to various levels of activation of monocytes from different donors) each peptide was tested with monocytes from at least three different donors and results were expressed relative to CD1 lb expression after stimulation with PMA.

Expression of VCAM in mouse endothelial cells. Svec4-10 cells, a mouse endothelial cell line, was stably transfected with the Pgl3 plasmid, containing the cDNA for firefly luciferase, with 1.5 kBp or 2.27 kBp of the proximal promoter of the human VCAM gene. Cells were co-transfected with PsvNeo and selected with 100 μg/mL of G418. Transfected SVEC4 cells were seeded into 96 well plates at the final density of 0.25xl0⁶ cells per well. After 24 hours cells were washed and apoA-I, HDL or apoA-I mimetic peptides were added at the final concentration of 0.75 mg/ml. After 18 hours of incubation, cells were washed and tissue necrosis factor (TNF-a) was added in serum-free medium to the final concentration of 10 ng/ml. Cells were incubated for 5 hours and luciferase activity was measured using Bright-Glo Assay (Promega, Madison, WI). Data were expressed per milligram of cellular protein and related to the luciferase activity in cells incubated with a vehicle instead of the peptides. All peptides were tested in the one study done in

quadruplicates and repeated twice, inter-assay variability was <10 .

Oxidation ofLDL. The capacity of the peptides to inhibit LDL oxidation was assessed as described by Kontush et al. (Arterioscler. Thromb. Vase. Biol. 23: 1881- 1888, 2003). In brief, freshly isolated LDL (final concentration 100 g/ml) was incubated at 25°C for the indicated periods of time with CuS0₄ (final concentration 15 μΜοΙ/L) in the presence of the peptides or apoA-I (final concentration of 100 μg/ml) in the cells of a multi-cell spectrophotometer (Beckman, DU800)

continuously measuring absorption at 234 nm. Rate of oxidation was calculated as maximum absorbance divided by the length of the lag period according to the published model of LDL oxidation (Pinchuk et ah, Biochim. Biophys. Acta 1389: 155-172, 1998). Each peptide was tested twice with different batches of LDL, apoA-I and peptide 5A were included in each assay, inter-assay variability was <15%.

Statistics. All studies were reproduced at least 2-4 times and representative experiments are shown. Unless otherwise indicated, experimental groups consisted of quadruplicates; means ± SEM are presented. In some of the studies, the Student's i-test was used to determine statistical significance of the differences. In some of the studies, differences between groups were analyzed by ANOVA with t values modified by the step down Bonferroni procedure; the differences considered statistically significant when p<0.05. Correlations were calculated using Pearson Product Moment Correlations or, when indicated, using Spearman Rank Order Correlations.

Example 2

Identification of non-cytotoxic peptides that promote ABCAl-dependent lipid efflux

This example illustrates a method for identifying non-cytotoxic peptides that promote ABCAl-dependent lipid efflux from cells. Peptide Design: Based on the principals and procedures described in the present application, an amino acid sequence can be designed for a multi-domain peptide that contains two or more amphipathic a- helices.

Peptide production: Peptides to be tested can be produced synthetically or by recombinant DNA methods, as described in the present application, and purified by reverse phase HPLC or other suitable techniques well known to one of skill in the art.

Peptide Cytotoxicity Testing: Peptides can be tested for cytotoxicity by any number of methods well known to one of skill in the art, such as the release of intracellular LDH or the release of hemoglobin from red blood cells. Such studies are performed by incubating various concentrations of the peptides with a cell line, a vesicle or red blood cells, as described herein.

Peptide ABCAl -specificity for Lipid Efflux: Peptides to be tested can be added to serum- free cell culture media in the approximate concentration range of 1- 20 micromolar and incubated with a control cell line that does not express the

ABCAl transporter and the same cell line after transfection with human cDNA for the ABCAl transporter, as described herein. Alternatively, cells, such as macrophages, that either express or do not express the ABCAl transporter depending on their cholesterol content and/or exposure to agents that induce the ABCAl transporter {e.g., cAMP and LXR agonists) can also be used. After a suitable period of approximately 4 to 24 hours, the conditioned media can be removed from the cells and the amount of cholesterol and or phospholipid effluxed can be quantified, as described herein. ABCAl -specific lipid efflux is calculated by subtracting the total lipid efflux from the ABCAl expressing cell line from the results obtained from the cell line that does not express the ABCAl transporter.

Example 3

Structure of synthesized apoA-I mimetic peptides

This example describes the characteristics of twenty-two synthesized apoA-I mimetic peptides.

Twenty two apoA-I mimetic peptides were synthesized and their sequences, physicochemical properties and features are shown in Table 1. Two peptides were used as benchmarks to introduce changes in their structures. Peptide 5 A (SEQ ID NO: 1) was described previously in U.S. Patent 7,572,771 issued August 11, 2009 (which is hereby incorporated by reference in its entirety); it includes two type A amphipathic a-helices connected through proline; hydrophobicity of the second helix was reduced by substituting hydrophobic amino acids with alanine. Four derivatives of 5 A were synthesized (SEQ ID NOs: 19-22) to test the impact of introduction of two anti-oxidant amino-acids, cysteine and histidine. ELK peptide (SEQ ID NO: 2) includes two identical canonical type A amphipathic α-helices with 180 degree hydrophobic face and neutral net charge; helices were connected with a proline residue. ELK peptide was used to design sixteen modifications (SEQ ID NOs: 3-18) testing the role of net charge, hydrophobicity, size of hydrophobic face, type of helix, asymmetry and configuration of the proline bridge between the two helices. The peptides were tested in lipid- free form to exclude the confounding effects of lipid-binding properties of the peptides and resulting size of "rHDL" particle.

Example 4

Efficiency of cholesterol efflux from human monocyte cell line THP-1

This example shows the efficiency of cholesterol efflux from human monocyte cell line THP- 1.

To test the capacity of cholesterol efflux by the apoA-I mimetic peptides, human monocytic cells THP-1 were differentiated into macrophages, activated or not with LXR agonist TO-901317, labeled with [ H]cholesterol and incubated with various concentrations of peptides for 4 hours. THP-1 cells not activated with LXR agonist contained low levels of ABC transporters. Therefore the efflux from non- activated cells was considered to represent the component of the efflux that was not mediated by these transporters. Activation of LXR resulted in considerable elevation of the abundance of ABC transporters and the efflux from cells activated with LXR agonist was considered to represent a combination of the efflux mediated and not mediated by the ABC transporters. The difference between the efflux in the presence and absence of LXR agonist was therefore defined as ABC-mediated cholesterol efflux. The dose-dependencies of the efflux from THP-1 cells are presented in FIGS. 7-9. FIG. 7 shows the efflux from the cells activated with TO- 901317, FIG. 8 shows the efflux from cells not-activated with TO-901317, and FIG. 9 shows a difference between the effluxes from activated and non-activated cells, i.e. ABC-dependent efflux. To quantitate cholesterol efflux, the areas under the dose dependence curves (AUC) were calculated. The contribution of ABC transporters to efflux at a non-saturating concentration of 20 g/ml was also determined. These parameters are shown in Table 2. Analysis of structure-function relationships as related to the capacity of the peptides to support ABC-dependent cholesterol efflux indicated the following:

(1) The relationship between mean hydrophobicity and the capacity of the peptides to support cholesterol efflux is shown in FIG. 1A. It appeared that the relationship is characterized by a sharp peak around a value of mean hydrophobicity of -0.5. Adding charges inevitably changes hydrophobicity making it difficult to investigate the effect of the charge independently of hydrophobicity. However, it appears that peptides carrying positive (triangles) and negative (squares) had lesser capacity to support cholesterol efflux and an overall neutral charge was optimal. The peptide ELK-C4 was synthesized post analysis creating a peptide with optimal charge and hydrophobicity. Indeed this peptide showed exceptional capacity to support cholesterol efflux, even exceeding this of apoA-I, supporting our

conclusions. Four peptides had average hydrophobicity around -0.5, but still failed to support cholesterol efflux (FIG. 1A); all had other features strongly detrimental for the efflux capacity (inclusion of histidine and/or cysteine residues or asymmetry in ELK peptides or both - see below).

(2) Decreasing the size of hydrophobic face was found to be detrimental for the capacity of the peptides to support cholesterol efflux. Increasing the size of hydrophobic face was beneficial as long as overall hydrophobicity and charge were maintained.

(3) Changing type of helix from type A to type G and Y, if anything, had a small beneficial effect on the capacity of the peptides to support cholesterol efflux.

(4) Substitution of Ala for Pro in the bridge was detrimental for the efflux capacity, however, substitution of Ala- Ala for Pro (which generated half of the angle generated by proline) restored the efflux capacity. (5) Asymmetry had a significant beneficial effect for the peptide 5A as compared to the parent symmetrical peptide L37PA (Sethi et ah, J. Biol. Chem. 283: 32273-32282, 2008). However, the same feature tested on ELK peptides had a strong detrimental effect (ELKA versus ELK).

(6) Inclusion of Cys and His was tested on the derivatives of asymmetrical peptide 5A (SEQ ID NO: 1). Inclusion of Cys or His and especially Cys+His in the first (hydrophobic) helix was detrimental for the efflux, while inclusion of these amino-acids in the second (less hydrophobic) helix was beneficial. With few exceptions, the contribution of ABC transporters into efflux was proportional to the overall capacity of the peptides to support ABC-dependent cholesterol efflux (Table 2 and FIG. 10), there was a statistically significant correlation between these two parameters (r=0.66, p<0.001), (FIG. IB). This finding confirms that changes in the peptide structure affected specifically the ABC-dependent component of the efflux. Example 5

Specificity of cholesterol efflux from human monocyte cell line THP-1

This example shows the specificity of cholesterol efflux from human monocyte cell line THP- 1.

The amphipathic nature of the peptides facilitates their capacity to support cellular cholesterol efflux and form lipoprotein particles; however, it can potentially cause cytotoxicity by damaging the plasma membrane. To analyze the contribution of the potentially cytotoxic "non-specific" efflux, cholesterol efflux to the disclosed peptides at saturating concentration (80 g/ml) from live THP-1 cells was measured and cells were fixed with paraformaldehyde (Sethi et ah, J. Biol. Chem. 283: 32273- 32282, 2008). The data for the efflux are shown in FIG. 11, the absolute values of non-specific efflux {i.e. efflux from fixed cells) is shown in FIG. 2A and the "specificity" of the efflux {i.e. efflux from live cells minus efflux from fixed cells divided by the efflux from live cells xl00 ) is shown in FIG. 2B. Peptides with low overall capacity to support efflux from live cells (marked with arrows in FIG. 2) were excluded from the analysis. The rationale for this was that analyzing efflux properties of the peptides that do not in fact support total cholesterol efflux would not provide meaningful information about specificity of the efflux. Two features of the peptides associated with the high level of non-specific efflux were:

1. Net positive charge of the peptide (peptides with charge > +2 are shown in open bars in FIG. 2); and

2. Replacement of proline a bridge with a single alanine (shown in cross- hatched bars in FIG. 2).

Thus, these two features should be avoided to avert toxicity of the peptides. The analysis also pointed to the two peptides with exceptional specificity, ELK-C4 and ELK-E; their specificity surpassing that of apoA-I. However, while the former peptide was very active in ABCA1 -dependent cholesterol efflux, the latter had a modest capacity for the ABC-dependent efflux indicating that peptide ELK-E may interact with a transporter other than ABC transporters.

Example 6

Cholesterol efflux from BHK and BHK/ABCA1 cell

This example shows cholesterol efflux from BHK and BHK/ ABC A 1 cells.

BHK cells do not have ABC transporters. Therefore, the capacity of the disclosed peptides to support cholesterol efflux from BHK cells was compared to cholesterol efflux measured in BHK cells stably transfected with ABCA1; the difference between the two was defined as ABCA1 -dependent cholesterol efflux.

With two exceptions, peptides ELK-D and C12/H12, the efficiency of the peptides toward specifically ABCA1 -dependent efflux was following that of ABC- dependent efflux from THP-1 cells (FIG. 3A). There was a significant correlation between the ABCA1 -dependent efflux from BHK cells and ABC-dependent efflux from THP-1 cells (rank order correlation: r=0.75, p<0.0001) (FIG. 3B). The relationships between structural features of the peptides and efficiency of the efflux from BHK-ABCAl cells was similar to that of THP-1 cells, except that relationship between efflux and mean hydrophobicity peaked at a slightly higher value of -0.4. Thus, cholesterol efflux to lipid-free peptides reflects ABCA1 -dependent efflux.

Example 7

Anti-inflammatory properties of the disclosed peptides This example shows the capacity of the disclosed peptides to mimic the property of apoA-I reduction in expression of CDl lb in human monocytes in response to activation with a number of pro-inflammatory stimuli.

Isolated resting human monocytes were activated with PMA in the presence of a disclosed peptide or apoA-I (final concentration 40 μg/ml). The expression of CDl lb was assessed by flow cytometry and is shown in FIG. 4. All peptides with the exception of ELK-F2 and ELK-D inhibited the expression of CDl lb on activated human monocytes; there was a 6-fold difference in the magnitude of inhibition between the most and the least efficient peptides. Analysis of structure- function relationships as related to the inhibition of CDl lb expression indicated the following:

1. The structural feature having a significant impact on the capacity of the peptides to inhibit CDl lb expression was the size of the hydrophobic face. Increase of the size of hydrophobic face over 180° was detrimental for the anti-inflammatory property of the peptides.

2. A second feature affecting this anti-inflammatory property was charge. Two additional positive charges were detrimental; however, additional negative charges were neither detrimental not beneficial.

3. Changing the type of the helix to type G or Y was detrimental for the antiinflammatory capacity of the peptides.

4. Introduction of Cys and His residues, asymmetry or manipulation with a proline bridge between the two helices or asymmetry had little impact on the anti-inflammatory properties of the peptides.

Thus, these studies indicate that an effective anti-inflammatory peptide has a pair of type A cc-helices, with a hydrophobic face less than 180° and with a neutral or negative charge.

Example 8

Anti-inflammatory properties of the disclosed peptides towards the

endothelium

This example shows the capacity of the disclosed peptides to mimic the antiinflammatory function of apoA-I towards endothelium. Mouse endothelial cells were stably transfected with luciferase under control of human VCAM promoter. It was originally suggested that while lipidated apoA-I or HDL were potent inhibitors of VCAM expression, lipid- free apoA-I may not be as effective ascribing endothelial anti-inflammatory property of HDL to its lipid constituencies (Baker et ah, J. Lipid Res. 40:345-353, 1999). Contrary to the previous studies, lipid-free apoA-I was as effective inhibitor of VCAM expression as HDL by inhibiting 90% of VCAM expression (FIG. 5). Peptides were tested in lipid-free form to avoid confounding effects of lipid binding properties and size of lipidated particles. Cells were activated with TNF and incubated with apoA-I, HDL or the peptides at the final concentration of 0.75 mg/ml. Results of inhibition of VCAM expression by the peptides are presented in FIG. 5. Analysis of structure- function relationships as related to the inhibition of VCAM expression led to the following conclusions:

1. Increased size of hydrophobic face was beneficial for the inhibition of the VCAM expression.

2. Negative charge was detrimental for the inhibition of VCAM expression.

3. Inclusion of a combination of Cys+His residue was detrimental

independently of their location, while inclusion of Cys residue into the first helix of asymmetrical peptide was beneficial.

4. Hydrophobicity, changing helix type, disruption of the proline bridge and asymmetry had limited impact on the capacity of the peptides to inhibit VCAM expression.

Thus, to be effective in inhibition of VCAM expression a desirable peptide has a larger hydrophobic face, positive or neutral charge, and may contain a Cys residue.

Example 9

Anti-oxidant properties of disclosed peptides

This example shows the anti-oxidant properties of the disclosed peptides.

The anti-oxidant properties of the peptides were assessed in an LDL oxidation assay. Human plasma LDL was incubated in the presence of Cu⁺⁺ and apoA-I mimetic peptides or apoA-I (final concentration 100 μg/ml), time-course of diene formation was monitored by measuring absorption at 234 nm. Duration of lag phase and maximum diene formation were used to quantitate the rate of LDL oxidation as described by Pinchuk et al. (Pinchuk et ah, Biochim. Biophys. Acta 1389: 155-172, 1998). The time-course curves for LDL oxidation are shown in FIG. 12, and the rates of LDL oxidation are shown in FIG. 6. All peptides, with the exception of ELK-D2, ELK-F2 and ELK-B2 inhibited oxidation of LDL by Cu⁺⁺. There was a 5-fold difference in the magnitude of inhibition between the most and least effective peptides. Analysis of structure-function relationships as related to the inhibition of LDL oxidation led to the following conclusions:

1. The presence of Cys and/or His residue significantly increased the antioxidant capacity of the peptides. The exact position of the residues had limited impact.

2. Unexpectedly, changes affecting secondary structure of the peptides had beneficial effect: asymmetrical peptides, peptides comprising type G and type Y helices as well as peptides with modification of the proline bridge were all better anti-oxidants. The majority of the disclosed peptides possessed better anti-oxidant properties than apoA-I.

3. Charge, hydrophobicity and size of hydrophobic face had limited impact on anti-oxidant capacity of the peptides.

Thus, an effective anti-oxidant peptide contains a Cys residue and preferably, does not include a type A cc-helix, but a type G or type Y.

Example 10

Relationships between different anti-atherogenic properties of the peptides

This example shows the relationships between the different anti-atherogenic properties of the peptides.

The finding that different apoA-I mimetic peptides have a wide range of efficiencies towards various anti-atherogenic properties made it possible to investigate if these functions were related to each other. No correlation between quantitative measures of peptide activities in various assays was found. Analysis of structural features showed that features beneficial for one function of apoA-I may be detrimental for another (Table 3). For example, increased size of hydrophobic face was beneficial for the efflux, but detrimental for the monocyte anti-inflammatory function. Further, maintaining the proline bridge was necessary for efflux, detrimental for the anti-oxidant function and does not affect monocyte antiinflammatory function. While several peptides demonstrated the activity toward individual functions that was better than that of apoA-I, none of them was better than apoA-I in all tested functional assays. These findings are consistent with a suggestion that various functions of apoA-I may have different structural

requirements and are determined by different regions of the protein.

Summary of Findings provided in Examples 2-10:

The presented studies illustrate the structural features responsible for the various anti-atherogenic functions of apoA-I mimetic peptides. The features of the peptides responsible for cholesterol efflux capacity and specificity were:

hydrophobicity, size of the hydrophobic face and charge along with requirement for an angle between two helices. Some of these features, such as requirement for a proline bridge or size of hydrophobic face, were investigated before and the presented data are consistent with the results of these studies. However, this is the first systematic head to head analysis of the effect of modifications of several physicochemical features on the efflux. Following this analysis one is able to synthesize a peptide combining all beneficial features required for cholesterol efflux, ELK-C4. This peptide was more effective than apoA-I in capacity and specificity of cholesterol efflux supporting the disclosed findings. The likely mechanism is that these features are required for the interaction of the peptides with ABC1

transporters, and for triggering the events leading to specific cholesterol efflux a suggestion supported by the results of studies with BHK/ABCA1 cells.

An unresolved issue among factors affecting cholesterol efflux is

requirement for an asymmetry of the peptides. It has previously been shown that introduction of asymmetry in bi-helical peptide L37PA resulted in dramatic improvement of specificity with only modest reduction in overall efflux capacity (Sethi et al, J. Biol. Chem. 283: 32273-32282, 2008). The introduction of such asymmetry into ELK-C4 peptide (peptide ELKA) was evaluated with the

expectation that a peptide with even better cholesterol efflux specificity would result. Unexpectedly this peptide had very low overall capacity to support cholesterol efflux.

Anti-oxidant property of the peptides strongly depended on the presence of anti-oxidant amino acids, histidine and cysteine, a finding consistent with this of Jia et al. (Biochem. Biophys. Res. Comm. 297: 206-213, 2002). Presence of these amino-acids and enhanced anti-oxidant capacity might be behind anti-atherogenic properties of apoA-lMiiano and apoA-Ipari_S. Physico-chemical properties of the peptides had limited impact on anti-oxidant capacity, but unexpectedly changes disrupting "apoA-I - like" secondary structure, such as changing helix type or removing the proline bridge were beneficial. Possibly these changes benefit binding of these peptides to LDL. Anti-inflammatory properties of the peptides were investigated in two models showing effects on expression of adhesion molecules on monocytes and endothelium. The effects of HDL on inflammatory response of these two cell types is very different: while the response of monocytes was fast, short lived and requires low concentration of apoA-I, the response of endothelial cells was slow, long lasting and requires high levels of HDL. Further, the structural requirements for anti-inflammatory function were almost opposite for monocytes and endothelium. The peptides active in inhibiting monocyte CD1 lb were those with smaller hydrophobic face and negative charge. The peptides active in inhibiting endothelial VCAM were those with larger hydrophobic face and positive charge.

Another finding of this study was that different athero-protective functions of the peptides were determined by different structural features. No correlation was observed between the capacities of the peptides to support various functions. No specific structural feature equally benefitted all functions. Furthermore, time and dose dependencies of the effects of the peptides and apoA-I on specific functions varied dramatically from one function to another: it took under 15 minutes to inhibit the expression of CD1 lb and almost 24 hours to inhibit expression of VCAM; the effects of most peptides required concentrations of 20 μg/ml for cholesterol efflux, 100 μg/ml for anti-oxidant capacity and 0.75 mg/ml for inhibition of VCAM expression in endothelium. These findings suggest that different anti-atherogenic functions of apoA-I have different mechanisms. Although a number of peptides were better than apoA-I in supporting individual functions, none of them could match versatility of apoA-I when all the functions were taken into consideration. Possibly, different parts of apoA-I are responsible for different anti- atherogenic functions and mimicking just one or two features of apoA-I through peptide structure is insufficient to create a peptide active in many anti-atherogenic facets of apoA-I.

The discovery of peptides that are active in one anti-atherogenic function, but not in another, offers a unique opportunity to investigate the relative contribution of different anti-atherogenic activities of HDL into overall anti-atherogenic potency. HDL constituents other than apoA-I contribute to the anti-atherogenic properties of HDL. Size of HDL affects its ability to support cholesterol efflux, paraoxonase has a significant contribution to the anti-oxidant function, phospholipids may contribute to the anti-inflammatory effects of HDL to endothelium and various pro- and antiinflammatory factors carried on HDL may contribute to the HDL anti-inflammatory effects. When introduced into circulation apoA-I mimetic peptides may bind to

(mostly) HDL and therefore can potentially modulate the abundance and activities of various HDL constituencies, as well as distribution of HDL among sub-fractions of different size. Furthermore, a number of HDL functions, such as anti-thrombotic activity, suppression of apoptosis, regulation of endothelial function, insulin secretion and glucose oxidation may contribute to the anti-atherogenic properties of HDL. However, most available data suggest that involvement of HDL in cholesterol efflux, inflammation and oxidation are the major determinants of its atheroprotective potential and therefore are the main target of "HDL therapy".

Peptides in this study were tested in lipid-free form. That was a deliberate strategy as physico-chemical properties of the peptides will have a significant impact on lipid content of rHDL particles assembled on the peptides, the size of these particles and number of peptide molecules per particle. These features may have a significant confounding influence on many anti-atherogenic properties of the peptides. When the disclosed apoA-I mimetic peptides are introduced into circulation they may acquire lipids forming "rHDL-like" particles or, more likely, bind to the existing HDL particles, both types of particles will be a subject for further remodeling. Lipidated peptides (rHDL) would have a similar fate and therefore would not be a better representation n of the in vivo situation than lipid- free apoA-I. It is recognized that in vivo apoA-I mimetic peptides are likely to exist in mainly lipidated form.

Example 11

In vivo effects of ELK peptides in mice

This example provides representative methods for evaluating the anti- atherogenic properties of the disclosed peptides using in vivo models.

The studies detailed above demonstrated that the disclosed peptides can be effective when administered intraperitoneally (IP). Unless stated otherwise, one of the disclosed peptides is administered intraperitoneally daily into mice at a concentrations of 30 mg/kg for 3 days prior to the conducting the studies described below. This is the concentration that had a profound effect on development of atherosclerosis in vitro as described herein.

To evaluate cholesterol efflux, RAW 264.7 mouse macrophages are radiolabeled and cholesterol-enriched by incubation in medium containing 5μΟ/ιη1 [ H]cholesterol and 100 g/ml of acetylated LDL for 48 hours. Cells are then injected intraperitoneally into C57BL/6J mice. Feces are collected after 24 hours when mice are euthanized. Blood and liver are harvested and analyzed for

[ H]cholesterol or its derivatives. Lipids are extracted from tissues, separated by TLC and radioactivity counted. An increase in cholesterol efflux by a sample having increased level of radioactivity as compared to the radioactivity level in a control sample (such as a C57BL/6J mice injected with vehicle alone).

To evaluate anti-inflammatory and anti-oxidant properties of the disclosed peptides, adhesion molecule expression and presence of oxidized LDL is analyzed in vivo in 10- week old apoE K/O mice fed a normal chow diet and treated with vehicle compared to the same mice treated with apoA-I disclosed mimetic peptides. Mice are treated IP three times a week with peptides for 4 weeks (30 mg/kg) and then culled and the aorta harvested, fixed and paraffin embedded. Cross sections of aorta are stained with antibodies against the endothelial adhesion molecules P-selectin, E- selectin, VCAM-1 and ICAM-1 and for macrophage infiltration (F480) using standard immunohistochemistry techniques. In addition, the aorta is stained with antibodies against oxLDL. To quantitate the abundance of adhesion molecules as well as presence of oxLDL, an infrared fluorescent dye conjugated to secondary antibodies is used and abundance then quantitated by using the Odyssey Infrared Imaging Scanner. An alteration in anti-inflammatory or anti-oxidant activity is indicated in test samples by detecting an alteration in the abundance of the evaluated adhesion molecules or oxLDL in test samples as compared to control samples. For example, a decrease in adhesion molecules expression, such as VCAM-1 expression indicates that the peptide has anti-inflammatory properties in vivo.

The effect of HDL peptides on platelet adhesion and subsequent thrombus formation in vivo are analyzed using a FeCl₃ induced injury model. Briefly under anesthesia, the mouse carotid artery is exposed and incubated with topical 6% FeCl₃ for 10 minutes. The occlusion time will be measured using a flow probe placed over the carotid injury site. An alteration in the occlusion time in the test sample as compared to the occlusion time in a control sample indicates that the disclosed peptide modulates platelet adhesion and subsequent thrombus formation in vivo. For example, a decrease in occlusion time indicates that the peptide increases platelet adhesion and subsequent thrombus formation in vivo. Alternatively, an increase in occlusion time in the test sample as compared to the control sample indicates that the peptide decreases platelet adhesion and subsequent thrombus formation in vivo.

The disclosed peptides are evaluated for anti-atherogenic efficacy in an in vivo model of atherosclerosis by utilizing an established model of atherosclerosis (such as, monitoring plaque development in apoE K/O mice). In these studies, the effect of a disclosed peptide on plaque development is compared to the peptide 5A, vehicle and an unrelated peptide. Four groups of apoE K/O mice (10 weeks old) are kept on a high fat diet (21 fat, 0.15% cholesterol) for 16 weeks to produce aortic atherosclerosis. Peptides are injected intraperitoneally three times a week at a concentration of 30 mg/kg. Lipoprotein profiles are analyzed by agarose gel electrophoresis every four weeks in venous blood. Mice are euthanized at 0

(common point for all groups) 4, 8 and 16 weeks (10 mice in each cull/group). The lipid accumulation and distribution and size (area) of atherosclerotic lesions predominantly in aorta arch are quantitatively assessed using en face analysis (size and distribution of lesions) after staining with Oil Red O. The mean lesion area is used as a measure of atherosclerosis. The cholesterol content of carotid arteries is assessed after lipid extraction.

At the conclusion of these studies, the in vivo effects, including the effects on cholesterol efflux, inflammation and oxidation of the disclosed peptides will be known.

Example 12

Associating a peptide capable of promoting lipid efflux and activating lipoprotein lipase with an implant

According to the teachings herein, one or more peptides that promote ABCA1 specific lipid efflux and/or activate LPL, or a combination thereof, can be placed in a suitable container, such as a tissue microcapsule implant, and placed within a subject to allow continuous, slow release of one or more of the disclosed peptides. Such peptides can either be used in the free state or after complexation with lipid.

Example 13

Method of treating or inhibiting a dyslipidemic, vascular or inflammatory disorder in a subject

According to the teachings herein, one or more of the disclosed peptides capable of stimulating cholesterol efflux and/or activating lipoprotein lipase can be used to prevent, treat or inhibit a dyslipidemic or vascular disorder in a subject without causing a substantial cytotoxic effect. A method of treating or inhibiting a dyslipidemic or vascular disorder in a subject includes administering to the subject a therapeutically effective amount of the pharmaceutical composition including one or more of the peptides disclosed herein. In one example, a therapeutically effective amount of the pharmaceutical composition is provided by injecting intraperitoneally 30 mg/kg of one or more of the disclosed peptides three times a week. In another particular example, the pharmaceutical composition includes at least two peptides, one that is capable of causing lipid efflux and another that is capable of activating lipoprotein lipase. In a certain example, a pharmaceutical composition includes a peptide with an amino acid sequence set forth in any one of SEQ ID NOs: 3-22. In one example, the pharmaceutical composition includes at least two peptides, such as a first isolated peptide with the amino acid sequence set forth in SEQ ID NO: 12 and a second isolated peptide with the amino acid sequence set forth in SEQ ID NO: 21.

In another particular example, a method of inhibiting an inflammatory disorder or a disorder associated with antioxidants is disclosed in which a pharmaceutical composition includes a peptide with an amino acid sequence set forth in any one of SEQ ID NOs: 3-22 and such composition is administered intraperitoneally at 100 mg/kg.

Example 14

Anti-inflammatory, anti-oxidant and cholesterol efflux properties of mixtures of apoA-I mimetic peptides

This example illustrates the effects of two disclosed apoA-I mimetic peptides either alone or in combination, on inflammation, oxidation and cholesterol efflux.

The effects of ELK-2A2K2E (SEQ ID NO: 12) and 5A-C1 (SEQ ID NO: 21) either alone or in combination on inflammation, oxidation and cholesterol efflux were determined by the methods described in the above examples. FIGS. 13A-13D are a series of bar graphs illustrating the anti-inflammatory, anti-oxidant and cholesterol efflux properties of peptides with an amino acid sequence set forth by SEQ ID NOs: 12 and 21 alone and in combination. As illustrated in FIG. 13A, the combination of the two peptides evoked cholesterol efflux at lower concentrations than compared to cholesterol efflux measured in the presence of either peptide alone. FIG. 13B shows the combination of the two peptides resulted in significantly greater reduction in inflammation in monocytes than either peptide alone. Further, the combination of the two peptides had similar anti-inflammatory (in endothelium) and anti-oxidant properties as observed with 5A-C1 (FIGS. 13C and 13D, respectively). These studies indicate that the combination of the two peptides ELK-2A2K2E (SEQ ID NO: 12) and 5A-C1 (SEQ ID NO: 21) result in greater cholesterol efflux as well as inhibition of inflammation in monocytes than when given alone suggesting a combination of the two peptides may be of therapeutic value.

Table 1. Sequences and structural features ofapoA-I mimetic peptides. Dashes in sequences indicate where one peptide ends and a second peptide begins, between which is a linker (such as proline). Returns in sequences are not material, but due to formatting.

SEQ Mean

Old

ID Name Sequence Hydrop Charge Key features name

NO# hobicity

2 ELK ELK EKLKELLEKLLEKLKELL- -0.4 0 Canonical Type A helix

P- with 180 degree

EKLKELLEKLLEKLKELL hydrophobic face and 0 net charge

3 ELK-C ELK- EELKEKLEELKEKLEEKL - -1.1 -6 3 x (K-E, L-K)

3E3K P- substitutions. Decreased

EELKEKLEELKEKLEEKL hydrophobic face and 3 additional negative charges per helix

4 ELK-C1 ELK- EELKAKLEELKAKLEEKL- -0.76 -2 3 x (K-E, E-A, L-K)

3E3K P- substitutions. 3A EELKAKLEELKAKLEEKL Decreased hydrophobic face and 1 additional negative charges per helix

5 ELK-D ELK- EKLKALLEKLLAKLKELL 0.12 +4 2 x E-A substitutions.

2A P- Increased hydrophobic

EKLKALLEKLLAKLKELL face and 2 negative charges per helix less

6 ELK-E ELK- EWLKELLEKLLEKLKELL- -0.19 -2 K-W substitution.

1W P- 1 positive charges per

EWLKELLEKLLEKLKELL helix less

7 ELK-F ELK- EKFKELLEKFLEKFKELL- -0.43 0 2x (L-F) substitutions.

2F P- Increased hydrophobic

EKFKELLEKFLEKFKELL face

8 ELK-B2 ELK- EKLKELLEKLLELLKKLL- -0.01 +2 K-L and E-K

1L1K P- substitutions.

EKLKELLEKLLELLKKLL 1 negative charge per helix less

9 ELK-C3 ELK- EKLKELLEKLKAKLEELL- -0.39 0 L-K, E-A and K-E

1K1A P- substitutions. IE EKLKELLEKLKAKLEELL Decreased hydrophobic face

10 ELK- ELK- EKLKELLEKLLAKLKELL- -0.1 +2 E-A substitution.

D2 1A P- 1 negative charge per

EKLKELLEKLLAKLKELL helix less

11 ELK-F2 ELK- EKFKELLEKLLEKLKELL- -0.35 0 L-F substitution

1F P- Increased hydrophobic

EKFKELLEKLLEKLKELL face

12 ELK-C4 ELK- EKLKAKLEELKAKLEELL- -0.47 0 2 x (E-A, L-K and K-E)

2A2K P- substitutions, optimal 2E EKLKAKLEELKAKLEELL hydrophobicity and charge

13 ELK-G ELK- EELKELLKELLKKLEKLL- -0.31 0 3 x (K-E, E-K)

3E3K P- substitutions.

ELKELLKELLKKLEKLL G-helix

14 ELK-H ELK- EELKKLLEELLKKLKELL- -0.31 0 2 x (K-E, E-K)

2E2K P- substitutions.

EELKKLLEELLKKLKELL Y-Helix

15 ELK-I ELK- EKLKELLEKLLEKLKELL- -0.2 0 A-P substitution in the

PA A- link

EKLKELLEKLLEKLKELL SEQ Mean

Old

ID Name Sequence Hydrop Charge Key features name

NO# hobicity

16 ELK-J ELK- EKLKELLEKLLEKLKELL- -0.16 0 2A-P substitution in the

P2A AA- link

EKLKELLEKLLEKLKELL

17 ELKA ELK EKLKAKLEELKAKLEELL- -0.49 0 ELK-2A2K2E peptide

A P- with 5 A substitution in

EKAKAALEEAKAKAEELA second helix.

Asymmetrical

18 ELKAS ELK EKLKAKLEELKAKLEELL- -0.46 0 ELKA peptide with C+H

A- P- substitution in the

CH2 EHAKAALEEAKCKAEELA second helix.

19 P-5A 5A- DHLKAFYDKVACKLKEAF- -0.47 0 C+H substitution in the

CH1 P- first helix

DWAKAAYDKAAEKAKEA

A

20 P- 5A- DWLKAFYDKVAEKLKEAF- -0.52 0 C+H substitution in the

5A(C12 CH2 P- second helix /H2) DHAKAAYDKAACKAKEAA

21 K 5A- DWLKAFYDKVACKLKEAF- -0.44 0 C substitution in the first

Cl P- helix

DWAKAAYNKAAEKAKEA

A

22 L 5A- DHLKAFYDKVAEKLKEAF- -0.61 +1 H substitution in the first

Hl P- helix

DWAKAAYDKAAEKAKEA A

Table 2. Efficiency of cholesterol efflux from THP-1 cells and contribution of ABC transporters.

Table 3. Structural features responsible and individual anti-atherogenic properties of the peptides.

It will be apparent that the precise details of the constructs, compositions, and methods described herein may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

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

Claims

We claim:

1. A purified or isolated peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 12, 21, 1-11, 13-20 or 22.

2. The isolated peptide of claim 1, comprising an additional peptide domain comprising 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, an apoC-II sequence or a combination of two or more thereof.

3. An isolated nucleic acid molecule encoding any one of the peptides according to claim 1 or 2.

4. A pharmaceutical composition, comprising one or more of the isolated peptides of claim 1 or 2 or one or more isolated nucleic acid molecule of claim 3, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the one or more of the isolated peptides of claim 1 or 2 comprises a first isolated peptide having the amino acid sequence set forth in SEQ ID NO: 12 and a second isolated peptide having the amino acid sequence set forth in SEQ ID NO: 21.

6. A method of treating or inhibiting a dyslipidemic or vascular disorder in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 4 or 5.

7. The method of claim 6, 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 a combination of two or more thereof.

8. The method of claim 7, wherein the dyslipidemic or vascular disorder is hypercholesterolemia.

9. The method of any one of claims 6-8, further comprising

administering an additional lipid lowering composition or other agent for raising HDL.

10. The method of any one of claims 6-9, wherein the pharmaceutical composition is delivered on an implant.

11. An implant coated with or impregnated with at least one isolated peptide of claim 1 or 2.

12. The implant of claim 11, wherein the at least one isolated peptide comprises a first isolated peptide having the amino acid sequence set forth in SEQ ID NO: 12 and a second isolated peptide having the amino acid sequence set forth in SEQ ID NO: 21.

13. The implant of claim 11 or 12, which, when implanted in a heart or peripheral vasculature, treats or inhibits a dyslipidemic or vascular disorder.

14. The peptide of claim 1 or 2 for use in the manufacture of an implant for treating or inhibiting a dyslipidemic or vascular disorder.