WO2011031460A2 - Novel anti-inflammatory peptides that bind oxidized phospholipids - Google Patents

Novel anti-inflammatory peptides that bind oxidized phospholipids Download PDF

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Publication number
WO2011031460A2
WO2011031460A2 PCT/US2010/046534 US2010046534W WO2011031460A2 WO 2011031460 A2 WO2011031460 A2 WO 2011031460A2 US 2010046534 W US2010046534 W US 2010046534W WO 2011031460 A2 WO2011031460 A2 WO 2011031460A2
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peptide
stent
peptides
trp
oxp
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PCT/US2010/046534
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French (fr)
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WO2011031460A3 (en
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Piotr P. Ruchala
Robert I. Lehrer
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the field of atherosclerosis and other conditions characterized by inflammation and/or the formation of various oxidized species.
  • this invention pertains to the identification of a class of peptides, the oxpholipins, that bind cholesterol and/or oxidized phospholipids and that ameliorate one or more symptoms of conditions (e.g., atherosclerosis) characterized by an inflammatory response and/or the formation of various oxidized species.
  • CVD cardiovascular disease
  • statins are the most widely prescribed lipid-modifying agent. They are also known as 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, which is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis de novo. Inhibition of this enzyme in the liver stimulates LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream and a i decrease in blood cholesterol levels. To date, it is believed that statins have provided the most effective approach to lowering the LDL ("bad"cholesterol).
  • HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors
  • Apolipoprotein A-I is a major protein component of high density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion.
  • ApoA-I Milano Naturally occurring mutant of ApoA-I, called ApoA-I Milano which contains an extra cysteine bridge, causing it to exist as a homodimer or as a heterodimer with ApoA-II proved to be an effective drug in clinical trials.
  • ApoA-I Milano possessed a significant disadvantage - it was be administered intravenously and its further development was recently halted by Pfizer.
  • D-4F also dramatically reduces the ability of the human aortic endothelial cells to oxidize LDL to a form that could stimulate the induction of monocyte chemoattractant protein- 1 (MCP-1), which is important factor in early stage of atherogenesis.
  • MCP-1 monocyte chemoattractant protein- 1
  • oxpholipin (phospholipid-binding) peptides described herein are a family of peptides that have potent anti-inflammatory and lipid binding properties.
  • Oxpholipin peptides may be administered to remove cholesterol and noxious oxidized phospholipids from the serum, and from vascular and nonvascular tissue sites.
  • oxpholipin peptides are administered in combination with a second agent, e.g. a statins.
  • a pharmaceutical composition comprising one or a cocktail of oxpholipin peptides as an active agent is administered to a patient suffering from hypercholesterolemia.
  • Oxpholipin peptides may be administered alone, or in combination with other agents.
  • Oxpholipin-mediated binding of oxidized lipids is also useful for modeling and screening candidate therapeutic agents.
  • an isolated oxpholipin peptide that consists of or comprises an amino acid sequence according to the formula:
  • n, and p are independently 0 or 1; X 1 when present is Aib, Ach, or bA; X 2 is Arg or Trp; X 3 is Glu or Ala; X 4 is Cys, Ctb, Arg, Ser, Chg, Cbl, PhF, Trp, Bip, Dpa, Ant, or Ctb; X 5 is Thr, Val, Chg, Ctb, Ach, or Aib; X 6 is Gly, Val, or Arg; X 7 is Leu, Trp, Cha, or Ctb; X 8 is Ala, Gly, Val, Chg, Ach, or Aib; X 9 is Trp, Arg, or Nal; X 10 is Glu, or Leu; X 11 is Trp, Ctb, or Nal; X 12 is Trp, Glu, or Nal; X 13 is Arg, or Thr; X 14 is Thr, or Glu; X 15 is Val, Trp, Ch
  • peptides of this formula range f from about 14, 15, 16, or 17 amino acids up to about 40, 50, 75, 100, 150, 200, 250, or 300 amino acids in length.
  • the peptide comprises at least one non- natural amino acid and/or "D" amino acid.
  • an isolated oxpholipin peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46), the retro amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46), an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of an amino acid sequence found in Table 6 (SEQ ID NOs: 13-46); and an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of a retro form of an amino acid sequence found in Table 6 (SEQ ID NOs: 13-46); where the peptide binds cholesterol and/or an oxidized phospholipid.
  • the peptide comprises the amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46) or the inverse of the amino acid sequence. In certain embodiments the peptide ranges in length up to about 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In certain embodiments the peptide is shorter than the cholesterol binding domain of a cholesterol dependent cytolysin. In certain embodiments the peptide comprises all "L” amino acids. In certain embodiments the peptide comprises one or more "D" amino acids (e.g., all "D” amino acids).
  • one or more peptide bonds are replaced by an a-ester, a ⁇ -ester, a thioamide, phosphonamide, carbomate, or a hydroxylate.
  • the peptide comprises a peptide backbone, a polyethylene oxide (PEG/PEO) backbone, a polypropylene oxide (PPO) backbone, an aliphatic backbone, an ester backbone, or an ether backbone.
  • the peptide comprises one or more protecting groups (e.g., a carboxyl protecting group on the carboxyl terminus and/or an amino protecting group on the amino terminus).
  • the carboxyl protecting group and/or the amino protecting group is independently selected from the group consisting of acetyl, amide, 3 to 20 carbon alkyl group, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methylbenzy
  • the carboxyl protecting group is nicotinic acid. In certain embodiments the carboxyl protecting group comprises a group selected from the group consisting of an amide, (Lys-Arg ⁇ -Lys-NHCFbCFbSH, and TMEA.
  • the peptide is formulated for administration via a route selected from the group consisting of oral administration, nasal administration, administration by inhalation, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, intraocular injection, and intramuscular injection.
  • a pharmaceutical formulation comprising one or more oxpholipin peptides as described and/or claimed herein and a pharmaceutically acceptable carrier.
  • the formulation is a unit dosage formulation.
  • the formulation is suitable for administration via a route selected from the group consisting of oral administration, nasal administration, administration by inhalation, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, intraocular injection, and intramuscular injection.
  • the formulation is a sterile formulation.
  • methods are provided for mitigating one or more symptoms of atherosclerosis in a mammal (e.g. in a human or a non-human mammal), the method comprising administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of atherosclerosis.
  • methods for mitigating one or more symptoms of a pathology characterized by an inflammatory response in a mammal (e.g. in a human or a non-human mammal).
  • the methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of the pathology.
  • the pathology is selected from the group consisting of the inflammatory pathology is a pathology selected from the group consisting of atherosclerosis, rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple sclerosis, and a viral illnesses.
  • methods for mitigating one or more symptoms of macular degeneration in a mammal (e.g. in a human or a non-human mammal).
  • the methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of the macular degeneration.
  • the administration is via eye drops or intraocular injection.
  • Methods of treating cancer e.g., ovarian cancer
  • a mammal e.g., a human or a non-human mammal
  • the methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein.
  • the cancer is a cancer selected from the group consisting of myeloma or multiple myeloma, ovarian cancer, breast cancer, colon cancer, bone cancer, cervical cancer, brain cancer, and prostate cancer.
  • Methods for sequestering cholesterol in a mammal are also provided.
  • the methods typically involve comprising: administering an effective dose of one or more oxpholipin peptides as described and/or claimed herein to the mammal.
  • Methods for sequestering lipid in a mammal are also provided.
  • the methods typically involve comprising: administering an effective dose of one or more oxpholipin peptides as described and/or claimed herein to the mammal.
  • Methods of treating a vascular condition and/or a condition characterized by an inflammatory response and/or a condition characterized by the formation of oxidized reactive species in a mammal are provided.
  • the methods typically involve administering to a mammal in need thereof one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to ameliorate one or more symptoms of the condition.
  • a mammal e.g., a human or a non-human mammal
  • administration is by a route selected from the group consisting of oral administration, nasal administration, rectal administration, intraperitoneal injection, and intravascular injection, subcutaneous injection, transcutaneous administration, and intramuscular injection.
  • the peptide is administered in conjunction with a drug selected from the group consisting of a CETP inhibitor, FTY720, Certican, DPP4 inhibitors, Calcium channel blockers, ApoAl derivative or mimetic or agonist, PPAR agonists , Steroids, Gleevec, Cholesterol Absorption blockers (Zetia) , Vytorin, Any Renin Angiotensin pathway blockers, Angiotensi II receptor antagonist (Diovan etc), ACE inhibitors, Renin inhibitors, MR antagonist and Aldosterone synthase inhibitor, Beta-blockers, Alpha- adrenergic antagonists, LXR agonist, FXR agonist, Scavenger Receptor Bl agonist, ABCA1 agonist, Adiponectic receptor agonist
  • Clopidigrel Factor Xa inhibitor, GPIIb/IIIa inhibitor, Factor Vila inhibitor, Warfarin, Low molecular weight heparin, Tissue factor inhibitor, Anti -inflammatory drugs:, Probucol and derivative e.g. AGI-1067 etc, CCR2 antagonist, CX3CR1 antagonist, IL-1 antagonist, Nitrates and NO donors, and Phosphodiesterase inhibitors.
  • a stent for delivering drugs to a vessel in a body.
  • the stent can comprise a stent framework including a plurality of reservoirs formed therein, and one or more oxpholipin peptides as described and/or claimed herein.
  • the peptide is contained within a polymer.
  • the stent framework comprises one of a metallic base or a polymeric base.
  • the stent framework base comprises a material selected from the group consisting of stainless steel, nitinol, tantalum, MP35N alloy, platinum, titanium, a suitable biocompatible alloy, a suitable biocompatible polymer, and a combination thereof.
  • the reservoirs comprise micropores.
  • the micropores have a diameter of about 20 microns or less. In certain embodiments the micropores have a diameter in the range of about 20 microns to about 50 microns. In certain embodiments the micropores have a depth in the range of about 10 to about 50 microns. In certain
  • the micropores have a depth of about 50 microns. In certain embodiments the micropores extend through the stent framework having an opening on an interior surface of the stent and an opening on an exterior surface of the stent. In various embodiments the stent further comprises a cap layer disposed on the interior surface of the stent framework, the cap layer covering at least a portion of the through-holes and providing a barrier characteristic to control an elution rate of a the peptide in the drug polymer from the interior surface of the stent framework. In certain embodiments the reservoirs comprise channels along an exterior surface of the stent framework.
  • the polymer comprises a first layer of a first drug polymer having a first pharmaceutical characteristic and the polymer layer comprises a second drug polymer having a second pharmaceutical characteristic.
  • the stent further comprises a catheter coupled to the stent framework.
  • the catheter includes a balloon used to expand the stent.
  • the catheter includes a sheath that retracts to allow expansion of the stent.
  • methods of manufacturing a drug-polymer stent typically involve providing a stent framework; cutting a plurality of reservoirs in the stent framework; applying a composition comprising one or more oxpholipin peptides as described and/or claimed herein to the reservoirs and drying the composition.
  • the method further comprises applying a polymer layer to the dried composition; and drying the polymer layer.
  • methods of treating a vascular condition typically involve positioning a stent according as described and/or claimed herein within a vessel of a body; expanding the stent; and eluting at least one oxpholipin peptide as described and/or claimed herein from at least a surface of the stent.
  • treat when used with reference to treating, e.g. a pathology or disease refers to the mitigation and/or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
  • isolated refers to material that is substantially or essentially free from
  • nucleic acids and/or polypeptides the term can refer to nucleic acids or polypeptides that are no longer flanked by the sequences typically flanking them in nature.
  • Chemically synthesized polypeptides are "isolated” because they are not found in a native state (e.g. in blood, serum, etc.). In certain embodiments, the term “isolated” indicates that the polypeptide is not found in nature.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term “peptide” refers to a polymer of amino acid residues typically ranging in length from 2 to about 50 or about 60 residues. In certain embodiments the peptide ranges in length from about 4, 5, 6, 7, 8, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues.
  • the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues.
  • the amino acid residues comprising the peptide are "L-form" amino acid residues, however, it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptide or the peptide can be all "D” amino acids.
  • Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" (e.g., where the peptide bond is replaced by an a-ester, a ⁇ -ester, a thioamide, phosphonamide, carbomate, hydroxylate, and the like (see, e.g., Spatola (1983) Chem. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et ah, U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).
  • modified linkages e.g., where the peptide bond is replaced by an a-ester, a ⁇ -ester, a thio
  • amino acid analogues include, but are not limited to 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine (beta-aminopropionic acid), 2-aminobutyric acid, 4- aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2- aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n- ethylasparagine, hydroxy lysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n
  • ⁇ -peptides comprise of " ⁇ amino acids”, which have their amino group bonded to the ⁇ carbon rather than the a-carbon as in the 20 standard biological amino acids.
  • the only commonly naturally occurring ⁇ amino acid is ⁇ -alanine.
  • Peptoids or N-substituted glycines, are a specific subclass of
  • peptidomimetics They are closely related to their natural peptide counterparts, but differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the a-carbons (as they are in natural amino acids).
  • L-, D-, or beta amino acid versions of the sequence are also contemplated as well as retro, inversion, and retro- inversion isoforms.
  • conservative substitutions e.g., in the binding peptide, and/or antimicrobial peptide, and/or linker peptide
  • Non-protein backbones such as PEG, alkane, ethylene bridged, ester backbones, and other backbones are also contemplated.
  • fragments ranging in length from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids up to the full length minus one amino acid of the peptide are contemplated where the fragment retains at least 50%, preferably at least 60% 70% or 80%, more preferably at least 90%, 95%, 98%, 99%, or at least 100% of the activity (e.g., binding specificity and/or avidity, antimicrobial activity, etc.) of the full length peptide are contemplated.
  • compound peptides comprising repeats of one or more oxpholipins are also contemplated.
  • the oxpholipins can be linked directly together or joined by a linker ⁇ e.g., a peptide linker or chemically conjugated).
  • oxpholipin peptides compromising at least 80%, preferably at least 85% or 90%>, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated.
  • the terms "identical” or percent “identity,” refer to two or more sequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence identity is determined over the full length of the peptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson & Lipman (1988) Proc. Natl. Acad. Sci., USA, 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.
  • ameliorating when used with respect to "ameliorating one or more symptoms of atherosclerosis” refers to a reduction, prevention, or elimination of one or more symptoms characteristic of atherosclerosis and/or associated pathologies. Such a reduction includes, but is not limited to a reduction or elimination of oxidized
  • phospholipids a reduction in atherosclerotic plaque formation and rupture, a reduction in clinical events such as heart attack, angina, or stroke, a decrease in hypertension, a decrease in inflammatory protein biosynthesis, reduction in plasma cholesterol, and the like.
  • enantiomeric amino acids refers to amino acids that can exist in at least two forms that are nonsuperimposable mirror images of each other. Most amino acids (except glycine) are enantiomeric and exist in a so-called L-form (L amino acid) or D- form (D amino acid). Most naturally occurring amino acids are "L” amino acids.
  • L amino acid L amino acid
  • D amino acid L amino acid
  • L amino acid L amino acid
  • L amino acid are used to refer to absolute configuration of the amino acid, rather than a particular direction of rotation of plane-polarized light. The usage herein is consistent with standard usage by those of skill in the art. Amino acids are designated herein using standard 1 -letter or three-letter codes, e.g. as designated in Standard ST.25 in the Handbook On Industrial Property Information and Documentation.
  • protecting group refers to a chemical group that, when attached to a functional group in an amino acid ⁇ e.g. a side chain, an alpha amino group, an alpha carboxyl group, etc.) blocks or masks the properties of that functional group.
  • Preferred amino-terminal protecting groups include, but are not limited to acetyl, or amino groups.
  • Other amino-terminal protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl and others.
  • Preferred carboxyl terminal protecting groups include, but are not limited to groups that form amides or esters.
  • the phrase "protect a phospholipid from oxidation by an oxidizing agent” refers to the ability of a compound to reduce the rate of oxidation of a phospholipid (or the amount of oxidized phospholipid produced) when that phospholipid is contacted with an oxidizing agent (e.g. hydrogen peroxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE, HETE, etc.).
  • an oxidizing agent e.g. hydrogen peroxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE, HETE, etc.
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • Group I HDL refers to a high density lipoprotein or components thereof (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in low density lipoproteins) or that protect oxidized lipids from oxidation by oxidizing agents.
  • the term "Group II HDL” refers to an HDL that offers reduced activity or no activity in protecting lipids from oxidation or in repairing (e.g. reducing) oxidized lipids.
  • the term "HDL component” refers to a component (e.g. molecules) that comprises a high density lipoprotein (HDL).
  • Assays for HDL that protect lipids from oxidation or that repair (e.g. reduce oxidized lipids) also include assays for components of HDL (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that display such activity.
  • human apo A-I peptide refers to a full-length human apo A-I peptide or to a fragment or domain thereof comprising a class A amphipathic helix.
  • a “monocytic reaction” as used herein refers to monocyte activity characteristic of the "inflammatory response” associated with atherosclerotic plaque formation.
  • the monocytic reaction is characterized by monocyte adhesion to cells of the vascular wall (e.g. cells of the vascular endothelium), and/or chemotaxis into the subendothelial space, and/or differentiation of monocytes into macrophages.
  • the term "absence of change" when referring to the amount of oxidized phospholipid refers to the lack of a detectable change, more preferably the lack of a statistically significant change (e.g. at least at the 85%, preferably at least at the 90%, more preferably at least at the 95%, and most preferably at least at the 98% or 99% confidence level).
  • the absence of a detectable change can also refer to assays in which oxidized phospholipid level changes, but not as much as in the absence of the protein(s) described herein or with reference to other positive or negative controls.
  • conservative amino acid substitution is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity (e.g. for lipoproteins)) or binding affinity (e.g. for lipids or lipoproteins)) of the molecule.
  • conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or
  • the following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)
  • the phrase "in conjunction with” when used in reference to the use of one or more drugs in conjunction with one or more active agents described herein indicates that the drug(s) and the active agent(s) are administered so that there is at least some chronological overlap in their physiological activity on the organism.
  • the drug(s) and active agent(s) can be administered simultaneously and/or sequentially. In sequential administration there may even be some substantial delay (e.g., minutes or even hours or days) before administration of the second moiety as long as the first administered drug/agent has exerted some physiological alteration on the organism when the second administered agent is administered or becomes active in the organism.
  • amino acid abbreviations shown in Table 1 are used herein. Table 1. Amino acid abbreviations.
  • Figure 1 shows results of a monocyte chemotaxis assay of OxP peptides.
  • Results were standardized to LDL and are expressed as Inflammatory Index (II). Agents with an II ⁇ 1 are considered to be inactive. Agents with an II>1 are considered to be pro- inflammatory and those with an II ⁇ 1 are considered to be anti-inflammatory. OxP-11, OxP- 13 and D-4F showed very similar activity.
  • FIG. 2 illustrates results of dose response experiments. OxP-11 and OxP-
  • Figure 3A-3C show in vivo activity of selected OxP peptides.
  • Results were normalized to LDL and are expressed as Inflammatory Indices. An index below 1.0 indicates anti-inflammatory activity in this test.
  • Figure 3B compares L-4F and OxP-11 both containing L-amino acids exclusively. Both peptides were injected
  • Figure 3C compares the in vivo activity of D-4F, OxP-1 ID and a 1 : 1 mixture (by weight), all administered at 1 mg/kg. D-4F and OxP-1 ID were equally effective alone and in combination.
  • FIG. 4 panels A-C and A'-C illustrate binding of OxP- 1 ID to cholesterol
  • FIG. 5 panels A-D, illustrates binding of oxidized lipids to OxP-1 ID.
  • 13(S)-HODE 13(S)-hydroperoxyoctadecadienoic acid
  • PEIPC 1-palmitoyl- 2-(5,6-deoxyisoprostane E2)-sn-glycero-3-phosphoryl choline
  • 12(S)-HPETE 12(S)- hydroperoxyeicosatetraenoic acid
  • 5(S)-HPETE 5(S)-hydroperoxyeicosatetraenoic acid.
  • FIG. 6 panels A, B, C, and D show circular dichroism (CD) and Fourier-
  • Transform Infrared (FTIR) spectra Panel A shows CD spectra of D-4F and Panel B shows the spectra of OxP-1 ID. Spectra were obtained in two solvent systems: 10 mM phosphate buffer, pH 7.4 (dashed line ), HFIP:Buffer, (10 mM, pH 7.4) 4:6, v:v (solid line— ).
  • the peptide concentration was 100 ⁇
  • the cuvette light path was 0.01 cm
  • the temperature was 25°C
  • the spectra are the average of 8 scans.
  • Panel C shows FTIR spectra of D-4F
  • Panel D shows FTIR spectra of OxP-1 ID.
  • the spectra were obtained in D 2 0 Buffer, TFE: deuterium Buffer 4:6, v:v, HFIP: deuterium Buffer, 4:6, v:v, deuterium vapor hydrated DMPC multilayers (peptide: lipid, 1 : 10, mole:mole), and deuterium vapor hydrated
  • DMPC:CHO (DMPC:CHO, 1.2: 1, mole:mole with a peptide to lipid ratio of 1 : 10, mole:mole).
  • FIG 7 panels A and B shows a molecular illustration of the structure of D- 4F (panel A) and OxP-1 ID (panel B) after 83 nsec of molecular dynamics in HFIP:aqueous buffer 6:4, v:v environment.
  • Alpha helical segments are in ribbon, disordered and turn segments are in highlight.
  • the N-terminus is at the lower left and the adjacent C-terminus is in the upper right of the figure.
  • FIG. 8 panels A, B, C, and D show time dependent stability studies.
  • Panel A shows Root Mean Square Deviation (RMSD) of the C-alpha carbons for D-4F.
  • Panel B shows these RMSD values for OxP-1 ID.
  • Panel C shows the radius of gyration for D-4F and Panel D shows the radius of gyration for OxP-1 ID. All values are shown as a function of time (in nanoseconds) and come from molecular dynamic studies in a simulated
  • novel oxpholipin peptides are provided. These peptides are effective to bind cholesterol and/or oxidized phospholipids and are believed to have utility in mitigating one or more symptoms of atherosclerosis and/or preventing the progression of atherosclerosis. The peptides also show anti-inflammatory activity are effective to mitigate one or more symptoms of a pathology characterized by an
  • the peptides are effective in the treatment of a mammal suffering from undesirable levels of cholesterol.
  • the level of total cholesterol considered to be undesirable may be greater than 150 mg/dl, greater than 200 mg/dl, or greater than 250 mg/dl.
  • the level of LDL cholesterol considered to be undesirable may be greater than 100 mg/dl, greater than 125 mg/dl, greater than 150 mg/dl, greater than 175 mg/dl.
  • the oxpholipins may be administered alone or in conjunction with other cholesterol lowering agents, including, but not limited to statins, e.g. PRAVACHOL, MEVACOR®, LIPITOR®, LESCOL®, CRESTOR®, ZOCOR®; nicotinic acid, e.g. Niacin, NIASPAN®, SLO-NIACIN®; fibric acid, e.g. LOPID®, TRICOR®; bile acid sequestrants, e.g. QUESTRAN®, WELCHOL®, COLESTID®; cholesterol absorption inhibitors, e.g. ZETIA®; and the like.
  • statins e.g. PRAVACHOL, MEVACOR®, LIPITOR®, LESCOL®, CRESTOR®, ZOCOR®
  • nicotinic acid e.g. Niacin, NIASPAN®, SLO-NIACIN®
  • fibric acid e.g. LOP
  • oxpholipin peptides can be any suitable oxpholipin peptide.
  • the oxpholipin peptides can be any suitable oxpholipin peptide.
  • illustrative oxpholipin peptide include peptides consisting of, or comprising, an amino acid sequence according to the formula:
  • n, and p are independently 0 or 1; X 1 when present is Aib, Ach, or bA; X 2 is Arg or Trp; X 3 is Glu or Ala; X 4 is Cys, Ctb, Arg, Ser, Chg, Cbl, PhF, Trp, Bip, Dpa, Ant, or Ctb; X 5 is Thr, Val, Chg, Ctb, Ach, or Aib; X 6 is Gly, Val, or Arg; X 7 is Leu, Trp, Cha, or Ctb; X 8 is Ala, Gly, Val, Chg, Ach, or Aib; X 9 is Trp, Arg, or Nal; X 10 is Glu,
  • peptides of this formula range f from about 14, 15, or 16, amino acids up to about 40, 50, 75, 100, 150, 200, 250, or 300 amino acids in length. In certain embodiments the peptide comprises at least one non-natural amino acid and/or "D" amino acid.
  • Nic is nicotinic acid
  • PhF is 1,2,3,4,5-pentafluoro-phenyl-alanine
  • Aib is aminoisobutyric acid
  • Bip is biphenyl- alanine
  • bA is ⁇ -alanine
  • Dpa is 3,3'-diphenyl-alanine
  • Ach is 1 -amino- 1-cyclohexane carboxylic acid
  • Ant is 3-(9-anthryl)-alanine
  • Ctb is S-tbutyl-cysteine
  • Cha is cyclohexyl- alanine
  • Cbl is S-(4-methyl)benzyl-cysteine
  • Nal is 3-(l-naphthyl)-alanine; and is cyclohexyl-glycine.
  • Illustrative novel oxpholipin peptides are shown herein in Table 6. While certain peptides in the table are shown with protecting groups and/or with D amino acids, it is contemplated that such peptides can be provided with or without protecting groups, can comprise one or more D amino acids, and/or can be all "L" amino acid peptides, and/or can comprise one or more substitutions of naturally occurring for non-naturally occurring amino acids.
  • this invention also contemplates retro and retro-inverso forms of each of these peptides.
  • retro forms the direction of the sequence is reversed.
  • inverse forms the chirality of the constituent amino acids is reversed (i.e., L form amino acids become D form amino acids and D form amino acids become L form amino acids).
  • retro- inverso form both the order and the chirality of the amino acids is reversed.
  • sequence can be viewed as representing the amino acid sequence in the amino to carboxyl orientation, the retro form (i.e., the amino acid sequence in the carboxyl to amino orientation), the retro form where L amino acids are replaced with D amino acids or D amino acids are replaced with L amino acids, and the retro-inverso form where both the order is reversed and the amino acid chirality is reversed.
  • dimmers, trimers, tetramers or larger polymers of these sequences are contemplated.
  • the dimmers, trimers, tetramers, or larger polymers can be attached terminally as a conjugates or expressed as fusion proteins (attached directly to each other or via a linker (e.g.
  • sequence are chemically conjugated (e.g., through the N or carboxyl termini, or through side chains (e.g., via a disulfide linkage at a cysteine).
  • amino acid sequence of the oxpholipin polypeptides shown in Table 6 may be altered in various ways known in the art to generate targeted changes in sequence.
  • the polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by one amino acid, and may differ by two amino acids.
  • the sequence changes may be substitutions, insertions or deletions.
  • Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, carboxylation, pegylation, and the like. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine,
  • the peptide consists essentially of a polypeptide sequence set forth in Table 6 or its retro form.
  • consisting essentially of in the context of a polypeptide described herein it is meant that the polypeptide is composed of the sequence set forth in the table, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) (e.g., cholesterol and/or lipid binding) of the polypeptide.
  • amino and/or carboxyl terminal flanking sequences independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. II. Peptide preparation.
  • the oxpholipin peptides described herein can be chemically synthesized using standard chemical peptide synthesis techniques or, particularly where the peptide does not comprise "D" amino acid residues, can be recombinantly expressed. In certain embodiments, even peptides comprising "D" amino acid residues are recombinantly expressed.
  • a host organism e.g. bacteria, plant, fungal cells, etc.
  • Recombinantly expressed peptides in such a system then incorporate those D amino acids.
  • the peptides are chemically synthesized by any of a number of fluid or solid phase peptide synthesis techniques known to those of skill in the art.
  • Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the polypeptides of this invention.
  • Techniques for solid phase synthesis are well known to those of skill in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111.
  • cysteines can be used to make thioethers
  • histidines can be used for linking to a metal ion complex
  • carboxyl groups can be used for forming amides or esters
  • amino groups can form amides, and the like.
  • polypeptides may also be isolated and purified in accordance with conventional methods of chemical or recombinant synthesis.
  • the peptides are synthesized by the solid phase peptide synthesis procedure using , for example, a SYMPHONY ® automated peptide synthesizer (Protein Technologies Inc., Arlington, AZ) or a CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), using 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields and Noble (1990) Int. J. Pept. Protein Res. 35: 161-214).
  • SYMPHONY ® automated peptide synthesizer Protein Technologies Inc., Arlington, AZ
  • CEM Liberty automatic microwave peptide synthesizer CEM Corporation Inc., Matthews, NC
  • the peptides After cleaving the peptides from the resin with modified reagent K (TFA 94% (v/v); phenol, 2% (w/v); water, 2% (v/v); TIS, 2% (v/v); 2 hours) the peptides can be precipitated with ice-cold diethyl ether and purified to >95% homogeneity by preparative reverse-phase high performance liquid chromatography (RP-HPLC).
  • modified reagent K modified reagent K
  • Peptide purity can be evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RP-HPLC, using, for example, a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA).
  • Suitable mobile phases are: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile.
  • Analytic assessments can use, for example, a reversed- phase, 4.6 X 250 mm C18 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 mL/min.
  • any of the peptides described herein can bear, e.g. an acetyl group protecting the amino terminus and/or an amide group protecting the carboxyl terminus.
  • Double protected peptide is OxP-14 (Nic-RE-Ctb-Val-R-Leu-Val-Trp-E-Trp-Trp-RE-Val-NH 2 , SEQ ID NO: 31) having nicotinic acid at the amino terminus and an amide at the carboxyl terminus. Either or both of these protecting groups can be eliminated and/or substituted with another protecting group as described herein.
  • blockage, particularly of the amino and/or carboxyl termini of the subject peptides of this invention can greatly improve oral delivery and/or significantly increase serum half-life.
  • a wide number of protecting groups are suitable for this purpose.
  • Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection.
  • the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others.
  • Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups.
  • an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus.
  • Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula: CH 3 -(CH 2 ) n -CO- where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
  • the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others.
  • Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups.
  • an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus.
  • These blocking groups enhance the helix-forming tendencies of the peptides.
  • Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula:
  • n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
  • Other protecting groups include, but are not limited to Fmoc, t- butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9- florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl
  • Trityl Trityl
  • Mtt 4-methyltrityl
  • Mmt 4-methoxytrityl
  • Mtr 4-methoxy-2,3,6-trimethyl- benzenesulphonyl
  • Mts Mesitylene-2-sulphonyl
  • Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the oxpholipin peptides described herein (see, e.g., Greene et al, (1991) Protective Groups in Organic Synthesis,
  • acetylation is accomplished during the synthesis when the peptide is on the resin using acetic anhydride.
  • Amide protection can be achieved by the selection of a proper resin for the synthesis.
  • rink amide resin can be used.
  • the semipermanent protecting groups on acidic bifunctional amino acids such as Asp and Glu and basic amino acid Lys, hydroxyl of Tyr are all simultaneously removed.
  • the peptides released from such a resin using acidic treatment comes out with the n-terminal protected as acetyl and the carboxyl protected as NH 2 and with the simultaneous removal of all of the other protecting groups.
  • the peptides comprise one or more D-form (dextro rather than levo) amino acids as described herein.
  • at least two enantiomeric amino acids, more preferably at least 4 enantiomeric amino acids and most preferably at least 8 or 10 enantiomeric amino acids are "D" form amino acids.
  • every other, ore even every amino acid (e.g. every enantiomeric amino acid) of the peptides described herein is a D-form amino acid.
  • At least 50% of the enantiomeric amino acids are "D" form, more preferably at least 80% of the enantiomeric amino acids are “D” form, and most preferably at least 90% or even all of the enantiomeric amino acids are "D" form amino acids.
  • the oxpholipin peptides described herein are circularized/cyclized to produce cyclic peptides.
  • Cyclic peptides include head/tail, head/side chain, tail/side chain, and side chain/side chain cyclized peptides.
  • peptides contemplated herein include homodet, containing only peptide bonds, and heterodet containing in addition disulfide, ester, thioester-bonds, or other bonds.
  • the cyclic peptides can be prepared using virtually any art-known technique for the preparation of cyclic peptides.
  • the peptides can be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries.
  • the chemistry used to cyclize the peptide will be sufficiently mild so as to avoid substantially degrading the peptide.
  • Suitable procedures for synthesizing the peptides described herein as well as suitable chemistries for cyclizing the peptides are well known in the art.
  • cyclization can be achieved via direct coupling of the N- and C-terminus to form a peptide (or other) bond, but can also occur via the amino acid side chains.
  • other functional groups including but not limited to amino, hydroxy, sulfhydryl, halogen, sulfonyl, carboxy, and thiocarboxy. These groups can be located at the amino acid side chains or be attached to their N- or C-terminus.
  • the chemical linkage used to covalently cyclize the peptides of the invention need not be an amide linkage.
  • Such linkages include, by way of example and not limitation amide, ester, thioester, CH 2 --NH, etc. Techniques and reagents for synthesizing peptides having modified termini and chemistries suitable for cyclizing such modified peptides are well- known in the art.
  • linkers may be desirable to attach linkers to the N- and/or C-termini to facilitate peptide cyclization.
  • linkers will bear reactive groups capable of forming covalent bonds with the termini of the peptide. Suitable linkers and chemistries are well- known in the art and include those previously described.
  • Cyclic peptides and depsipeptides heterodetic peptides that include ester
  • immobilized activated intermediate where activation of the immobilized peptide is carried out with N-protecting group intact and subsequent removal leading to cyclization;
  • peptidomimetics are also contemplated.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (Fauchere
  • polypeptide ⁇ e.g. oxpholipin 1 ID (OxP-1 ID) shown in Table 6
  • One particularly preferred non-peptide linkage is -CH 2 NH-.
  • Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced
  • circularly permutations of the peptides described herein or constrained peptides (including cyclized peptides) comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61 : 387); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • oxpholipin peptides or formulations comprising oxpholipin peptides are administered to a mammal at risk for, or having, undesirably high cholesterol levels (e.g., undesirably high levels of LDL) and/or at risk for atherosclerosis or having one more symptoms of atherosclerosis.
  • undesirably high cholesterol levels e.g., undesirably high levels of LDL
  • the oxpholipin peptides described herein are effective for mitigating one or more symptoms and/or reducing the rate of onset and/or severity of one or more indications described herein (e.g., elevated cholesterol, atherosclerosis, inflammatory pathologies, etc.).
  • the oxpholipin peptides described herein are believed to be effective for mitigating one or more symptoms of atherosclerosis.
  • the peptides can bind the "seeding molecules" required for the formation of pro-inflammatory oxidized phospholipids such as Ox-PAPC, POVPC, PGPC, and PEIPC.
  • HDL from mice that were fed an atherogenic diet and injected with PBS failed to inhibit the oxidation of human LDL and failed to inhibit LDL-induced monocyte chemotactic activity in human artery wall cocultures.
  • HDL from mice fed an atherogenic diet and injected daily with peptides described herein was as effective in inhibiting human LDL oxidation and preventing LDL-induced monocyte chemotactic activity in the cocultures as was normal human.
  • this invention provides methods for ameliorating and/or preventing one or more symptoms of atherosclerosis by administering one or more of the active agents described herein.
  • c-reactive protein a marker for inflammation
  • congestive heart failure there is an accumulation of reactive oxygen species and vasomotion abnormalities. Because of their effects in preventing/reducing the formation of various oxidized species and/or because of their effect in improving vasoreactivity and/or arteriole function (see below) the oxpholipin peptides described herein will be effective in treating congestive heart failure.
  • oxpholipin peptides described herein can function to improve arteriole structure and function and/or to slow the rate and/or severity of arteriole dysfunction. Without being bound to a particular theory, it is believed that arteriole dysfunction is a causal factor in various brain and kidney disorders. Use of the oxpholipin peptides described herein thus provides a method to improve the structure and function of arterioles and preserve the function of end organs such as the brain and kidney.
  • administration of one or more of the oxpholipin peptides described herein is expected to reduce one or more symptoms or to slow the onset or severity of arteriolar disease associated with aging, and/or Alzheimer's disease, and/or Parkinson's disease, and/or with multi-infarct dementia, and/or subarachnoid hemorrhage, and the like.
  • administration of one or more agents described herein is expected to mitigate one or more symptoms and/or to slow the onset and/or severity of chronic kidney disease, and/or hypertension.
  • oxpholipin peptides are believed to improve vasoreactivity.
  • the oxpholipin peptides described herein are believed to be suitable for the treatment of peripheral vascular disease, erectile dysfunction, and the like.
  • oxpholipin peptides described herein are also believed to be suitable for treatment of a variety of pulmonary indications. These include, but are not limited to chronic obstructive pulmonary disease (COPD), emphysema, pulmonary disease, asthma, idiopathic pulmonary fibrosis, and the like.
  • COPD chronic obstructive pulmonary disease
  • emphysema pulmonary disease
  • asthma idiopathic pulmonary fibrosis
  • Ox-PAPC oxidized l-palmitoyl-2- arachidonoyl-sft-glycero-3-phosphorylcholine
  • isoprostane 8- iso prostaglandin E 2
  • PAPC unoxidized phospholipid
  • the osteon resembles the artery wall in that the osteon is centered on an endothelial cell-lined lumen surrounded by a subendothelial space containing matrix and fibroblast-like cells, which is in turn surrounded by preosteoblasts and osteoblasts occupying a position analogous to smooth muscle cells in the artery wall (Id.).
  • Trabecular bone osteoblasts also interface with bone marrow subendothelial spaces (Id.). Parhami et al.
  • oxpholipin peptides described herein are useful for mitigation one or more symptoms of osteoporosis (e.g., for inhibiting decalcification) or for inducing recalcification of osteoporotic bone. It is believed the oxpholipin peptides are also useful as prophylactics to prevent the onset of symptom(s) of osteoporosis in a mammal (e.g., a patient at risk for osteoporosis).
  • this invention contemplates the use of the oxpholipin peptides described herein to inhibit or prevent a symptom of a disease such as coronary calcification, calcific aortic stenosis, osteoporosis, and the like.
  • Chronic inflammatory and/or autoimmune conditions are also characterized by the formation of a number of reactive oxygen species and are amenable to treatment using one or more of the active agents described herein.
  • the oxpholipin peptides described herein are useful, prophylactically or therapeutically, to mitigate the onset and/or more or more symptoms of a variety of other conditions including, but not limited to rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, polymyalgia rheumatica, scleroderma, multiple sclerosis, and the like.
  • the oxpholipin peptides are useful in mitigating one or more symptoms caused by, or associated with, an inflammatory response in these conditions. [0115] Also, in certain embodiments, the oxpholipin peptides are useful in mitigating one or more symptoms caused by or associated with an inflammatory response associated with AIDS.
  • oxpholipin peptides described herein can be administered (e.g. orally or by injection) to patients (including, for example with known coronary artery disease during influenza infection or other events that can generate an acute phase inflammatory response, e.g. due to viral infection, bacterial infection, trauma, transplant, various autoimmune conditions, etc.) and thus we can prevent by this short term treatment the increased incidence of heart attack and stroke associated with pathologies that generate such inflammatory states.
  • the oxpholipin peptides are believed to be useful in the treatment of infection (e.g., viral infection, bacterial infection, fungal infection) and/or the inflammatory pathologies associated with infection (e.g. meningitis) and/or trauma.
  • infection e.g., viral infection, bacterial infection, fungal infection
  • inflammatory pathologies associated with infection e.g. meningitis
  • the oxpholipin peptides described herein are also useful in the treatment of a wound or other trauma, mitigating adverse effects associated with organ or tissue transplant, and/or organ or tissue transplant rejection, and/or implanted prostheses, and/or transplant atherosclerosis, and/or biofilm formation.
  • oxpholipin peptides described herein are also useful in mitigating the effects of spinal cord injuries. G) Diabetes and associated conditions.
  • the oxpholipin peptides described herein can be used in methods of treating (therapeutically and/or prophylactically) diabetes and/or associated pathologies (e.g., Type I diabetes, Type II diabetes, juvenile onset diabetes, diabetic nephropathy, nephropathy, diabetic neuropathy, diabetic retinopathy, and the like).
  • diabetes and/or associated pathologies e.g., Type I diabetes, Type II diabetes, juvenile onset diabetes, diabetic nephropathy, nephropathy, diabetic neuropathy, diabetic retinopathy, and the like.
  • the oxpholipin peptides can also be used to improve insulin sensitivity.
  • NFKB is a transcription factor that is normally activated in response to proinflammatory cytokines and that regulates the expression of more than 200 genes.
  • Many tumor cell lines show constitutive activation of NFKB signaling.
  • Various studies of mouse models of intestinal, and mammary tumors conclude that activation of the NFKB pathway enhances tumor development and may act primarily in the late stages of tumorigenesis (see, e.g., ( 2004) Cell 1 18 : 285; (2004) J. Clin. Invest., 114: 569). Inhibition of NFKB signaling suppressed tumor development.
  • mechanisms for this suppression are believed to include an increase in tumor cell apoptosis, reduced expression of tumor cell growth factors supplied by surrounding stromal cells, and/or abrogation of a tumor cell dedifferentiation program that is critical for tumor
  • this invention provides methods of treating a pathology characterized by elevated NFKB by administering one or more active agents described herein.
  • this invention contemplates inhibiting NFKB activation associated with cancer by administering one ore more active agents described herein, optionally in combination with appropriate cancer therapeutics.
  • inhibition of the onset, progression or severity of a cancer by administration of an oxpholipin is contemplated.
  • Illustrative cancers include, but are not limited to myeloma/multiple myeloma, ovarian cancer, breast cancer, colon cancer, bone cancer, cervical cancer, prostate cancer, skin cancer, liver cancer, glioma or other brain cancers, and the like.
  • cardiovascular complications e.g., atherosclerosis, stroke, etc.
  • cardiovascular complications e.g., atherosclerosis, stroke, etc.
  • an acute phase inflammatory response e.g., such as that associated with a recurrent inflammatory disease, a viral infection (e.g., influenza), a bacterial infection, a fungal infection, an organ transplant, a wound or other trauma, and so forth.
  • this invention contemplates administering one or more of the oxpholipin peptides described herein to a subject at risk for, or incurring, an acute inflammatory response and/or at risk for or incurring a symptom of atherosclerosis and/or an associated pathology (e.g., stroke).
  • a symptom of atherosclerosis and/or an associated pathology e.g., stroke
  • a person having or at risk for coronary disease may prophylactically be administered a one or more oxpholipin peptides of this invention during flu season.
  • a person (or animal) subject to a recurrent inflammatory condition e.g., rheumatoid arthritis, various autoimmune diseases, etc.
  • a person (or animal) subject to trauma e.g., acute injury, tissue transplant, etc.
  • Such methods will entail a diagnosis of the occurrence or risk of an acute inflammatory response.
  • the acute inflammatory response typically involves alterations in metabolism and gene regulation in the liver. It is a dynamic homeostatic process that involves all of the major systems of the body, in addition to the immune, cardiovascular and central nervous system. Normally, the acute phase response lasts only a few days; however, in cases of chronic or recurring inflammation, an aberrant continuation of some aspects of the acute phase response may contribute to the underlying tissue damage that accompanies the disease, and may also lead to further complications, for example cardiovascular diseases or protein deposition diseases such as amyloidosis.
  • An important aspect of the acute phase response is the radically altered biosynthetic profile of the liver.
  • APRs acute phase reactants
  • APPs acute phase proteins
  • SAA serum amyloid A
  • CRP C-reactive protein
  • SAP serum amyloid P component
  • the acute phase response, or risk therefore is evaluated by measuring one or more APPs. Measuring such markers is well known to those of skill in the art, and commercial companies exist that provide such measurement (e.g., AGP measured by Cardiotech Services, Louisville, KY).
  • the oxpholipin peptides described herein are useful in the treatment (e.g. mitigation and/or prevention) of corneal ulcers, endothelial sloughing, Crohn's disease, acute and chronic dermatitis (including, but not limited to eczema and/or psoriasis), macular degeneration, neuropathy, scleroderma, and ulcerative colitis.
  • COPD chronic obstructive pulmonary disease
  • CNSV central nervous system vasculitis
  • influenza (including avian flu)
  • sepsis syndrome (clinical syndrome where it appears that the patient is septic but no organisms are recovered from the blood)
  • reperfusion injury prevent and/or treat
  • renal failure e.g., acute renal failure, chronic renal failure, end stage renal failure
  • the oxpholipin peptide(s) will be administered to a mammal (e.g., a human) in need thereof.
  • a mammal e.g., a human
  • Such a mammal will typically include a mammal (e.g. a human) having or at risk for one or more of the pathologies described herein.
  • the active agent(s) can be administered, as described herein, according to any of a number of standard methods including, but not limited to injection, suppository, nasal spray, time-release implant, transdermal patch, and the like.
  • the peptide(s) are administered orally (e.g. as a syrup, capsule, or tablet).
  • the methods involve the administration of a single oxpholipin peptide described herein or the administration of two or more different oxpholipin peptides.
  • the oxpholipin peptides can be provided as monomers (e.g., in separate or combined
  • the multimeric forms may comprise associated monomers (e.g., ionically or hydrophobically linked) while certain other multimeric forms comprise covalently linked monomers (directly linked or through a linker).
  • While the invention is described with respect to use in humans, it is also suitable for animal, e.g. veterinary use.
  • animal e.g. veterinary use.
  • certain preferred organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, largomorphs, and the like.
  • the methods of this invention are not limited to humans or non-human animals showing one or more symptom(s) of the pathologies described herein, but are also useful in a prophylactic context.
  • the active agents of this invention can be
  • Atherosclerosis e.g., atherosclerosis, stroke, etc.
  • Particularly preferred subjects in this context are subjects showing one or more risk factors for the pathology.
  • atherosclerosis risk factors include family history, hypertension, obesity, high alcohol consumption, smoking, high blood cholesterol, high blood triglycerides, elevated blood LDL, VLDL, IDL, or low HDL, diabetes, or a family history of diabetes, high blood lipids, heart attack, angina or stroke, etc.
  • one more oxpholipin peptides are administered, e.g. to an individual diagnosed as having one or more symptoms of atherosclerosis, or as being at risk for atherosclerosis, and/or diagnosed as having high cholesterol or being at risk for high cholesterol, and or the various other pathologies described herein.
  • the oxpholipin peptide(s) can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the present method.
  • Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.
  • acid addition salts are prepared from the free base using conventional methodology, that typically involves reaction with a suitable acid.
  • a suitable acid such as methanol or ethanol
  • the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto.
  • the resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent.
  • Suitable acids for preparing acid addition salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • organic acids e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic
  • An acid addition salt may be reconverted to the free base by treatment with a suitable base.
  • Particularly preferred acid addition salts of the active agents herein are halide salts, such as may be prepared using hydrochloric or hydrobromic acids.
  • preparation of basic salts of the active agents of this invention are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like.
  • Particularly preferred basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.
  • the pKa of the counterion is preferably at least about 2 pH lower than the pKa of the drug.
  • the pKa of the counterion is preferably at least about 2 pH higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base.
  • the generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable.
  • the counterion is a pharmaceutically acceptable counterion.
  • Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, t
  • esters typically involves functionalization of hydroxyl and/or carboxyl groups that may be present within the molecular structure of the drug.
  • the esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl.
  • Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.
  • Amides and prodrugs can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Prodrugs are typically prepared by covalent attachment of a moiety that results in a compound that is therapeutically inactive until modified by an individual's metabolic system.
  • the oxpholipin peptide(s) identified herein are useful for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., atherosclerosis and/or symptoms thereof, pathologies characterized by an inflammatory response, etc.).
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • Suitable unit dosage forms include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, lipid complexes, etc.
  • a pharmaceutically acceptable carrier excipient
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • pharmaceutically acceptable carrier(s) including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).
  • the excipients are preferably sterile and generally free of undesirable matter.
  • compositions may be sterilized by conventional, well-known sterilization techniques.
  • the oxpholipin peptide(s) are administered to a patient suffering from one or more symptoms of one or more pathologies described herein, or at risk for one or more of the pathologies described herein in an amount sufficient to prevent and/or cure and/or or at least partially prevent, slow the progression, or arrest the disease and/or its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health.
  • Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient.
  • the concentration of oxpholipin peptide(s) can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosages range from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about 7.2 mg/kg/day to about 1 1.0 mg/kg/day, and most preferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day.
  • dosages range from about 10 mg/kg/day to about 50 mg/kg/day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.
  • the oxpholipin peptide(s) described herein are administered orally (e.g. via a tablet) or as an injectable in accordance with standard methods well known to those of skill in the art.
  • the peptides may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent(s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin.
  • the drug composition is typically contained in a layer, or "reservoir,” underlying an upper backing layer.
  • reservoir in this context refers to a quantity of "active ingredient(s)" that is ultimately available for delivery to the surface of the skin.
  • the “reservoir” may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art.
  • the patch may contain a single reservoir, or it may contain multiple reservoirs.
  • the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
  • suitable skin contact adhesive materials include, but are not limited to, poly ethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
  • the backing layer in these laminates which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility.
  • the material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.
  • Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives.
  • Creams containing the selected active agent are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil.
  • Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
  • the specific ointment or cream base to be used is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.
  • D-form amino acids can be administered, even orally, without protection against proteolysis by stomach acid, etc.
  • peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome.
  • protective excipients This is typically accomplished either by complexing the polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome.
  • Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Patent 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).
  • Elevated serum half-life can be maintained by the use of sustained-release protein "packaging" systems.
  • sustained release systems are well known to those of skill in the art.
  • the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy (1998) Biotechnol. Prog., 14: 108; Johnson et al. (1996) Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the active agent in a polymer matrix that can be compounded as a dry formulation with or without other agents.
  • the ProLease microsphere fabrication process was specifically designed to achieve a high encapsulation efficiency while maintaining integrity of the active agent.
  • the process consists of (i) preparation of freeze-dried drug particles from bulk by spray freeze- drying the drug solution with stabilizing excipients, (ii) preparation of a drug-polymer suspension followed by sonication or homogenization to reduce the drug particle size, (iii) production of frozen drug-polymer microspheres by atomization into liquid nitrogen, (iv) extraction of the polymer solvent with ethanol, and (v) filtration and vacuum drying to produce the final dry-powder product.
  • the resulting powder contains the solid form of the active agents, which is homogeneously and rigidly dispersed within porous polymer particles.
  • the polymer most commonly used in the process poly(lactide-co-glycolide) (PLG), is both biocompatible and biodegradable.
  • Encapsulation can be achieved at low temperatures (e.g., -40°C).
  • the protein is maintained in the solid state in the absence of water, thus minimizing water-induced conformational mobility of the protein, preventing protein degradation reactions that include water as a reactant, and avoiding organic-aqueous interfaces where proteins may undergo denaturation.
  • a preferred process uses solvents in which most proteins are insoluble, thus yielding high encapsulation efficiencies (e.g., greater than 95%).
  • one or more components of the solution can be provided as a "concentrate”, e.g., in a storage container (e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water.
  • the oxpholipin peptide(s) described herein are administered in conjunction with one or more lipids.
  • the lipids can be formulated as an excipient to protect and/or enhance transport/uptake of the active agents or they can be administered separately.
  • the lipids can be formed into liposomes that encapsulate the oxpholipin peptide(s) described herein and/or they can be complexed/admixed with the oxpholipin peptides and/or they can be covalently coupled to the oxpholipin peptide(s).
  • Methods of making liposomes and encapsulating reagents are well known to those of skill in the art (see, e.g., Martin and Papahadjopoulos (1982) J. Biol. Chem., 257: 286-288;
  • Illustrative phospholipids for use in these methods have fatty acids ranging from about 4 carbons to about 24 carbons in the sn-1 and sn-2 positions.
  • the fatty acids are saturated.
  • the fatty acids can be unsaturated.
  • Various fatty acids are illustrated in Table 3.
  • the fatty acids in these positions can be the same or different. Certain suitable
  • phospholipids have phosphorylcholine at the sn-3 position.
  • Restenosis the reclosure of a previously stenosed and subsequently dilated peripheral or coronary vessel occurs at a significant rate (e.g., 20-50% for these procedures) and is dependent on a number of clinical and morphological variables. Restenosis may begin shortly following an angioplasty procedure, but usually ceases at the end of approximately six (6) months.
  • Stents are typically devices that are permanently implanted (expanded) in coronary and peripheral vessels.
  • the goal of these stents is to provide a long-term "scaffolding" or support for the diseased (stenosed) vessels. The theory being, if the vessel is supported from the inside, it will not close down or restenose.
  • Known stent designs include, but are not limited to monofilament wire coil stents (see, e.g., U.S. Patent 4,969,458); welded metal cages (see, e.g., U.S. Patents 4,733,665 and 4,776,337), thin-walled metal cylinders with axial slots formed around the circumference (see, e.g., U.S. Patents 4,733,665, 4,739,762, 4,776,337, and the like).
  • stents can be covered and/or impregnated with one or more pharmaceutical, e.g., in controlled release formulations to inhibit cell proliferation associated with rest enosis. Most commonly such "drug-eluting" stents are designed to deliver various cancer drugs (cytotoxins).
  • this invention contemplates stents having one or more of the oxpholipin peptide(s) described herein coated on the surface and/or retained within cavities or microcavities in the surface of the stent.
  • the active agents are contained within biocompatible matrices (e.g. biocompatible polymers such as urethane, silicone, and the like). Suitable biocompatible materials are described, for example, in U.S. Patent Publications
  • the polymers include, but are not limited to silicone-urethane copolymer, a polyurethane, a phenoxy, ethylene vinyl acetate, polycaprolactone, poly(lactide-co-glycolide), polylactide, polysulfone, elastin, fibrin, collagen, chondroitin sulfate, a biocompatible polymer, a biostable polymer, a biodegradable polymer
  • this invention provides a stent for delivering drugs to a vessel in a body.
  • the stent typically comprises stent framework including a plurality of reservoirs formed therein.
  • the reservoirs typically include an active agent and/or active agent-containing polymer positioned in the reservoir and/or coated on the surface of the stent.
  • the stent is a metallic base or a polymeric base.
  • Certain preferred stent materials include, but are not limited to stainless steel, nitinol, tantalum, MP35N alloy, platinum, titanium, a suitable biocompatible alloy, a suitable biocompatible polymer, and/or a combination thereof.
  • the pores can include micropores (e.g., having a diameter that ranges from about 10 to about 50 ⁇ , preferably about 20 ⁇ or less). In various embodiments the micropores have a depth in the range of about 10 ⁇ to about 50 ⁇ . In various embodiments the micropores extend through the stent framework having an opening on an interior surface of the stent and an opening on an exterior surface of the stent. In certain embodiments the stent can, optionally comprise a cap layer disposed on the interior surface of the stent framework, the cap layer covering at least a portion of the through-holes and providing a barrier
  • the reservoirs comprise channels along an exterior surface of the stent framework.
  • the stent can optionally have multiple layers of polymer where different layers of polymer carry different active agent(s) and/or other drugs.
  • the stent comprises: an adhesion layer positioned between the stent framework and the polymer.
  • Suitable adhesion layers include, but are not limited to a polyurethane, a phenoxy, poly(lactide-co-glycolide)- , polylactide, polysulfone, polycaprolactone, an adhesion promoter, and/or a combination thereof.
  • the oxpholipin peptide(s) can be coated on or contained within essentially any implantable medical device configured for implantation in a extravascular and/or intravascular location.
  • the methods involve providing a stent framework; cutting a plurality of reservoirs in the stent framework, e.g., using a high power laser; applying one or more of the oxpholipin peptide(s) and/or a drug polymer to at least one reservoir; drying the drug polymer; applying a polymer layer to the dried drug polymer; and drying the polymer layer.
  • the active agent(s) and/or polymer(s) can be applied by any convenient method including but not limited to spraying, dipping, painting, brushing and dispensing.
  • the methods typically involve positioning a stent or other implantable device as described above within the body (e.g. within a vessel of a body) and eluting at least one oxpholipin peptide from at least one surface of the implant.
  • Vascular grafts can be classified as either biological or synthetic. There are two commonly used types of biological grafts. An autograft is one taken from another site in the patient. In peripheral vascular surgery by far the most commonly used such graft is the long saphenous vein. This can be used in situ with the valves surgically destroyed with an intraluminal cutting valvutome.
  • the vein can be removed and reversed but this typically produces a discrepancy between the anastomotic size of the artery and vein.
  • thoracic surgery the use of internal mammary artery for coronary artery bypass surgery is another example of an autograft.
  • An allograft is one taken from another animal of the same species.
  • Externally supported umbilical vein is rarely used but is an example of such a graft.
  • Synthetic grafts are most commonly made from Dacron or
  • PTFE polytetrafluroethylene
  • vascular grafts The commonest complications associated with the use of vascular grafts include Graft occlusion, Graft infection, true and false aneurysms at the site of anastomosis, distal embolization, and erosion in to adjacent structures - e.g. Aorto-enteric fistulae. Many of these conditions are associated with an inflammatory response, macrophage migration into the site, and/or the formation of reactive oxygen species ⁇ e.g., oxidized lipids). To reduce such complications, the graft (synthetic or biological can be soaked, or otherwise coated, with one or more of the oxpholipin peptide(s) described herein.
  • implantable tissues or materials can similarly be impregnated or coated with one or more oxpholipin peptide(s) described herein.
  • this invention contemplates the use of impregnated sutures to minimize inflammation and/or infection and/or tissue rejection.
  • one or more oxpholipin peptides described herein are administered alone or in combination with other therapeutics as described herein in implantable ⁇ e.g., subcutaneous) matrices.
  • implantable ⁇ e.g., subcutaneous matrices A major problem with standard drug dosing is that typical delivery of drugs results in a quick burst of medication at the time of dosing, followed by a rapid loss of the drug from the body. Most of the side effects of a drug occur during the burst phase of its release into the bloodstream. Secondly, the time the drug is in the bloodstream at therapeutic levels is very short, most is used and cleared during the short burst.
  • Drugs e.g., the oxpholipin peptides described herein
  • Drugs embedded, for example, in polymer beads or in polymer wafers have several advantages. First, most systems allow slow release of the drug, thus creating a continuous dosing of the body with small levels of drug. This typically prevents side effects associated with high burst levels of normal injected or pill based drugs. Secondly, since these polymers can be made to release over hours to months, the therapeutic span of the drug is markedly increased. Often, by mixing different ratios of the same polymer components, polymers of different degradation rates can be made, allowing remarkable flexibility depending on the agent being used. A long rate of drug release is beneficial for people who might have trouble staying on regular dosage, such as the elderly, but is also an ease of use
  • polymers can be made to degrade and be cleared by the body over time, so they will not remain in the body after the therapeutic interval.
  • Another advantage of polymer based drug delivery is that the polymers often can stabilize or solubilize proteins, peptides, and other large molecules that would otherwise be unusable as medications.
  • drug/polymer mixes can be placed directly in the disease area, allowing specific targeting of the medication where it is needed without losing drug to the "first pass" effect. This is certainly effective for treating the brain, which is often deprived of medicines that can't penetrate the blood/brain barrier.
  • sustained release systems include, but are not limited to Re- Gel®, SQ2Gel®, and Oligosphere® by MacroMed, ProLease® and Medisorb® by
  • Other "specialty" delivery systems include, but are not limited to lipid based oral mist that allows absorption of drugs across the oral mucosa, developed by Generex Biotechnology, the oral transmucosal system (OTSTM) by Anesta Corp., the inhalable dry powder and PulmoSpheres technology by Inhale Therapeutics, the AERx® Pulmonary Drug Delivery System by Aradigm, the AIR mechanism by Alkermes, and the like.
  • DST Drug Sipping Technology
  • oxpholipins in combination with other active agents is contemplated in the treatment of the various pathologies/indications described herein.
  • the use of combinations of active agents can alter pharmacological activity, bioavailability, and the like.
  • oxpholipins described herein in combination with a salicylanilide e.g., niclosamide
  • a salicylanilide e.g., niclosamide
  • Additional pharmacologically active materials can be delivered in conjunction with one or more of the oxpholipin peptide(s) described herein.
  • agents include, but are not limited to agents that reduce the risk of atherosclerotic events and/or complications thereof.
  • agents include, but are not limited to beta blockers, beta blocker and thiazide diuretic combinations, statins, aspirin, ace inhibitors, ace receptor inhibitors (ARBs), and the like.
  • the agents can be administered in conjunction with the oxpholipin (e.g., before, after, or at the same time as the oxpholipin(s)), or they can be administered as a combined formulation.
  • methods involve administering one or more of the oxpholipins described herein in conjunction with one or more statins.
  • the oxpholipin(s) and/or the statins when administered in conjunction with each other the oxpholipin(s) and/or the statins can be administered at significantly lower dosages thereby avoiding various harmful side effects (e.g., muscle wasting) associated with high dosage statin use and/or the anti-inflammatory properties of statins at any given dose are significantly enhanced.
  • Suitable statins include, but are not limited to atorvastatin (LIPITOR®,
  • TORVAST® cerivastatin (e.g., LIPOBAY®, BAYCOL®) fluvastatin (e.g., LESCOL®, LESCOL XL®), lovastatin (e.g. , MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin, pitavastatin (e.g., LIVALO®, PITAVA®), pravastatin (e.g., PRAVACHOL®,
  • SELEKTINE® rosuvastatin
  • CRESTOR® rosuvastatin
  • simvastatin e.g., ZOCOR®, LIPEX®
  • simvastatin+ezetimibe e.g. , VYTORIN®
  • therapy lovastatin+niacin extended-release e.g. , ADVICOR®
  • atorvastatin+amlodipine besylate e.g., CADUET®
  • simvastatin+niacin extended-release e.g., SIMCOR®
  • the oxpholipin(s) described herein are administered in conjunction with one or more beta blockers.
  • Suitable beta blockers include, but are not limited to cardioselective (selective beta 1 blockers), e.g., acebutolol (SECTRAL®), atenolol (TENORMIN®), betaxolol (KERLONE®), bisoprolol (ZEBETA®), metoprolol (LOPRESSOR®), and the like.
  • Suitable non-selective blockers include, but are not limited to carteolol (CARTROL®), nadolol (CORGARD®), penbutolol (LEVATOL®), pindolol (VISKEN®), propranolol (INDERAL®), timolol (BLOCKADREN®), labetalol (NORMODYNE®, TRANDATE®), and the like.
  • Suitable beta blocker thiazide diuretic combinations include, but are not limited to LOPRESSOR HCT®, ZIAC®, TENORETIC®, CORZIDE®, TIMOLIDE®, INDERAL LA 40/25®, INDERIDE®, NORMOZIDE®, and the like.
  • Suitable ace inhibitors include, but are not limited to captopril (e.g.
  • CAPOTEN® by Squibb benazepril (e.g., LOTENSIN® by Novartis), enalapril (e.g., VASOTEC® by Merck), fosinopril (e.g., MONOPRIL® by Bristol-Myers), lisinopril (e.g. PRINIVIL® by Merck or ZESTRIL® by Astra-Zeneca), quinapril (e.g. ACCUPRIL® by Parke -Davis), ramipril (e.g., ALT ACE® by Hoechst Marion Roussel, King
  • Suitable ARBS include but are not limited to losartan (e.g. COZAAR® by Merck), irbesartan (e.g., AVAPRO® by Sanofi), candesartan (e.g., ATACAND® by Astra Merck), valsartan (e.g., DIOVAN® by Novartis), and the like.
  • one or oxpholipin peptide(s) described herein are administered with one or more of the drugs identified below.
  • one or more oxpholipin(s) are administered in conjunction with cholesteryl ester transfer protein (CETP) inhibitors (e.g., torcetrapib, JTT- 705. CP-529414) and/or acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitors (e.g., Avasimibe (CI-1011), CP 113818, F-1394, and the like), and/or immunomodulators (e.g., FTY720 (sphingosine-1 -phosphate receptor agonist), THALOMID® (thalidomide), IMURAN® (azathioprine), COPAXONE® (glatiramer acetate), CERTICAN®
  • CETP cholesteryl ester transfer protein
  • ACAT acyl-CoA:cholesterol O-acyltransferase
  • immunomodulators e.g., FTY720 (sphingosine-1 -phosphate receptor agonist), THALOMID® (thal
  • DPP4 inhibitors e.g., 2-Pyrrolidinecarbonitrile, l-[[[2-[(5-cyano-2-pyridinyl)
  • Tomelukast Prostaglandin A 2
  • Prostaglandin J 2 Tetradecylthioacetic acid
  • TTA Tetradecylthioacetic acid
  • CS-045 Troglitazone
  • WY-14643 Wirinixic acid
  • one or more oxpholipin(s) are administered in conjunction with fibrates (e.g., clofibrate (atromid), gemfibrozil (lopid), fenofibrate (tricor), etc.), bile acid sequestrants ⁇ e.g., cholestyramine, colestipol, etc.), cholesterol absorption blockers ⁇ e.g., ezetimibe (Zetia), etc.), VYTORIN® ((ezetimibe/simvastatin combination), and/or steroids, warfarin, and/or aspirin, and/or Bcr-Abl inhibitors/antagonists ⁇ e.g., Gleevec (Imatinib Mesylate), AMN107, STI571 (CGP57148B), ON 012380, PLX225, and the like), and/or renin angiotensin pathway blockers ⁇ e.g., Losartan (COZAAR®),
  • fibrates e.
  • losartan (Cozaar), valsartan (Diovan), irbesartan (Avapro), candesartan (Atacand) and telmisartan (Micardis), etc.
  • PKC inhibitors e.g., Calphostin C, Chelerythrine chloride, Chelerythrine . chloride, Copper bis-3, 5- diisopropylsalicylate, Ebselen, EGF Receptor (human) (651-658) (N-Myristoylated), Go 6976, H-7 .
  • D- erytAro-Sphingosine isolated
  • D-erytAro-Sphingosine synthetic
  • Sphingosine N,N- dimethyl
  • D-erytAro-Sphingosine Dihydro-
  • D-erytAro-Sphingosine ⁇ , ⁇ -Dimethyl-
  • D- erytAro-Sphingosine chloride ⁇ , ⁇ , ⁇ -Trimethyl-, Staurosporine, Bisindolylmaleimide I, G- 6203, and the like.
  • one or more of the oxpholipins are administered in conjunction with ApoAI, Apo A-I derivatives and/or agonists ⁇ e.g., L-4F, D-4F, see, e.g., PCT/US2001/026497 (WO 2002/015923), PCT/US2003/032442 (WO 2004/034977), PCT/US2004/026288 (WO/2005/016280), PCT/US2003/09988 ((WO 2003/086326), and the like).
  • ApoAI milano see, e.g., U.S.
  • Patent 5,639,726, and the like and/or low molecular weight heparin, and/or AGE inhibitors/breakers (e.g., Benfotiamine, aminoguanidine, pyridoxamine, Tenilsetam, Pimagedine, and the like) and/or ADP receptor blockers (e.g., Clopidigrel, AZD6140, and the like), and/or ABCA1 agonists, and/or scavenger receptor Bl agonists, and/or
  • AGE inhibitors/breakers e.g., Benfotiamine, aminoguanidine, pyridoxamine, Tenilsetam, Pimagedine, and the like
  • ADP receptor blockers e.g., Clopidigrel, AZD6140, and the like
  • ABCA1 agonists e.g., scavenger receptor Bl agonists, and/or
  • Adiponectic receptor agonist or adiponectin inducers and/or stearoyl-CoA Desaturase I (SCD1) inhibitors, and/or Cholesterol synthesis inhibitors (non-statins), and/or
  • DGAT1 Diacylglycerol Acyltransferase I (DGAT1) inhibitors, and/or Acetyl CoA Carboxylase 2 inhibitors, and/or LP-PLA2 inhibitors, and/or GLP-1, and/or glucokinase activator, and/or CB-1 agonists, and/or anti-thrombotic/coagulants, and/or Factor Xa inhibitors, and/or GPIIb/IIIa inhibitors, and/or Factor Vila inhibitors, and/or Tissue factor inhibitors, and/or anti-inflammatory drugs, and/or Probucol and derivatives (e.g.
  • CCR2 antagonists and/or CX3CR1 antagonists, and/or IL-1 antagonists, and/or nitrates and NO donors, and/or phosphodiesterase inhibitors, and the like.
  • the oxpholipin(s) described herein can be administered in conjunction with niacin or extended release niacin.
  • Niacin (nicotinic acid) lowers lipids by inhibiting very-low-density lipoprotein (VLDL) production in the liver and reducing the level of VLDL that can be converted into low-density lipoprotein (LDL).
  • VLDL very-low-density lipoprotein
  • LDL low-density lipoprotein
  • Niacin can lower LDL cholesterol by 10 to 25 percent and triglyceride levels by 20 to 50 percent, and can raise levels of high density lipoprotein (HDL) cholesterol by 15 to 35 percent.
  • HDL high density lipoprotein
  • niacin can be in a form for immediate delivery (e.g., unmodified niacin), and/or intermediate release niacin (IR niacin, and/or extended release niacin (ER niacin), and/or niacin sustained release (niacin SR), and/or niacin preparations that are modified to avoid interactions with the receptor that mediates the flushing associated with niacin.
  • IR niacin intermediate release niacin
  • ER niacin extended release niacin
  • niacin sustained release niacin sustained release
  • niacin dosages range from about 300 mg/day up to 3,000 mg/day, more preferably from about 500 mg/day to 1500 mg/day.
  • the niacin is provided as a combined formulation with a statin (e.g. , ADVICOR® is a combination product containing both extended-release niacin and lovastatin) and/or with one or more of the active agents described herein (e.g., - HD (OxP-l lD), etc.).
  • a statin e.g. , ADVICOR® is a combination product containing both extended-release niacin and lovastatin
  • active agents described herein e.g., - HD (OxP-l lD), etc.
  • kits for amelioration of one or more symptoms of atherosclerosis or for the prophylactic treatment of a subject (human or animal) at risk for atherosclerosis and/or the treatment or prophylaxis of one or more of the conditions described herein preferably comprise a container containing one or more of the oxpholipin peptide(s) described herein.
  • the oxpholipin(s) can be provided in a unit dosage formulation (e.g. suppository, tablet, caplet, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable carriers/excipients.
  • the kit can, optionally, further comprise one or more other agents used in the treatment of the condition/pathology of interest.
  • agents include, but are not limited to, beta blockers, vasodilators, aspirin, statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like, e.g. as described above.
  • kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the
  • therapeutics or “prophylactics” of this invention.
  • Preferred instructional materials describe the use of one or more oxpholipin peptide(s) described herein to mitigate one or more symptoms of atherosclerosis (or other pathologies described herein) and/or to prevent the onset or increase of one or more of such symptoms in an individual at risk for atherosclerosis (or other pathologies described herein).
  • the instructional materials can also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • Oxpholipin IIP An Anti-Inflammatory Peptide that Binds Cholesterol and Oxidized
  • Oxpholipin- 1 ID contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3.
  • OxP-1 ID bound oxidized (phospho) lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I- mimetic peptide with anti-inflammatory properties.
  • D-4F which adopts a stable a-helical structure in solution
  • the OxP-1 ID structure was flexible and contained multiple turn-like features. Given the substantial evidence that oxidized phopholipids are pro-inflammatory in vivo, OxP-1 ID and other Oxpholipins are believed to have therapeutic potential.
  • Drugs, 8: 201-212 that also have anti-inflammatory properties, and act to sequester oxysterols and oxidized lipids (Van Lenten et al. (2008) J. Lipid Res., 49: 2302-231 1). Moreover, these peptides have similar properties to apoA-I Milano (. Riccioni et al. (2002) Int. J. Immunopathol.
  • OxP-1 ID a novel 14-residue peptide whose sequence resembles a cholesterol-binding domain found in a family of pore-forming bacterial exotoxins, and related oxpholipins.
  • OxP-1 ID reduced the release of monocyte chemotactic factors from LDL-stimulated human aortic endothelial cells, another property of D-4F.
  • Solid phase peptide synthesis was done with a Symphony® automated peptide synthesizer (Protein Technologies Inc., Arlington, AZ) or a CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), using 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields and Noble (1990) Int. J. Pept. Protein Res., 35 : 161-214). Amino acid derivatives and reagents were from EMD
  • Peptide purity was evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RPHPLC, using a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA).
  • the mobile phases were: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile.
  • Analytic assessments used a reversed-phase, 4.6(250 mm CI 8 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 ml/min.
  • OxP-3, OxP-3D and OxP-21 were obtained by dimerizing the appropriate monomer (10 mg/ml) in a 50% aqueous solution of DMSO at room temperature over 48 h.
  • OxP-22 was obtained by trimerizing OxP20 in 50% aqueous solution of DMF using Tris-[2- maleimidoethyl] amine and the manufacturer's protocol (Pierce Biotechnology, Rockford, IL, Cat#33043). The progress of the reaction was monitored by mass spectrometry. All peptides were lyophilized for storage. Peptide stock solutions were made in HPLC grade water containing 0.01% acetic acid, and peptide concentrations were determined by absorbance at 280 nm (Pace et al. (1995) Protein Sci., 4: 2411-2423). For analytical details concerning synthesized peptides see Supporting Information (Table 4). Lipids
  • PAPC L-a-l-palmitoyl-2-arachidonoyl-5/7-glycero-3-phosphorylcholine
  • PAPE l-palmitoyl-2-arachidonoyl-5n-glycero-3-phosphatidylethanolamine
  • POVPC 1- palmitoyl-2-(5-oxovaleroyl)-5/7-glycero-3-phosphorylcholine
  • PGPC 1-palmitoyl-2- glutaroyl-sn-glycero-3-phosphorylcholine
  • POPC l-Palmitoyl-2-oleoyl-s/?-glycero- 3 -phosphoryl choline
  • PEIPC l-palmitoyl-2- (5,6-deoxyisoprostane E2)-sft-glycero-3-phosphorylcholine
  • BIAcore 3000 system (BiaCore AB, Piscataway, NJ). Peptide ligands and apoA-I were immobilized on a BIAcore CM5 sensor chip activated per the manufacturer's protocol with N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide. After achieving adequate immobilization, the activated sensor surface was blocked with ethanolamine.
  • Lipid stock solutions were prepared in absolute ethanol and then diluted into a standard BIAcore buffer (HBS-EP), containing 10 mM HEPES, pH 7.4, 150 mM
  • Lipid concentrations used in the binding studies were selected to give binding responses of 30-500 resonance units.
  • Lipid stock solutions were prepared at 1 mg/ml in ethanol. Since the highest analyte lipid concentrations did not exceed 10 ⁇ , the highest ethanol concentration in any analyte solutions was 1 %, and for most lipids the ethanol concentration was
  • Lipid binding was measured by observing the change in the SPR angle as 150 ml of lipid analyte (various concentrations) in HBS-EP buffer flowed over the biosensor for 3 min at 50 ⁇ /min. Biosensors were washed with 25 or 50% ethanol to regenerate them between binding studies. SPR data were corrected for background binding to the matrix of the chip ("blank"channel) and analyzed with BIAevaluation 4.1 software (Biacore, Piscataway, NJ). Human monocytes and aortic endothelial cells
  • HAEC cells were treated with native LDL (250 ⁇ g/ml) in the absence or presence of HDL or tested peptides for 8 h. After these cultures were washed, the medium was replaced by fresh Medium 199 and the cultures were incubated for an additional 8 h. This culture medium was collected and assayed for monocyte chemotactic activity using chambers purchased from Neuroprobe (Cabin John, MD). After monocytes were added to the upper compartment, the chamber was incubated for 60 min at 37°C and subsequently disassembled.
  • the membrane was rinsed, air dried, fixed with 1%> glutaraldehyde, stained with 0.1 %> crystal violet dye, and 12 standardized high power fields were examined microscopically.
  • the number of migrated monocytes was expressed as the mean ⁇ SD of monocytes counted. Values obtained in the absence of HDL were normalized to a value of 1.0. Normalized values >1.0 after HDL addition were considered to be pro-inflammatory, and values ⁇ 1.0 as being anti- inflammatory.
  • mice were maintained in a breeding colony in the Department of Laboratory and Animal Medicine at the David Geffen School of Medicine at UCLA. The mice were maintained on a chow diet (Ralston Purina, St. Louis, MO).
  • ABCT buffer 50 mM NH4HC03, 0.1% Tween 20
  • OxP peptides in ABCT buffer.
  • blood was removed from the retro-orbital sinus under mild isoflurane anesthesia, and anticoagulated with heparin (2.5 U/ml).
  • CD spectra from 190-260 nm of D-4F and OxP-1 ID were examined in different solution environments using a JASCO 715 spectropolarimeter (Jasco Inc., Easton, MD) that was calibrated for wavelength and optical rotation with 10-camphorsulphonic acid (Johnson (1990) Proteins 7: 205-214; Miles et al. (2003) Spectroscopy 17: 653-661). Peptides were scanned at 20 nm per minute in 0.01 cm path-length cells at 25°C with a sample interval of 0.2 nm. Peptide concentration was determined by UV absorbance at 280 nm.
  • Infrared spectra were recorded at 25°C using a Bruker Vector 22TM FTIR spectrometer with a deuterated triglycine sulfate (DTGS) detector, and averaged over 256 scans at a gain of 4 with a resolution of 2 cm-1. Lipid and peptide samples were initially freeze-dried several times from 10 mM HCl in D20 to remove any interfering counter ions and residual H20.
  • DTGS deuterated triglycine sulfate
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • Lipid-peptide films were prepared by air-drying mixtures of DMPC and DMPC: cholesterol (1.2: 1, mole:mole) in chloroform with D-4F or OxP-1 ID in TFE onto a 50 x 20 x2 mm, 45° ATR crystal (Pike Technologies, Madison, WI) fitted to the Bruker spectrometer (Gordon et al. (1996) Protein Sci., 5 : 1662-1675) to form a multilayer film (lipid:peptide, 10: 1, mole:mole). After evaporation the solvent lipid:peptide film was hydrated by passaging deuterium-saturated nitrogen gas through the sample chamber for one hour prior to spectroscopy (Yamaguchi et al. (2001) Biophys.
  • Minimized monomeric D-4F or OxP-1 ID ensembles were ported to the Gromacs program suite, version 4.0.4 (www.gromacs.org), and subjected to the steepest descent method using the OPLS AA option (Hess et al. (2008) J. Chem. Theory Comput. , 4: 435-447).
  • Peptide purity was evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RP-HPLC, using a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA).
  • the mobile phases were: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile.
  • Analytic assessments used a reversed-phase, 4.66250 mm C18 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 ml/min.
  • Analytical data for OxP peptides is shown in Table 4.
  • OxP oxpholipin
  • Table 5 shows the highly conserved, cholesterol-binding domain found in seven different cholesterol-dependent cytolysins, and Table 6 shows the analogs included in this study.
  • modifying cysteine-3 with a bulky hydrophobic group (OxP-2) enhanced the peptide's ability to inhibit CDC-mediated hemolysis (data not shown).
  • OxPs-5 to 12 were designed to introduce other substitutions at position-3, and OxPs-13 to 20 were designed to vary the separation of hydrophobic and ionic residues.
  • ALO anthrolysin O, from Bacillus anthracis
  • PFO perfringolysin O, from Clostridium perfringens
  • LLO listeriolysin O, from Listeria monocytogenes
  • ALV ALV
  • alveolysin from Bacillus alvi ; SLO: streptolysin O, from Group A streptococcus; IVL: ivanolysin from Listeria ivanovi; PLY: pneumolysin from S. pneumoniae.
  • OxP-1
  • Oxpholipin-1 (this example).
  • OxP- -4 WA- -Arg- ⁇ Thr- -V- ⁇ Trp- -Gly- ⁇ Arg- -L- -Ctb- ⁇ Glu- -TE- -Trp- -NH 2 18
  • OxP- -4D WA-Arg- ⁇ Thr- -V- ⁇ Trp- -Gly- ⁇ Arg- -L- -Ctb- ⁇ Glu- -TE- -Trp- -NH 2 19
  • OxP- -7 RE- -Cbl- ⁇ Thr- -G- ⁇ Leu- -Ala- ⁇ Trp- -E- -Trp- ⁇ Trp- -RT- -Val- -NH 2 23
  • OxP- -11 RE- -Dpa- ⁇ Thr- -G- ⁇ Leu- -Ala- ⁇ Trp- -E- -Trp- ⁇ Trp- -RT- -Val- -NH 2 27
  • OxP- -12 RE-Ant- ⁇ Thr--G-Leu- ⁇ Ala-- rp--E-Trp- ⁇ Trp-RT-Val--NH 2 28
  • OxP- -15 Nic- -bA- ⁇ RE-( tb-Val .-R-: Leu-Val- 1 Trp >-E-' Trp- Trp- -RE- 33
  • OxP- -15D Nic- -bA- ⁇ RE-( tb-Val .-R-: Leu-Val- 1 Trp >-E-' Trp- Trp- -RE- 34
  • Analogues whose identifiers end with a D are composed of D-amino acids.
  • any of these peptides can have just a carboxyl terminal protecting group, just an amino terminal protecting group, protecting groups at both termini, protecting groups on one or more side chains, or no terminal and/or side chain protecting groups.
  • LDL containing oxidized phospholipids derived from arachidonic acid induces the production of monocyte chemoattractant protein- 1 (MCP- 1) by HAEC, an effect that may contribute to atherogenesis (Id.).
  • MCP- 1 monocyte chemoattractant protein- 1
  • ApoA-I mimetic peptides such as D-4F and ApoJ inhibit LDL-induced chemotaxis in vitro and in vivo, by
  • panels A-D show binding isotherms for OxP-1 ID to 13(S)- HODE), PEIPC, 12(S)-HPETE, and 5(S)-HPETE.
  • OxP-1 ID bound five of the lipids with higher affinity than D-4F, including palmitic acid, 5(S)-HPETE, 12(S)-HPETE, 15(S)- HPETE, 13(S) and 13(S)-HODE.
  • lipids were bound with higher affinity by D-4F, including arachidonic and linoleic acids, PGPC, POVPC, PEIPC, Kodia-PC, 13(S)- HPODE, 12(S)-HETE, 15(S)-HETE and 9(S)-HODE (Table 7)).
  • D-4F seems to bind with higher affinity to oxidized lipids containing palmitoyl moiety: PGPC, POVPC, PEIPC and KOdiA-PC.
  • OxP-1 ID on the other hand seems to be more selective toward certain sterols: 20(S)-hydroxycholesterol, 22(S)- hydroxycholesterol, 24(S)-hydroxycholesterol and certain arachi-'donic acid derivatives: 5(S)-HPETE, 12(S)-HPETE, 15(S)-HPETE and 13(S)-HODE.
  • PGPC l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine
  • POVPC 1- palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine
  • PEIPC l-palmitoyl-2-(5,6- epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine
  • KOdiA-PC-1 palmitoyl-2-(5-keto- 6-octene-dioyl)-sn-glycero-phosphatidylcholine
  • HPETE hydroperoxyeicosatetraenoic acid
  • HPODE hydroperoxyoctadecadienoic acid
  • HETE hydroxyeicosatetraenoic acid
  • HODE hydroxyoctadecadienoic acid.
  • OxP-l lD in HFIP :Buffer 25.0 33.0 14.0 28.0 peptides ( 100 uM ) in 10 mM phosphate buffer pH 7.4 or HFIP: 10 mM phosphate buffer pH 7.4 were analyzed for secondary conformation based as described in the Methods section.
  • OxP-1 ID displayed similar FTIR characteristics, but with a slightly higher helical signal and lower contributions from turn and beta sheet elements (Table 9).
  • Table 9 Secondary structural composition of D-4F and OxP-1 ID in different solvent systems, inferred from infrared (IR) spectroscopy. The peptides (1 mM) were studied in the following solvent systems: deuterated 10 mM phosphate buffer pD 7.4 ("DPB");
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • OxP-1 ID bound 12(S)-HPETE hydro ⁇ peroxyeicosatetraenoic acid
  • D-4F hydroxyeicosatetraenoic acid
  • OxP-1 ID bound 13(S)-HODE hydroxyoctadecadienoic acid
  • D-4F had greater affinity for 9(S)-HODE.
  • OxP-1 ID Changes in the secondary structure of OxP-1 ID in lipid environments were noted in our structural studies, and may correlate with our functional measurements in vitro and in vivo. CD, FTIR and molecular dynamics studies showed the peptide to have considerable conformational freedom. OxP-1 ID had a less defined conformation in more polar environments, however in hydrophobic solvent systems and in lipid multilayer ensembles, including these with cholesterol, the peptide showed highly ordered helix and turn structures.
  • 5(S)-HPETE which is derived from arachidonic acid by the actions of 5- lipoxygenase, is a direct precursor of leukotrienes A4, B4 and C4.
  • the KD values show that D-4F binds arachidonic acid, 5(S)-HPETE and 12(S)-HPETE with similar affinities.
  • the affinity of OxP-1 ID for 5(S)-HPETE was approximately 57-fold higher than its affinity for arachidonic acid, and its affinity for 12(S)- HPETE was approximately 28.5-fold higher.
  • OxP-1 ID and structurally related peptides are interesting lead

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Abstract

Oxpholipin peptides are a family of peptides that have potent properties attributable to their lipid binding properties. Oxpholipin peptides may be administered orally or parenterally to remove cholesterol and/or oxidized phospholipids from the serum, and from vascular and nonvascular tissue sites. In certain embodiments oxpholipin peptides are administered in combination with a second agent, e.g. statins, ApoAI peptides, etc. The peptides also possess also anti-inflammatory properties as determined by in vitro monocyte chemotactic assay (MCA) and their activity is carried on into relevant in vivo system of ApoE deficient mice. Surface plasmon resonance (SPR) studies revealed that the most potent anti-inflammatory compound, OxP-11D binds with high affinity to various oxidized phospholipids and sterols.

Description

NOVEL ANTI-INFLAMMATORY PEPTIDES THAT BIND OXIDIZED
PHOSPHOLIPIDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of USSN 61/310, 187, filed on March 3, 2010 and USSN 61/236,659, filed on August 25, 2009, both of which are incorporated herein by reference in their entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[ Not Applicable ]
FIELD OF THE INVENTION
[0002] This invention relates to the field of atherosclerosis and other conditions characterized by inflammation and/or the formation of various oxidized species. In particular, this invention pertains to the identification of a class of peptides, the oxpholipins, that bind cholesterol and/or oxidized phospholipids and that ameliorate one or more symptoms of conditions (e.g., atherosclerosis) characterized by an inflammatory response and/or the formation of various oxidized species.
BACKGROUND
[0003] Cardiovascular Disease (CVD) is the leading cause of death worldwide. An estimated 17.5 million people died from CVDs in 2005, representing 30% of all global deaths. Annually more people die from CVDs than from any other cause and World Health Organization (WHO) estimates rise in the occurrence of CVD in the close future: by 2015, an estimated 20 million people will die from cardiovascular disease every year reaching 23.4 million in 2030.
[0004] Widespread occurrence of CVD created high demand for drugs that could be used for its treatment. Currently, statins are the most widely prescribed lipid-modifying agent. They are also known as 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, which is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis de novo. Inhibition of this enzyme in the liver stimulates LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream and a i decrease in blood cholesterol levels. To date, it is believed that statins have provided the most effective approach to lowering the LDL ("bad"cholesterol). Other approaches to the treatment of CVD are based on the use of ApoA-I mimetic peptides. Apolipoprotein A-I (ApoA-I) is a major protein component of high density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion. Naturally occurring mutant of ApoA-I, called ApoA-I Milano which contains an extra cysteine bridge, causing it to exist as a homodimer or as a heterodimer with ApoA-II proved to be an effective drug in clinical trials. Despite its desirable properties ApoA-I Milano possessed a significant disadvantage - it was be administered intravenously and its further development was recently halted by Pfizer.
[0005] Similar therapeutic effects to those observed upon administration of ApoA-I
Milano can be also seen for certain other peptides that mimic ApoA-I function. So far the most prominent ApoA-I mimetic peptide is believed to be D-4F, an 18-amino acid analogue (Ac-DWFKAFYDKVAEKFKEAF-NH2, (SEQ ID NO: l)) consisting of D-amino acids that can be taken orally. The mechanism of action of D-4F appears to rely mostly on its ability preferentially bind oxidized phospholipids thereby promoting their subsequent removal, which may unblock the anti-oxidant enzymes associated with HDL. D-4F also dramatically reduces the ability of the human aortic endothelial cells to oxidize LDL to a form that could stimulate the induction of monocyte chemoattractant protein- 1 (MCP-1), which is important factor in early stage of atherogenesis.
[0006] Administration of the D-4F peptide to mice that possess inflammatory HDL resulted in "conversion" of HDL to an "anti-inflammatory" form that has inhibitory activity against LDL-induced monocyte chemotaxis. The D-4F peptide also appeared to promote ATP -binding cassette transporter (ABC A Independent cholesterol efflux of cellular cholesterol and phospholipids to HDL apolipoproteins as well as enhance formation of pre- β HDL which may relate to its ability to separate cholesterol from phospholipids.
[0007] To date, only a few sequences of peptides having such activities are known.
SUMMARY
[0008] Here we report the discovery of new family of peptides that possess similar properties to D-4F. The novel oxpholipin (phospholipid-binding) peptides described herein are a family of peptides that have potent anti-inflammatory and lipid binding properties. Oxpholipin peptides may be administered to remove cholesterol and noxious oxidized phospholipids from the serum, and from vascular and nonvascular tissue sites. In some embodiments of the invention, oxpholipin peptides are administered in combination with a second agent, e.g. a statins.
[0009] In certain embodiments a pharmaceutical composition comprising one or a cocktail of oxpholipin peptides as an active agent is administered to a patient suffering from hypercholesterolemia. Oxpholipin peptides may be administered alone, or in combination with other agents. Oxpholipin-mediated binding of oxidized lipids is also useful for modeling and screening candidate therapeutic agents.
[0010] Accordingly, in certain embodiments, an isolated oxpholipin peptide is provided that consists of or comprises an amino acid sequence according to the formula:
where n, and p are independently 0 or 1; X 1 when present is Aib, Ach, or bA; X 2 is Arg or Trp; X3 is Glu or Ala; X4 is Cys, Ctb, Arg, Ser, Chg, Cbl, PhF, Trp, Bip, Dpa, Ant, or Ctb; X5 is Thr, Val, Chg, Ctb, Ach, or Aib; X6 is Gly, Val, or Arg; X7 is Leu, Trp, Cha, or Ctb; X8 is Ala, Gly, Val, Chg, Ach, or Aib; X9 is Trp, Arg, or Nal; X10 is Glu, or Leu; X11 is Trp, Ctb, or Nal; X12 is Trp, Glu, or Nal; X13 is Arg, or Thr; X14 is Thr, or Glu; X15 is Val, Trp, Chg, Ctb, Ach, or Aib; and X16 when present is Lys and the peptide binds cholesterol and/or an oxidized lipid. In certain embodiments peptides of this formula range f from about 14, 15, 16, or 17 amino acids up to about 40, 50, 75, 100, 150, 200, 250, or 300 amino acids in length. In certain embodiments the peptide comprises at least one non- natural amino acid and/or "D" amino acid. In certain embodiments an isolated oxpholipin peptide is provided comprising an amino acid sequence selected from the group consisting of the amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46), the retro amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46), an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of an amino acid sequence found in Table 6 (SEQ ID NOs: 13-46); and an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of a retro form of an amino acid sequence found in Table 6 (SEQ ID NOs: 13-46); where the peptide binds cholesterol and/or an oxidized phospholipid. In certain embodiments the peptide comprises the amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46) or the inverse of the amino acid sequence. In certain embodiments the peptide ranges in length up to about 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In certain embodiments the peptide is shorter than the cholesterol binding domain of a cholesterol dependent cytolysin. In certain embodiments the peptide comprises all "L" amino acids. In certain embodiments the peptide comprises one or more "D" amino acids (e.g., all "D" amino acids). In certain embodiments one or more peptide bonds are replaced by an a-ester, a β-ester, a thioamide, phosphonamide, carbomate, or a hydroxylate. In certain embodiments the peptide comprises a peptide backbone, a polyethylene oxide (PEG/PEO) backbone, a polypropylene oxide (PPO) backbone, an aliphatic backbone, an ester backbone, or an ether backbone. In certain embodiments the peptide comprises one or more protecting groups (e.g., a carboxyl protecting group on the carboxyl terminus and/or an amino protecting group on the amino terminus). In certain embodiments the carboxyl protecting group and/or the amino protecting group is independently selected from the group consisting of acetyl, amide, 3 to 20 carbon alkyl group, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6- diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2- chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), Trifluoroacetyl (TFA), nicotinic acid, (Lys-Arg)3-Lys- NHCH2CH2SH, and TMEA. In certain embodiments the carboxyl protecting group is nicotinic acid. In certain embodiments the carboxyl protecting group comprises a group selected from the group consisting of an amide, (Lys-Arg^-Lys-NHCFbCFbSH, and TMEA. [0011] In various embodiments the peptide is formulated for administration via a route selected from the group consisting of oral administration, nasal administration, administration by inhalation, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, intraocular injection, and intramuscular injection. [0012] Also provided is a pharmaceutical formulation comprising one or more oxpholipin peptides as described and/or claimed herein and a pharmaceutically acceptable carrier. In certain embodiments the formulation is a unit dosage formulation. In certain embodiments the formulation is suitable for administration via a route selected from the group consisting of oral administration, nasal administration, administration by inhalation, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, intraocular injection, and intramuscular injection. In certain embodiments the formulation is a sterile formulation. [0013] In certain embodiments methods are provided for mitigating one or more symptoms of atherosclerosis in a mammal (e.g. in a human or a non-human mammal), the method comprising administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of atherosclerosis. [0014] In certain embodiments methods are provided for mitigating one or more symptoms of a pathology characterized by an inflammatory response in a mammal (e.g. in a human or a non-human mammal). The methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of the pathology. In certain embodiments the pathology is selected from the group consisting of the inflammatory pathology is a pathology selected from the group consisting of atherosclerosis, rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple sclerosis, and a viral illnesses.
[0015] In certain embodiments methods are provided for mitigating one or more symptoms of macular degeneration in a mammal (e.g. in a human or a non-human mammal). The methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to mitigate one or more symptoms of the macular degeneration. In certain embodiments the administration is via eye drops or intraocular injection. [0016] Methods of treating cancer (e.g., ovarian cancer) in a mammal (e.g., a human or a non-human mammal) are also provided. The methods typically involve administering to the mammal one or more oxpholipin peptides as described and/or claimed herein. In certain embodiments the cancer is a cancer selected from the group consisting of myeloma or multiple myeloma, ovarian cancer, breast cancer, colon cancer, bone cancer, cervical cancer, brain cancer, and prostate cancer.
[0017] Methods for sequestering cholesterol in a mammal (e.g., a human or a non- human mammal) are also provided. The methods typically involve comprising: administering an effective dose of one or more oxpholipin peptides as described and/or claimed herein to the mammal.
[0018] Methods for sequestering lipid in a mammal (e.g., a human or a non-human mammal) are also provided. The methods typically involve comprising: administering an effective dose of one or more oxpholipin peptides as described and/or claimed herein to the mammal.
[0019] Methods of treating a vascular condition and/or a condition characterized by an inflammatory response and/or a condition characterized by the formation of oxidized reactive species in a mammal (e.g., a human or a non-human mammal) are provided. The methods typically involve administering to a mammal in need thereof one or more oxpholipin peptides as described and/or claimed herein in an amount sufficient to ameliorate one or more symptoms of the condition. In certain embodiments the
administration is by a route selected from the group consisting of oral administration, nasal administration, rectal administration, intraperitoneal injection, and intravascular injection, subcutaneous injection, transcutaneous administration, and intramuscular injection. In certain embodiments the peptide is administered in conjunction with a drug selected from the group consisting of a CETP inhibitor, FTY720, Certican, DPP4 inhibitors, Calcium channel blockers, ApoAl derivative or mimetic or agonist, PPAR agonists , Steroids, Gleevec, Cholesterol Absorption blockers (Zetia) , Vytorin, Any Renin Angiotensin pathway blockers, Angiotensi II receptor antagonist (Diovan etc), ACE inhibitors, Renin inhibitors, MR antagonist and Aldosterone synthase inhibitor, Beta-blockers, Alpha- adrenergic antagonists, LXR agonist, FXR agonist, Scavenger Receptor Bl agonist, ABCA1 agonist, Adiponectic receptor agonist or adiponectin inducers, Stearoyl-CoA Desaturase I (SCD1) inhibitor, Cholesterol synthesis inhibitors (non-statins), Diacylglycerol Acyltransferase I (DGATl) inhibitor, Acetyl CoA Carboxylase 2 inhibitor, PAI-1 inhibitor, LP-PLA2 inhibitor, GLP-1, Glucokinase activator, CB-1 agonist, AGE inhibitor/breaker, PKC inhibitors, Anti-thrombotic/coagulants:, Aspirin, ADP receptor blockers e.g.
Clopidigrel, Factor Xa inhibitor, GPIIb/IIIa inhibitor, Factor Vila inhibitor, Warfarin, Low molecular weight heparin, Tissue factor inhibitor, Anti -inflammatory drugs:, Probucol and derivative e.g. AGI-1067 etc, CCR2 antagonist, CX3CR1 antagonist, IL-1 antagonist, Nitrates and NO donors, and Phosphodiesterase inhibitors.
[0020] In various embodiments a stent is provided for delivering drugs to a vessel in a body. The stent can comprise a stent framework including a plurality of reservoirs formed therein, and one or more oxpholipin peptides as described and/or claimed herein. In certain embodiments the peptide is contained within a polymer. In certain embodiments the stent framework comprises one of a metallic base or a polymeric base. In certain embodiments the stent framework base comprises a material selected from the group consisting of stainless steel, nitinol, tantalum, MP35N alloy, platinum, titanium, a suitable biocompatible alloy, a suitable biocompatible polymer, and a combination thereof. In certain embodiments the reservoirs comprise micropores. In certain embodiments the micropores have a diameter of about 20 microns or less. In certain embodiments the micropores have a diameter in the range of about 20 microns to about 50 microns. In certain embodiments the micropores have a depth in the range of about 10 to about 50 microns. In certain
embodiments the micropores have a depth of about 50 microns. In certain embodiments the micropores extend through the stent framework having an opening on an interior surface of the stent and an opening on an exterior surface of the stent. In various embodiments the stent further comprises a cap layer disposed on the interior surface of the stent framework, the cap layer covering at least a portion of the through-holes and providing a barrier characteristic to control an elution rate of a the peptide in the drug polymer from the interior surface of the stent framework. In certain embodiments the reservoirs comprise channels along an exterior surface of the stent framework. In certain embodiments the polymer comprises a first layer of a first drug polymer having a first pharmaceutical characteristic and the polymer layer comprises a second drug polymer having a second pharmaceutical characteristic. In certain embodiments the stent further comprises a catheter coupled to the stent framework. In certain embodiments the catheter includes a balloon used to expand the stent. In certain embodiments the catheter includes a sheath that retracts to allow expansion of the stent.
[0021] In various embodiments methods of manufacturing a drug-polymer stent are provided. The methods typically involve providing a stent framework; cutting a plurality of reservoirs in the stent framework; applying a composition comprising one or more oxpholipin peptides as described and/or claimed herein to the reservoirs and drying the composition. In certain embodiments the method further comprises applying a polymer layer to the dried composition; and drying the polymer layer.
[0022] In certain embodiments methods method of treating a vascular condition are provided. The methods typically involve positioning a stent according as described and/or claimed herein within a vessel of a body; expanding the stent; and eluting at least one oxpholipin peptide as described and/or claimed herein from at least a surface of the stent.
DEFINITIONS
[0023] The term "treat" when used with reference to treating, e.g. a pathology or disease refers to the mitigation and/or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
[0024] The terms "isolated", "purified", or "biologically pure" when referring to an isolated polypeptide refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state. With respect to nucleic acids and/or polypeptides the term can refer to nucleic acids or polypeptides that are no longer flanked by the sequences typically flanking them in nature. Chemically synthesized polypeptides are "isolated" because they are not found in a native state (e.g. in blood, serum, etc.). In certain embodiments, the term "isolated" indicates that the polypeptide is not found in nature.
[0025] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In certain embodiments the term "peptide" refers to a polymer of amino acid residues typically ranging in length from 2 to about 50 or about 60 residues. In certain embodiments the peptide ranges in length from about 4, 5, 6, 7, 8, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues. In certain embodiments the amino acid residues comprising the peptide are "L-form" amino acid residues, however, it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptide or the peptide can be all "D" amino acids. Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" (e.g., where the peptide bond is replaced by an a-ester, a β-ester, a thioamide, phosphonamide, carbomate, hydroxylate, and the like (see, e.g., Spatola (1983) Chem. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et ah, U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).
[0026] The term "residue"" as used herein refers to natural, synthetic, or modified amino acids. Various amino acid analogues include, but are not limited to 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine (beta-aminopropionic acid), 2-aminobutyric acid, 4- aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2- aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n- ethylasparagine, hydroxy lysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n- methyllysine, n-methylvaline, norvaline, norleucine, ornithine, and the like. These modified amino acids are illustrative and not intended to be limiting.
[0027] "β-peptides" comprise of "β amino acids", which have their amino group bonded to the β carbon rather than the a-carbon as in the 20 standard biological amino acids. The only commonly naturally occurring β amino acid is β-alanine.
[0028] Peptoids, or N-substituted glycines, are a specific subclass of
peptidomimetics. They are closely related to their natural peptide counterparts, but differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the a-carbons (as they are in natural amino acids).
[0029] Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated as well as retro, inversion, and retro- inversion isoforms. In addition, conservative substitutions (e.g., in the binding peptide, and/or antimicrobial peptide, and/or linker peptide) are contemplated. Non-protein backbones, such as PEG, alkane, ethylene bridged, ester backbones, and other backbones are also contemplated. Also fragments ranging in length from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids up to the full length minus one amino acid of the peptide are contemplated where the fragment retains at least 50%, preferably at least 60% 70% or 80%, more preferably at least 90%, 95%, 98%, 99%, or at least 100% of the activity (e.g., binding specificity and/or avidity, antimicrobial activity, etc.) of the full length peptide are contemplated. In addition compound peptides comprising repeats of one or more oxpholipins are also contemplated. The oxpholipins can be linked directly together or joined by a linker {e.g., a peptide linker or chemically conjugated).
[0030] In certain embodiments, oxpholipin peptides compromising at least 80%, preferably at least 85% or 90%>, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated. The terms "identical" or percent "identity," refer to two or more sequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. With respect to the peptides of this invention sequence identity is determined over the full length of the peptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson & Lipman (1988) Proc. Natl. Acad. Sci., USA, 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.
[0031] The term "ameliorating" when used with respect to "ameliorating one or more symptoms of atherosclerosis" refers to a reduction, prevention, or elimination of one or more symptoms characteristic of atherosclerosis and/or associated pathologies. Such a reduction includes, but is not limited to a reduction or elimination of oxidized
phospholipids, a reduction in atherosclerotic plaque formation and rupture, a reduction in clinical events such as heart attack, angina, or stroke, a decrease in hypertension, a decrease in inflammatory protein biosynthesis, reduction in plasma cholesterol, and the like.
[0032] The term "enantiomeric amino acids" refers to amino acids that can exist in at least two forms that are nonsuperimposable mirror images of each other. Most amino acids (except glycine) are enantiomeric and exist in a so-called L-form (L amino acid) or D- form (D amino acid). Most naturally occurring amino acids are "L" amino acids. The terms "D amino acid" and "L amino acid" are used to refer to absolute configuration of the amino acid, rather than a particular direction of rotation of plane-polarized light. The usage herein is consistent with standard usage by those of skill in the art. Amino acids are designated herein using standard 1 -letter or three-letter codes, e.g. as designated in Standard ST.25 in the Handbook On Industrial Property Information and Documentation.
[0033] The term "protecting group" refers to a chemical group that, when attached to a functional group in an amino acid {e.g. a side chain, an alpha amino group, an alpha carboxyl group, etc.) blocks or masks the properties of that functional group. Preferred amino-terminal protecting groups include, but are not limited to acetyl, or amino groups. Other amino-terminal protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl and others. Preferred carboxyl terminal protecting groups include, but are not limited to groups that form amides or esters.
[0034] The phrase "protect a phospholipid from oxidation by an oxidizing agent" refers to the ability of a compound to reduce the rate of oxidation of a phospholipid (or the amount of oxidized phospholipid produced) when that phospholipid is contacted with an oxidizing agent (e.g. hydrogen peroxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE, HETE, etc.).
[0035] The terms "low density lipoprotein" or "LDL" is defined in accordance with common usage of those of skill in the art. Generally, LDL refers to the lipid-protein complex which when isolated by ultracentrifugation is found in the density range d = 1.019 to d = 1.063.
[0036] The terms "high density lipoprotein" or "HDL" is defined in accordance with common usage of those of skill in the art. Generally "HDL" refers to a lipid-protein complex which when isolated by ultracentrifugation is found in the density range of d = 1.063 to d = 1.21.
[0037] The term "Group I HDL" refers to a high density lipoprotein or components thereof (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in low density lipoproteins) or that protect oxidized lipids from oxidation by oxidizing agents.
[0038] The term "Group II HDL" refers to an HDL that offers reduced activity or no activity in protecting lipids from oxidation or in repairing (e.g. reducing) oxidized lipids. [0039] The term "HDL component" refers to a component (e.g. molecules) that comprises a high density lipoprotein (HDL). Assays for HDL that protect lipids from oxidation or that repair (e.g. reduce oxidized lipids) also include assays for components of HDL (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that display such activity.
[0040] The term "human apo A-I peptide" refers to a full-length human apo A-I peptide or to a fragment or domain thereof comprising a class A amphipathic helix.
[0041] A "monocytic reaction" as used herein refers to monocyte activity characteristic of the "inflammatory response" associated with atherosclerotic plaque formation. The monocytic reaction is characterized by monocyte adhesion to cells of the vascular wall (e.g. cells of the vascular endothelium), and/or chemotaxis into the subendothelial space, and/or differentiation of monocytes into macrophages.
[0042] The term "absence of change" when referring to the amount of oxidized phospholipid refers to the lack of a detectable change, more preferably the lack of a statistically significant change (e.g. at least at the 85%, preferably at least at the 90%, more preferably at least at the 95%, and most preferably at least at the 98% or 99% confidence level). The absence of a detectable change can also refer to assays in which oxidized phospholipid level changes, but not as much as in the absence of the protein(s) described herein or with reference to other positive or negative controls.
[0043] The term "conservative substitution" is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity (e.g. for lipoproteins)) or binding affinity (e.g. for lipids or lipoproteins)) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or
hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0044] The phrase "in conjunction with" when used in reference to the use of one or more drugs in conjunction with one or more active agents described herein indicates that the drug(s) and the active agent(s) are administered so that there is at least some chronological overlap in their physiological activity on the organism. Thus the drug(s) and active agent(s) can be administered simultaneously and/or sequentially. In sequential administration there may even be some substantial delay (e.g., minutes or even hours or days) before administration of the second moiety as long as the first administered drug/agent has exerted some physiological alteration on the organism when the second administered agent is administered or becomes active in the organism.
Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.
[0045] In various embodiments the amino acid abbreviations shown in Table 1 are used herein. Table 1. Amino acid abbreviations.
Figure imgf000015_0001
carboxylic acid
Lys(N(epsilon)-trifluoroacetyl) K[TFA]
a-aminoisobutyric acid Aib B
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Figure 1 shows results of a monocyte chemotaxis assay of OxP peptides.
Results were standardized to LDL and are expressed as Inflammatory Index (II). Agents with an II ~1 are considered to be inactive. Agents with an II>1 are considered to be pro- inflammatory and those with an II<1 are considered to be anti-inflammatory. OxP-11, OxP- 13 and D-4F showed very similar activity.
[0047] Figure 2 illustrates results of dose response experiments. OxP-11 and OxP-
13, the two most active oxpholipins in the monocyte chemotaxis assay were re-tested at three concentrations: ^g/ml, 0. ^g/ml and 0.0^g/ml. A 10-fold reduction, from 1.0 to 0.1 μg/ml caused a relatively small decrease in activity, but a 100-fold reduction to 10 ng/ml reduced activity substantially.
[0048] Figure 3A-3C show in vivo activity of selected OxP peptides. Figure 3A:
Fasting female apoE deficient mice received 1.0 mg/kg of OxP peptides subcutaneously in 200 of ABCT buffer. After 6 hours of additional fasting, blood was obtained and HDL was isolated and tested in the monocyte chemotaxis assay as described in Example 1.
Results were normalized to LDL and are expressed as Inflammatory Indices. An index below 1.0 indicates anti-inflammatory activity in this test. Figure 3B compares L-4F and OxP-11 both containing L-amino acids exclusively. Both peptides were injected
subcutaneously at three different amounts, 1.0, 0.5 and 0.1 mg/kg. Figure 3C compares the in vivo activity of D-4F, OxP-1 ID and a 1 : 1 mixture (by weight), all administered at 1 mg/kg. D-4F and OxP-1 ID were equally effective alone and in combination.
[0049] Figure 4, panels A-C and A'-C illustrate binding of OxP- 1 ID to cholesterol,
20(S)- and 24(S)-hydroxycholesterols. In panels A', B' and C the dashed line shows binding of 3 mg/ml of the lipid to the biosensor's matrix and the line decorated with square symbols shows its binding to the sensor presenting OxPl 1-D. In panels A, B, and C, the results show binding isotherms corrected for background binding to the matrix.
[0050] Figure 5, panels A-D, illustrates binding of oxidized lipids to OxP-1 ID.
Abbreviations: 13(S)-HODE: 13(S)-hydroperoxyoctadecadienoic acid, PEIPC: 1-palmitoyl- 2-(5,6-deoxyisoprostane E2)-sn-glycero-3-phosphoryl choline, 12(S)-HPETE: 12(S)- hydroperoxyeicosatetraenoic acid, 5(S)-HPETE: 5(S)-hydroperoxyeicosatetraenoic acid. The biosensor presented 4686 RU of OxP-1 ID.
[0051] Figure 6, panels A, B, C, and D show circular dichroism (CD) and Fourier-
Transform Infrared (FTIR) spectra. Panel A shows CD spectra of D-4F and Panel B shows the spectra of OxP-1 ID. Spectra were obtained in two solvent systems: 10 mM phosphate buffer, pH 7.4 (dashed line ), HFIP:Buffer, (10 mM, pH 7.4) 4:6, v:v (solid line— ).
The peptide concentration was 100 μΜ, the cuvette light path was 0.01 cm, the temperature was 25°C, and the spectra are the average of 8 scans. Panel C shows FTIR spectra of D-4F and Panel D shows FTIR spectra of OxP-1 ID. The spectra were obtained in D20 Buffer, TFE: deuterium Buffer 4:6, v:v, HFIP: deuterium Buffer, 4:6, v:v, deuterium vapor hydrated DMPC multilayers (peptide: lipid, 1 : 10, mole:mole), and deuterium vapor hydrated
DMPC:CHO (DMPC:CHO, 1.2: 1, mole:mole with a peptide to lipid ratio of 1 : 10, mole:mole).
[0052] Figure 7, panels A and B shows a molecular illustration of the structure of D- 4F (panel A) and OxP-1 ID (panel B) after 83 nsec of molecular dynamics in HFIP:aqueous buffer 6:4, v:v environment. Alpha helical segments are in ribbon, disordered and turn segments are in highlight. The N-terminus is at the lower left and the adjacent C-terminus is in the upper right of the figure.
[0053] Figure 8, panels A, B, C, and D show time dependent stability studies. Panel A shows Root Mean Square Deviation (RMSD) of the C-alpha carbons for D-4F. Panel B shows these RMSD values for OxP-1 ID. Panel C shows the radius of gyration for D-4F and Panel D shows the radius of gyration for OxP-1 ID. All values are shown as a function of time (in nanoseconds) and come from molecular dynamic studies in a simulated
HFIP:buffer (6:4) environment. [0054] Figure 9, panels A and B show DSSP plots of secondary structure of D-4F
(Panel A) and OxP-1 ID (Panel B) as a function of time in a simulated HFIP:buffer 6:4 environment.
DETAILED DESCRIPTION
[0055] In various embodiments, novel oxpholipin peptides are provided. These peptides are effective to bind cholesterol and/or oxidized phospholipids and are believed to have utility in mitigating one or more symptoms of atherosclerosis and/or preventing the progression of atherosclerosis. The peptides also show anti-inflammatory activity are effective to mitigate one or more symptoms of a pathology characterized by an
inflammatory response (e.g., associated with a pathology such as atherosclerosis, rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple sclerosis, a viral illnesses, and the like). In addition, the peptides are effective in the treatment of a mammal suffering from undesirable levels of cholesterol. In various embodiments the level of total cholesterol considered to be undesirable may be greater than 150 mg/dl, greater than 200 mg/dl, or greater than 250 mg/dl. The level of LDL cholesterol considered to be undesirable may be greater than 100 mg/dl, greater than 125 mg/dl, greater than 150 mg/dl, greater than 175 mg/dl.
[0056] In various embodiments the oxpholipins may be administered alone or in conjunction with other cholesterol lowering agents, including, but not limited to statins, e.g. PRAVACHOL, MEVACOR®, LIPITOR®, LESCOL®, CRESTOR®, ZOCOR®; nicotinic acid, e.g. Niacin, NIASPAN®, SLO-NIACIN®; fibric acid, e.g. LOPID®, TRICOR®; bile acid sequestrants, e.g. QUESTRAN®, WELCHOL®, COLESTID®; cholesterol absorption inhibitors, e.g. ZETIA®; and the like.
[0057] Similarly in various embodiments the oxpholipin peptides can be
administered therapeutically or prophylactically to mitigate the onset and/or one or more symptoms of an inflammatory response or to mitigate the onset and/or one or more symptoms of a pathology characterized by an inflammatory response.
I. Oxpholipin peptides.
[0058] In certain embodiments illustrative oxpholipin peptide include peptides consisting of, or comprising, an amino acid sequence according to the formula:
χ1η.χ2.χ3.χ4.χ5.χ6.χ7_χ8_χ9_χ10_χ11 _χ12_χ13_χ14_χ15_χ16ρ j where n, and p are independently 0 or 1; X 1 when present is Aib, Ach, or bA; X 2 is Arg or Trp; X3 is Glu or Ala; X4 is Cys, Ctb, Arg, Ser, Chg, Cbl, PhF, Trp, Bip, Dpa, Ant, or Ctb; X5 is Thr, Val, Chg, Ctb, Ach, or Aib; X6 is Gly, Val, or Arg; X7 is Leu, Trp, Cha, or Ctb; X8 is Ala, Gly, Val, Chg, Ach, or Aib; X9 is Trp, Arg, or Nal; X10 is Glu, or Leu; X11 is Trp, Ctb, or Nal; X12 is Trp, Glu, or Nal; X13 is Arg, or Thr; X14 is Thr, or Glu; X15 is Val, Trp, Chg, Ctb, Ach, or Aib; and X16 when present is Lys. In certain embodiments peptides of this formula range f from about 14, 15, or 16, amino acids up to about 40, 50, 75, 100, 150, 200, 250, or 300 amino acids in length. In certain embodiments the peptide comprises at least one non-natural amino acid and/or "D" amino acid. In formula I, Nic is nicotinic acid; PhF is 1,2,3,4,5-pentafluoro-phenyl-alanine, Aib is aminoisobutyric acid; Bip is biphenyl- alanine; bA is β-alanine; Dpa is 3,3'-diphenyl-alanine; Ach is 1 -amino- 1-cyclohexane carboxylic acid; Ant is 3-(9-anthryl)-alanine; Ctb is S-tbutyl-cysteine; Cha is cyclohexyl- alanine; Cbl is S-(4-methyl)benzyl-cysteine; Nal is 3-(l-naphthyl)-alanine; and is cyclohexyl-glycine.
[0059] Illustrative novel oxpholipin peptides are shown herein in Table 6. While certain peptides in the table are shown with protecting groups and/or with D amino acids, it is contemplated that such peptides can be provided with or without protecting groups, can comprise one or more D amino acids, and/or can be all "L" amino acid peptides, and/or can comprise one or more substitutions of naturally occurring for non-naturally occurring amino acids.
[0060] It will also be appreciated in addition to the peptide sequences expressly illustrated herein, this invention also contemplates retro and retro-inverso forms of each of these peptides. In retro forms, the direction of the sequence is reversed. In inverse forms, the chirality of the constituent amino acids is reversed (i.e., L form amino acids become D form amino acids and D form amino acids become L form amino acids). In the retro- inverso form, both the order and the chirality of the amino acids is reversed.
[0061] Where reference is made to a sequence and orientation is not expressly indicated, the sequence can be viewed as representing the amino acid sequence in the amino to carboxyl orientation, the retro form (i.e., the amino acid sequence in the carboxyl to amino orientation), the retro form where L amino acids are replaced with D amino acids or D amino acids are replaced with L amino acids, and the retro-inverso form where both the order is reversed and the amino acid chirality is reversed.
[0062] In addition to the peptide sequences expressly illustrated herein, circular permutations of such sequences are also contemplated.
[0063] Also contemplated are any of these sequences comprising one, two, three, four, five, 6, seven, eight, nine, or ten conservative substitutions. In addition deletions of one, two, three, or four amino acids are contemplated.
[0064] Peptides or peptide conjugates comprising multiple oxpholipin domains
(e.g., one or more of the sequences listed in Table 6 the retro forms, inverso forms or retro inverso forms) are also contemplated. In various embodiments the same domain can be repeated, in other embodiments, two or more different oxpholipin domains can be incorporated into the peptide. Thus, in certain embodiments, dimmers, trimers, tetramers or larger polymers of these sequences are contemplated. The dimmers, trimers, tetramers, or larger polymers can be attached terminally as a conjugates or expressed as fusion proteins (attached directly to each other or via a linker (e.g. , a proline, a Gly3 linker, a (Gly4Ser)3 (SEQ ID NO:2) linker, etc.). In certain embodiments the sequence are chemically conjugated (e.g., through the N or carboxyl termini, or through side chains (e.g., via a disulfide linkage at a cysteine).
[0065] In various embodiments the amino acid sequence of the oxpholipin polypeptides shown in Table 6 may be altered in various ways known in the art to generate targeted changes in sequence. The polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by one amino acid, and may differ by two amino acids. The sequence changes may be substitutions, insertions or deletions.
[0066] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, carboxylation, pegylation, and the like. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine,
phosphoserine, or phosphothreonine.
[0067] In certain embodiments the peptide consists essentially of a polypeptide sequence set forth in Table 6 or its retro form. By "consisting essentially of in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the sequence set forth in the table, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) (e.g., cholesterol and/or lipid binding) of the polypeptide. In certain embodiments amino and/or carboxyl terminal flanking sequences independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. II. Peptide preparation.
[0068] The oxpholipin peptides described herein can be chemically synthesized using standard chemical peptide synthesis techniques or, particularly where the peptide does not comprise "D" amino acid residues, can be recombinantly expressed. In certain embodiments, even peptides comprising "D" amino acid residues are recombinantly expressed. Where the polypeptides are recombinantly expressed, a host organism (e.g. bacteria, plant, fungal cells, etc.) in cultured in an environment where one or more of the amino acids is provided to the organism exclusively in a D form. Recombinantly expressed peptides in such a system then incorporate those D amino acids.
[0069] In certain embodiments the peptides are chemically synthesized by any of a number of fluid or solid phase peptide synthesis techniques known to those of skill in the art. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are well known to those of skill in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111.
[0070] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allows for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines can be used for linking to a metal ion complex, carboxyl groups can be used for forming amides or esters, amino groups can form amides, and the like.
[0071] The polypeptides may also be isolated and purified in accordance with conventional methods of chemical or recombinant synthesis.
[0072] In certain embodiments, the peptides are synthesized by the solid phase peptide synthesis procedure using , for example, a SYMPHONY® automated peptide synthesizer (Protein Technologies Inc., Tucson, AZ) or a CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), using 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields and Noble (1990) Int. J. Pept. Protein Res. 35: 161-214). After cleaving the peptides from the resin with modified reagent K (TFA 94% (v/v); phenol, 2% (w/v); water, 2% (v/v); TIS, 2% (v/v); 2 hours) the peptides can be precipitated with ice-cold diethyl ether and purified to >95% homogeneity by preparative reverse-phase high performance liquid chromatography (RP-HPLC).
[0073] Peptide purity can be evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RP-HPLC, using, for example, a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). Suitable mobile phases are: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile. Analytic assessments can use, for example, a reversed- phase, 4.6 X 250 mm C18 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 mL/min.
III. Blocking groups and D residues.
[0074] While the various peptides and/or amino acid pairs described herein may be shown with no protecting groups, in certain embodiments {e.g. particularly for oral administration), they can bear one, two, three, four, or more protecting groups. The protecting groups can be coupled to the C- and/or N-terminus of the peptide(s) and/or to one or more internal residues comprising the peptide(s) {e.g., one or more R-groups on the constituent amino acids can be blocked). Thus, for example, in certain embodiments, any of the peptides described herein can bear, e.g. an acetyl group protecting the amino terminus and/or an amide group protecting the carboxyl terminus. One example of such a "dual protected peptide is OxP-14 (Nic-RE-Ctb-Val-R-Leu-Val-Trp-E-Trp-Trp-RE-Val-NH2, SEQ ID NO: 31) having nicotinic acid at the amino terminus and an amide at the carboxyl terminus. Either or both of these protecting groups can be eliminated and/or substituted with another protecting group as described herein.
[0075] Without being bound by a particular theory, it is believed that blockage, particularly of the amino and/or carboxyl termini of the subject peptides of this invention can greatly improve oral delivery and/or significantly increase serum half-life.
[0076] A wide number of protecting groups are suitable for this purpose. Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. In certain particularly preferred embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups. In one preferred embodiment, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. These blocking groups enhance the helix-forming tendencies of the peptides. Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula: CH3-(CH2)n-CO- where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
[0077] In certain particularly embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups. In one preferred embodiment, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. These blocking groups enhance the helix-forming tendencies of the peptides. Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula:
CH3-(CH2)n-CO- where n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
[0078] Other protecting groups include, but are not limited to Fmoc, t- butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9- florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl
(Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl- benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl
(Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3- nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2- bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO),t- butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), Trifluoroacetyl (TFA), nicotinic acid, (Lys-Arg)3-Lys-NHCH2CH2SH, and TMEA.
[0079] Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the oxpholipin peptides described herein (see, e.g., Greene et al, (1991) Protective Groups in Organic Synthesis,
2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). In one embodiment, for example, acetylation is accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection can be achieved by the selection of a proper resin for the synthesis. During the synthesis of the peptide, rink amide resin can be used. After the completion of the synthesis, the semipermanent protecting groups on acidic bifunctional amino acids such as Asp and Glu and basic amino acid Lys, hydroxyl of Tyr are all simultaneously removed. The peptides released from such a resin using acidic treatment comes out with the n-terminal protected as acetyl and the carboxyl protected as NH2 and with the simultaneous removal of all of the other protecting groups.
[0080] In certain particularly preferred embodiments, the peptides comprise one or more D-form (dextro rather than levo) amino acids as described herein. In certain embodiments at least two enantiomeric amino acids, more preferably at least 4 enantiomeric amino acids and most preferably at least 8 or 10 enantiomeric amino acids are "D" form amino acids. In certain embodiments every other, ore even every amino acid (e.g. every enantiomeric amino acid) of the peptides described herein is a D-form amino acid. [0081] In certain embodiments at least 50% of the enantiomeric amino acids are "D" form, more preferably at least 80% of the enantiomeric amino acids are "D" form, and most preferably at least 90% or even all of the enantiomeric amino acids are "D" form amino acids.
IV. Peptide circularization.
[0082] In certain embodiments the oxpholipin peptides described herein are circularized/cyclized to produce cyclic peptides. Cyclic peptides, as contemplated herein, include head/tail, head/side chain, tail/side chain, and side chain/side chain cyclized peptides. In addition, peptides contemplated herein include homodet, containing only peptide bonds, and heterodet containing in addition disulfide, ester, thioester-bonds, or other bonds.
[0083] The cyclic peptides can be prepared using virtually any art-known technique for the preparation of cyclic peptides. For example, the peptides can be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries. Preferably, the chemistry used to cyclize the peptide will be sufficiently mild so as to avoid substantially degrading the peptide. Suitable procedures for synthesizing the peptides described herein as well as suitable chemistries for cyclizing the peptides are well known in the art. [0084] In various embodiments cyclization can be achieved via direct coupling of the N- and C-terminus to form a peptide (or other) bond, but can also occur via the amino acid side chains. Furthermore it can be based on the use of other functional groups, including but not limited to amino, hydroxy, sulfhydryl, halogen, sulfonyl, carboxy, and thiocarboxy. These groups can be located at the amino acid side chains or be attached to their N- or C-terminus.
[0085] Accordingly it is to be understood that the chemical linkage used to covalently cyclize the peptides of the invention need not be an amide linkage. In many instances it may be desirable to modify the N- and C-termini of the linear or non-cyclized peptide so as to provide, for example, reactive groups that may be cyclized under mild reaction conditions. Such linkages include, by way of example and not limitation amide, ester, thioester, CH2 --NH, etc. Techniques and reagents for synthesizing peptides having modified termini and chemistries suitable for cyclizing such modified peptides are well- known in the art.
[0086] Alternatively, in instances where the ends of the peptide are
conformationally or otherwise constrained so as to make cyclization difficult, it may be desirable to attach linkers to the N- and/or C-termini to facilitate peptide cyclization. Of course, it will be appreciated that such linkers will bear reactive groups capable of forming covalent bonds with the termini of the peptide. Suitable linkers and chemistries are well- known in the art and include those previously described.
[0087] Cyclic peptides and depsipeptides (heterodetic peptides that include ester
(depside) bonds as part of their backbone) have been well characterized and show a wide spectrum of biological activity. The reduction in conformational freedom brought about by cyclization often results in higher receptor-binding affinities. Frequently in these cyclic compounds, extra conformational restrictions are also built in, such as the use of D- and N- alkylated-amino acids, α,β-dehydro amino acids or α,α-disubstituted amino acid residues.
[0088] Methods of forming disulfide linkages in peptides are well known to those of skill in the art {see, e.g., Eichler and Houghten (1997) Protein Pept. Lett. 4: 157-164).
[0089] Reference may also be made to Marlowe (1993) Biorg. Med. Chem. Lett. 3: 437-44 who describes peptide cyclization on TFA resin using trimethylsilyl (TMSE) ester as an orthogonal protecting group; Pallin and Tarn (1995) J. Chem. Soc. Chem. Comm. 2021-2022) who describe the cyclization of unprotected peptides in aqueous solution by oxime formation; Algin et al. (1994) Tetrahedron Lett. 35: 9633-9636 who disclose solid- phase synthesis of head-to-tail cyclic peptides via lysine side-chain anchoring; Kates et al. (1993) Tetrahedron Lett. 34: 1549-1552 who describe the production of head-to-tail cyclic peptides by three-dimensional solid phase strategy; Tumelty et al. (1994) J. Chem. Soc. Chem. Comm. 1067-1068, who describe the synthesis of cyclic peptides from an
immobilized activated intermediate, where activation of the immobilized peptide is carried out with N-protecting group intact and subsequent removal leading to cyclization;
McMurray et al. (1994) Peptide Res. 7: 195-206) who disclose head-to-tail cyclization of peptides attached to insoluble supports by means of the side chains of aspartic and glutamic acid; Hruby et al. (1994) Reactive Polymers 22: 231-241) who teach an alternate method for cyclizing peptides via solid supports; and Schmidt and Langer (1997) J. Peptide Res. 49: 67-73, who disclose a method for synthesizing cyclotetrapeptides and cyclopentapeptides.
[0090] These methods of peptide cyclization are illustrative and non-limiting. Using the teaching provide herein, other cyclization methods will be available to one of skill in the art.
V. Peptide Mimetics.
[0091] In addition to the peptides described herein, peptidomimetics are also contemplated. Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere
(1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al.
(1987) J. Med. Chem. 30: 1229) and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
[0092] Generally, peptidomimetics are structurally similar to a paradigm
polypeptide {e.g. oxpholipin 1 ID (OxP-1 ID) shown in Table 6), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: - CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, - CH2SO-, etc. by methods known in the art and further described in the following references: Spatola (1983) p. 267 in Chemistry and Biochemistry of Amino Acids, Peptides, and
Proteins, B. Weinstein, eds., Marcel Dekker, New York,; Spatola (1983) Vega Data 1(3) Peptide Backbone Modifications, (general review); Morley (1980) Trends Pharm Sci pp. 463-468 (general review); Hudson et al. (1979) Int JPept Prot Res 14: 177-185 (-CH2NH-, CH2CH2-); Spatola et al. (1986) Life Sci 38: 1243-1249 (-CH2-S); Hann, (1982) J Chem Soc Perkin Trans I 307-314 (-CH-CH-, cis and trans); Almquist et al. (1980) J Med Chem. 23: 1392-1398 (-COCH2-); Jennings- White et al. (1982) Tetrahedron Lett. 23: 2533 (- COCH2-); Szelke et al. (1982) European Appln. EP 45665 CA: 97:39405 (1982) (-
CH(OH)CH2-); Holladay et al. (1983) Tetrahedron Lett 24:4401-4404 (-C(OH)CH2-); and Hruby (1982) Life Sci., 31 : 189-199 (-CH2-S-)).
[0093] One particularly preferred non-peptide linkage is -CH2NH-. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced
pharmacological properties (half- life, absorption, potency, efficacy, etc.), reduced antigenicity, and others.
[0094] In addition, circularly permutations of the peptides described herein or constrained peptides (including cyclized peptides) comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61 : 387); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
VI. Uses of oxpholipin peptides.
[0095] In various embodiments, oxpholipin peptides or formulations comprising oxpholipin peptides are administered to a mammal at risk for, or having, undesirably high cholesterol levels (e.g., undesirably high levels of LDL) and/or at risk for atherosclerosis or having one more symptoms of atherosclerosis.
[0096] The oxpholipin peptides described herein are effective for mitigating one or more symptoms and/or reducing the rate of onset and/or severity of one or more indications described herein (e.g., elevated cholesterol, atherosclerosis, inflammatory pathologies, etc.). In particular, the oxpholipin peptides described herein are believed to be effective for mitigating one or more symptoms of atherosclerosis. Without being bound to a particular theory, it is believed that the peptides can bind the "seeding molecules" required for the formation of pro-inflammatory oxidized phospholipids such as Ox-PAPC, POVPC, PGPC, and PEIPC. [0097] In addition, since many inflammatory conditions and/or other pathologies are mediated at least in part by oxidized lipids, we believe that the peptides of this invention are effective in ameliorating conditions that are characterized by the formation of biologically active oxidized lipids. In addition, there are a number of other conditions for which the oxpholipin peptides described are believed to be efficacious.
[0098] A number of pathologies for which the active agents described herein appear to be a palliative and/or a preventative are described below.
A) Atherosclerosis and associated pathologies.
[0099] It is believed the oxpholipin peptides described herein can function in a manner similar to the peptides L-4F and D-4F described in PCT/US2001/026497 (WO
2002/015923), and in U.S. Patents 6,664,230 and 6,933,279. HDL from mice that were fed an atherogenic diet and injected with PBS failed to inhibit the oxidation of human LDL and failed to inhibit LDL-induced monocyte chemotactic activity in human artery wall cocultures. In contrast, HDL from mice fed an atherogenic diet and injected daily with peptides described herein was as effective in inhibiting human LDL oxidation and preventing LDL-induced monocyte chemotactic activity in the cocultures as was normal human.
[0100] The in vitro responses of human artery wall cells to HDL and LDL from mice fed the atherogenic diet and injected with a peptide according to this invention are consistent with the protective action shown by such peptides in vivo. The peptides described herein are believed to prevent progression of atherosclerotic lesions, e.g., in mice fed an atherogenic diet.
[0101] Thus, in one embodiment, this invention provides methods for ameliorating and/or preventing one or more symptoms of atherosclerosis by administering one or more of the active agents described herein.
[0102] It is also noted that c-reactive protein, a marker for inflammation, is elevated in congestive heart failure. Also, in congestive heart failure there is an accumulation of reactive oxygen species and vasomotion abnormalities. Because of their effects in preventing/reducing the formation of various oxidized species and/or because of their effect in improving vasoreactivity and/or arteriole function (see below) the oxpholipin peptides described herein will be effective in treating congestive heart failure. B) Arteriole/vascular indications.
[0103] Vessels smaller than even the smallest arteries (i.e., arterioles) thicken, become dysfunctional and cause end organ damage to tissues as diverse as the brain and the kidney. It is believed the oxpholipin peptides described herein can function to improve arteriole structure and function and/or to slow the rate and/or severity of arteriole dysfunction. Without being bound to a particular theory, it is believed that arteriole dysfunction is a causal factor in various brain and kidney disorders. Use of the oxpholipin peptides described herein thus provides a method to improve the structure and function of arterioles and preserve the function of end organs such as the brain and kidney.
[0104] Thus, for example, administration of one or more of the oxpholipin peptides described herein is expected to reduce one or more symptoms or to slow the onset or severity of arteriolar disease associated with aging, and/or Alzheimer's disease, and/or Parkinson's disease, and/or with multi-infarct dementia, and/or subarachnoid hemorrhage, and the like. Similarly, administration of one or more agents described herein is expected to mitigate one or more symptoms and/or to slow the onset and/or severity of chronic kidney disease, and/or hypertension.
[0105] Similarly, the oxpholipin peptides are believed to improve vasoreactivity.
Because of the improvement of vasoreactivity and/or arteriole function, the oxpholipin peptides described herein are believed to be suitable for the treatment of peripheral vascular disease, erectile dysfunction, and the like.
C) Pulmonary indications.
[0106] The oxpholipin peptides described herein are also believed to be suitable for treatment of a variety of pulmonary indications. These include, but are not limited to chronic obstructive pulmonary disease (COPD), emphysema, pulmonary disease, asthma, idiopathic pulmonary fibrosis, and the like.
D) Mitigation of a symptom or condition associated with coronary calcification and osteoporosis.
[0107] Vascular calcification and osteoporosis often co-exist in the same subjects
(Ouchi et al. (1993) Ann NY Acad Sci., 676: 297-307; Boukhris and Becker f 1972) JAMA, 219: 1307-1311; Banks et al. (1994) Eur J Clin Invest., 24: 813-817; Laroche et al. (1994) Clin Rheumatol., 13: 611-614; Broulik and Kapitola (1993) Endocr Regul., 27: 57-60; Frye et al. (1992) Bone Mine., 19: 185-194; Barengolts et al. (1998) Calcif Tissue Int., 62: 209- 213; Burnett and Vasikaran (2002) Ann Clin Biochem., 39: 203-210. Parhami et al. (1997) Arterioscl Thromb Vase Biol., 17: 680-687, demonstrated that mildly oxidized LDL (MM- LDL) and the biologically active lipids in MM-LDL [i.e. oxidized l-palmitoyl-2- arachidonoyl-sft-glycero-3-phosphorylcholine) (Ox-PAPC)], as well as the isoprostane, 8- iso prostaglandin E2,but not the unoxidized phospholipid (PAPC) or isoprostane 8-iso progstaglandin F2a induced alkaline phosphatase activity and osteoblastic differentiation of calcifying vascular cells (CVCs) in vitro, but inhibited the differentiation of MC3T3-E1 bone cells.
[0108] The osteon resembles the artery wall in that the osteon is centered on an endothelial cell-lined lumen surrounded by a subendothelial space containing matrix and fibroblast-like cells, which is in turn surrounded by preosteoblasts and osteoblasts occupying a position analogous to smooth muscle cells in the artery wall (Id.). Trabecular bone osteoblasts also interface with bone marrow subendothelial spaces (Id.). Parhami et al. (1997) Arterioscl Thromb Vase Biol., 17: 680-687, postulated that lipoproteins could cross the endothelium of bone arteries and be deposited in the subendothelial space where they could undergo oxidation as in coronary arteries (Id.). Based on their in vitro data they predicted that LDL oxidation in the subendothelial space of bone arteries and in bone marrow would lead to reduced osteoblastic differentiation and mineralization which would contribute to osteoporosis (Id.). Their hypothesis further predicted that LDL levels would be positively correlated with osteoporosis as they are with coronary calcification (Pohle et al. (2001) Circulation, 104: 1927-1932), but HDL levels would be negatively correlated with osteoporosis (Parhami et al. (1997) supra.).
[0109] In vitro, the osteoblastic differentiation of the marrow stromal cell line M2- 10B4 was inhibited by MM-LDL but not native LDL (Parhami et al. (1999) J Bone Miner Res., 14: 2067-2078). When marrow stromal cells from atherosclerosis susceptible
C57BL/6 (BL6) mice fed a low fat chow diet were cultured there was robust osteogenic differentiation (Id.). In contrast, when the marrow stromal cells taken from the mice after a high fat, atherogenic diet were cultured they did not undergo osteogenic differentiation (Id.). This observation provides a possible explanation for the decreased osteogenic potential of marrow stromal cells in the development of osteoporosis (Nuttall and Gimble (2000) Bone, 27: 177-184). In vivo the decrease in osteogenic potential is accompanied by an increase in adipogenesis in osteoporotic bone (Id.). [0110] It is believed that in a manner analogous to D-4F, addition of one or more oxpholipin peptides described herein to the drinking water of apoE null will dramatically increase trabecular bone mineral density and it is believed that the oxpholipin peptides described herein will act similarly. [0111] Osteoporosis can be regarded as an "atherosclerosis of bone". It appears to be a result of the action of oxidized lipids. HDL destroys these oxidized lipids and promotes osteoblastic differentiation. Accordingly, administering oxpholipin peptides described herein to a mammal (e.g., in the drinking water of apoE null mice) is expected to dramatically increase trabecular bone. Accordingly, it is believed that the oxpholipin peptides described herein are useful for mitigation one or more symptoms of osteoporosis (e.g., for inhibiting decalcification) or for inducing recalcification of osteoporotic bone. It is believed the oxpholipin peptides are also useful as prophylactics to prevent the onset of symptom(s) of osteoporosis in a mammal (e.g., a patient at risk for osteoporosis).
[0112] We believe similar mechanisms are a cause of coronary calcification, e.g., calcific aortic stenosis. Thus, in certain embodiments, this invention contemplates the use of the oxpholipin peptides described herein to inhibit or prevent a symptom of a disease such as coronary calcification, calcific aortic stenosis, osteoporosis, and the like.
E) Inflammatory and Autoimmune Indications.
[0113] Chronic inflammatory and/or autoimmune conditions are also characterized by the formation of a number of reactive oxygen species and are amenable to treatment using one or more of the active agents described herein. Thus, without being bound to a particular theory, we also believe the oxpholipin peptides described herein are useful, prophylactically or therapeutically, to mitigate the onset and/or more or more symptoms of a variety of other conditions including, but not limited to rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, polymyalgia rheumatica, scleroderma, multiple sclerosis, and the like.
[0114] In certain embodiments, the oxpholipin peptides are useful in mitigating one or more symptoms caused by, or associated with, an inflammatory response in these conditions. [0115] Also, in certain embodiments, the oxpholipin peptides are useful in mitigating one or more symptoms caused by or associated with an inflammatory response associated with AIDS.
F) Infections/trauma/transplants.
0116] It has been observed that a consequence of influenza infection and other infections is the diminution in paraoxonase and platelet activating acetylhydrolase activity in the HDL. Without being bound by a particular theory, it is believed that, as a result of the loss of these HDL enzymatic activities and also as a result of the association of pro- oxidant proteins with HDL during the acute phase response, HDL is no longer able to prevent LDL oxidation and is no longer able to prevent the LDL-induced production of monocyte chemotactic activity by endothelial cells.
[0117] It is believed that the oxpholipin peptides described herein can be administered (e.g. orally or by injection) to patients (including, for example with known coronary artery disease during influenza infection or other events that can generate an acute phase inflammatory response, e.g. due to viral infection, bacterial infection, trauma, transplant, various autoimmune conditions, etc.) and thus we can prevent by this short term treatment the increased incidence of heart attack and stroke associated with pathologies that generate such inflammatory states.
[0118] In addition, by restoring and/or maintaining paroxonase levels and/or monocyte activity, the oxpholipin peptides are believed to be useful in the treatment of infection (e.g., viral infection, bacterial infection, fungal infection) and/or the inflammatory pathologies associated with infection (e.g. meningitis) and/or trauma.
[0119] In certain embodiments, because of the combined anti-inflammatory activity and anti -infective activity, the oxpholipin peptides described herein are also useful in the treatment of a wound or other trauma, mitigating adverse effects associated with organ or tissue transplant, and/or organ or tissue transplant rejection, and/or implanted prostheses, and/or transplant atherosclerosis, and/or biofilm formation. In addition, we believe oxpholipin peptides described herein are also useful in mitigating the effects of spinal cord injuries. G) Diabetes and associated conditions.
[0120] In various embodiments, it is believed the oxpholipin peptides described herein can be used in methods of treating (therapeutically and/or prophylactically) diabetes and/or associated pathologies (e.g., Type I diabetes, Type II diabetes, juvenile onset diabetes, diabetic nephropathy, nephropathy, diabetic neuropathy, diabetic retinopathy, and the like).
[0121] In certain embodiments it is believed the oxpholipin peptides can also be used to improve insulin sensitivity.
H) Cancer.
[0122] NFKB is a transcription factor that is normally activated in response to proinflammatory cytokines and that regulates the expression of more than 200 genes. Many tumor cell lines show constitutive activation of NFKB signaling. Various studies of mouse models of intestinal, and mammary tumors conclude that activation of the NFKB pathway enhances tumor development and may act primarily in the late stages of tumorigenesis (see, e.g., ( 2004) Cell 1 18 : 285; (2004) J. Clin. Invest., 114: 569). Inhibition of NFKB signaling suppressed tumor development. Without being bound to a particular theory, mechanisms for this suppression are believed to include an increase in tumor cell apoptosis, reduced expression of tumor cell growth factors supplied by surrounding stromal cells, and/or abrogation of a tumor cell dedifferentiation program that is critical for tumor
invasion/metastasis.
[0123] Without being bound by a particular theory, it is believed the administration of one or more oxpholipin peptides described herein will inhibit expression and/or secretion, and/or activity of NFKB or otherwise interfere with the progression or maintenance of a cancer. Thus, in certain embodiments, this invention provides methods of treating a pathology characterized by elevated NFKB by administering one or more active agents described herein. Thus, in various embodiments this invention contemplates inhibiting NFKB activation associated with cancer by administering one ore more active agents described herein, optionally in combination with appropriate cancer therapeutics. In certain embodiments, inhibition of the onset, progression or severity of a cancer by administration of an oxpholipin is contemplated. Illustrative cancers include, but are not limited to myeloma/multiple myeloma, ovarian cancer, breast cancer, colon cancer, bone cancer, cervical cancer, prostate cancer, skin cancer, liver cancer, glioma or other brain cancers, and the like.
I) Mitigation of a symptom of atherosclerosis associated with an acute inflammatory response.
[0124] The oxpholipin peptides described herein are believed to be useful in a number of contexts. For example, cardiovascular complications (e.g., atherosclerosis, stroke, etc.) frequently accompany or follow the onset of an acute phase inflammatory response, e.g., such as that associated with a recurrent inflammatory disease, a viral infection (e.g., influenza), a bacterial infection, a fungal infection, an organ transplant, a wound or other trauma, and so forth.
[0125] Thus, in certain embodiments, this invention contemplates administering one or more of the oxpholipin peptides described herein to a subject at risk for, or incurring, an acute inflammatory response and/or at risk for or incurring a symptom of atherosclerosis and/or an associated pathology (e.g., stroke).
[0126] Thus, for example, a person having or at risk for coronary disease may prophylactically be administered a one or more oxpholipin peptides of this invention during flu season. A person (or animal) subject to a recurrent inflammatory condition, e.g., rheumatoid arthritis, various autoimmune diseases, etc., can be treated with a one or more agents described herein to mitigate or prevent the development of atherosclerosis or stroke. A person (or animal) subject to trauma, e.g., acute injury, tissue transplant, etc. can be treated with a polypeptide of this invention to mitigate the development of atherosclerosis or stroke.
[0127] In certain instances such methods will entail a diagnosis of the occurrence or risk of an acute inflammatory response. The acute inflammatory response typically involves alterations in metabolism and gene regulation in the liver. It is a dynamic homeostatic process that involves all of the major systems of the body, in addition to the immune, cardiovascular and central nervous system. Normally, the acute phase response lasts only a few days; however, in cases of chronic or recurring inflammation, an aberrant continuation of some aspects of the acute phase response may contribute to the underlying tissue damage that accompanies the disease, and may also lead to further complications, for example cardiovascular diseases or protein deposition diseases such as amyloidosis. [0128] An important aspect of the acute phase response is the radically altered biosynthetic profile of the liver. Under normal circumstances, the liver synthesizes a characteristic range of plasma proteins at steady state concentrations. Many of these proteins have important functions and higher plasma levels of these acute phase reactants (APRs) or acute phase proteins (APPs) are required during the acute phase response following an inflammatory stimulus. Although most APRs are synthesized by hepatocytes, some are produced by other cell types, including monocytes, endothelial cells, fibroblasts and adipocytes. Most APRs are induced between 50% and several-fold over normal levels. In contrast, the major APRs can increase to 1000-fold over normal levels. This group includes serum amyloid A (SAA) and either C-reactive protein (CRP) in humans or its homologue in mice, serum amyloid P component (SAP). So-called negative APRs are decreased in plasma concentration during the acute phase response to allow an increase in the capacity of the liver to synthesize the induced APRs.
[0129] In certain embodiments, the acute phase response, or risk therefore is evaluated by measuring one or more APPs. Measuring such markers is well known to those of skill in the art, and commercial companies exist that provide such measurement (e.g., AGP measured by Cardiotech Services, Louisville, KY).
J) Other indications.
[0130] In various embodiments it is contemplated that the oxpholipin peptides described herein are useful in the treatment (e.g. mitigation and/or prevention) of corneal ulcers, endothelial sloughing, Crohn's disease, acute and chronic dermatitis (including, but not limited to eczema and/or psoriasis), macular degeneration, neuropathy, scleroderma, and ulcerative colitis.
[0131] A summary of indications/conditions for which the active agents (i.e., oxpholipin peptides described herein) are believed to be effective is shown in Table 2.
Table 2. Summary of conditions in which the active agents (e.g., OxP-1 ID) are believed to be effective.
atherosclerosis/ symptoms/ consequences thereof
plaque formation
lesion formation
myocardial infarction
stroke congestive heart failure
vascular function:
arteriole function
arteriolar disease
associated with aging
associated with alzheimer's disease associated with chronic kidney disease associated with hypertension associated with multi-infarct dementia associated with subarachnoid hemorrhage peripheral vascular disease
pulmonary disease:
chronic obstructive pulmonary disease (COPD), emphysema
asthma
idiopathic pulmonary fibrosis
pulmonary fibrosis
adult respiratory distress syndrome
osteoporosis
Paget's disease
coronary calcification
autoimmune:
rheumatoid arthritis
polyarteritis nodosa
polymyalgia rheumatica
lupus erythematosus
multiple sclerosis
Wegener's granulomatosis central nervous system vasculitis (CNSV)
Sjogren's syndrome
Scleroderma
polymyositis.
AIDS inflammatory response
infections:
bacterial
fungal
viral
parasitic
influenza (including avian flu)
viral pneumonia
endotoxic shock syndrome
sepsis
sepsis syndrome (clinical syndrome where it appears that the patient is septic but no organisms are recovered from the blood)
trauma/wound:
organ transplant
transplant atherosclerosis
transplant rejection
corneal ulcer
chronic/non-healing wound
ulcerative colitis
reperfusion injury (prevent and/or treat)
ischemic reperfusion injury (prevent and/or treat)
spinal cord injuries (mitigating effects)
cancers
myeloma/multiple myeloma
ovarian cancer
breast cancer
colon cancer
bone cancer
cervical cancer
prostate cancer
osteoarthritis
inflammatory bowel disease
allergic rhinitis
cachexia
diabetes
Alzheimer's disease
implanted prosthesis
biofilm formation
Crohns' disease
renal failure (e.g., acute renal failure, chronic renal failure, end stage renal failure)
sickle cell disease, sickle cell crisis
amelioration of adriamycin toxicity
amelioration of anthracylin toxicity
to improve insulin sensitivity
to treat the metabolic syndrome
to increase adiponectin
to reduce abdominal fat
dermatitis, acute and chronic
eczema
psoriasis
contact dermatitis
scleroderma diabetes and related conditions
Type I Diabetes
Type II Diabetes
Juvenile Onset Diabetes
Prevention of the onset of diabetes
Diabetic Nephropathy
Diabetic Neuropathy
Diabetic Retinopathy
erectile dysfunction
macular degeneration
multiple sclerosis
nephropathy
neuropathy
Parkinson's Disease
peripheral vascular disease
meningitis
Specific biological activities:
increase Heme Oxygenase 1
increase extracellular superoxide dismutase
prevent endothelial sloughing
prevent the association of myeloperoxidase with ApoA-I
prevent the nitrosylation of tyrosine in ApoA-I
render HDL anti-inflammatory
improve vasoreactivity
increase the formation of pre-beta HDL
promote reverse cholesterol transport
promote reverse cholesterol transport from macrophages
synergize the action of statins
[0132] It is noted that the conditions listed in Table 2 are intended to be illustrative and not limiting.
K) Administration.
[0133] Typically the oxpholipin peptide(s) will be administered to a mammal (e.g., a human) in need thereof. Such a mammal will typically include a mammal (e.g. a human) having or at risk for one or more of the pathologies described herein.
[0134] The active agent(s) can be administered, as described herein, according to any of a number of standard methods including, but not limited to injection, suppository, nasal spray, time-release implant, transdermal patch, and the like. In one particularly preferred embodiment, the peptide(s) are administered orally (e.g. as a syrup, capsule, or tablet).
[0135] The methods involve the administration of a single oxpholipin peptide described herein or the administration of two or more different oxpholipin peptides. The oxpholipin peptides can be provided as monomers (e.g., in separate or combined
formulations), or in dimeric, oligomeric or polymeric forms. In certain embodiments, the multimeric forms may comprise associated monomers (e.g., ionically or hydrophobically linked) while certain other multimeric forms comprise covalently linked monomers (directly linked or through a linker).
[0136] While the invention is described with respect to use in humans, it is also suitable for animal, e.g. veterinary use. Thus certain preferred organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, largomorphs, and the like.
[0137] The methods of this invention are not limited to humans or non-human animals showing one or more symptom(s) of the pathologies described herein, but are also useful in a prophylactic context. Thus, the active agents of this invention can be
administered to organisms to prevent the onset/development of one or more symptoms of the pathologies described herein (e.g., atherosclerosis, stroke, etc.). Particularly preferred subjects in this context are subjects showing one or more risk factors for the pathology. Thus, for example, in the case of atherosclerosis risk factors include family history, hypertension, obesity, high alcohol consumption, smoking, high blood cholesterol, high blood triglycerides, elevated blood LDL, VLDL, IDL, or low HDL, diabetes, or a family history of diabetes, high blood lipids, heart attack, angina or stroke, etc.
VII. Pharmaceutical formulations and devices. A) Pharmaceutical formulations.
[0138] In various embodiments one more oxpholipin peptides are administered, e.g. to an individual diagnosed as having one or more symptoms of atherosclerosis, or as being at risk for atherosclerosis, and/or diagnosed as having high cholesterol or being at risk for high cholesterol, and or the various other pathologies described herein. The oxpholipin peptide(s) can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the present method. Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.
[0139] For example, acid addition salts are prepared from the free base using conventional methodology, that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt may be reconverted to the free base by treatment with a suitable base. Particularly preferred acid addition salts of the active agents herein are halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the active agents of this invention are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Particularly preferred basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.
[0140] For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate (i.e., break down into the individual entities of drug and counterion) in an aqueous environment. [0141] Preferably, the counterion is a pharmaceutically acceptable counterion.
Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.
[0142] Preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups that may be present within the molecular structure of the drug. The esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.
[0143] Amides and prodrugs can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Prodrugs are typically prepared by covalent attachment of a moiety that results in a compound that is therapeutically inactive until modified by an individual's metabolic system.
[0144] The oxpholipin peptide(s) identified herein are useful for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., atherosclerosis and/or symptoms thereof, pathologies characterized by an inflammatory response, etc.). The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, lipid complexes, etc. [0145] In various embodiments the oxpholipin peptide(s) described herein can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
[0146] Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).
[0147] The excipients are preferably sterile and generally free of undesirable matter.
These compositions may be sterilized by conventional, well-known sterilization techniques.
[0148] In therapeutic applications, the oxpholipin peptide(s) are administered to a patient suffering from one or more symptoms of one or more pathologies described herein, or at risk for one or more of the pathologies described herein in an amount sufficient to prevent and/or cure and/or or at least partially prevent, slow the progression, or arrest the disease and/or its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient.
[0149] The concentration of oxpholipin peptide(s) can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosages range from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about 7.2 mg/kg/day to about 1 1.0 mg/kg/day, and most preferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferred embodiments, dosages range from about 10 mg/kg/day to about 50 mg/kg/day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.
[0150] In certain embodiments, the oxpholipin peptide(s) described herein are administered orally (e.g. via a tablet) or as an injectable in accordance with standard methods well known to those of skill in the art. In other preferred embodiments, the peptides, may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent(s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an upper backing layer. It will be appreciated that the term
"reservoir" in this context refers to a quantity of "active ingredient(s)" that is ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs.
[0151] In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, poly ethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.
[0152] Other formulations for topical drug delivery include, but are not limited to, ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent, are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil. Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.
[0153] In various embodiments the oxpholipin peptides described herein comprising
D-form amino acids can be administered, even orally, without protection against proteolysis by stomach acid, etc. Nevertheless, in certain embodiments, peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Patent 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).
[0154] Elevated serum half-life can be maintained by the use of sustained-release protein "packaging" systems. Such sustained release systems are well known to those of skill in the art. In one preferred embodiment, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy (1998) Biotechnol. Prog., 14: 108; Johnson et al. (1996) Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the active agent in a polymer matrix that can be compounded as a dry formulation with or without other agents. [0155] The ProLease microsphere fabrication process was specifically designed to achieve a high encapsulation efficiency while maintaining integrity of the active agent. The process consists of (i) preparation of freeze-dried drug particles from bulk by spray freeze- drying the drug solution with stabilizing excipients, (ii) preparation of a drug-polymer suspension followed by sonication or homogenization to reduce the drug particle size, (iii) production of frozen drug-polymer microspheres by atomization into liquid nitrogen, (iv) extraction of the polymer solvent with ethanol, and (v) filtration and vacuum drying to produce the final dry-powder product. The resulting powder contains the solid form of the active agents, which is homogeneously and rigidly dispersed within porous polymer particles. The polymer most commonly used in the process, poly(lactide-co-glycolide) (PLG), is both biocompatible and biodegradable.
[0156] Encapsulation can be achieved at low temperatures (e.g., -40°C). During encapsulation, the protein is maintained in the solid state in the absence of water, thus minimizing water-induced conformational mobility of the protein, preventing protein degradation reactions that include water as a reactant, and avoiding organic-aqueous interfaces where proteins may undergo denaturation. A preferred process uses solvents in which most proteins are insoluble, thus yielding high encapsulation efficiencies (e.g., greater than 95%).
[0157] In another embodiment, one or more components of the solution can be provided as a "concentrate", e.g., in a storage container (e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water.
[0158] The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised. B) Lipid-based formulations.
[0159] In certain embodiments, the oxpholipin peptide(s) described herein are administered in conjunction with one or more lipids. The lipids can be formulated as an excipient to protect and/or enhance transport/uptake of the active agents or they can be administered separately.
[0160] Without being bound by a particular theory, it is believed that that administration (e.g. oral administration, nasal administration, etc.) of certain phospholipids can significantly increase HDL/LDL ratios. In addition, it is believed that certain medium- length phospholipids are transported by a process different than that involved in general lipid transport. Thus, co-administration of certain medium-length phospholipids with the oxpholipin peptide(s) described herein can confer a number of advantages: They protect the active agents from digestion or hydrolysis, they can improve uptake, and they may improve HDL/LDL ratios.
[0161] The lipids can be formed into liposomes that encapsulate the oxpholipin peptide(s) described herein and/or they can be complexed/admixed with the oxpholipin peptides and/or they can be covalently coupled to the oxpholipin peptide(s). Methods of making liposomes and encapsulating reagents are well known to those of skill in the art (see, e.g., Martin and Papahadjopoulos (1982) J. Biol. Chem., 257: 286-288;
Papahadjopoulos et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 11460-11464; Huang et al. (1992) Cancer Res., 52:6774-6781; Lasic et al. (1992) FEBS Lett., 312: 255-258., and the like).
[0162] Illustrative phospholipids for use in these methods have fatty acids ranging from about 4 carbons to about 24 carbons in the sn-1 and sn-2 positions. In certain preferred embodiments, the fatty acids are saturated. In other preferred embodiments, the fatty acids can be unsaturated. Various fatty acids are illustrated in Table 3.
Table 3. Illustrative fatty acids in the sn-1 and/or sn-2 position of phospholipids for administration of oxpholipin peptide(s) described herein.
Carbon No. Common Name IUPAC Name
3:0 Propionoyl Trianoic
4:0 Butanoyl Tetranoic
5:0 Pentanoyl Pentanoic
6:0 Caproyl Hexanoic
7:0 Heptanoyl Heptanoic
8:0 Capryloyl Octanoic
9:0 Nonanoyl Nonanoic
10:0 Capryl Decanoic
11 :0 Undcanoyl Undecanoic
12:0 Lauroyl Dodecanoic
13:0 Tridecanoyl Tridecanoic
14:0 Myristoyl Tetradecanoic
15:0 Pentadecanoyl Pentadecanoic
16:0 Palmitoyl Hexadecanoic
17:0 Heptadecanoyl Heptadecanoic 18:0 Stearoyl Octadecanoic
19:0 Nonadecanoyl Nonadecanoic
20:0 Arachidoyl Eicosanoic
21 :0 Heniecosanoyl Heniecosanoic
22:0 Behenoyl Docosanoic
23:0 Trucisanoyl Trocosanoic
24:0 Lignoceroyl Tetracosanoic
14: 1 Myristoleoyl (9-cis)
14: 1 Myristelaidoyl (9-trans)
16: 1 Palmitoleoyl (9-cis)
16: 1 Palmitelaidoyl (9-trans)
The fatty acids in these positions can be the same or different. Certain suitable
phospholipids have phosphorylcholine at the sn-3 position.
C) Specialized delivery/devices. 1. Drug-eluting stents.
[0163] Restenosis, the reclosure of a previously stenosed and subsequently dilated peripheral or coronary vessel occurs at a significant rate (e.g., 20-50% for these procedures) and is dependent on a number of clinical and morphological variables. Restenosis may begin shortly following an angioplasty procedure, but usually ceases at the end of approximately six (6) months.
[0164] A recent technology that has been developed to address the problem of restenosis in intravascular stents. Stents are typically devices that are permanently implanted (expanded) in coronary and peripheral vessels. The goal of these stents is to provide a long-term "scaffolding" or support for the diseased (stenosed) vessels. The theory being, if the vessel is supported from the inside, it will not close down or restenose.
[0165] Known stent designs include, but are not limited to monofilament wire coil stents (see, e.g., U.S. Patent 4,969,458); welded metal cages (see, e.g., U.S. Patents 4,733,665 and 4,776,337), thin-walled metal cylinders with axial slots formed around the circumference (see, e.g., U.S. Patents 4,733,665, 4,739,762, 4,776,337, and the like).
Known construction materials for use in stents include, but are not limited to polymers, organic fabrics and biocompatible metals, such as, stainless steel, gold, silver, tantalum, titanium, and shape memory alloys such as Nitinol. [0166] To further prevent restenosis, stents can be covered and/or impregnated with one or more pharmaceutical, e.g., in controlled release formulations to inhibit cell proliferation associated with rest enosis. Most commonly such "drug-eluting" stents are designed to deliver various cancer drugs (cytotoxins). [0167] However, because of their activity in mitigating inflammatory responses, reducing and/or eliminated oxidized lipids and/or other oxidized species, inhibiting macrophage chemotactic activity and the like, the active agents described herein are well suited to prevent restenosis. Thus, in certain embodiments, this invention contemplates stents having one or more of the oxpholipin peptide(s) described herein coated on the surface and/or retained within cavities or microcavities in the surface of the stent.
[0168] In certain embodiments the active agents are contained within biocompatible matrices (e.g. biocompatible polymers such as urethane, silicone, and the like). Suitable biocompatible materials are described, for example, in U.S. Patent Publications
20050084515, 200500791991 , 20050070996, and the like. In various embodiments the polymers include, but are not limited to silicone-urethane copolymer, a polyurethane, a phenoxy, ethylene vinyl acetate, polycaprolactone, poly(lactide-co-glycolide), polylactide, polysulfone, elastin, fibrin, collagen, chondroitin sulfate, a biocompatible polymer, a biostable polymer, a biodegradable polymer
[0169] Thus, in certain embodiments this invention provides a stent for delivering drugs to a vessel in a body. The stent typically comprises stent framework including a plurality of reservoirs formed therein. The reservoirs typically include an active agent and/or active agent-containing polymer positioned in the reservoir and/or coated on the surface of the stent. In various embodiments the stent is a metallic base or a polymeric base. Certain preferred stent materials include, but are not limited to stainless steel, nitinol, tantalum, MP35N alloy, platinum, titanium, a suitable biocompatible alloy, a suitable biocompatible polymer, and/or a combination thereof.
[0170] In various embodiments where the stent comprises pores (e.g. reservoirs), the pores can include micropores (e.g., having a diameter that ranges from about 10 to about 50 μιη, preferably about 20 μιη or less). In various embodiments the micropores have a depth in the range of about 10 μιη to about 50 μιη. In various embodiments the micropores extend through the stent framework having an opening on an interior surface of the stent and an opening on an exterior surface of the stent. In certain embodiments the stent can, optionally comprise a cap layer disposed on the interior surface of the stent framework, the cap layer covering at least a portion of the through-holes and providing a barrier
characteristic to control an elution rate of the active agent(s) in the polymer from the interior surface of the stent framework. In various embodiments the reservoirs comprise channels along an exterior surface of the stent framework. The stent can optionally have multiple layers of polymer where different layers of polymer carry different active agent(s) and/or other drugs.
[0171] In certain embodiments the stent comprises: an adhesion layer positioned between the stent framework and the polymer. Suitable adhesion layers include, but are not limited to a polyurethane, a phenoxy, poly(lactide-co-glycolide)- , polylactide, polysulfone, polycaprolactone, an adhesion promoter, and/or a combination thereof.
[0172] In addition to stents, the oxpholipin peptide(s) can be coated on or contained within essentially any implantable medical device configured for implantation in a extravascular and/or intravascular location.
[0173] Also provided are methods of manufacturing a drug-polymer stent, comprising. The methods involve providing a stent framework; cutting a plurality of reservoirs in the stent framework, e.g., using a high power laser; applying one or more of the oxpholipin peptide(s) and/or a drug polymer to at least one reservoir; drying the drug polymer; applying a polymer layer to the dried drug polymer; and drying the polymer layer. The active agent(s) and/or polymer(s) can be applied by any convenient method including but not limited to spraying, dipping, painting, brushing and dispensing.
[0174] Also provided are methods of treating a vascular condition and/or a condition characterized by an inflammatory response and/or a condition characterized by the formation of oxidized reactive species. The methods typically involve positioning a stent or other implantable device as described above within the body (e.g. within a vessel of a body) and eluting at least one oxpholipin peptide from at least one surface of the implant.
2. Impregnated grafts and transplants.
[0175] Vascular grafts can be classified as either biological or synthetic. There are two commonly used types of biological grafts. An autograft is one taken from another site in the patient. In peripheral vascular surgery by far the most commonly used such graft is the long saphenous vein. This can be used in situ with the valves surgically destroyed with an intraluminal cutting valvutome.
[0176] Alternatively, the vein can be removed and reversed but this typically produces a discrepancy between the anastomotic size of the artery and vein. In thoracic surgery the use of internal mammary artery for coronary artery bypass surgery is another example of an autograft. An allograft is one taken from another animal of the same species. Externally supported umbilical vein is rarely used but is an example of such a graft.
[0177] Synthetic grafts are most commonly made from Dacron or
polytetrafluroethylene (PTFE). Dacron grafts are frequently used in aortic and aorto-iliac surgery. Below the inguinal ligament the results of all synthetic grafts are inferior to those obtained with the use of vein grafts. Suitable vein is not always available and in this situation PTFE is typically used. It can be used in conjunction with vein as a composite graft. Neointimal hyperplasia at the distal anastomosis can be reduced by the incorporation of a segment of vein as either a Millar Cuff or Taylor Patch to improve the long-term patency of the grafts.
[0178] The commonest complications associated with the use of vascular grafts include Graft occlusion, Graft infection, true and false aneurysms at the site of anastomosis, distal embolization, and erosion in to adjacent structures - e.g. Aorto-enteric fistulae. Many of these conditions are associated with an inflammatory response, macrophage migration into the site, and/or the formation of reactive oxygen species {e.g., oxidized lipids). To reduce such complications, the graft (synthetic or biological can be soaked, or otherwise coated, with one or more of the oxpholipin peptide(s) described herein.
[0179] In addition, it is contemplated that other implantable tissues or materials can similarly be impregnated or coated with one or more oxpholipin peptide(s) described herein. Thus, for example, in certain embodiments this invention contemplates the use of impregnated sutures to minimize inflammation and/or infection and/or tissue rejection.
3. Subcutaneous matrices.
[0180] In certain embodiments, one or more oxpholipin peptides described herein are administered alone or in combination with other therapeutics as described herein in implantable {e.g., subcutaneous) matrices. [0181] A major problem with standard drug dosing is that typical delivery of drugs results in a quick burst of medication at the time of dosing, followed by a rapid loss of the drug from the body. Most of the side effects of a drug occur during the burst phase of its release into the bloodstream. Secondly, the time the drug is in the bloodstream at therapeutic levels is very short, most is used and cleared during the short burst.
[0182] Drugs (e.g., the oxpholipin peptides described herein) imbedded in various matrix materials for sustained release provides some solution to these problems. Drugs embedded, for example, in polymer beads or in polymer wafers have several advantages. First, most systems allow slow release of the drug, thus creating a continuous dosing of the body with small levels of drug. This typically prevents side effects associated with high burst levels of normal injected or pill based drugs. Secondly, since these polymers can be made to release over hours to months, the therapeutic span of the drug is markedly increased. Often, by mixing different ratios of the same polymer components, polymers of different degradation rates can be made, allowing remarkable flexibility depending on the agent being used. A long rate of drug release is beneficial for people who might have trouble staying on regular dosage, such as the elderly, but is also an ease of use
improvement that everyone can appreciate. Most polymers can be made to degrade and be cleared by the body over time, so they will not remain in the body after the therapeutic interval. [0183] Another advantage of polymer based drug delivery is that the polymers often can stabilize or solubilize proteins, peptides, and other large molecules that would otherwise be unusable as medications. Finally, many drug/polymer mixes can be placed directly in the disease area, allowing specific targeting of the medication where it is needed without losing drug to the "first pass" effect. This is certainly effective for treating the brain, which is often deprived of medicines that can't penetrate the blood/brain barrier.
[0184] A number of implantable matrix (sustained release) systems are know to those of skill and can readily be adapted for use with one or more of the active agents described herein. Suitable sustained release systems include, but are not limited to Re- Gel®, SQ2Gel®, and Oligosphere® by MacroMed, ProLease® and Medisorb® by
Alkermes, Paclimer® and Gliadel® Wafer by Guilford pharmaceuticals, the Duros implant by Alza, acoustic bioSpheres by Point Biomedical, the Intelsite capsule by Scintipharma, Inc., and the like. 4. Other "specialty delivery systems".
[0185] Other "specialty" delivery systems include, but are not limited to lipid based oral mist that allows absorption of drugs across the oral mucosa, developed by Generex Biotechnology, the oral transmucosal system (OTS™) by Anesta Corp., the inhalable dry powder and PulmoSpheres technology by Inhale Therapeutics, the AERx® Pulmonary Drug Delivery System by Aradigm, the AIR mechanism by Alkermes, and the like.
[0186] Another approach to delivery developed by Alkermes is a system targeted for elderly and pediatric use, two populations for which taking pills is often difficult is known as Drug Sipping Technology (DST). The medication is placed in a drinking straw device, prevented from falling out by filters on either end of it. The patient merely has to drink clear liquid (water, juice, soda) through the straw. The drug dissolves in the liquid as it is pulled through and is ingested by the patient. The filter rises to the top of the straw when all of the medication is taken. This method has the advantage in that it is easy to use, the liquid often masks the medication's taste, and the drug is pre-dissolved for more efficient absorption.
[0187] It is noted that these uses and delivery systems are intended to be illustrative and not limiting. Using the teachings provided herein, other uses and delivery systems will be known to those of skill in the art.
VIII. Additional pharmacologically active agents,
[0188] In various embodiments, the use of oxpholipins in combination with other active agents is contemplated in the treatment of the various pathologies/indications described herein. The use of combinations of active agents can alter pharmacological activity, bioavailability, and the like.
[0189] For example, it is believed that administration of one or more oxpholipins described herein in combination with a salicylanilide (e.g., niclosamide) as described in PCT Publication; Application No: PCT/US2007/017551 (WO 2008/021088) can increase bioavailability and/or activity of an orally administered peptide.
[0190] Additional pharmacologically active materials (i.e., drugs) can be delivered in conjunction with one or more of the oxpholipin peptide(s) described herein. In certain embodiments, such agents include, but are not limited to agents that reduce the risk of atherosclerotic events and/or complications thereof. Such agents include, but are not limited to beta blockers, beta blocker and thiazide diuretic combinations, statins, aspirin, ace inhibitors, ace receptor inhibitors (ARBs), and the like.
[0191] In certain embodiments the agents can be administered in conjunction with the oxpholipin (e.g., before, after, or at the same time as the oxpholipin(s)), or they can be administered as a combined formulation.
[0192] In certain embodiments methods are provided that involve administering one or more of the oxpholipins described herein in conjunction with one or more statins. In certain embodiments, when administered in conjunction with each other the oxpholipin(s) and/or the statins can be administered at significantly lower dosages thereby avoiding various harmful side effects (e.g., muscle wasting) associated with high dosage statin use and/or the anti-inflammatory properties of statins at any given dose are significantly enhanced.
[0193] Suitable statins include, but are not limited to atorvastatin (LIPITOR®,
TORVAST®), cerivastatin (e.g., LIPOBAY®, BAYCOL®) fluvastatin (e.g., LESCOL®, LESCOL XL®), lovastatin (e.g. , MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin, pitavastatin (e.g., LIVALO®, PITAVA®), pravastatin (e.g., PRAVACHOL®,
SELEKTINE®), rosuvastatin (e.g., CRESTOR®), simvastatin (e.g., ZOCOR®, LIPEX®), simvastatin+ezetimibe (e.g. , VYTORIN®), therapy lovastatin+niacin extended-release (e.g. , ADVICOR®), atorvastatin+amlodipine besylate (e.g., CADUET®), simvastatin+niacin extended-release (e.g., SIMCOR®), and the like.
[0194] In various embodiments the oxpholipin(s) described herein are administered in conjunction with one or more beta blockers. Suitable beta blockers include, but are not limited to cardioselective (selective beta 1 blockers), e.g., acebutolol (SECTRAL®), atenolol (TENORMIN®), betaxolol (KERLONE®), bisoprolol (ZEBETA®), metoprolol (LOPRESSOR®), and the like. Suitable non-selective blockers (block beta 1 and beta 2 equally) include, but are not limited to carteolol (CARTROL®), nadolol (CORGARD®), penbutolol (LEVATOL®), pindolol (VISKEN®), propranolol (INDERAL®), timolol (BLOCKADREN®), labetalol (NORMODYNE®, TRANDATE®), and the like.
[0195] Suitable beta blocker thiazide diuretic combinations include, but are not limited to LOPRESSOR HCT®, ZIAC®, TENORETIC®, CORZIDE®, TIMOLIDE®, INDERAL LA 40/25®, INDERIDE®, NORMOZIDE®, and the like. [0196] Suitable ace inhibitors include, but are not limited to captopril (e.g.
CAPOTEN® by Squibb), benazepril (e.g., LOTENSIN® by Novartis), enalapril (e.g., VASOTEC® by Merck), fosinopril (e.g., MONOPRIL® by Bristol-Myers), lisinopril (e.g. PRINIVIL® by Merck or ZESTRIL® by Astra-Zeneca), quinapril (e.g. ACCUPRIL® by Parke -Davis), ramipril (e.g., ALT ACE® by Hoechst Marion Roussel, King
Pharmaceuticals), imidapril, perindopril erbumine (e.g., ACEON® by Rhone-Polenc Rorer), trandolapril (e.g., MAVIK® by Knoll Pharmaceutical), and the like. Suitable ARBS (Ace Receptor Blockers) include but are not limited to losartan (e.g. COZAAR® by Merck), irbesartan (e.g., AVAPRO® by Sanofi), candesartan (e.g., ATACAND® by Astra Merck), valsartan (e.g., DIOVAN® by Novartis), and the like.
[0197] In various embodiments, one or oxpholipin peptide(s) described herein are administered with one or more of the drugs identified below.
[0198] Thus, in certain embodiments one or more oxpholipin(s) are administered in conjunction with cholesteryl ester transfer protein (CETP) inhibitors (e.g., torcetrapib, JTT- 705. CP-529414) and/or acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitors (e.g., Avasimibe (CI-1011), CP 113818, F-1394, and the like), and/or immunomodulators (e.g., FTY720 (sphingosine-1 -phosphate receptor agonist), THALOMID® (thalidomide), IMURAN® (azathioprine), COPAXONE® (glatiramer acetate), CERTICAN®
(everolimus), NEORAL® (cyclosporine), antd the like), and/or dipeptidyl-peptidase-4 (DPP4) inhibitors (e.g., 2-Pyrrolidinecarbonitrile, l-[[[2-[(5-cyano-2-pyridinyl)
amino] ethyl] amino ]acetyl ], see also U.S. Patent Publication 2005-0070530), and/or calcium channel blockers (e.g., ADALAT®, ADALAT CC®, CALAN®, CALAN SR®, CARDENE®, CARDIZEM®, CARDIZEM CD®, CARDIZEM SR®, DILACOR-XR®, DYNACIRC®, ISOPTIN®, ISOPTIN SR®, NIMOTOP®, NORVASC®, PLENDIL®, PROCARDIA®, PROCARDIA XL®, VASCOR®, VERELAN®), and/or peroxisome proliferator-activated receptor (PPAR) agonists for, e.g., α, γ; δ receptors (e.g., Azelaoyl PAF, 2-Bromohexadecanoic acid, CIGLITIZONE®, CLOFIBRATE®, 15-Deoxy-512'14- prostaglandin J2, Fenofibrate, Fmoc-Leu-OH, GW1929, GW7647, 8(S)-Hydroxy- (5Z,9E,1 lZ,14Z)-eicosatetraenoic acid (8(S)-HETE), Leukotriene B4, LY-171,883
(Tomelukast), Prostaglandin A2, Prostaglandin J2, Tetradecylthioacetic acid (TTA), Troglitazone (CS-045), WY-14643 (Pirinixic acid)), and the like.
[0199] In certain embodiments one or more oxpholipin(s) are administered in conjunction with fibrates (e.g., clofibrate (atromid), gemfibrozil (lopid), fenofibrate (tricor), etc.), bile acid sequestrants {e.g., cholestyramine, colestipol, etc.), cholesterol absorption blockers {e.g., ezetimibe (Zetia), etc.), VYTORIN® ((ezetimibe/simvastatin combination), and/or steroids, warfarin, and/or aspirin, and/or Bcr-Abl inhibitors/antagonists {e.g., Gleevec (Imatinib Mesylate), AMN107, STI571 (CGP57148B), ON 012380, PLX225, and the like), and/or renin angiotensin pathway blockers {e.g., Losartan (COZAAR®), Valsartan (Diovan®), Irbesartan (Avapro®), Candesartan (Atacand®), and the like), and/or angiotensin II receptor antagonists {e.g,. losartan (Cozaar), valsartan (Diovan), irbesartan (Avapro), candesartan (Atacand) and telmisartan (Micardis), etc.), and/or PKC inhibitors {e.g., Calphostin C, Chelerythrine chloride, Chelerythrine . chloride, Copper bis-3, 5- diisopropylsalicylate, Ebselen, EGF Receptor (human) (651-658) (N-Myristoylated), Go 6976, H-7 . dihydrochloride, l-O-Hexadecyl-2-O-methyl-rac-glycerol, Hexadecyl- phosphocholine (Ci6:o); Miltefosine, Hypericin, Melittin (natural), Melittin (synthetic), ML- 7 . hydrochloride, ML-9 . hydrochloride, Palmitoyl-DL-carnitine . hydrochloride, Protein Kinase C (19-31), Protein Kinase C (19-36), Quercetin dihydrate, Quercetin . dihydrate, D- erytAro-Sphingosine (isolated), D-erytAro-Sphingosine (synthetic), Sphingosine, N,N- dimethyl, D-erytAro-Sphingosine, Dihydro-, D-erytAro-Sphingosine, Ν,Ν-Dimethyl-, D- erytAro-Sphingosine chloride, Ν,Ν,Ν-Trimethyl-, Staurosporine, Bisindolylmaleimide I, G- 6203, and the like).
[0200] In certain embodiments, one or more of the oxpholipins are administered in conjunction with ApoAI, Apo A-I derivatives and/or agonists {e.g., L-4F, D-4F, see, e.g., PCT/US2001/026497 (WO 2002/015923), PCT/US2003/032442 (WO 2004/034977), PCT/US2004/026288 (WO/2005/016280), PCT/US2003/09988 ((WO 2003/086326), and the like). ApoAI milano, see, e.g., U.S. Patent Publications 20050004082, 20040224011, 20040198662, 20040181034, 20040122091, 20040082548, 20040029807, 20030149094, 20030125559, 20030109442, 20030065195, 20030008827, and 20020071862, and U.S. Patents 6,831,105, 6,790,953, 6,773,719, 6,713,507, 6,703,422, 6,699,910, 6,680,203, 6,673,780, 6,646,170, 6,617,134, 6,559,284, 6,506,879, 6,506,799, 6,459,003, 6,423,830, 6,410,802, 6,376,464, 6,367,479, 6,329,341, 6,287,590, 6,090,921, 5,990,081, and the like), renin inhibitors {e.g., SPP630 and SPP635, SPP100, Aliskiren, and the like), and/or MR antagonist {e.g., spironolactone, aldosterone glucuronide, and the like), and/or aldosterone synthase inhibitors, and/or alpha-adrenergic antagonists {e.g., ALDOMET® (Methyldopa), CARDURA® (Doxazosin), CATAPRES®; CATAPRES-TTS®; Duraclon™ (Clonidine), DIBENZYLINE® (Phenoxybenzamine), Hylorel® (Guanadrel), Hytrin® (Terazosin), MINIPRESS® (Prazosin), TENEX® (Guanfacine), Guanabenz, Phentolamine, Reserpine, and the like), and/or liver X receptor (LXR) agonists (e.g., T0901317, GW3965, ATI-829, acetyl-podocarpic dimer (APD), and the like), and/or farnesoid X receptor (FXR) agonists (e.g., GW4064, 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), T0901317, and the like), and/or plasminogen activator- 1 (PAI-1) inhibitors (see, e.g., oxime-based PAI-1 inhibitors, see also U.S. Patent 5,639,726, and the like), and/or low molecular weight heparin, and/or AGE inhibitors/breakers (e.g., Benfotiamine, aminoguanidine, pyridoxamine, Tenilsetam, Pimagedine, and the like) and/or ADP receptor blockers (e.g., Clopidigrel, AZD6140, and the like), and/or ABCA1 agonists, and/or scavenger receptor Bl agonists, and/or
Adiponectic receptor agonist or adiponectin inducers, and/or stearoyl-CoA Desaturase I (SCD1) inhibitors, and/or Cholesterol synthesis inhibitors (non-statins), and/or
Diacylglycerol Acyltransferase I (DGAT1) inhibitors, and/or Acetyl CoA Carboxylase 2 inhibitors, and/or LP-PLA2 inhibitors, and/or GLP-1, and/or glucokinase activator, and/or CB-1 agonists, and/or anti-thrombotic/coagulants, and/or Factor Xa inhibitors, and/or GPIIb/IIIa inhibitors, and/or Factor Vila inhibitors, and/or Tissue factor inhibitors, and/or anti-inflammatory drugs, and/or Probucol and derivatives (e.g. AGI-1067, etc.), and/or CCR2 antagonists, and/or CX3CR1 antagonists, and/or IL-1 antagonists, and/or nitrates and NO donors, and/or phosphodiesterase inhibitors, and the like.
[0201] In certain embodiments the oxpholipin(s) described herein can be administered in conjunction with niacin or extended release niacin. Niacin (nicotinic acid) lowers lipids by inhibiting very-low-density lipoprotein (VLDL) production in the liver and reducing the level of VLDL that can be converted into low-density lipoprotein (LDL). Niacin can lower LDL cholesterol by 10 to 25 percent and triglyceride levels by 20 to 50 percent, and can raise levels of high density lipoprotein (HDL) cholesterol by 15 to 35 percent. These effects can be enhanced by administering niacin in conjunction with one or more of the active agents described herein. In certain embodiments, it is believed that administration with one or more of the agents described herein can reduce liver toxicity associated with niacin administration. The niacin can be in a form for immediate delivery (e.g., unmodified niacin), and/or intermediate release niacin (IR niacin, and/or extended release niacin (ER niacin), and/or niacin sustained release (niacin SR), and/or niacin preparations that are modified to avoid interactions with the receptor that mediates the flushing associated with niacin. ER, IR, and SR forms of niacin are known to those of skill in the art. For example, intermediate release (IR) niacin formulations are described, for in U.S. Patent 6,746,691, which is incorporated herein by reference. Inositol hexanicotinate is one form of a sustained release (SR) niacin. One form of extended release niacin is marketed as the drug Niaspan®, while others include, but are not limited to Nicobid, and Slo-Niacin. In various embodiments niacin dosages range from about 300 mg/day up to 3,000 mg/day, more preferably from about 500 mg/day to 1500 mg/day.
[0202] In certain embodiments the niacin is provided as a combined formulation with a statin (e.g. , ADVICOR® is a combination product containing both extended-release niacin and lovastatin) and/or with one or more of the active agents described herein (e.g., - HD (OxP-l lD), etc.). IX. Kits for the treatment of one or more indications.
[0203] In another embodiment this invention provides kits for amelioration of one or more symptoms of atherosclerosis or for the prophylactic treatment of a subject (human or animal) at risk for atherosclerosis and/or the treatment or prophylaxis of one or more of the conditions described herein. The kits preferably comprise a container containing one or more of the oxpholipin peptide(s) described herein. The oxpholipin(s) can be provided in a unit dosage formulation (e.g. suppository, tablet, caplet, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable carriers/excipients.
[0204] The kit can, optionally, further comprise one or more other agents used in the treatment of the condition/pathology of interest. Such agents include, but are not limited to, beta blockers, vasodilators, aspirin, statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like, e.g. as described above.
[0205] In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the
"therapeutics" or "prophylactics" of this invention. Preferred instructional materials describe the use of one or more oxpholipin peptide(s) described herein to mitigate one or more symptoms of atherosclerosis (or other pathologies described herein) and/or to prevent the onset or increase of one or more of such symptoms in an individual at risk for atherosclerosis (or other pathologies described herein). The instructional materials can also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
[0206] While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES
[0207] The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Oxpholipin IIP: An Anti-Inflammatory Peptide that Binds Cholesterol and Oxidized
Phospholipids
[0208] Many Gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT, SEQ ID NO:3) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C -terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind lipids including cholesterol. Several of them manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin- 1 ID (OxP-1 ID), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-1 ID bound oxidized (phospho) lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I- mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable a-helical structure in solution, the OxP-1 ID structure was flexible and contained multiple turn-like features. Given the substantial evidence that oxidized phopholipids are pro-inflammatory in vivo, OxP-1 ID and other Oxpholipins are believed to have therapeutic potential.
Introduction
[0209] Evidence gathered over past two decades has shown that inflammation is a key player in pathophysiology of atherogenic cardiovascular disease (CVD) (Hansson and Libby (2006) Nat. Rev. Immunol. 6: 508-519; Klingenberg and Hansson (2009) Eur. Heart
J. 30(23):2838-2844; Montecucco and Mach (2009) Semin. Immunopathol. 31 : 1-3). Consequently, therapies targeting inflammatory response have already been implemented in clinical practice of CVD (Montecucco and Mach (2009) Best Pract. Res. Clin. Endocrinol. Metab. 23: 389-400; Montecucco and Mach (2009) Semin. Immunopathol. 31 : 127-142; Moubayed et al. (2007) Curr. Opin. Lipidol, 18: 638-644) and further strategies and anti- inflammatory treatment regimens are being investigated (Klingenberg and Hansson (2009) Eur. Heart J. 30(23):2838-2844). An emerging approach in the treatment of cardiovascular disease is based on apoA-I mimetic peptides (Bloedon et al. (2008) J. Lipid Res. 49: 1344- 1352; Navab et al. (2005) Arterioscler. Thromb. Vase. Biol., 25 : 1325-1331; Navab et al. (2006) Nat. Clin. Pract. Cardiovasc. Med., 3 : 540-547; Navab et al. (2008) Curr. Drug Targets, 9: 204-209; Sethi et al. (2007) Curr. Opin. Investig. Drugs, 8: 201-212) that also have anti-inflammatory properties, and act to sequester oxysterols and oxidized lipids (Van Lenten et al. (2008) J. Lipid Res., 49: 2302-231 1). Moreover, these peptides have similar properties to apoA-I Milano (. Riccioni et al. (2002) Int. J. Immunopathol.
Pharmacol. 15: 171-182; Zhu et al. (2005) J. Lipid Res., 46: 1303-131 1). This naturally occurring apoA-I mutant containing an extra cysteine disulfide bridge, reduced atheromas by up to 30% (Chiesa and Sirtori (2003) Curr. Opin. Lipidol., 14: 159-163) and ameliorated plaque build-up in arterial walls (Nissen et al. (2003) JAMA 290: 2292-2300). In contrast to apoA-I Milano, which requires intravenous administration, the most prominent member of apoA-I-mimetic peptide family, D-4F (Ac- DWFKAFYDKVAEKFKEAF-NH2, SEQ ID NO:4), can be taken orally (. Bloedon et al. (2008) J. Lipid Res. 49: 1344-1352; Navab et al. (2006) Nat. Clin. Pract. Cardiovasc. Med., 3 : 540-547; Navab et al. (2004) Circulation 109: 3215-3220; Navab et al. (2005) Circ. Res. 97: 524-532; Navab et al. (2009) J. Lipid Res. 50: \5?>%-\54Ί ;39;46-48).
[0210] This example describes OxP-1 ID, a novel 14-residue peptide whose sequence resembles a cholesterol-binding domain found in a family of pore-forming bacterial exotoxins, and related oxpholipins. The ability of OxP-1 ID to bind oxidized phospholipids and sterols resemble those of D-4F (Van Lenten et al. (2008) J. Lipid Res., 49: 2302-231 1). In addition, OxP-1 ID reduced the release of monocyte chemotactic factors from LDL-stimulated human aortic endothelial cells, another property of D-4F. Materials and Methods
Ethics Statement
[0211] The experiments were performed using protocols approved by the Animal
Research Committee at UCLA. Peptide synthesis and characterization
[0212] Solid phase peptide synthesis was done with a Symphony® automated peptide synthesizer (Protein Technologies Inc., Tucson, AZ) or a CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), using 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields and Noble (1990) Int. J. Pept. Protein Res., 35 : 161-214). Amino acid derivatives and reagents were from EMD
Biosciences (San Diego, CA) or Chem-Impex International (Wood Dale, IL). After cleaving the peptides from the resin with modified reagent K (TFA 94% (v/ v); phenol, 2%> (w/v); water, 2%> (v/v); TIS, 2%> (v/v); 2 hours) they were precipitated with ice-cold diethyl ether and purified to .95% homogeneity by preparative reverse-phase high performance liquid chromatography (RP-HPLC).
[0213] Peptide purity was evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RPHPLC, using a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). The mobile phases were: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile. Analytic assessments used a reversed-phase, 4.6(250 mm CI 8 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 ml/min. Peptides OxP-3, OxP-3D and OxP-21 were obtained by dimerizing the appropriate monomer (10 mg/ml) in a 50% aqueous solution of DMSO at room temperature over 48 h. OxP-22 was obtained by trimerizing OxP20 in 50% aqueous solution of DMF using Tris-[2- maleimidoethyl] amine and the manufacturer's protocol (Pierce Biotechnology, Rockford, IL, Cat#33043). The progress of the reaction was monitored by mass spectrometry. All peptides were lyophilized for storage. Peptide stock solutions were made in HPLC grade water containing 0.01% acetic acid, and peptide concentrations were determined by absorbance at 280 nm (Pace et al. (1995) Protein Sci., 4: 2411-2423). For analytical details concerning synthesized peptides see Supporting Information (Table 4). Lipids
[0214] PAPC (L-a-l-palmitoyl-2-arachidonoyl-5/7-glycero-3-phosphorylcholine),
PAPE (l-palmitoyl-2-arachidonoyl-5n-glycero-3-phosphatidylethanolamine), POVPC (1- palmitoyl-2-(5-oxovaleroyl)-5/7-glycero-3-phosphorylcholine, PGPC (1 -palmitoyl-2- glutaroyl-sn-glycero-3-phosphorylcholine), and POPC (l-Palmitoyl-2-oleoyl-s/?-glycero- 3 -phosphoryl choline) were from Avanti Polar Lipids (Alabaster, AL). Cholesterol, >99% pure, 20(5)-hydroxycholesterol, 22(5)- and 25(5)-hydroxycholesterol, arachidonic acid, linoleic acid, palmitic acid and bovine serum albumin were from Sigma-Aldrich (St. Louis, MO). 13(5)-HPODE (hydroperoxyoctadecadienoic acid), 5(S)-, 12(5)-, and 15(5)-HPETE (hydroperoxyeicosatetraenoic acid), 12(5)- and 15(5)-HETE (hydroxyeicosatetraenoic acid), were from BioMol, Plymouth Meeting, PA. 9(5)- and 13(5)-HODE
(hydroxyoctadecadienoic acid). KOdiA-PC was from Cayman Chemical US (Ann Arbor, MI). 24(5)-hydroxycholesterol was from Steraloids (Newport, RI). PEIPC (l-palmitoyl-2- (5,6-deoxyisoprostane E2)-sft-glycero-3-phosphorylcholine) was prepared as previously described (Subbanagounder et al. (2002) J. Biol. Chem., 277: 7271-7281; Gharavi et al. (2007) Arterioscler. Thromb. Vase. Biol, 27: 1346-1353).
Binding studies
[0215] Binding experiments were done by surface plasmon resonance (SPR) on a
BIAcore 3000 system (BiaCore AB, Piscataway, NJ). Peptide ligands and apoA-I were immobilized on a BIAcore CM5 sensor chip activated per the manufacturer's protocol with N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide. After achieving adequate immobilization, the activated sensor surface was blocked with ethanolamine.
[0216] Lipid stock solutions were prepared in absolute ethanol and then diluted into a standard BIAcore buffer (HBS-EP), containing 10 mM HEPES, pH 7.4, 150 mM
NaCl, 3 mM EDTA and 0.005% (v/v) surfactant P20. Lipid concentrations used in the binding studies were selected to give binding responses of 30-500 resonance units.
Lipid stock solutions were prepared at 1 mg/ml in ethanol. Since the highest analyte lipid concentrations did not exceed 10 μ^ιηΐ, the highest ethanol concentration in any analyte solutions was 1 %, and for most lipids the ethanol concentration was
considerably lower. Ethanol-free HBS-EP buffer was used during the dissociation phase.
Lipid binding was measured by observing the change in the SPR angle as 150 ml of lipid analyte (various concentrations) in HBS-EP buffer flowed over the biosensor for 3 min at 50 μΐ/min. Biosensors were washed with 25 or 50% ethanol to regenerate them between binding studies. SPR data were corrected for background binding to the matrix of the chip ("blank"channel) and analyzed with BIAevaluation 4.1 software (Biacore, Piscataway, NJ). Human monocytes and aortic endothelial cells
[0217] Normal human monocytes were isolated as previously described (Fogelman et al. (1988) J. Lipid Res., 29: 1243-1247). Human aortic endothelial cells (HAEC) were isolated and maintained as previously reported (Navab et al. (1991) J. Clin. Invest., 88: 2039-2046). Acquisition and use of these cells was in accordance with protocols approved by the UCLA Human Research Subject Protection Committee.
Monocyte chemotaxis assays
[0218] As previously described (Fogelman et al. (1988) J. Lipid Res., 29: 1243-
1247; Navab et al. (1991) J. Clin. Invest., 88: 2039-2046), HAEC cells were treated with native LDL (250 μg/ml) in the absence or presence of HDL or tested peptides for 8 h. After these cultures were washed, the medium was replaced by fresh Medium 199 and the cultures were incubated for an additional 8 h. This culture medium was collected and assayed for monocyte chemotactic activity using chambers purchased from Neuroprobe (Cabin John, MD). After monocytes were added to the upper compartment, the chamber was incubated for 60 min at 37°C and subsequently disassembled. The membrane was rinsed, air dried, fixed with 1%> glutaraldehyde, stained with 0.1 %> crystal violet dye, and 12 standardized high power fields were examined microscopically. The number of migrated monocytes was expressed as the mean ±SD of monocytes counted. Values obtained in the absence of HDL were normalized to a value of 1.0. Normalized values >1.0 after HDL addition were considered to be pro-inflammatory, and values < 1.0 as being anti- inflammatory.
Mouse experiments
[0219] ApoE null mice on a C57BL/6J background, originally from Jackson
Laboratories (Bar Harbor, ME), were maintained in a breeding colony in the Department of Laboratory and Animal Medicine at the David Geffen School of Medicine at UCLA. The mice were maintained on a chow diet (Ralston Purina, St. Louis, MO). [0220] Groups of 6 fasting female apoE deficient mice, 4-6 months of age, were injected subcutaneously with 200 μΐ. of either ABCT buffer (50 mM NH4HC03, 0.1% Tween 20) or 1.0 mg/kg of OxP peptides in ABCT buffer. Six hours later, and with continued fasting, blood was removed from the retro-orbital sinus under mild isoflurane anesthesia, and anticoagulated with heparin (2.5 U/ml). Plasma was obtained and fractionated by FPLC. HDL-containing fractions were tested in HAEC cultures for their ability to inhibit LDL-induced monocyte chemotactic activity. All procedures conformed to regulations of the UCLA Animal Research Committee. An HDL inflammatory index was determined using the standard monocyte chemotaxis assay. Indices >1 were interpreted as pro-inflammatory, and indices <1 as anti-inflammatory.
Circular dichroism (CD) analyses of secondary structure
[0221] CD spectra from 190-260 nm of D-4F and OxP-1 ID were examined in different solution environments using a JASCO 715 spectropolarimeter (Jasco Inc., Easton, MD) that was calibrated for wavelength and optical rotation with 10-camphorsulphonic acid (Johnson (1990) Proteins 7: 205-214; Miles et al. (2003) Spectroscopy 17: 653-661). Peptides were scanned at 20 nm per minute in 0.01 cm path-length cells at 25°C with a sample interval of 0.2 nm. Peptide concentration was determined by UV absorbance at 280 nm. After baseline correction, the spectra were expressed as the Mean Residue Ellipticity [0]MRE- Quantitative estimates of the secondary structural contributions were made with VARSLC (Johnson (1999) Proteins 35 : 307-312) using the spectral basis set for proteins implemented in the Olis Global WorksTM software package (Olis Inc., Bogart, GA).
Fourier transform infrared (FTIR) spectroscopy
[0222] Infrared spectra were recorded at 25°C using a Bruker Vector 22™ FTIR spectrometer with a deuterated triglycine sulfate (DTGS) detector, and averaged over 256 scans at a gain of 4 with a resolution of 2 cm-1. Lipid and peptide samples were initially freeze-dried several times from 10 mM HCl in D20 to remove any interfering counter ions and residual H20. Solution spectra of peptides were made in deuterated 10 mM phosphate buffer, pD 7.4 (pD = pH+0.4) and in structure promoting mixed solvent-buffer solutions (trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) at a sample concentration of 1 mM. [0223] Spectra were acquired using a temperature controlled, demountable liquid cell with calcium fluoride windows fitted with a 50 μιη thick spacer (Harrick Scientific, Pleasantville, NY). Lipid-peptide films were prepared by air-drying mixtures of DMPC and DMPC: cholesterol (1.2: 1, mole:mole) in chloroform with D-4F or OxP-1 ID in TFE onto a 50 x 20 x2 mm, 45° ATR crystal (Pike Technologies, Madison, WI) fitted to the Bruker spectrometer (Gordon et al. (1996) Protein Sci., 5 : 1662-1675) to form a multilayer film (lipid:peptide, 10: 1, mole:mole). After evaporation the solvent lipid:peptide film was hydrated by passaging deuterium-saturated nitrogen gas through the sample chamber for one hour prior to spectroscopy (Yamaguchi et al. (2001) Biophys. J., 81 : 2203-2214; Ulrich and Watts (1994) Biophys. J., 66: 1441 -1449; Bechinger and Seelig (1991) Chem. Phys. Lipids, 58: 1-5). The relative proportions of a-helix, β-turn, β-sheet, and disordered conformations of solution and multilayer IR spectra were determined by Fourier self- deconvolution for band narrowing and area calculations of component peaks of the FTIR spectra using curve-fitting software supplied by Galactic Software (GRAMS/AI, version 8.0; Thermo Electron Corp., Waltham, MA). The frequency limits for the different structures were: a-helix (1662-1645 cm"1), β-sheet (1637-1613 and 1710-1682 cm"1), turns (1682-1662 cm"1), and disordered or random (1650-1637 cm"1) (Byler and Susi (1986) Biopolymers 25: 469-487).
Molecular dynamics modeling
[0224] Monomeric starting structures for D-4F and OxP-1 ID were obtained by using Hyperchem 7.5 (www.hyper.com) to build the peptides in a helical conformation. These structures were placed in a periodic 56 A3 box of TIP4P water or HFIP:TIP4P water (4:6, v:v) and the ensemble was neutralized with counter-ions to simulate the environment used for our CD measurements (equilibrated HFIP solvent box and topology files courtesy of D. Roccatano) (Roccatano et al. (2005) Protein Sci., 14: 2582-2589). The peptide in the solution box was conjugate-gradient- minimized using the Polak-Ribiere approach implemented in Hyperchem. Minimized monomeric D-4F or OxP-1 ID ensembles were ported to the Gromacs program suite, version 4.0.4 (www.gromacs.org), and subjected to the steepest descent method using the OPLS AA option (Hess et al. (2008) J. Chem. Theory Comput. , 4: 435-447).
[0225] The system was subjected to 20 psec of pre-run molecular dynamics at
300°K allowing the solvent to equilibrate while restraining the peptide. After pre-run solvent equilibration the peptides were subjected to 100 nsec of free MD simulations at 300°K without any experimental constraints, utilizing Berendsen temperature and pressure coupling and the Particle Mesh Ewald method for evaluating long-range electrostatic interactions. T he time-dependent evolution of the root mean square deviations (RMSD) for the peptide C-a carbons, radius of gyration and secondary structure (i.e., analyzed using the DSSP criteria (Kabsch and Sander (1983) Biopolymers, 22: 2577-2637) for the peptide in the HFIP -water environment indicated when equilibrium was reached. Molecular model illustrations were rendered using PyMOL v0.99 (www.pymol.org).
Supporting Information
[0226] Peptide purity was evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) and by analytical RP-HPLC, using a ProStar 210 HPLC system with a ProStar 325 Dual Wavelength detector set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). The mobile phases were: Solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile. Analytic assessments used a reversed-phase, 4.66250 mm C18 column (Vydac 218TP54) and a linear 0 to 100% gradient of solvent B applied over 100 min at 1 ml/min. Analytical data for OxP peptides is shown in Table 4.
Table 4. Analytical data for OxP peptides.
MW (g/mole)
Peptide Composition RT (min)
Calc / Found
OxP-1 C82H118N24O20S1 1792.07 / 1792.53 37.703
OxP-2 C86H126N24O20S1 1848.18 / 1848.08 39.619
OxP-3 C164H234 48O40S2 3582.12 / 3583.93 41.561
OxP-3D C164H234 48O40S2 3582.12 / 3582.93 41.200
OxP-4 C86H126N24O20S1 1848.18 / 1849.27 39.494
OxP-4D C86H126N24O20S1 1848.18 / 1848.80 39.305
OxP-5 C82H118N24O21 1776.00 / 1776.17 36.927
OxP-5D C82H118N24O21 1776.00 / 1776.01 37.030
OxP-6 C87H126N24O20 1828.12 / 1828.99 39.550
OxP-7 C 0H126N24O20S1 1896.22 / 1897.16 41.630
OxP-8 C88H117N24O20F5 1926.05 / 1926.44 41.061
OxP-9 C 0H123N25O20 1875.14 / 1876.37 39.847
OxP- 10 C 4H126N24O20 1912.20 / 1912.56 42.860
OxP- 11 C 4H126N24O20 1912.20 / 1912.78 41.157
OxP-l lD C94H126N24O20 1912.20 / 1913.05 41.428
OxP- 12 C96H126N24O20 1936.22 / 1936.14 42.980
OxP- 13 C98H148N28O21S1 2086.51 / 2087.29 44.044
OxP- 13D C98H148N28O21S1 2086.51 / 2086.80 43.624
OxP- 14 C100H144N28O21 Si 2106.50 / 2107.52 48.715 OxP-14D C100H144N28O21 Si 2106.50 / 2106.81 48.445
OxP-15 C103H14 N2 O22 Si 2177.58 / 2178.86 47.310
OxP-15D C103H149N29O22 Si 2177.58 / 2178.19 47.311
OxP-16 C110H164N28O21 Si 2246.77 / 2247.06 52.401
OxP-16D C110H164N28O21S1 2246.77 / 2246.88 51.936
OxP-17 C113H168N28O21S1 2286.84 / 2287.19 55.466
OxP-18 C116H176N30O22S1 2374.95 / 2375.54 46.691
OxP-18D C116H176N30O22S1 2374.95 / 2375.49 48.480
OxP-19 C105H162N28O21S5 2312.96 / 2312.74 48.030
OxP-20 C160H255 45O28S2 3321.23 / 3323.91 52.558
OxP-21 C320H508N90O56S4 6640.44 / 6642.01 57.240
OxP-22 C4 8H783N139O90S6 10350.05 / 10351.83 56.788
OxP-23 C107H158N28O21S1 2204.69 / 2204.78 50.457
OxP-24 C95H142N28O21S1 2044.43 / 2045.18 40.947
OxP-25 C101H150N28O21S1 2124.56 / 2124.28 45.826
Results
Design and synthesis of the "oxpholipin" ( OxP) peptides.
[0227] Table 5 shows the highly conserved, cholesterol-binding domain found in seven different cholesterol-dependent cytolysins, and Table 6 shows the analogs included in this study. We noticed that modifying cysteine-3 with a bulky hydrophobic group (OxP-2) enhanced the peptide's ability to inhibit CDC-mediated hemolysis (data not shown).
Accordingly, OxPs-5 to 12 were designed to introduce other substitutions at position-3, and OxPs-13 to 20 were designed to vary the separation of hydrophobic and ionic residues. We also synthesized three disulfide-linked dimers (OxP-3, OxP-3D and OxP-21) and created one trimer (OxP -22) by S-alkylation with TMEA. We completed this panel with three analogues (OxP-23 to 25) that contained α,α-di-substituted amino acids, intended to enhance resistance to proteolysis (Yamaguchi et al. (2003) Biosci. Biotechnol. Biochem., 67: 2269-2272).
Table 5. Comparison of the cholesterol binding domains of the CDC toxins.
Toxin Sequence SEQ
ID NO
ALO q"GSGKDKTAHYSTVIPLPPNSKNIKIVARECTGLAWEWWRTIINE 5
QNVPLTNE494
PFO 401GNYQDKTAHYSTVI PLEA AR IRIKARECTGLAWEWWRDVI SE g
YDVPLTNN452 LLO ENNKSKLAHFTSSIYLPGNAR INVYAKECTGLAWEWWRTVIDD
RNLPLVKN506
ALV 432GNWRDRSAHFSTEIPLPPNAK IRIFARECTGLAWEWWRTVVDE 8
YNVPLASD483
SL0 481NNWYSKTSPFST IPLGA SR IRIMARECTGLAWEWWRKVI g
DERDVKLSKE532
IVL 454ENDKDKLAHFTTSIYLPGNARNINIHAKECTGLAWEWWRTVV 1Q
DDRNLPLVKN505
pLY 399RNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEK n
TDLPLVRK450
Oxp-i RECTGLAWEWWRTV 1 2
Abbreviations: ALO: anthrolysin O, from Bacillus anthracis; PFO: perfringolysin O, from Clostridium perfringens; LLO: listeriolysin O, from Listeria monocytogenes; ALV;
alveolysin, from Bacillus alvi ; SLO: streptolysin O, from Group A streptococcus; IVL: ivanolysin from Listeria ivanovi; PLY: pneumolysin from S. pneumoniae. OxP-1,
Oxpholipin-1 (this example).
Table 6. List of synthesized OxP peptides. Analogues denoted with capital D at the end of name are composed exclusively from D-amino acids.
Peptide Sequence SEQ
ID NO
OxP- -11D RE- -Dpa- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 13
OxP- -1 RE- -Cys- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 14
OxP- -2 RE- -Ctb- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 15
OxP- -3 RE- -Cys- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 16
1
RE- -Cys- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2
OxP- -3D RE- -Cys- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 17
RE- -Cys- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2
OxP- -4 WA- -Arg- ■Thr- -V- ■Trp- -Gly- ■Arg- -L- -Ctb- ■Glu- -TE- -Trp- -NH2 18
OxP- -4D WA-Arg- ■Thr- -V- ■Trp- -Gly- ■Arg- -L- -Ctb- ■Glu- -TE- -Trp- -NH2 19
OxP- -5 RE- -Ser- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 20
OxP- -5D RE- -Ser- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 21
OxP- -6 RE- -Chg- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 22
OxP- -7 RE- -Cbl- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 23
OxP- -8 RE- -PhF- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 24
OxP- -9 RE- - rp- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 25
OxP- -10 RE- -Bip- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 26
OxP- -11 RE- -Dpa- ■Thr- -G- ■Leu- -Ala- ■Trp- -E- -Trp- ■Trp- -RT- -Val- -NH2 27 OxP- -12 RE-Ant-■Thr--G-Leu-■Ala-- rp--E-Trp-■Trp-RT-Val--NH2 28
OxP- -13 Aib- -RE- ■Ctb- -Val- -R- ■Leu- -Val- - rp -E- ■Trp -Trp -RE- -Val- -NH2 29
OxP- -13D Aib- -RE- ■Ctb- -Val- -R- ■Leu- -Val- - rp -E- ■Trp -Trp -RE- -Val- -NH2 30
OxP- -14 Nic- -RE- ■Ctb- -Val- -R- ■Leu- -Val- - rp -E- ■Trp -Trp -RE- -Val- -NH2 31
OxP- -14D Nic- -RE- ■Ctb- -Val- -R- ■Leu- -Val- -Trp -E- ■Trp -Trp -RE- -Val- -NH2 32
OxP- -15 Nic- -bA- ■RE-( tb-Val .-R-: Leu-Val-1 Trp >-E-' Trp- Trp- -RE- 33
Val--NH2
OxP- -15D Nic- -bA- ■RE-( tb-Val .-R-: Leu-Val-1 Trp >-E-' Trp- Trp- -RE- 34
Val--NH2
OxP- -16 Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Trp -E- ■Trp -Trp -RE- -Chg- -NH2 35
OxP- -16D Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Trp -E- ■Trp -Trp -RE- -Chg- -NH2 36
OxP- -17 Ach- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Trp -E- ■Trp -Trp -RE- -Chg- -NH2 37
OxP- -18 Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Trp -E- ■Trp -Trp -RE- -Chg- -K- 38
NH2
OxP- -18D Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Trp -E- ■Trp -Trp -RE- -Chg- -K- 39
NH2
OxP- -19 Aib- -RE- ■Ctb- -Ctb- -R- ■Ctb- -Ctb- -Trp -E- ■Trp -Trp -RE- -Ctb- -NH2 40
OxP- -20 Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Nal -E- ■Nal -Nal -RE- -Chg- -X 41
OxP- -21 Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Nal -E- ■Nal -Nal -RE- -Chg- -x2 42
OxP- -22 Aib- -RE- ■Ctb- -Chg- -R- ■Cha- -Chg- -Nal -E- ■Nal -Nal -RE- -Chg- -x3 43
OxP- -23 Ach- -RE- ■Ctb- -Ach- -R- ■Leu- -Ach- -Trp -E- ■Trp -Trp -RE- -Ach- -NH2 44
OxP- -24 Aib- -RE- ■Ctb- -Aib- -R- ■Leu- -Aib- -Trp -E- ■Trp -Trp -RE- -Aib- -NH2 45
OxP- -25 Aib- -RE- ■Ctb- -Ach- -R- ■Leu- -Ach- -Trp -E- ■Trp -Trp -RE- -Aib- -NH2 46
Analogues whose identifiers end with a D are composed of D-amino acids. Abbreviations: Nic: nicotinic acid; PhF: 1 ,2,3,4,5-pentafluoro-phenyl-alanine, Aib: aminoisobutyric acid; Bip: biphenyl-alanine; bA: β-alanine; Dpa: 3,3'-diphenyl-alanine; Ach: 1 -amino- 1- cyclohexane carboxylic acid; Ant: 3-(9-anthryl)-alanine; Ctb: S-tbutyl-cysteine; Cha:
cyclohexyl-alanine; Cbl: S-(4-methyl)benzyl-cysteine; Nal: 3-(l-naphthyl)-alanine; Chg: cyclohexyl-glycine; X: (Lys-Arg)3-Lys-NHCH2CH2SH; X2-dimerized via disulphide bond, X3-trimerized with TMEA: tris-[2-maleimidoethyl]amine hydrochloride. While particular peptide are identified above in Table 6, it will be recognized that in certain embodiments, all "D" forms of any of these peptides, all "L" forms of any of these peptides, dimmers, trimers, tetramers of any of these peptides, combinations of two or more of the above-peptides (e.g., as a fusion protein with or without linkers) or as a chemical conjugate), the above peptides comprising modified linkages, and the like, are also contemplated. In addition, while protecting groups are identified on some peptides in Table 6 it will be appreciated that any of these peptides can have just a carboxyl terminal protecting group, just an amino terminal protecting group, protecting groups at both termini, protecting groups on one or more side chains, or no terminal and/or side chain protecting groups.
Anti-inflammatory activity of OxP peptides.
[0228] Because monocyte chemotactic assays had predictive value in assessing prospective apoA-I mimetic peptides (Navab et al. (2005) Arterioscler. Thromb. Vase. Biol, 25: 1325-1331), we tested the ability of the Oxpholipins to suppress LDL-induced monocyte chemotaxis in cultured human aortic endothelial cells (HAEC) (Navab et al. (2000) J. Lipid Res., 41 : 1495-1508). LDL containing oxidized phospholipids derived from arachidonic acid induces the production of monocyte chemoattractant protein- 1 (MCP- 1) by HAEC, an effect that may contribute to atherogenesis (Id.). ApoA-I mimetic peptides such as D-4F and ApoJ inhibit LDL-induced chemotaxis in vitro and in vivo, by
sequestering or otherwise removing lipid hydroperoxides (Navab et al. (1997) J. Clin. Invest., 99: 2005-2019; Datta et al. (2001) J. Lipid Res., 42: 1096-1 104).
[0229] When tested at 1 mg/ml, several Oxpholipins reduced the monocyte - chemotactic activity of LDL (Figure 1). The most active peptides, OxP-11 and 13, had activity comparable to D-4F. Tests performed at lower concentrations: (0.01, 0.1 and 1 mg/ml) showed their effects to be dose-dependent (Figure 2). Based on these initial monocyte chemotaxis assays, we synthesized eight additional peptides (OxPs-3D, 5D, 1 ID, 13D, 14D, 15D, 16D and 18D), all composed exclusively of D-amino acids, hoping to obtain analogs that would be effective after oral administration.
[0230] When the analogues were administered subcutaneously to apoE deficient mice at 1 mg/kg (Figure 3A), several showed anti-inflammatory activity, including OxP- 1 ID whose activity rivaled D-4F. Given subcutaneously, L-4F (the all L-amino acid version of D-4F) is also active in this apoE-deficient mouse model. Accordingly, we gave three different concentrations (0.1, 0.5 and 1 mg/kg) of all-L OxP- 11 subcutaneously to apoE deficient mice (Figure 3B). L-4F and OxP-11 had similar efficacy at 1 mg/kg, but OxP-11 was considerably less active than L-4F at the lower concentrations.
[0231] We speculated that if the anti-inflammatory effects of D-4F and OxP- 1 ID on monocyte chemotaxis arose from different mechanisms, synergism might ensue when the peptides were given in combination. However, we saw no apparent in vivo synergy between D-4F and OxP-1 ID at 1 mg/kg dose (Figure 3C). Lower doses were not tested in this experiment. OxP-llD binds oxidized lipids and sterols.
[0232] We used surface plasmon resonance (SPR) to examine binding of OxP-1 ID to various sterols, oxidized and non-oxidized lipids. Binding of some of our peptides to cholesterol, including OxP-1 ID, was suggested by our early experiments showing that peptides can prevent hemolysis of human RBCs mediated by CDCs in concentration dependent manner (data not shown, separate manuscript in preparation) and we decided to investigate this phenomenon in greater detail. OxP-1 ID binds to cholesterol (Figure 4, panel A) and several oxysterols, including 20(S)-hydroxycholesterol (Figure 4, panel B) and 24(S)-hydroxycholesterol (Figure 4, panel C). D-4F and OxP-1 ID bound these sterols similarly (Table 7). Figure 5, panels A-D show binding isotherms for OxP-1 ID to 13(S)- HODE), PEIPC, 12(S)-HPETE, and 5(S)-HPETE. OxP-1 ID bound five of the lipids with higher affinity than D-4F, including palmitic acid, 5(S)-HPETE, 12(S)-HPETE, 15(S)- HPETE, 13(S) and 13(S)-HODE. Ten lipids were bound with higher affinity by D-4F, including arachidonic and linoleic acids, PGPC, POVPC, PEIPC, Kodia-PC, 13(S)- HPODE, 12(S)-HETE, 15(S)-HETE and 9(S)-HODE (Table 7)).
[0233] Generally, D-4F seems to bind with higher affinity to oxidized lipids containing palmitoyl moiety: PGPC, POVPC, PEIPC and KOdiA-PC. OxP-1 ID on the other hand seems to be more selective toward certain sterols: 20(S)-hydroxycholesterol, 22(S)- hydroxycholesterol, 24(S)-hydroxycholesterol and certain arachi-'donic acid derivatives: 5(S)-HPETE, 12(S)-HPETE, 15(S)-HPETE and 13(S)-HODE. The position of oxidation appeared to influence binding affinity, as OxP-1 ID bound tightly to cholesterol derivatives hydroxylated in positions 20(S)-, 22(S)- and 24(S)-, but not to 25- hydroxycholesterol or 43 -hydroxycholesterol which were preferentially bound by D-4F.
Table 7. Binding of OxP-1 ID and D-4F to oxidized and nonoxidized (phospho)lipids and sterols. The mean KD values for apoA-I and D-4F are from (42), and those for OxP-1 ID are from this study. The ratio (KD D 4f)/ KD°xP 11d) is also shown. Ratios below 1 indicate that D- 4F binds the lipid with higher affinity (lower KD) than OxP-1 ID. Ratios greater than 1 indicate that OxP-1 ID binds the lipid with higher affinity than D-4F.
KD (M) D-4F/OxP-llD
Ligands
apoA-I D-4F OxP-llD KD Ratio
Nonoxidized lipids
Arachidonic acid 1.0 10~8 1.4 X 10~8 9.7 X 10"7 0.014
Linoleic acid 1.1 X 10"8 1.4 X 10"8 6.0 X 10"7 0.023 Palmitic acid 2.0 10"6 6.7 X 10"7 3.3 X lo-7 2.030
Oxidized lipids
PGPC 1.0 10"6 7.3 X 10~8 3.2 X 10"6 0.023
POVPC 1.0 x 10"5 1.1 X 10~9 3.0 X lo 7 0.004
PEIPC 2.1 X 10"5 2.3 X 10"11 1.2 X 10"8 0.002
KOdiA-PC 6.0 X 10"7 4.9 X 10"10 1.9 X lo 7 0.003
5(5 HPETE 1.1 X 10~2 3.5 X 10~8 1.7 X 10"8 2.059
12(S)-HPETE 1.3 X 10"2 7.2 X 10"7 3.4 X 10"8 21.176
15(S)-HPETE 1.7 X 10"2 9.1 X 10~9 4.5 X 10"8 0.202
13(5)-HPODE 3.5 X 10"2 4.8 X 10"8 3.1 X 10"8 1.548
12(S)-HETE 1.4 X 10"2 1.3 X 10"7 4.8 X 10"8 2.708
15(S)-HETE 1.6 X 10"3 5 .8 X 10"10 9.3 X 10"8 0.006
9(5)-HODE 1.2 X 10"2 2.2 X 10"9 2.1 X 10"8 0.105
13(5)-HODE 6.8 X 10"3 9.9 X 10"8 3.8 X lo 9 26.053
Sterols
Cholesterol 1.0 x 10"2 9.4 X 10"7 3.6 X lo 7 2.611
20(iS)-Hydroxycholesterol BND 8.1 X 10"6 5.1 X lo 7 15.882
22(5)-Hydroxycholesterol BND 1.2 X 10"6 4.5 X lo 7 2.667
24(5)-Hydroxycholesterol BND 1.2 X 10"6 2.4 X lo 7 5.000
25 -Hydroxycholesterol BND 6.6 X 10"10 BND —
4B-Hydroxycholesterol BND 3.4 X 10"5 BND —
Control
Bovine albumin 4.7 X 10"9 1.8 X 10"6 6.9 X 10"7 2.609
Abbreviations: PGPC: l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; POVPC: 1- palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; PEIPC: l-palmitoyl-2-(5,6- epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine; KOdiA-PC-1 : palmitoyl-2-(5-keto- 6-octene-dioyl)-sn-glycero-phosphatidylcholine; HPETE: hydroperoxyeicosatetraenoic acid; HPODE: hydroperoxyoctadecadienoic acid; HETE: hydroxyeicosatetraenoic acid; HODE: hydroxyoctadecadienoic acid.
CD and FTIR studies of secondary structure.
[0234] D-4F and OxP-1 ID assumed stable conformations (Figure 6, panels A and
B) in aqueous buffer and in the structure-promoting HFIP:buffer solvent system. CD spectra of D-4F showed a highly helical structure (Table 8) with the definitive maxima at 222 and 208 nm expected for a mostly a-helical, D-amino acid peptide. FTIR
measurements of D-4F, both in buffer and in structure -promoting solvent systems, revealed strong absorbance around 1656 cm-1 (Figure 6, panel C), indicating a predominantly helical secondary structure (Table 9) consistent with the CD studies. In contrast, the FTIR spectra of OxP-1 ID in aqueous buffer showed a mixture of helical (1662-1645 cm"1), turn (1682-1662 cm"1), disordered (1650-1637 cm"1) and beta-sheet (1637-1613 and 1710-1682 cm"1) structural elements. In "structure-promoting" solvents, OxP-1 ID showed more turn and helix propensity, suggesting it might assume a more highly ordered structure in these more amphipathic and hydrophobic environments (Figure 6, panel D).
Table 8. Proportions of different components of secondary structure for D-4F and OxP- 1 ID peptides solvent systems of buffer and HFIP -buffer based on Circular Dichroic spectroscopic analysis.
Sample * % Conformation
a-helix turns β-sheet disordered
D-4F in Buffer 65.0 12.0 13.0 10.0
D-4F in HFIP :Buffer 74.0 9.0 11.0 6.0
OxP-1 ID in Buffer 15.0 19.0 30.0 36.0
OxP-l lD in HFIP :Buffer 25.0 33.0 14.0 28.0 peptides ( 100 uM ) in 10 mM phosphate buffer pH 7.4 or HFIP: 10 mM phosphate buffer pH 7.4 were analyzed for secondary conformation based as described in the Methods section.
[0235] To characterize the secondary structures of both peptides in lipid
environments, we performed FTIR in hydrated lipid multilayers of DMPC ±cholesterol. In multilayers composed of DMPC ± cholesterol, D-4F assumed a strong helical conformation, manifested by a dominant absorption peek at 1656 cm"1 similar to its spectrum in the aqueous solvent systems. OxP-1 ID displayed a more complex conformational signature in the multilayers. In phospholipid alone (DMPC) there were contributions from helix (1662- 1645 cm"1) followed by turn (1682-1662 cm"1), disordered (1650-1637 cm"1) and beta sheet (1637-1613 and 1710-1682 cm"1) conformations respectively (Table 8). In multilayers of DMPC with cholesterol, OxP-1 ID displayed similar FTIR characteristics, but with a slightly higher helical signal and lower contributions from turn and beta sheet elements (Table 9). Table 9 Secondary structural composition of D-4F and OxP-1 ID in different solvent systems, inferred from infrared (IR) spectroscopy. The peptides (1 mM) were studied in the following solvent systems: deuterated 10 mM phosphate buffer pD 7.4 ("DPB");
trifluoroethanol (TFE):DPB; or hexafluoroisopropanol (HFIP): DPB. Measurements were done ans spectra were analyzed as described in the Methods. The peak area error for these estimates is ± 4%.
Structural Conformation (%)
Peptide/ Solvent System disordere a-helix turns β-sheet
d
D-4F / Buffer 63.0 11.4 11.8 13.7
D-4F / TFE :Buffer 67.2 8.8 11.3 12.7
D-4F / HFIP :Buffer 71.5 8.0 11.9 8.6
D-4F / DMPC (1 : 10 mole:mole) 57.8 16.4 1.9 23.9
D-4F / DMPC:CHO (1 : 10
58.1 15.2 4.1 22.6 mole:mole)
OxP-1 ID / Buffer 20.1 24.8 33.7 21.4
OxP-l lD / TFE:Buffer 32.9 31.0 22.9 13.2
OxP-l lD / HFIP :Buffer 32.4 37.3 10.3 20.0
OxP-l lD / DMPC (1 : 10
26.5 24.4 24.9 24.2 mole:mole)
OxP-l lD/DMPC:CHO (1 : 10
29.5 27.7 22.6 20.2 mole: mole)
Secondary structure studies: Molecular dynamics simulations.
[0236] Simulations of D-4F and OxP-1 ID in the "structure-promoting"
HFIP:buffer, solvent system were consistent with the structures deduced from CD and FTIR measurements (Figure 7). D-4F maintained a highly helical structure in the simulated HFIP:buffer environment. After 40 nanoseconds, the radius of gyration and RMSD of its C-alpha carbon backbone atoms indicated a stable conformation with an uninterrupted helix between residues 2 to 15. In contrast, within 20 nanoseconds, OxP-1 ID changed its initially helical structure to a more flexible conformation with multiple turn-like features, evident from both the C-alpha carbons and the radius of gyration (Figure 8). The DSSP plot of OxP-1 ID after 20 nanoseconds revealed a stable turn (residues 9 to 13), some helical propensity (residues 4-6), and more disordered-coil conformations elsewhere (Figure 9).
Discussion
[0237] This study was undertaken to investigate the properties of peptides derived from a highly conserved, cholesterol-binding domain found in over 20 bacterial exotoxins ("cholesterol dependent cytolysins") secreted by Gram-positive bacteria. Initially, we hoped only to design peptides or peptidomimetic analogs that, by binding cholesterol, would protect human cells from the lytic effects of these bacterial exotoxins. Indeed, several of the peptides in Table 6 protected human erythrocytes from lysis by anthrolysin O, listeriolysin O, and pneumolysin. [0238] Because peptides that bind cholesterol lipids have potential as antiinflammatory and/or anti-atherosclerosis agents we also examined the binding of other lipids by the "Oxpholipin"peptides, as well as their activity in a monocyte chemotaxis assay that guided the initial development of L-4F and related apoA-I mimetic peptides. The results of these studies caused us to focus on OxP-1 ID, which is the principal subject of this example.
[0239] In vitro and in studies with apoE deficient mice, the effects of OxP-1 ID on monocyte chemotaxis were dose-dependent and similar in magnitude to those of D-4F. Surface plasmon resonance studies revealed additional similarities to D-4F. Both peptides bound normal and oxidized cholesterol, but OxP-1 ID did so with higher affinity. Five of the 15 nonsteroid lipids in Table 7 were appare ntly bound with higher affinity by OxP-1 ID and ten were bound more effectively by D-4F. For example, the affinity of D-4F for arachidonic and linoleic acid, POVPC, PEIPC and KODiaPC appear to be considerably higher than that of OxP-1 ID.
[0240] Certain studies suggest that the factors responsible for the different binding preferences of D-4F and OxP-1 ID reside in the fine structure of the lipids as well as in those of the peptides (Datta et al. (2001) J. Lipid Res., 42: 1096-1 104; Anantharamaiah et al. (2006) Curr. Opin. Lipidol, 17: 233-237; Datta et al. (2004) J. Biol. Chem. 279: 26509-26517; Epand et al. (2004) J. Biol. Chem., 279: 51404-51414; Epand et al.
(2004) Biochemistry 43 : 50735083; Venkatachalapathi et al. (1993) Proteins 15 : 349- 359). For example, OxP-1 ID bound 12(S)-HPETE (hydro^peroxyeicosatetraenoic acid) with higher affinity than D-4F, andyet D-4F bound 12(S)-HETE (hydroxyeicosatetraenoic acid) with much higher affinity than OxP-1 ID. Similarly, OxP-1 ID bound 13(S)-HODE (hydroxyoctadecadienoic acid) with higher affinity than D-4F, yet D-4F had greater affinity for 9(S)-HODE.
[0241] Changes in the secondary structure of OxP-1 ID in lipid environments were noted in our structural studies, and may correlate with our functional measurements in vitro and in vivo. CD, FTIR and molecular dynamics studies showed the peptide to have considerable conformational freedom. OxP-1 ID had a less defined conformation in more polar environments, however in hydrophobic solvent systems and in lipid multilayer ensembles, including these with cholesterol, the peptide showed highly ordered helix and turn structures.
[0242] Recently, L-4F and D-4F were shown to bind oxidized lipids with much higher affinity than human apoA-I (Van Lenten et al. (2008) J. Lipid Res., 49: 2302-231 1) and it was suggested that this property might contribute to its anti-inflammatory activity by sequestering strongly pro-inflammatory oxidized sterols and lipids (Torocsik et al. (2009) Mol. Aspects. Med., 30: 134-152; Olkkonen and Hynynen (2009) Mol. Aspects. Med., 30: 123-133; Calder (2008) Mol. Nutr. Food. Res. 52: 885-897; Vejux and Lizard (2009) Mol. Aspects. Med. 30: 153-170; Poli et al. (2009) Mol. Aspects. Med. 30: 180-189). How do the lipid binding properties of D-4F and OxP-1 ID compare? If we can use the KD values and ratios shown in Table 7 as a basis for comparison, there are similarities and differences. Both peptides bind cholesterol and its oxidized derivatives, with the advantage going to OxP-1 ID. This is hardly surprising, given that 13 of the 14 residues (92.7%) in OxP-1 ID are identical to the library's cholesterol-binding design template.
[0243] Their respective binding of 5(S)-HPETE and 12(S)-HPETE is also instructive. 5(S)-HPETE, which is derived from arachidonic acid by the actions of 5- lipoxygenase, is a direct precursor of leukotrienes A4, B4 and C4. The KD values (Table 7) show that D-4F binds arachidonic acid, 5(S)-HPETE and 12(S)-HPETE with similar affinities. In contrast, based on the KD ratios, the affinity of OxP-1 ID for 5(S)-HPETE was approximately 57-fold higher than its affinity for arachidonic acid, and its affinity for 12(S)- HPETE was approximately 28.5-fold higher.
[0244] OxP-1 ID bound oxidized lipids and sterols preferentially, and with much higher affinity than apoA-I. Therefore it may function as oxidized-compounds'
sequestering agent influencing lipid metabolism on molecular and cellular level. For these and other reasons, OxP-1 ID and structurally related peptides are interesting lead
compounds for the development of novel peptide and peptidomimetic therapeutics.
[0245] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:
1. An isolated oxpholipin peptide, said peptide comprising an amino acid sequence according to the formula: wherein
n, and p are independently 0 or 1;
X1 when present is Aib, Ach, or bA;
X2 is Arg or Trp;
X3 is Glu or Ala;
X4 is Cys, Ctb, Arg, Ser, Chg, Cbl, PhF, Trp, Bip, Dpa, Ant, or Ctb;
X5 is Thr, Val, Chg, Ctb, Ach, or Aib;
X6 is Gly, Val, or Arg;
X7 is Leu, Trp, Cha, or Ctb;
X8 is Ala, Gly, Val, Chg, Ach, or Aib;
X9 is Trp, Arg, or Nal;
X10 is Glu, or Leu;
Xu is Trp, Ctb, or Nal;
X12 is Trp, Glu, or Nal;
X13 is Arg, or Thr;
X14 is Thr, or Glu;
X15 is Val, Trp, Chg, Ctb, Ach, or Aib; and
X16 when present is Lys; and
said peptide ranges in length from 14 up to about 100 amino acids and said peptide binds cholesterol and/or an oxidized phospholipid.
2. An isolated oxpholipin peptide, said peptide comprising an amino acid sequence selected from the group consisting of:
the amino acid sequence of a peptide found in Table 6 (SEQ ID
NOs: 13-46),
the retro amino acid sequence of a peptide found in Table 6 (SEQ ID
NOs: 13-46), an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of an amino acid sequence found in Table 6 (SEQ ID NOs: 13-46);
a dimer, trimer, or tetramer of an amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46), and
an amino acid sequence comprising 1, 2, 3, 4, 5, or 6 conservative substitutions of a retro form of an amino acid sequence found in Table 6 (SEQ ID NOs: 13- 46);
wherein said peptide binds cholesterol and/or an oxidized
phospholipid.
3. The peptide of claim 2, wherein said peptide comprises the amino acid sequence of a peptide found in Table 6 (SEQ ID NOs: 13-46) or the inverse of said amino acid sequence.
4. The peptide of claim 2, wherein said peptide comprises the amino acid sequence Arg-Glu-Dpa-Thr-Gly-Leu-Ala-Trp-Glu-Trp-Trp-Arg-Thr-Val (SEQ ID NO: 13, Oxp-1 ID) or the retro or retro inverso form of the sequence.
5. The peptide according to any one of claims 1-4, wherein said peptide has an inflammatory index less than 1.
6. The peptide according to any one of claims 1-4, wherein said peptide ranges in length up to about 50 amino acids.
7. The peptide according to any one of claims 1-4, wherein said peptide is shorter than the cholesterol binding domain of a cholesterol dependent cytolysin.
8. The peptide according to any one of claims 1-4, wherein said peptide comprises all "L" amino acids.
9. The peptide according to any one of claims 1-4, wherein said peptide comprises one or more "D" amino acids.
10. The peptide according to any one of claims 1-4, wherein said peptide comprises all "D" amino acids.
11. The peptide according to any one of claims 1-10, wherein one or more peptide bonds are replaced by an a-ester, a β-ester, a thioamide, phosphonamide, carbomate, or a hydroxylate.
12. The peptide according to any one of claims 1-10, wherein said peptide comprises a peptide backbone, a polyethylene oxide (PEG/PEO) backbone, a polypropylene oxide (PPO) backbone, an aliphatic backbone, an ester backbone, or an ether backbone.
13. The peptide according to any one of claims 1-12, wherein said peptide comprises one or more protecting groups.
14. The peptide of claim 13, wherein said peptide comprises a carboxyl protecting group on the carboxyl terminus and/or an amino protecting group on the amino terminus.
15. The peptide of claim 13, wherein said carboxyl protecting group and/or said amino protecting group is independently selected from the group consisting of acetyl, amide, 3 to 20 carbon alkyl group, Fmoc, Tboc, 9-fluoreneacetyl group, 1- fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6- diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2- chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), Trifluoroacetyl (TFA), nicotinic acid, (Lys-Arg)3-Lys- NHCH2CH2SH, and TMEA.
16. The peptide of claim 13, wherein said amino protecting group is nicotinic acid.
17. The peptide according to any one of claims 13 and 16, wherein said carboxyl protecting group comprises a group selected from the group consisting of an amide, (Lys-Arg)3-Lys-NHCH2CH2SH, and TMEA.
18. The peptide according to any one of claims 1-17, wherein said peptide is formulated for administration via a route selected from the group consisting of oral administration, nasal administration, administration by inhalation, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, intraocular injection, and intramuscular injection.
19. A pharmaceutical formulation comprising one or more oxpholipin peptides according to any one of claims 1-17 and a pharmaceutically acceptable carrier.
20. A method of mitigating one or more symptoms of atherosclerosis in a mammal, said method comprising administering to said mammal a peptide according to any one of claims 1-18 in an amount sufficient to mitigate one or more symptoms of
atherosclerosis.
21. A method of mitigating one or more symptoms of a pathology characterized by an inflammatory response in a mammal, said method comprising administering to said mammal a peptide according to any one of claims 1-18 in an amount sufficient to mitigate one or more symptoms of said pathology.
22. The method of claim 21 , wherein said pathology is selected from the group consisting of said inflammatory pathology is a pathology selected from the group consisting of atherosclerosis, rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple sclerosis, and a viral illnesses.
23. A method of mitigating one or more symptoms of macular degeneration in a mammal, said method comprising administering to said mammal a peptide according to any one of claims 1-18 in an amount sufficient to mitigate one or more symptoms of said macular degeneration.
24. The method of claim 23, wherein said administration is via eye drops or intraocular injection.
25. A method of treating cancer in a mammal, said method comprising administering to said mammal a peptide according to any one of claims 1-18.
26. The method of claim 25, wherein said cancer is a cancer selected form the group consisting of myeloma or multiple myeloma, ovarian cancer, breast cancer, colon cancer, bone cancer, cervical cancer, brain cancer, and prostate cancer.
27. A method for sequestering cholesterol in a mammal, the method comprising: administering an effective dose of an oxpholipin peptide according to any one of claims 1-18 to said mammal.
28. A method for sequestering lipid in a mammal, the method
comprising: administering an effective dose of an oxpholipin peptide according to any one of claims 1-18 to said mammal.
29. A method of treating a vascular condition and/or a condition characterized by an inflammatory response and/or a condition characterized by the formation of oxidized reactive species in a mammal, said method comprising:
administering to a mammal in need peptide according to any one of claims 1-18 in an amount sufficient to ameliorate one or more symptoms of said condition.
30. The method of claim 29, wherein said administration is by a route selected from the group consisting of oral administration, nasal administration, rectal administration, intraperitoneal injection, and intravascular injection, subcutaneous injection, transcutaneous administration, and intramuscular injection.
31. The method of claim 29, wherein said peptide is administered in conjunction with a drug selected from the group consisting of a CETP inhibitor, FTY720, Certican, DPP4 inhibitors, Calcium channel blockers, ApoAl derivative or mimetic or agonist, PPAR agonists , Steroids, Gleevec, Cholesterol Absorption blockers (Zetia) , Vytorin, Any Renin Angiotensin pathway blockers, Angiotensi II receptor antagonist (Diovan etc), ACE inhibitors, Renin inhibitors, MR antagonist and Aldosterone synthase inhibitor, Beta-blockers, Alpha-adrenergic antagonists, LXR agonist, FXR agonist,
Scavenger Receptor Bl agonist, ABCA1 agonist, Adiponectic receptor agonist or adiponectin inducers, Stearoyl-CoA Desaturase I (SCD1) inhibitor, Cholesterol synthesis inhibitors (non-statins), Diacylglycerol Acyltransferase I (DGAT1) inhibitor, Acetyl CoA Carboxylase 2 inhibitor, PAI-1 inhibitor, LP-PLA2 inhibitor, GLP-1, Glucokinase activator, CB-1 agonist, AGE inhibitor/breaker, PKC inhibitors, Anti-thrombotic/coagulants:, Aspirin, ADP receptor blockers e.g. Clopidigrel, Factor Xa inhibitor, GPIIb/IIIa inhibitor, Factor Vila inhibitor, Warfarin, Low molecular weight heparin, Tissue factor inhibitor, Anti- inflammatory drugs:, Probucol and derivative e.g. AGI-1067 etc, CCR2 antagonist,
CX3CR1 antagonist, IL-1 antagonist, Nitrates and NO donors, and Phosphodiesterase inhibitors.
32. A stent for delivering drugs to a vessel in a body comprising: a stent framework including a plurality of reservoirs formed therein, and peptide according to any one of claims 1-17.
33. The stent of claim 32, wherein said peptide is contained within a polymer.
34. The stent of claim 32, wherein the stent framework comprises one of a metallic base or a polymeric base.
35. The stent of claim 32, wherein the stent framework base comprises a material selected from the group consisting of stainless steel, nitinol, tantalum, MP35N alloy, platinum, titanium, a suitable biocompatible alloy, a suitable biocompatible polymer, and a combination thereof.
36. The stent of claim 32, wherein the reservoirs comprise micropores.
37. The stent of claim 36, wherein the micropores have a diameter of about 20 microns or less.
38. The stent of claim 36, wherein the micropores have a diameter in the range of about 20 microns to about 50 microns.
39. The stent of claim 36, wherein the micropores have a depth in the range of about 10 to about 50 microns.
40. The stent of claim 36, wherein the micropores have a depth of about
50 microns.
41. The stent of claim 36, wherein the micropores extend through the stent framework having an opening on an interior surface of the stent and an opening on an exterior surface of the stent.
42. The stent of claim 36, further comprising a cap layer disposed on the interior surface of the stent framework, the cap layer covering at least a portion of the through-holes and providing a barrier characteristic to control an elution rate of a said peptide in the drug polymer from the interior surface of the stent framework.
43. The stent of claim 32, wherein the reservoirs comprise channels along an exterior surface of the stent framework.
44. The stent of claim 33, wherein the polymer comprises a first layer of a first drug polymer having a first pharmaceutical characteristic and the polymer layer comprises a second drug polymer having a second pharmaceutical characteristic.
45. The stent of claim 32, further comprising a catheter coupled to the stent framework.
46. The stent of claim 45, wherein the catheter includes a balloon used to expand the stent.
47. The stent of claim 45, wherein the catheter includes a sheath that retracts to allow expansion of the stent.
48. A method of manufacturing a drug-polymer stent, comprising providing a stent framework; cutting a plurality of reservoirs in the stent framework;
applying a composition comprising one or more peptides according to any one of claims 1- 17 to the reservoirs; and drying the composition.
49. The method of claim 48, further comprising applying a polymer layer to the dried composition; and drying the polymer layer.
50. A method of treating a vascular condition, comprising:
positioning a stent according to claim 32 within a vessel of a body; expanding the stent; and eluting at least one peptide according to any one of claims 1-17 from at least a surface of the stent.
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