# Patent

## Title
Salicylanilides enhance oral delivery of therapeutic peptides

## Abstract

This invention pertains to the surprising discovery that salicylanilides, e.g., niclosamide and/or niclosamide analogues when orally administered in conjunction with a peptide pharmaceutical (e.g., a class A amphipathic helical peptide as described herein) significantly increases the bioavailability of that peptide. Methods of peptide delivery using such “delivery agents” and pharmaceutical formulations are provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and benefit of U.S. Ser. No. 60/836,501, filed on Aug. 8, 2006, and U.S. Ser. No. 60/868,845, filed on Dec. 6, 2006, both of which are incorporated herein by reference in their entirety for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This work was supported, in part, by USPHS Grant 2 P01 HL-030568. The government of the United States of America may possess certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to oral peptide pharmaceuticals where the active compounds include a plurality of amino acids and at least one peptide bond in their molecular structures, and to methods of enhancing bioavailability of such peptide compounds when administered orally.
BACKGROUND OF THE INVENTION
Numerous human hormones, neurotransmitters, or therapeutic antibodies are peptides or comprise peptides as a substantial part of their molecular structures. Therapeutically effective amounts of such biologically relevant peptides may be administered to patients in a variety of ways. Oral delivery of pharmacologically active agents is generally the delivery route of choice since it is convenient, self administration is relatively easy and generally painless, resulting in greater patient compliance as compared to other modes of delivery.
Biological, chemical and physical barriers such as varying pH in the gastrointestinal tract, powerful digestive enzymes in the stomach and intestine, and active agent impermeable gastrointestinal membranes, however, often makes the effective delivery of peptide pharmaceuticals problematic. For example, the oral delivery of calcitonins, has proven difficult due, at least in part, to the insufficient stability of calcitonin in the gastrointestinal tract as well as the inability of calcitonin to be readily transported through the intestinal walls into the blood stream.
Consequently, peptide pharmaceuticals used in the prior art frequently have been administered by injection or by nasal administration. Insulin is one example of a peptide pharmaceutical frequently administered by injection. Injection and nasal administration, however, are significantly less convenient than, and involve more patient discomfort than, oral administration. Often this inconvenience or discomfort results in substantial patient noncompliance with a treatment regimen. Thus, there is a need in the art for more effective and reproducible oral administration of peptide pharmaceuticals like insulin, calcitonin and others discussed in more detail herein.
SUMMARY OF THE INVENTION
This invention pertains to the surprising discovery that salicylanilides, e.g., niclosamide and/or niclosamide analogues when orally administered in conjunction with a peptide pharmaceutical (e.g., a class A amphipathic helical peptide as described herein) significantly increase the bioavailability of that peptide. Methods of peptide delivery using such “delivery agents” and pharmaceutical formulations are provided.
Thus, in certain embodiments, compositions (e.g., pharmaceutical formulations) are provided that comprise a therapeutic agent (e.g., a therapeutic peptide) in combination with a salicylanilide (e.g., niclosamide and/or a niclosamide analogue). In certain embodiments the salicylanilide comprises niclosamide or niclosamide analogue such as 2′5-dichloro-4′-nitrosalicylanilide, 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt, 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt, and 5-chloro-salicyl-(2-chloro-4-nitro) anilide monohydrate. In certain embodiments the niclosamide analogue is a compound in FIGS. 2, 3, 4, 5, 6, 7, and/or Table 1. In various embodiments the peptide ranges in length from 3 amino acids to 300 amino acids, preferably from about 5 to about 200 amino acids, more preferably from about 5, 10, 15, 18, 20, 25, or amino acids to about 200, 150, 100, 90, 70, or 50 amino acids. In various embodiments the peptide comprises an amphipathic helix. In certain embodiments the peptide is an ApoJ peptide, ApoA-I, ApoA-I milano, or 18A. In certain embodiments the peptide comprises a class A amphipathic helix. In various embodiments the peptide consists of all “L” amino acids, or one or more “D” amino acids, or all “D” amino acids. In certain embodiments the peptide is a D or L peptide whose sequence is shown in any of Tables 2-11 and/or SEQ ID Nos:1-989. In certain embodiments the peptide comprises a protecting group at the amino and/or carboxyl terminus. In certain embodiments the protecting group is a protecting group selected from the group consisting of acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-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), 1-(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), and Trifluoroacetyl (TFA). In certain embodiments the amino protecting group is a protecting group selected from the group consisting of acetyl, propeonyl, and a 3 to 20 carbon alkyl and/or the carboxyl said second protecting group is an amide. In certain embodiments the salyclanalide (e.g., niclosamide or niclosamide analogue) and the therapeutic peptide are intermixed. In certain embodiments the salicylanilide (e.g. niclosamide or niclosamide analogue) and the therapeutic peptide are combined (e.g., under acidic conditions) to form an adduct. In certain embodiments the composition is a unit dosage formulation. In certain embodiments the peptide and the salicylanilide are segregated from each other. In certain embodiments the composition is formulated so that the niclosamide or niclosamide analogue is released or solubilized before the peptide.
In certain embodiments said salicylanilide is niclosamide or a niclosamide analogue; and the peptide is a D or L peptide comprising the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO:5) or the amino acid sequence FAEKFKEAVKDYFAKFWD (SEQ ID NO:104). In certain embodiments the peptide comprises a carboxyl terminal protecting group (e.g., an amide) and/or an amino terminal protecting group (e.g., acetyl). In certain embodiments the niclosamide or niclosamide analogue is niclosamide. In certain embodiments the niclosamide forms an adduct with the peptide.
In various embodiments methods are provided for enhancing the in vivo activity of a therapeutic peptide orally administered to a mammal (e.g., a human or a non-human mammal). The methods typically involve orally administering the peptide in conjunction with an amount of niclosamide or a niclosamide analogue sufficient to enhance the in vivo activity of the peptide. In various embodiments the peptide ranges in length from 3 amino acids to 300 amino acids, preferably from about 5 to about 200 amino acids, more preferably from about 5, 10, 15, 18, 20, 25, or 30 amino acids to about 200, 150, 100, 90, 70, or 50 amino acids. In various embodiments the peptide comprises an amphipathic helix. In certain embodiments the peptide is an ApoJ peptide, ApoA-I, ApoA-I milano (Apolipoprotein M), or 18A. In certain embodiments the peptide comprises a class A amphipathic helix. In various embodiments the peptide consists of all “L” amino acids, or one or more “D” amino acids, or all “D” amino acids. In certain embodiments the peptide is a D or L peptide whose sequence is shown in any of Tables 2-11 and/or SEQ ID Nos:1-989. In certain embodiments the peptide is a D or L peptide comprising the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO:5) or the amino acid sequence FAEKFKEAVKDYFAKFWD (SEQ ID NO:104). In certain embodiments the peptide comprises a carboxyl terminal protecting group (e.g., an amide) and/or an amino terminal protecting group (e.g., acetyl). In various embodiments the peptide used in this method are protected with a carboxyl and/or an amino protecting group as described herein (e.g., a protecting group selected from the group consisting of acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-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), 1-(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), and Trifluoroacetyl (TFA)). In various embodiments the niclosamide or niclosamide analogue is administered before administration of said peptide. In various embodiments the niclosamide or niclosamide analogue is administered at the same time as said peptide. In certain embodiments the niclosamide or niclosamide analogue is combined with the peptide to form an adduct. In certain embodiments the niclosamide analogue is a compound in FIGS. 2, 3, 4, 5, 6, 7, and/or Table 1.
In another embodiment pharmaceutical formulations are provided. The formulations typically comprise an orally administered pharmacologically active agent (e.g., a therapeutic peptide); and a salicylanilide (e.g., niclosamide and/or a niclosamide analogue). In certain embodiments the pharmacologically active agent is a therapeutic peptide and the peptide and the salicylanilide form an adduct. In certain embodiments the pharmaceutically active agent is not a non-peptide antiproliferative agent and/or not a non-peptide anti-cancer drug. In certain embodiments the pharmaceutically active agent is a peptide antiproliferative agent. In certain embodiments the formulation comprises a therapeutic amphipathic helical peptide; and niclosamide and/or a niclosamide analogue, where the niclosamide and/or niclosamide analogue in the formulation shows substantially greater solubility in an aqueous solution than the niclosamide and/or niclosamide analogue in an aqueous solution absent the amphipathic helical peptide. In certain embodiments the peptide is selected from the group consisting of ApoJ, ApoA-I, ApoA-I milano, or 18A. In certain embodiments the peptide forms a class A amphipathic helix. In certain embodiments the peptide consists of all “L” amino acids, or at least one “D” amino acid, or all “D” amino acids. In various embodiments the peptide consists of all “L” amino acids, or one or more “D” amino acids, or all “D” amino acids. In certain embodiments the peptide is a D or L peptide whose sequence is shown in any of Tables 2-11 and/or SEQ ID Nos:1-989. In certain embodiments the peptide is a D or L peptide comprising the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO:5) or the amino acid sequence FAEKFKEAVKDYFAKFWD (SEQ ID NO:104). In certain embodiments the peptide comprises a carboxyl terminal protecting group (e.g., an amide) and/or an amino terminal protecting group (e.g., acetyl). In various embodiments the peptides used in this method are protected with a carboxyl and/or an amino protecting group as described herein (e.g., a protecting group selected from the group consisting of acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-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), 1-(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), and Trifluoroacetyl (TFA)). In various embodiments the niclosamide or niclosamide analogue is administered before administration of said peptide. In various embodiments the niclosamide or niclosamide analogue is administered at the same time as said peptide. In certain embodiments the niclosamide or niclosamide analogue is combined with the peptide to form an adduct. In certain embodiments the niclosamide or niclosamide analogue is a compound in FIGS. 2, 3, 4, 5, 6, 7, and/or Table 1.
Also provided are methods of mitigating one or more symptoms of a pathology characterized by an inflammatory response in a mammal (e.g., a human, a non-human primate, a feline, an equine, a porcine, a bovine, a rodent, etc.). The methods typically involve orally administering to the mammal an amphipathic helical peptide that mitigates one or more symptoms of atherosclerosis or other pathology characterized by an inflammatory response in conjunction with niclosamide or a niclosamide analogue, whereby the oral delivery provides in vivo activity of the peptide to mitigate one or more symptoms of the pathology. In certain embodiments the niclosamide or niclosamide analogue is administered before the peptide, or administered simultaneously with peptide. In certain embodiments the niclosamide or niclosamide analogue and the peptide are administered as a single formulation. In certain embodiments the niclosamide or niclosamide analogue and the peptide are combined to form an adduct prior to administration. In certain embodiments the niclosamide or niclosamide analogue is selected from the group consisting of 2′5-dichloro-4′-nitrosalicylanilide, 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt, 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt, and 5-chloro-salicyl-(2-chloro-4-nitro) anilide monohydrate. In certain embodiments the niclosamide or niclosamide analogue is a compound in FIGS. 2, 3, 4, 5, 6, 7, and/or Table 1. In certain embodiments the niclosamide or niclosamide analogue and/or the peptide are administered as a unit dosage formulation. In certain embodiments the niclosamide or niclosamide analogue and the peptide are administered as a unit dosage formulation formulated so that the niclosamide or niclosamide analogue is released or solubilized simultaneously with, or before the peptide. In certain embodiments the pathology is selected from the group consisting of atherosclerosis, rheumatoid arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple sclerosis, chronic obstructive pulmonary disease, asthma, diabetes, and a viral illnesses. In various embodiments the peptide ranges in length from 3 amino acids to 300 amino acids, preferably from about 5 to about 200 amino acids, more preferably from about 5, 10, 15, 18, 20, 25, or 30 amino acids to about 200, 150, 100, 90, 70, or 50 amino acids. In various embodiments the peptide comprises an amphipathic helix. In certain embodiments the peptide is an ApoJ peptide, ApoA-I, ApoA-I milano (Apolipoprotein M), or 18A. In certain embodiments the peptide comprises a class A amphipathic helix. In various embodiments the peptide consists of all “L” amino acids, or one or more “D” amino acids, or all “D” amino acids. In certain embodiments the peptide is a D or L peptide whose sequence is shown in any of Tables 2-11 and/or SEQ ID Nos:1-989. In certain embodiments the peptide is a D or L peptide comprising the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO:5) or the amino acid sequence FAEKFKEAVKDYFAKFWD (SEQ ID NO:104). In certain embodiments the peptide comprises a carboxyl terminal protecting group (e.g., an amide) and/or an amino terminal protecting group (e.g., acetyl). In various embodiments the peptide used in this method are protected with a carboxyl and/or an amino protecting group as described herein (e.g., a protecting group selected from the group consisting of acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-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), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1-Z),2-bromobenzyloxycarbonyl (2-Br—Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA)).
In various embodiments kits are provided. In certain embodiments the kits comprise a container containing a salicylanilide and a therapeutic peptide. The salicylanilide and the peptide can be in separate containers or combined in a single container. In certain embodiments the salicylanilide and the peptide are combined to form an adduct. In various embodiments the salicylanilide is niclosamide or a niclosamide analogue as described herein. In certain embodiments the peptide is a therapeutic peptide as described herein (e.g., ApoJ, ApoA-I, ApoA-I milano, and 18A, D-4F, L-4F, retro D-4F, retro L-4F, etc.).
In certain embodiments this invention also contemplates formulations and methods where the salicylanilide (e.g., niclosamide and/or a niclosamide analogue) is replaced or used in combination with any one or more of the other “delivery agents” described herein (e.g., N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2-hydroxybenzoyl]aminodecanoic acid (SNAD), and N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC) and various salts thereof (e.g., disodium salts), any one or more of the delivery agents disclosed in U.S. Pat. No. 5,866,536, U.S. Pat. No. 5,773,647, and WO 00/059863, and the like).
In certain embodiments this invention excludes formulations and/or methods utilizing any one or more of the delivery agents disclosed in U.S. Pat. No. 5,866,536, U.S. Pat. No. 5,773,647, WO 00/059863.
In certain embodiments niclosamide analogs used in the methods and compositions described herein include, but are not limited to those defined by Formula I, where substituents R1, R2, R5, R6, R7, R8, R9, R10, R11 and R12 are as described herein. In certain embodiments these substituents do not comprise one or more of the following moieties: carboxylic acid, and/or alkyl carboxylates, and/or hydroxamic acid and/or alkyl hydroxamates, and/or sulfonic acid and/or alkyl sulfones, and/or phosphoric acid and/or alkyl phosphates, and/or tetrazole.
DEFINITIONS
The phrase “enhancing the in vivo activity” or “enhancing the apparent activity” when referring to the agents described herein indicates that the agents, when administered in conjunction with an orally delivered pharmaceutical produce a greater biological response in the organism than the same dosage orally administered without the agent. Without being bound to a particular theory, the in vivo activity can be enhanced by any of a number of mechanisms including, but not limited to increased absorption, decreased degradation, a combination of increased absorption and decreased degradation, enhanced active transport, and the like.
The terms “coadministration” or “administration in conjunction with” when used in reference to the use of a delivery agent (e.g., niclosamide, niclosamide analogue or other delivery agent described herein) in conjunction with an orally administered pharmaceutical (e.g., a therapeutic peptide such as L-4F) indicates that the delivery agent and the orally administered pharmaceutical are administered so that there is at least some chronological overlap in the activity of the delivery agent and administration of the pharmaceutical such that the delivery agent enhances in vivo activity (e.g., via increased uptake and/or bioavailability) of the pharmaceutical. In sequential administration there may even be some substantial delay (e.g., minutes or even hours) between administration of the delivery agent and the pharmaceutical as long as the delivery agent is present in a manner that enhances in vivo activity of the pharmaceutical.
The term mammal includes essentially any mammal including, but not limited to dogs, cats, sheep, cattle, horses, goats, mice, rabbits, hamsters, pigs, monkeys and other non-human primates, and humans. Thus, veterinary as well as medical applications of this invention are contemplated.
The term “oral bioavailability” refers to the bioavailablity (e.g., plasma concentration) of an active agent when administered orally (e.g., in an oral formulation).
The term “L form peptide” refers to a peptide comprising all L form amino acids.
The term “D form peptide” refers to a peptide comprising at least one D amino acid. In certain embodiments at least half, and preferably all of the amino acids are D amino acids.
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.
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.
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. Where the amino acid sequence of a peptide is provided the description of that peptide includes L peptides, D peptides, inverse peptides, retro peptides, and retroinverse peptides.
The term “an amphipathic helical peptide” refers to a peptide comprising at least one amphipathic helix (amphipathic helical domain). Certain amphipathic helical peptides of this invention can comprise two or more (e.g., 3, 4, 5, etc.) amphipathic helices.
The term “class A amphipathic helix” refers to a protein structure that forms an α-helix producing a segregation of a polar and nonpolar faces with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face (see, e.g., Segrest et al. (1990) Proteins: Structure, Function, and Genetics 8: 103-117).
“Apolipoprotein J” (apo J) is known by a variety of names including clusterin, TRPM2, GP80, and SP 40 (see, e.g., Fritz (1995) Pp 112 In: Clusterin: Role in Vertebrate Development, Function, and Adaptation (Harmony JAK Ed.), R. G. Landes, Georgetown, Tex.). It was first described as a heterodimeric glycoprotein and a component of the secreted proteins of cultured rat Sertoli cells (see, e.g., Kissinger et al. (1982) Biol. Reprod.; 27: 233240). The translated product is a single-chain precursor protein that undergoes intracellular cleavage into a disulfide-linked 34 kDa α subunit and a 47 kDa β subunit (see, e.g., Collard and Griswold (1987) Biochem., 26: 3297-3303). It has been associated with cellular injury, lipid transport, apoptosis and it may be involved in clearance of cellular debris caused by cell injury or death. Clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate (Bailey et al. (2001) Biochem., 40: 11828-11840).
The class G amphipathic helix is found in globular proteins, and thus, the name class G. The feature of this class of amphipathic helix is that it possesses a random distribution of positively charged and negatively charged residues on the polar face with a narrow nonpolar face. Because of the narrow nonpolar face this class does not readily associate with phospholipid (see, e.g., Segrest et al. (1990) Proteins: Structure, Function, and Genetics. 8: 103-117; Erratum (1991) Proteins: Structure, Function and Genetics, 9: 79). Several exchangeable apolipoproteins possess similar but not identical characteristics to the G amphipathic helix. Similar to the class G amphipathic helix, this other class possesses a random distribution of positively and negatively charged residues on the polar face. However, in contrast to the class G amphipathic helix which has a narrow nonpolar face, this class has a wide nonpolar face that allows this class to readily bind phospholipid and the class is termed G* to differentiate it from the G class of amphipathic helix (see, e.g., Segrest et al. (1992) J. Lipid Res., 33: 141-166; Anantharamaiah et al. (1993) Pp. 109-142 In: The Amphipathic Helix, Epand, R. M. Ed CRC Press, Boca Raton, Fla.). Computer programs to identify and classify amphipathic helical domains have been described by Jones et al. (1992) J. Lipid Res. 33: 287-296) and include, but are not limited to the helical wheel program (WHEEL or WHEEL/SNORKEL), helical net program (HELNET, HELNET/SNORKEL, HELNET/Angle), program for addition of helical wheels (COMBO or COMBO/SNORKEL), program for addition of helical nets (COMNET, COMNET/SNORKEL, COMBO/SELECT, COMBO/NET), consensus wheel program (CONSENSUS, CONSENSUS/SNORKEL), and the like.
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.
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.
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. In certain embodiments 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. In certain embodiments, 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.).
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.
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.
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.
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.
The terms “human apo A-I peptide” or “human apo A-I protein” can refer 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 following abbreviations may be used herein: PAPC: L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC: 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC: 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine; ChCl8:2: cholesteryl linoleate; ChCl8:2-OOH: cholesteryl linoleate hydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine; PON: paraoxonase; HPF: Standardized high power field; PAPC: L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; BL/6: C57BL/6J; C3H:C3H/HeJ.
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).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides 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.
One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The phrases “adjacent to each other in a helical wheel diagram of a peptide” or “contiguous in a helical wheel diagram of a peptide” when referring to residues in a helical peptide indicates that in the helical wheel representation the residues appear adjacent or contiguous even though they may not be adjacent or contiguous in the linear peptide.
As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 6 ring carbon atoms, inclusive. Illustrative cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. The C1-10 alkyl group can be substituted or unsubstituted. Illustrative substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C1-10 alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclopropylethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, cyclobutyl, cyclobutylmethyl, cyclobutylethyl, n-pentyl, cyclopentyl, cyclopentylmethyl, cyclopentylethyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-timethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, cyclohexyl, and the like.
A “C2-10 alkenyl” refers to a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 10 carbon atoms. A C2-10 alkenyl can optionally include monocyclic or polycyclic rings, in which each ring has from three to six members. The C2-10 alkenyl group can be substituted or unsubstituted. Illustrative substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2-10 alkenyls include, but are not limited to, vinyl; allyl; 2-cyclopropyl-1-ethenyl; 1-propenyl; 1-butenyl; 2-butenyl; 3-butenyl; 2-methyl-1-propenyl; 2-methyl-2-propenyl; 1-pentenyl; 2-pentenyl; 3-pentenyl; 4-pentenyl; 3-methyl-1-butenyl; 3-methyl-2-butenyl; 3-methyl-3-butenyl; 2-methyl-1-butenyl; 2-methyl-2-butenyl; 2-methyl-3-butenyl; 2-ethyl-2-propenyl; 1-methyl-1-butenyl; 1-methyl-2-butenyl; 1-methyl-3-butenyl; 2-methyl-2-pentenyl; 3-methyl-2-pentenyl; 4-methyl-2-pentenyl; 2-methyl-3-pentenyl; 3-methyl-3-pentenyl; 4-methyl-3-pentenyl; 2-methyl-4-pentenyl; 3-methyl-4-pentenyl; 1,2-dimethyl-1-propenyl; 1,2-dimethyl-1-butenyl; 1,3-dimethyl-1-butenyl; 1,2-dimethyl-2-butenyl; 1,1-dimethyl-2-butenyl; 2,3-dimethyl-2-butenyl; 2,3-dimethyl-3-butenyl; 1,3-dimethyl-3-butenyl; 1,1-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, and the like.
A “C2-10 alkynyl” refers to a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 10 carbon atoms. A C2-10 alkynyl can optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring has five or six members. The C2-10 alkynyl group can be substituted or unsubstituted. Illustrative substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2-10 alkynyls include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butenyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 5-hexene-1-ynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl; 1-methyl-2-propynyl; 1-methyl-2-butenyl; 1-methyl-3-butynyl; 2-methyl-3-butynyl; 1,2-dimethyl-3-butynyl; 2,2-dimethyl-3-butynyl; 1-methyl-2-pentynyl; 2-methyl-3-pentynyl; 1-methyl-4-pentynyl; 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, and the like.
A “C2-6 heterocyclyl” refers to a stable 5- to 7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic ring that is saturated, partially unsaturated or unsaturated (aromatic), and that consists of 2 to 6 carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclyl group can be substituted or unsubstituted. Illustrative substituents include, but are not limited to alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. The nitrogen and sulfur heteroatoms can optionally be oxidized. The heterocyclic ring can be covalently attached via any heteroatom or carbon atom that results in a stable structure, e.g., an imidazolinyl ring can be linked at either of the ring-carbon atom positions or at the nitrogen atom. A nitrogen atom in the heterocycle can optionally be quaternized. In certain embodiments, when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. Heterocycles include, but are not limited to, 1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl, b-carbolinyl, chromanyl, chromenyl, cinolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred 5 to 10 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and isoquinolinyl. In certain embodiments, 5 to 6 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, and the like.
A “C6-12 aryl” refers to an aromatic group having a ring system comprised of carbon atoms with conjugated electrons (e.g., phenyl). The aryl group typically has from 6 to 12 carbon atoms. Aryl groups can optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring has five or six members. The aryl group can be substituted or unsubstituted. Illustrative substituents include, but are not limited to, alkyl, hydroxy, alkoxy, aryloxy, sulflhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, quaternary amino groups, and the like.
A “C7-14 alkaryl” refers to an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbon atoms.
A “C3-10 alkheterocyclyl” refers to an alkyl substituted heterocyclic group having from 3 to 10 carbon atoms in addition to one or more heteroatoms (e.g., 3-furanylmethyl, 2-furanylmethyl, 3-tetrahydrofuranylmethyl, 2-tetrahydrofuranylmethyl, and the like).
A “C1-10 heteroalkyl” refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 10 carbon atoms in addition to one or more heteroatoms, where one or more methylenes (CH2) or methines (CH) are replaced by nitrogen, oxygen, sulfur, carbonyl, thiocarbonyl, phosphoryl, or sulfonyl. Heteroalkyls include, but are not limited to, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl can optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring has three to six members. The heteroalkyl group can be substituted or unsubstituted. Illustrative substituents include, but are not limited to alkoxy, aryloxy, sulfhydryl, allylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, amino alkyl, disubstituted amino, quaternary amino, hydroxyalkyl, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
The term “acyl” refers to a chemical moiety with the formula R—C(O)—, where R is selected from C1-10 alkyl, C1-10 alkenyl, C1-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, C1-10 heteroalkyl, and the like.
A “halide” refers to meant bromine, chlorine, iodine, or fluorine.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, panels A-D, shows various forms of niclosamide. A: 2′5-dichloro-4′-nitrosalicylanilide; B: 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt; C: 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt; and D: 5-chloro-salicyl-(2-chloro-4-nitro)anilide monohydrate.
FIG. 2 illustrates various niclosamide analogues. A: Oxyclozanide (3,3′,5,5′,6-pentachloro-2′-hydroxy salicylanilide; 2,3,5-trichloro-N-(3,5-dichloro-2-hydroxyphenyl)-6-hydroxybenzamide); B: Closantel (5′-Chloro-alpha4-(p-chlorophenyl)-alpha4-cyano-3,5-diiodo-2′,4′-salicyloxylidide; N-[5-Choloro-4-[(4-Chlorophenyl) Cyanomethyl]-2-Methylphenyl]-2-Hydroxy-3-5-Diiodobenzamide); C: Rafoxanide (also known as Disalan; Flukanide; N-(3-chloro-4-(4-chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide; 3′-Chloro-4′-(p-chlorophenoxy)-3,5-diiodosalicylanilide); D: Flusalan (3,5-Dibromo-2-hydroxy-N-(3-trifluoromethyl-phenyl)-benzamide); E: Tribromsalan (3,5-Dibromo-N-(4-bromo-phenyl)-2-hydroxy-benzamide); F: Resorantel (N-(4-Bromo-phenyl)-2,6-dihydroxy-benzamide); G: Clioxanide (Acetic acid 2-(4-chloro-phenylcarbamoyl)-4,6-diiodo-phenyl ester).
FIG. 3 illustrates various niclosamide analogues and salts thereof.
FIG. 4 illustrates niclosamide analogues in which one halogen group is relocated within the same ring (see, e.g., compounds A-D) or both halogen groups are relocated within the same ring (see, e.g., compounds E-G).
FIG. 5 illustrates niclosamides in which the nitro group is relocated within the same ring (see, e.g., compounds A-C) and niclosamide analogues where the hydroxyl group is relocated within the same ring (see, e.g., compounds D-F).
FIG. 6 illustrates niclosamide analogues where both halogen and hydroxy and/or nitro groups are relocated while keeping the substituents within the aromatic ring (see, e.g., compounds A-F) and niclosamide analogues having a nitro- and a hydroxyl group relocation (see, e.g., compounds G-I).
FIG. 7 illustrates niclosamide analogues comprising a single halogen exchange (see, e.g., compounds A-D), niclosamide analogues comprising a double halogen exchange (see, e.g., compounds E-F), niclosamide analogues comprising an exchange of Cl— to Br— (see, e.g., compound G), and niclosamide analogs comprising an exchange of Cl— to F— (see, e.g., compound H).
FIG. 8 shows HDL inflammatory index for apoE null mice fed chow containing or not containing additions. C: Mice were given chow alone; D: Mice given chow supplemented with 8.0 micrograms of niclosamide; E: Mice given chow supplemented with 2.0 micrograms of L-4F; F: Mice given chow supplemented with 8.0 micrograms of Niclosamide together with 2.0 micrograms of L-4F (free base) per gram of chow. The mouse HDL (C-J) was also compared to a standard human HDL (B) that was added at the same concentrations as the mouse HDL. The resulting monocyte chemotactic activity was normalized to the standard control LDL added alone (A). The results are plotted as the HDL-inflammatory index, which is the result of dividing the monocyte chemotactic activity measured for each condition by the monocyte chemotactic activity obtained by the standard control LDL added alone, which was normalized to 1.0. G-I: A second experiment. G: Chow alone; H: chow supplemented with 100 micrograms of Niclosamide per gram of chow; I: Chow supplemented with 10 micrograms of L-4F (free base) per gram of mouse chow; J: Chow supplemented with 10 micrograms of L-4F (free base) together with 100 micrograms of Niclosamide per gram of chow. The data shown are the Mean±S.D.
FIG. 9 shows that administration of niclosamide as an oral bolus by gastric gavage (stomach tube) immediately followed by administration of L-4F as an oral bolus by stomach tube rendered apoE null mouse HDL anti-inflammatory. the HDL-containing fractions were tested for their ability to inhibit the induction of monocyte chemotactic activity by a standard control human LDL, which was added to cultures of human aortic endothelial cells. The values obtained after the addition of the standard control HDL or the mouse HDL were compared to the values obtained by the standard control LDL alone to give the HDL Inflammatory Index. The values shown are the Mean±S.D.
FIG. 10 shows that administration of Niclosamide as an oral bolus by stomach tube immediately followed by administration of L-4F as an oral bolus by stomach tube significantly reduced the ability of apoE null mouse LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells. The LDL fractions from the mice described in FIG. 9 were tested for their ability to induce monocyte chemotactic activity in cultures of human aortic endothelial cells and compared to a standard control human LDL whose values were normalized to 1.0 for the LDL-inflammatory index. The data shown are the Mean±S.D.
FIG. 11 shows that oral administration of niclosamide (5.0 mg/kg body weight) immediately followed by oral administration of L-4F (0.5 mg/kg/body weight) renders monkey HDL anti-inflammatory. The data shown are the Mean±S.D. for the HDL
FIG. 12 shows that oral administration of niclosamide (5.0 mg/kg body weight) immediately followed by oral administration of L-4F (0.5 mg/kg/body weight) significantly reduced the ability of monkey LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells. The LDL fractions from the monkey plasma described in FIG. 11 were tested as described in FIG. 10. The data shown are the Mean±S.D.
FIG. 13 shows that an amphipathic helical peptide (L-4F) increases the solubility of niclosamide in an aqueous system. Niclosamide at 10 mg per mL was added to water, or to water containing 1.0 mg/mL L-4F (free base) and was homogenized in a glass-glass homogenizer. The solutions were stored at 4° C. for ten days and photographed
FIG. 14 shows the HDL inflammatory index for female apoE null mice that were given by gastric gavage (stomach tube) 100 μL water alone or 100 μL water containing niclosamide or containing niclosamide in combination with L-4F at the doses shown on the X-axis. The solutions of niclosamide with or without L-4F shown in FIG. 13 were serially diluted and given by gastric gavage (stomach tube) to fasting seven month old female apoE null mice in a volume of 100 microliters per mouse (n=8 per group). Blood was collected 6 hours following treatment while the mice were still fasting and the plasma was separated by FPLC and the HDL fractions were tested as described in FIG. 8. The data shown are the Mean±S.D, h=human, m=mouse.
FIG. 15 LDL from the mice described in FIG. 14 was tested for its ability to induce human aortic endothelial cells to produce monocyte chemotactic activity. The data are plotted as the LDL-inflammatory index as described for FIG. 10. The values shown are the Mean±S.D.
FIG. 16 shows the HDL from mice that were given niclosamide in mouse chow at 250 μg per day per mouse with or without L-4F (free base). Seven month old female apoE null mice (n=8 per treatment group) were given niclosamide in mouse chow at 250 micrograms per day per mouse with or without L-4F (free base) at 25 micrograms per day per mouse in the drinking water or in mouse chow (food) with the niclosamide. After three days the mice were bled, their plasma was fractionated by FPLC and the ability of the mouse HDL (m) to inhibit LDL-induced monocyte chemotactic activity was determined in cultures of human aortic endothelial cells and calculated as the HDL-inflammatory index as described in FIG. 8. Normal anti-inflammatory human (h) HDL was included in the assays as a positive control. The values shown are the Mean±S.D.
FIG. 17 shows the results of LDL from the mice (m) in FIG. 16 tested for its ability to induce monocyte chemotactic activity in cultures of human aortic endothelial cells. The data is expressed as the LDL-inflammatory index by comparing the results to the monocyte chemotactic activity induced by a standard control human (h) LDL alone, which was normalized to 1.0. The values shown are the Mean±S.D; h=human, m=mouse.
FIG. 18 shows pre-beta HDL formation in mice administered niclosamide with L-4F compared to D-4F.
FIG. 19 shows the HDL-inflammatory index after oral administration of D-4F or L-4F. Niclosamide was homogenized with or without D-4F or L-4F (both as the free-base) in a ratio of 10:1 (nicolsamide:peptipde; wt:wt) in ABCT buffer pH 7.0 and incubated at 37° C. for 1 hour. The buffer without peptide or with the peptides at 2.5, 5.0, or 10 μg was administered to 3 month old fasting female apoE null mice (n=8 per group) in 100 μL by stomach tube. Six hours later the mice were bled and their plasma separated by FPLC and the HDL fractions from the mice were tested in cultures of human aortic endothelial cells exposed to normal human LDL to determine the HDL-inflammatory index as described in FIG. 8. In the absence of added HDL (0) the monocyte chemotactic activity obtained after addition of the normal control LDL was normalized to 1.0. The monocyte chemotactic activity after addition of the human LDL plus a normal control human HDL (h) or mouse HDL (m) was divided by the monocyte chemotactic activity obtained following addition of the human LDL without HDL to give the HDL-inflammatory index. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 20 shows the results of a cell-free assay of HDL taken from mice receiving oral D-4F or L-4F. The HDL from the mice described in FIG. 19 was tested in the cell-free assay. The data shown are the Mean±S.D.
FIG. 21 shows plasma paraoxonase activity from the mice described in FIG. 19. The data shown are the Mean±S.D.
FIG. 22 shows that co-administration of niclosamide with L-4F renders apoE null mouse HDL anti-inflammatory to a degree that is similar to normal human HDL. Free base D-4F or L-4F were homogenized with or without niclosamide in a ratio of 10:1 (niclosamide:peptide; wt:wt) in ABCT buffer adjusted to pH 8.0 using 0.1 NaOH. The buffer without the peptide or with the peptides at 10 μg in 100 μL was administered to 4-month-old fasting apoE null female mice (n=8 per group) by stomach tube. Seven hours later the mice were bled and their plasma separated by FPLC and the HDL fractions from the mice were tested in cultures of human aortic endothelial cells exposed to normal human LDL to determine the HDL-inflammatory index as described in FIG. 8. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 23. The LDL-inflammatory index from the mice described in FIG. 22 was determined. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 24 shows that new salicylanilides (BP-1001 and BP-1012) are more potent than niclosamide in improving the HDL-inflammatory index. Niclosamide (BP-124) or BP-1001, or BP-1012 were homogenized with or without D-4F or L-4F (both as the free base) in a ratio of 10:1 (wt:wt) in ABCT buffer. The buffer without peptide or with peptide at 5 μg in 100 μL was administered to 4-month-old fasting apoE null mice (n=4 per group) by stomach tube. Six hours later the mice were bled and their plasma separated by FPLC and the HDL fractions from the mice were tested in cultures of human aortic endothelial cells exposed to normal human LDL to determine the HDL-inflammatory index as described in FIG. 8. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 25. The LDL-inflammatory index for LDL taken from the mice described in FIG. 24 was determined as described in FIG. 10. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 26 shows a comparison of niclosamide (BP-124) with other salicylanilides. Niclosamide (BP-124) or the salicylanilides whose numbers (BP#) are shown on the X-axis were homogenized with L-4F (as the free base) in a ratio of 10:1 (salicylanilide:L-4F; wt:wt) in ABCT buffer which was adjusted to pH 8.0 with 0.1N NaOH. The buffer without peptide or salicylanilide or with salicylanilide at 100 μg together with L-4F at 10 μg in 100 μL was administered to 5-month-old fasting male apoE null mice (n=4 per group) by stomach tube. Eight hours later the mice were bled and their plasma separated by FPLC and the HDL fractions from the mice were tested in cultures of human aortic endothelial cells exposed to normal human LDL to determine the HDL-inflammatory index as described in FIG. 8. The data shown are the Mean±S.D; h=human, m=mouse.
FIG. 27 shows that niclosamide increases L-4F absorption in apoE null mice. Fasted apoE null mice 6-months of age (n=4 per group) were administered by stomach tube 14C-L-4F (21,000 dpm containing 10 micrograms of L-4F per mouse) with or without 100 micrograms of niclosamide in 200 microliters. Fasting was continued and the mice were bled at the time points shown on the X-axis and the dpm per mL plasma determined.
FIG. 28 demonstrates that the 14C-L-4F used in FIG. 27 was biologically active. The HDL inflammatory index was determined as described in FIG. 8 after administration of the compounds shown in FIG. 27.
FIG. 29 shows aortic sinus lesion score in apoE null mice receiving oral doses of niclosamide, L-4F, or niclosamide together with L-4F. Seventeen week old female apoE null mice who were on chow were divided into three groups and the following additions were made to the chow for each group: Group I: Niclosamide at 250 micrograms/mouse/day; Group II: L-4F at 25 micrograms/mouse/day; Group III: L-4F at 25 micrograms/mouse/day plus Niclosamide at 250 micrograms/mouse/day. All groups received 50 micrograms/mouse/day of pravastatin in their drinking water. After 14 weeks the mice were sacrificed and aortic sinus lesion area was determined as described previously (Navab et al. (2005) Arterioscler. Thromb. Vasc. Biol., 25: 1426-1432).
FIG. 30 shows the percent aortic surface area determined by en face analysis for the mice described in FIG. 29.
FIG. 31 shows the percent macrophage lesion area for the mice described in FIG. 29.
FIG. 32 shows that oral administration of L-4F together with niclosamide causes lesion regression in old apoE null mice. Ninety-five female apoE null mice age 9.5 months from the UCLA breeding colony were identified. Twenty-three were sacrificed at time Zero (Group I) to establish lesion area at the start of the experiment. The remainder of the mice were divided into three groups of 24 mice each and the following additions were made to the chow for each group: Group II: Niclosamide at 2,000 micrograms/mouse/day; Group III: L-4F at 200 micrograms/mouse/day; Group IV: L-4F at 200 micrograms/mouse/day plus Niclosamide at 2,000 micrograms/mouse/day. All groups received 50 micrograms/mouse/day of pravastatin in their drinking water. At the veterinarian's request because of fighting and/or ulcerative dermatitis mice were euthanized prior to the end of the experiment as follows: 6 mice from Group II; 5 mice from Group III; 4 mice from Group IV. After six months the remaining mice were sacrificed and aortic sinus lesion area was determined as described previously (Id.).
FIG. 33 shows the percent aortic surface lesion area determined by en face analysis for the mice described in FIG. 32.
FIG. 34 shows the percent macrophage lesion area for the mice described in FIG. 32.
FIG. 35 shows the HDL-inflammatory index determined for apoE-null mice administered L-[113-122]apoJ or L-4F with and without niclosamide. Ten month old apoE null mice (n=4 per group) were administered by stomach tube 2 mg of niclosamide or 200 micrograms of L-[113-122]apoJ or 2 mg of niclosamide plus 200 micrograms of L-[113-122]apoJ. Eight hours later the mice were bled, their plasma separated by FPLC and the HDL-inflammatory index determined as described in FIG. 8. The data shown are Mean±S.D.



DETAILED DESCRIPTION
This invention pertains to the surprising discovery that salicylanilides, including, but not limited to niclosamide and/or niclosamide analogues when orally administered in conjunction with a pharmaceutical (e.g., a peptide pharmaceutical such as a helical peptide (e.g., a class A amphipathic helical peptide, a G* helical peptide, etc.) as described herein) significantly increases the bioavailability and/or apparent in vivo activity of that peptide. Moreover, the increase in bioavailability or apparent activity is sufficient so that peptide pharmaceuticals previously formulated as “D” amino acid isomers and protected at both termini to permit oral administration can readily be formulated utilizing all L form amino acids with optionally protected termini for oral administration. This significantly reduces the cost to manufacture such peptides and increases the predictability of the peptide's behavior in mammalian systems since the biological activity of L peptides is generally better characterized and understood.
Moreover, it was a surprising discovery that when salicylanilides, including, but not limited to niclosamide and/or niclosamide analogues, are combined (e.g., under acidic conditions) with peptide or protein therapeutics (e.g., amphipathic helical peptides, e.g., apolipoprotein A-I [apoA-I] or portions of apoA-I, or ApoJ, etc.) the salicylanilide and the peptide form an adduct that increases the apparent solubility of the bioactive agent(s) and/or the bioavailablity of the agent(s).
Thus, in certain embodiments, this invention contemplates methods of enhancing the uptake and in vivo activity of a peptide orally administered to a mammal by orally administering the peptide in conjunction with an amount of niclosamide or a niclosamide analogue sufficient to enhance in vivo activity (e.g., via enhanced uptake and/or bioavailability) of the peptide. To facilitate such methods, in certain embodiments, pharmaceutical formulations are contemplated that comprise both the peptide pharmaceutical(s) along with niclosamide and/or a niclosamide analogue. In certain embodiments the result of the reaction between the salicylanilide (e.g., niclosamide or niclosamides analogue) with the peptide or protein will be achieved by chemical synthesis prior to administration of the peptide/protein comprising the salicylanilide-derived adduct.
It was also a surprising discovery that the amphipathic helical peptides described herein can increase the solubility of niclosamide and/or niclosamide analogues in aqueous systems thereby enhancing/facilitating the incorporation of niclosamide in a pharmaceutical formulation. Thus, in certain embodiments, this invention contemplates pharmaceutical formulations comprising a combination of a therapeutic amphipathic helical peptide (e.g., D-4F, L-4F, L-5F, etc.) and niclosamide or a niclosamide analogue, wherein said niclosamide in the formulation shows substantially greater solubility in an aqueous solution than niclosamide in an aqueous solution absent the amphipathic helical peptide.
In certain embodiments, this invention also pertains to the surprising discovery that agents such as N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2-hydroxybenzoyl]aminodecanoic acid (SNAD), and N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), and the like, can increase the oral bioavailability and/or apparent activity of L form peptides to therapeutically relevant levels. This permits the use of such L form peptides as orally delivered therapeutics where previously D form peptides were preferred. In certain preferred embodiments the L form peptides are the amphipathic helical peptides described herein (e.g., L-4F, L-5F, etc.).
In certain embodiments, when administered in conjunction niclosamide and/or niclosamide analogues as described herein (including, but not necessarily limited to those shown in Formula I and/or Table 1), L-form peptides, e.g., as described herein, do not even require amino or carboxyl terminal blocking/protecting groups. Peptides lacking such blocking groups can easily be synthesized using recombinant expression systems rather than chemical peptide synthesis methods. Bioreactors can thus readily be used to prepare such unprotected peptides at very low cost (as compared to chemically synthesized peptides).
In various embodiments formulations comprising one or more therapeutic peptides in combination with niclosamide and/or niclosamide analogues as described herein, are contemplated. The formulations are typically suitable for oral administration. In certain embodiments the formulations can provide for release of niclosamide and/or niclosamide analogues and/or permeability enhancer(s) before the peptide.
While niclosamide and niclosamide analogues and/or other “permeability” enhancers described herein are particularly useful for enhancing the oral bioavailability of L peptides as described herein, the uses of these agents is not so limited. Thus, in certain embodiments the use of such agents with protected L peptides and or protected or unprotected peptides comprising one or more D amino acid residues is also contemplated.
I. Salicylanilides to Enhance Pharmaceutical In Vivo Activity.
As indicated above, it is a surprising discovery that various salicylanilides including, but not limited to niclosamide and niclosamide analogues are effective to substantially increase the in vivo activity (e.g., bioavailability, bioactivity, etc.) of a pharmaceutical (e.g., a therapeutic peptide) orally administered to a mammal.
A) Niclosamide and Niclosamide Analogues
Niclosamide is a chloronitrophenol derivative (see compound A in FIG. 1) principally used against aquatic snails but also as an antiparasitic drug in human and veterinary medicine. Niclosamide is known by the IUPAC designation: 2′5-dichloro-4′-nitrosalicylanilide and by the CAS designation: CAS: 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide.
Niclosamide is not very water soluble, 5-8 mg/L at 20° C., sparingly soluble in ether, ethanol and chloroform, and soluble in acetone; the ethanolamine salt dissolves in distilled water 180-280 mg/L at 20° C. It was a surprising discovery, however, that the inclusion of an amphipathic helical peptide, e.g., as described herein, significantly increases the solubility of niclosamide and facilitates the preparation of pharmaceutical formulations.
In tablets niclosamide undergoes a biodegradation in moist environments but niclosamide itself is stable in an aqueous solution for several months. The ethanolamine salt is stable to heat, hydrolyzed by concentrated acid or alkali, and stable in aquatic environments.
Niclosamide is readily available in a number of formulations. These include, but are not limited to, the ethanolamine salt (see compound C in FIG. 1) known by the IUPAC designation 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt or the CAS designation 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide with 2-aminoethanol (1:1), the piperazine salt (see compound B in FIG. 1) known by the IUPAC designation 5-chloro-salicyl-(2-chloro-4-nitro) anilide piperazine salt or the CAS designation 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide with piperazine (2:1), and niclosamide monohydrate (see compound D in FIG. 1) known by the IUPAC designation 5-chloro-salicyl-(2-chloro-4-nitro) anilide monohydrate or the CAS designation 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide with monohydrate (1:1).
Niclosamide is commercially available in a number of formulations including, but not limited to BAYER 73®, BAYER 2353®, BAYER 25 648®, BAYLUSCID®, BAYLUSCIDE®, CESTOCID®, CLONITRALID, DICHLOSALE®, FENASAL®, HL 2447®, IOMESAN®, IOMEZAN®, LINTEX®, MANOSIL®, NASEMO®, NICLOSAMID®, PHENASAL®, TREDEMINE®, SULQUI®, VERMITID®, VERMITIN®, YOMESAN®, and the like.
This invention also contemplates the use of various niclosamide analogues to enhance the in vivo of orally administered pharmaceuticals (e.g., therapeutic peptides). Such analogues include, but are not limited to, compounds according to Formula I:




where X is N or CR10; Y is N or CR11; Z is N or CR12; and each of R1, R2, R5, R6, R7, R8, R9, R10, R11 and R12 is independently selected from H, halide (F, Cl, Br, or I), NO2, OH, OR13, SR14, NR15R16, CN, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, C1-10 heteroalkyl, or is described by one of the Formulas II-XIV:








In compounds of formula I, R3 and R4 are independently selected from the group consisting of C═O, C═S, C═NR42, NH, NR43, CHOR44, CH2, and the like. Groups R2 and R4; X and R4; R5 and R3; R9 and R3 may combine to form a six-membered ring, using connections described by one of the groups:




For compounds of formula I, each E1 is independently O, S, or NR42; each E2 is independently CR49R50, O or S; each E3 is independently CR51R52, O, S, or NR53; each Q is, independently, O, S, or NR54. R13 and R14 are each independently, acyl, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, C1-10 heteroalkyl; R18, R23, R28, R29, R30, R42, R54 are each, independently, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, C1-10 heteroalkyl; R15, R16, R17, R19, R20, R21, R22, R24, R25, R26, R27, R43, R44, R45, R46, R47, R48, R51, R52, and R53 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, C1-10 heteroalkyl; R31, R32, R33, R34, R35, R36, R37, R38, R39, R40, R41, R49, and R50 are each, independently, H, halide, NO2, CN, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C2-6 heterocyclyl, C6-12 aryl, C7-14 alkaryl, C3-10 alkheterocyclyl, or C1-10 heteroalkyl.

In certain embodiments, compounds of formula I are further described by any of formulas XVIII-XXI:




where X, Y, Z, E1, R1, R5, R6, R7, R8, R9, R47, and R48 are as defined above.

In certain embodiments compounds include compounds described by Formula XXII:




where R1, R2, R5, R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of H, halide, NO2, CF3, OH, acyl, CN, C1-C10 alkyl (preferably C1-C3 alkyl), C1-C10 heteroalkyl (preferably C1-C3 heteroalkyl); and wherein R3 and R4 are as defined above. In certain embodiments, R3 is C═O, while R4 is NH or R3 is NH while R4 is C═O. In these and certain other embodiments, only two of R1, R2, R10, R11, and R12 are present, and one is H or OH, while the other is halogen (e.g., Cl, Br, or F).

In these and certain other embodiments, only two of R5, R6, R7, R8, and R9 are present and these are NO2 and halogen (e.g., Cl, Br, or F).
In certain embodiments niclosamide analogues include, but are not limited to niclosamide analogues in which one halogen group is relocated within the same ring (see, e.g., compounds A-D in FIG. 4) or both halogen groups are relocated within the same ring (see, e.g., compounds E-G in FIG. 4), niclosamides in which the nitro group is relocated within the same ring (see, e.g., compounds A-C in FIG. 5), niclosamide analogues where the hydroxyl group is relocated within the same ring (see, e.g., compounds D-F in FIG. 5), niclosamide analogues where both halogen and hydroxy and/or nitro groups are relocated while keeping the substituents within the aromatic ring (see, e.g., compounds A-F in FIG. 6), compounds like A-F in FIG. 6, except having except (3-chloro-4-nitrophenyl) in place of (2-chloro-4-nitrophenyl), niclosamide analogues having a nitro- and a hydroxyl group relocation (see, e.g., compounds G-I in FIG. 6), niclosamide analogues comprising a single halogen exchange (see, e.g., compounds A-D in FIG. 7), niclosamide analogues comprising a double halogen exchange (see, e.g., compounds E-F in FIG. 7), niclosamide analogs comprising an exchange of Cl— to Br— (see, e.g., compound G in FIG. 7), niclosamide analogs comprising an exchange of Cl— to F— (see, e.g., compound H in FIG. 7), and the like.
In certain embodiments the niclosamide analogues include, but are not limited to compounds according to Formula XXIII:




where R1, R2, R3, R4, and R5, are independently present or absent, and when present are independently selected from the group consisting of Cl, Br, alkyl, methyl, hydroxyalkyl, and the like. These analogues are meant to be illustrative and not limiting. Using the teaching provided herein, other suitable niclosamide analogs will be recognized by one of skill in the art.

In certain embodiments the salicylanilides include, but are not limited to salicylanilides shown in Table 1.







TABLE 1







Illustrative salicylanilides.










Cmpd
Salicylanilide
Parent Acid
Parent Amine





BP 1001














 


BP 1002 














 


BP 1003














 


BP 1004














 


BP 1005














 


BP 1006














 


BP 1007














 


BP 1008














 


BP 1009














 


BP 1010














 


BP 1011














 


BP 1012














 


BP 1013














 


BP 1014














 


BP 1015














 


BP 1016














 


BP 1017














 


BP 1018














 


BP 1019














 


BP 1020














 


BP 1021














 


BP 1022














 


BP 1023














 


BP 1024














 


BP 1025














 


BP 1026














 


BP 1027














 


BP 1028














 


BP 1029














 


BP 1030














 


BP 1031














 


BP 1032














 


BP 1033














 


BP 1034














 


BP 1035














 


BP 1036














 


BP 1037














 


BP 1038














 


BP 1039














 


BP 1040














 


BP 1041














 


BP 1042














 


BP 1043














 


BP 1044














 


BP 1045














 


BP 1046














 


BP 1047














 


BP 1048














 


BP 1049














 


BP 1050














 


BP 1051














 


BP 1052














 


BP 1053














 


BP 1055














 


BP 1056














 


BP 1057














 


BP 1058














 


BP 1059














 


BP 1061














 


BP 1063














 


BP 1064














 


BP 1065














 


BP 1067














 


BP 1068














 


BP 1069














 


BP 1070














 


BP 1071














 


BP 1072














 


BP 1073





















B) Other Salicylanilides
Without being bound by a particular theory, it is believed that a number of other salicylanilides can act in a manner similar to niclosamide to enhance in vivo activity of orally administered pharmaceuticals (e.g., therapeutic peptides). Illustrative salicylanilides include, but are not limited to Closantel (CAS #: 57808-65-8, see, e.g., FIG. 2, compound A), Oxyclozanide (CAS #: 2277-92-1, see, e.g., FIG. 2, compound B), Rafoxanide (CAS #: 22662-39-1, see, e.g., FIG. 2, compound C), Flusalan (CAS #: 4776-06-1, see, e.g., FIG. 2, compound D), Tribromsalan (CAS #: 87-10-5, see, e.g., FIG. 2, compound E), Resorantel (CAS #: 20788-07-2, see, e.g., FIG. 2, compound F), Clioxanide (CAS #: 14437-41-3, see, e.g., FIG. 2, compound G) Other suitable salicylanilides include Brotianide (CAS #: 23233-88-7), 4′-chloro-3-nitrosalicylanilide, 4′-chloro-5-nitrosalicylanilide, 2′-chloro-5′-methoxy-3-nitrosalicylanilide, 2′-methoxy-3,4′-dinitrosalicylanilide, 2′,4′-dimethyl-3-nitrosalicylanilide, 4′,5-dibromo-3-nitrosalicylanilide, 2′-chloro-3,4′-dinitrosalicylanilide, 2′-ethyl-3-nitrosalicylanilide, 2′-bromo-3-nitrosalicylanilide, and the like. In certain embodiments the salicylanilides include one or more of the compounds shown in FIG. 3.
It is noted that these salicylanilides are intended to be illustrative and not limiting. Methods of making salicylanilides are well known to those of skill in the art (see, e.g., PCT/US2003/022026 (WO 2004/006906) which is herein incorporated by reference for all purposes).
C) Identifying Effective Salicylanilides.
Using the teaching provided herein, other suitable salicylanilides can readily be identified using only routine experimentation. Various salicylanilides can be purchased from commercial vendors (e.g., Sigma Chemical, Aldrich, etc.) and then screened for their ability to enhance the apparent in vivo activity of an orally administered pharmaceutical (e.g., a peptide such as L-4F). Such screening methods can include for example, administering the salicylanilide in question in conjunction with L-4F (SEQ ID NO:5) to an apoE null mouse with appropriate controls and evaluating HDL-containing blood fractions for their ability to inhibit monocyte chemotactic activity induced by a standard control human LDL in cultures of human aortic endothelial cells. Salicylanilides that, when administered with L-4F produce more protective HDL than L-4F alone are compounds that enhance the in vivo activity (apparent activity) of that peptide. Such assays are illustrated herein in Example 1.
II. Other Delivery Agents.
Without being bound to a particular theory, in view of the niclosamide data presented herein, it is also believed that number of other delivery agents are also capable of enhancing the in vivo activity (apparent activity) of therapeutic orally administered pharmaceuticals, including, but not limited to amphipathic helical peptides (e.g., ApoA-I, ApoA-I milano, 4F, D18A, etc.) such that the L form of the peptide achieves therapeutically relevant levels of bioavailability when administered with the delivery agent(s).
Such delivery agents include, but are not limited to agents such N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2-hydroxybenzoyl]aminodecanoic acid (SNAD), and N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC) and various salts (e.g., disodium salts) thereof. In certain embodiments such delivery agents include any one or more of the modified amino acids disclosed in aforementioned U.S. Pat. No. 5,866,536 or any one of the modified amino acids described in U.S. Pat. No. 5,773,647, which are incorporated herein by reference. Also included are various salts of such agents including, but not limited to the disodium salts described in WO 00/059863 which is incorporated herein by reference.
In certain embodiments the delivery agents comprise a compound selected from the group consisting of 4-[4-{N-(4-bromobenzoyl)aminophenyl]}butyric acid, 4-{4-N-(2-iodobenzoyl)aminophenyl]}butyric acid, 3-(4-(2,5-dimethoxybenzoyl)aminophenyl)propionic acid, 4-{n-[4-(3-iodobenzoyl)aminophenyl]}butyric acid, 4-(o-anisoyl)aminophenylacetic acid, 3-[4-(2,4-dimethoxybenzoyl)aminophenyl]prioionic acid, 4-{4-[N-(4-iodobenzoyl)]aminophenyl}butyric acid, 3-4-(2,3-dimethoxybenzoyl)aminophenyl]pripionic acid, 4-{N-2[N-2-bromobenzoyl)]aminophenyl}butyric acid, 4-{N-2[N-3-bromobenzoyl]aminophenyl}butyric acid, 4-{4-[N-(4-bromobenzoyl)aminophenyl]}butyric acid, 4-{N-[4-(2-methoxy-4-nitrobenzoyl)aminophenyl]}butyric acid, 4-(4-(2,3-dimethoxybenzoyl)aminophenyl)butyric acid, 4-[4-N-(4-methoxy-3-nitrobenzoyl)aminophenyl]butyric acid, and the like.
III. Therapeutic Peptides.
This invention pertains to the use of salicylanilides (e.g., niclosamide) as well as other delivery agents to facilitate/permit the oral delivery of therapeutic peptides even when the peptides are L-form peptides and/or are unprotected. A therapeutic peptide is a peptide that is used to mitigate one or more symptoms of a disease or pathology.
A wide variety of therapeutic peptides are known to those of skill in the art and can be use in the formulations and methods of this invention. Such peptides include, for example, growth hormone (e.g., isolated and/or human, porcine, or bovine growth hormones), natural, synthetic, or recombinant growth hormone releasing hormones (GHRH), interferons (e.g., alpha, beta, and gamma interferon), interleukins (e.g., interleukin-1, interleukin, 2, etc.), natural, synthetic or recombinant insulin (e.g., porcine, bovine, human insulins), insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF2, somatostatin), heparin, heparinoids, dermatans, chondroitins, calcitonin (e.g., natural, synthetic, or recombinant salmon, procine, eel, chicken, and human calcitonin), antigens (e.g., influenza antigen, hepatitis A, B, C antigen, HPV antigen, etc), antibodies (polyclonal and monoclonal) (e.g., HERCEPTIN®, RITUXAN®, AVASTIN®, ERBITUX®, etc.), oxytocin, leutinizing-hormone-releasing hormone (LHRH), follicle stimulating hormone (FSH); glucocerebrosidase, thrombopoietin; filgrastim; prostaglandins; vasopressin; cromolyn sodium (e.g., sodium or disodium chromoglycate), vancomycin, desferrioxamine (DFO); parathyroid hormone (PTH) including its fragments, antimicrobials (e.g., anti-bacterial agents, including anti-fungal agents, etc.), and the like. In addition, the therapeutic peptides include analogs, fragments, mimetics or modified derivatives of these compounds (e.g., polyethylene glycol (PEG)-modified derivatives, glycosylated derivatives, etc.), or any combination thereof.
In certain preferred embodiments, the therapeutic peptides are peptides that ameliorate one or more symptoms of a pathology associated with an inflammatory response (e.g., atherosclerosis). Such peptides include, but are not limited to ApoA-I (natural, synthetic, recombinant), ApoA-I milano, (natural, synthetic, recombinant), apolipoprotein M, 18A, and related peptides (see, e.g., U.S. Pat. No. 4,643,988, U.S. Pat. No. 6,037,323, and PCT Publication WO 97/36927 all of which are incorporated herein by reference).
In certain particularly preferred embodiments, the therapeutic peptides used in the methods and formulations described herein include one or more of the peptides described below.
A) Class A Amphipathic Helical Peptides.
In certain embodiments, the peptides for use in the method of this invention include class A amphipathic helical peptides, e.g., as described in U.S. Pat. No. 6,664,230, and PCT Publications WO 02/15923 and WO 2004/034977. It was discovered that peptides comprising a class A amphipathic helix (“class A peptides”), in addition to being capable of mitigating one or more symptoms of atherosclerosis are also useful in the treatment of one or more of the other indications described herein.
Class A peptides are characterized by formation of an α-helix that produces a segregation of polar and non-polar residues thereby forming a polar and a nonpolar face with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face (see, e.g., Anantharamaiah (1986) Meth. Enzymol., 128: 626-668). It is noted that the fourth exon of apo A-I, when folded into 3.667 residues/turn produces a class A amphipathic helical structure.
One class A peptide, designated 18A (see, e.g., Anantharamaiah (1986) Meth. Enzymol., 128: 626-668) was modified as described herein to produce peptides orally administrable and highly effective at inhibiting or preventing one or more symptoms of atherosclerosis and/or other indications described herein. Without being bound by a particular theory, it is believed that the peptides of this invention may act in vivo by picking up/sequestering seeding molecule(s) that mitigate oxidation of LDL.
We determined that increasing the number of Phe residues on the hydrophobic face of 18A would theoretically increase lipid affinity as determined by the computation described by Palgunachari et al. (1996) Arteriosclerosis, Thrombosis, & Vascular Biol. 16: 328-338. Theoretically, a systematic substitution of residues in the nonpolar face of 18A with Phe could yield six peptides. Peptides with an additional 2, 3 and 4 Phe would have theoretical lipid affinity (λ) values of 13, 14 and 15 units, respectively. However, the λ values jumped four units if the additional Phe were increased from 4 to 5 (to 19λ units). Increasing to 6 or 7 Phe would produce a less dramatic increase (to 20 and 21λ units, respectively).
A number of these class A peptides were made including, the peptide designated 4F (L-4F), D-4F, 5F (L-5F), and D-5F, and the like. Various class A peptides inhibited lesion development in atherosclerosis-susceptible mice. In addition, the peptides show varying, but significant degrees of efficacy in mitigating one or more symptoms of the various pathologies described herein. A number of such peptides are illustrated in Table 2.







TABLE 2







Illustrative class A amphipathic helical peptides


for use in this invention.









Pep-

SEQ


tide

ID


Name
Amino Acid Sequence
NO.












18A
   D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F
1


 


2F
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2
2


 


3F
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2
3


 


3F14
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
4


 


4F
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
5


 


5F
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
6


 


6F
Ac-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2
7


 


7F
Ac-D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2
8


 



Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH2
9


 



Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH2
10


 



Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH2
11


 



Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH2
12


 



Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
13


 



Ac-E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH2
14


 



Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
15


 



Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH2
16


 



Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH2
17


 



Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH2
18


 



Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH2
19


 



Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
20


 



        Ac-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2
21


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
22


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
23


 



        Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2
24


 



        Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2
25


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
26


 



        Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH2
27


 



        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH2
28


 



        Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH2
29


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH2
30


 



        Ac-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-NH2
31


 



        Ac-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH2
32


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
33


 



        Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH2
34


 



        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH2
35


 



        Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH2
36


 



        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH2
37


 



        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
38


 



Ac-D-W-L-K-A-L-Y-D-K-V-A-E-K-L-K-E-A-L-NH2
39


 



Ac-D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH2
40


 



Ac-D-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH2
41


 



Ac-E-W-L-K-A-L-Y-E-K-V-A-E-K-L-K-E-A-L-NH2
42


 



Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH2
43


 



Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH2
44


 



Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH2
45


 



Ac-E-W-L-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH2
46


 



Ac-E-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH2
47


 



Ac-D-F-L-K-A-W-Y-D-K-V-A-E-K-L-K-E-A-W-NH2
48


 



Ac-E-F-L-K-A-W-Y-E-K-V-A-E-K-L-K-E-A-W-NH2
49


 



Ac-D-F-W-K-A-W-Y-D-K-V-A-E-K-L-K-E-W-W-NH2
50


 



Ac-E-F-W-K-A-W-Y-E-K-V-A-E-K-L-K-E-W-W-NH2
51


 



Ac-D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-NH2
52


 



Ac-D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-NH2
53


 



Ac-E-K-L-K-A-F-Y-E-K-V-F-E-W-A-K-E-A-F-NH2
54


 



Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH2
55


 



Ac-D-W-L-K-A-F-V-D-K-F-A-E-K-F-K-E-A-Y-NH2
56


 



Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH2
57


 



Ac-D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-NH2
58


 



Ac-E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F-NH2
59


 



Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2
60


 



Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH2
61


 



Ac-D-W-L-K-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH2
62


 



Ac-E-W-L-K-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH2
63


 



Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH2
64


 



Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH2
65


 



Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH2
66


 



Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH2
67


 



Ac-D-W-L-K-A-F-Y-D-R-V-A-E-R-L-K-E-A-F-NH2
68


 



Ac-E-W-L-K-A-F-Y-E-R-V-A-E-R-L-K-E-A-F-NH2
69


 



Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH2
70


 



Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH2
71


 



Ac-D-W-L-R-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH2
72


 



Ac-E-W-L-R-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH2
73


 



Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-R-E-A-F-NH2
74


 



Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-R-E-A-F-NH2
75


 



Ac-D-W-L-R-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH2
76


 



Ac-E-W-L-R-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH2
77


 



D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W-
78



L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F



 



D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-P-D-W-
79



L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F



 



D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W-
80



F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F



 



D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-P-D-K-
81



L-K-A-F-Y-D-K-V-F-E-W-L-K-E-A-F



 



D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-P-D-K-
82



W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L



 



D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-P-D-W-
83



F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F



 



D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-P-D-W-
84



L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F



 



D-W-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F-P-D-W-
85



L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F



 



 Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F-NH2
86


 



 Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH2
87


 



 Ac-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH2
88


 



 Ac-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH2
89


 



NMA-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH2
90


 



NMA-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH2
91


 



NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
92


 



NMA-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F-NH2
93


 



NMA-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
94


 



NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH2
95


 



Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
96


 



NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2



 



 Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH2
97


 



NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH2



 



 Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2
98


 



NMA-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH2



 



 Ac-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH2
99


 



NMA-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH2



 



 Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH2
100


 



NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH2



 



 Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH2
101


 



NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH2



 



 Ac-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH2
102


 



NMA-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH2



 



 Ac-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH2
103


 



NMA-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH2





1Linkers are underlined.


NMA is N-Methyl Anthranilyl.






NMA is N-Methyl Anthranilyl.

In certain preferred embodiments, the peptides include variations of 4F ((SEQ ID NO:5 in Table 2), also known as L-4F, where all residues are L form amino acids) or D-4F where one or more residues are D form amino acids). In any of the peptides described herein, the C-terminus, and/or N-terminus, and/or internal residues can be blocked with one or more blocking groups as described herein. Also, with respect to any of the peptides disclosed herein this invention contemplates L-form peptides as well as D form peptides, retro-sequences, inverse-sequences, and retro-inverse sequences.
In addition, while various peptides of Table 2, are illustrated with an acetyl group or an N-methylanthranilyl group protecting the amino terminus and an amide group protecting the carboxyl terminus, any of these protecting groups may be eliminated and/or substituted with another protecting group as described herein. In particularly preferred embodiments, the peptides comprise one or more D-form amino acids as described herein. In certain embodiments, every amino acid (e.g., every enantiomeric amino acid) of the peptides of Table 2 is a D-form amino acid.
It is also noted that Table 2 is not fully inclusive. Using the teachings provided herein, other suitable class A amphipathic helical peptides can routinely be produced (e.g., by conservative or semi-conservative substitutions (e.g., D replaced by E), extensions, deletions, and the like). Thus, for example, one embodiment utilizes truncations of any one or more of peptides shown herein (e.g., peptides identified by SEQ ID Nos:2-20 and 39—in Table 2). Thus, for example, SEQ ID NO:21 illustrates a peptide comprising 14 amino acids from the C-terminus of 18A comprising one or more D amino acids, while SEQ ID NOS:22-38 illustrate other truncations.
Longer peptides are also suitable. Such longer peptides may entirely form a class A amphipathic helix, or the class A amphipathic helix (helices) can form one or more domains of the peptide. In addition, this invention contemplates multimeric versions of the peptides (e.g., concatamers). Thus, for example, the peptides illustrated herein can be coupled together (directly or through a linker (e.g., a carbon linker, or one or more amino acids) with one or more intervening amino acids). Illustrative polymeric peptides include 18A-Pro-18A and the peptides of SEQ ID NOs:78-85, in certain embodiments comprising one or more D amino acids, more preferably with every amino acid a D amino acid as described herein and/or having one or both termini protected.
It will also be appreciated in addition to the D-form and L-form 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. Thus, for example, a retro form of the 4F peptide (DWFKAFYDKVAEKFKEAF, SEQ ID NO:5), where the amino terminus is at the aspartate (D) and the carboxyl terminus is at the phenylalanine (F), has the same sequence, but the amino terminus is at the phenylalanine and the carboxy terminus is at the aspartate (i.e., FAEKFKEAVKDYFAKFWD, SEQ ID NO:104). Where the 4F peptide comprises all L amino acids, the retro-inverso form will have the sequence shown above (SEQ ID NO:104) and comprise all D form amino acids. As illustrated in the helical wheel diagrams shown in related application U.S. Ser. No. 11/407,390 and PCT/US2006/014389, which are incorporated herein by reference, 4F and retroinverso (Rev-4F) are mirror images of each other with identical segregation of the polar and nonpolar faces with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face. These mirror images of the same polymer of amino acids are identical in terms of the segregation of the polar and nonpolar faces with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face. Thus, 4F and Rev-4F are enantiomers of each other. For a discussion of retro- and retro-inverso peptides see, e.g., Chorev and Goodman, (1995) TibTech, 13: 439-445.
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.
B) Class A Amphipathic Helical Peptide Mimetics of apoA-I Having Aromatic or Aliphatic Residues in the Non-Polar Face.
In certain embodiments, this invention also provides modified class A amphipathic helix peptides. Certain preferred peptides incorporate one or more aromatic residues at the center of the nonpolar face, e.g., 3FCπ, (as present in 4F), or with one or more aliphatic residues at the center of the nonpolar face, e.g., 3FIπ, see, e.g., Table 3. Without being bound to a particular theory, we believe the central aromatic residues on the nonpolar face of the peptide 3FCπ, due to the presence of π electrons at the center of the nonpolar face, allow water molecules to penetrate near the hydrophobic lipid alkyl chains of the peptide-lipid complex, which in turn would enable the entry of reactive oxygen species (such as lipid hydroperoxides) shielding them from the cell surface. Similarly, we also believe the peptides with aliphatic residues at the center of the nonpolar face, e.g., 3FIπ, will act similarly but not quite as effectively as 3FCπ.
Preferred peptides will convert pro-inflammatory HDL to anti-inflammatory HDL or make anti-inflammatory HDL more anti-inflammatory, and/or decrease LDL-induced monocyte chemotactic activity generated by artery wall cells equal to or greater than D-4F or other peptides shown in Table 2.







TABLE 3







Examples of certain preferred peptides.











Name
Sequence
SEQ ID NO






(3FCπ)
Ac-DKWKAVYDKFAEAFKEFL-NH2
105


 



(3FIπ)
Ac-DKLKAFYDKVFEWAKEAF-NH2
106









C) Other Class A and Some Class Y Amphipathic Helical Peptides.
In certain embodiments this invention also contemplates class a amphipathic helical peptides that have an amino acid composition identical to one or more of the class a amphipathic helical peptides described above. Thus, for example, in certain embodiments this invention contemplates peptides having an amino acid composition identical to 4F. Thus, in certain embodiments, this invention includes peptides that comprise 18 amino acids, where the 18 amino acids consist of 3 alanines (A), 2 aspartates (D), 2 glutamates (E), 4 phenylalanines (F), 4 lysines (K), 1 valine (V), 1 tryptophan (W), and 1 tyrosine (Y); and where the peptide forms a class A amphipathic helix; and protects a phospholipid against oxidation by an oxidizing agent. In various embodiments, the peptides comprise least one “D” amino acid residue; and in certain embodiments, the peptides comprise all “D: form amino acid residues. A variety of such peptides are illustrated in Table 4. Reverse (retro-), inverse, retro-inverso-, and circularly permuted forms of these peptides are also contemplated.







TABLE 4







Illustrative 18 amino acid length class A amphipathic helical


peptides with the amino acid composition 3 alanines (A),


2 aspartates (D), 2 glutamates (E), 4 phenylalanines (F),


4 lysines (K), 1 valine (V), 1 tryptophan (W),


and 1 tyrosine (Y).











SEQ


Name
Sequence
ID NO





[Switch D-E]-4F analogs




[Switch D-E]-1-4F
Ac-EWFKAFYEKVADKFKDAF-NH2
107


[Switch D-E]-2-4F
Ac-EWFKAFYDKVADKFKEAF-NH2
108


[Switch D-E]-3-4F
Ac-DWFKAFYEKVADKFKEAF-NH2
109


[Switch D-E]-4-4F
Ac-DWFKAFYEKVAEKFKDAF-NH2
110


 


[W-2, F-3 positions reversed]




4F-2
Ac-DFWKAFYDKVAEKFKEAF-NH2
111


[Switch D-E]-1-4F-2
Ac-EFWKAFYEKVADKFKDAF-NH2
112


[Switch D-E]-2-4F-2
Ac-EFWKAFYDKVADKFKEAF-NH2
113


[Switch D-E]-3-4F-2
Ac-DFWKAFYEKVADKFKEAF-NH2
114


[Switch D-E]-4-4F-2
Ac-DFWKAFYEKVAEKFKDAF-NH2
115


 


[F-6 and Y-7 positions switched]




4F-3
Ac-DWFKAYFDKVAEKFKEAF-NH2
116


[Switch D-E]-1-4F-5
Ac-EWFKAYFEKVADKFKDAF-NH2
117


[Switch D-E]-2-4F-5
Ac-EWFKAYFDKVADKFKEAF-NH2
118


[Switch D-E]-3-4F-5
Ac-DWFKAYFEKVADKFKEAF-NH2
119


[Switch D-E]-4-4F-5
Ac-DWFKAYFEKVAEKFKDAF-NH2
120


 


[Y-7 and 10V positions switched]




4F-4
Ac-DWFKAFVDKYAEKFKEAF-NH2
121


[Switch D-E]-1-4F-4
Ac-EWFKAFVEKYADKFKDAF-NH2
122


[Switch D-E]-2-4F-4
Ac-EWFKAFVDKYADKFKEAF-NH2
123


[Switch D-E]-3-4F-4
Ac-DWFKAFVEKYADKFKEAF-NH2
124


[Switch D-E]-4-4F
Ac-DWFKAFVEKYAEKFKDAF-NH2
125


 


[V-10 and A-11 switched]




4-F-5
Ac-DWFKAFYDKAVEKFKEAF-NH2
126


[Switch D-E]-1-4F-5
Ac-EWFKAFYEKAVDKFKDAF-NH2
127


[Switch D-E]-2-4F-5
Ac-EWFKAFYDKAVDKFKEAF-NH2
128


[Switch D-E]-3-4F-5
Ac-DWFKAFYEKAVDKFKEAF-NH2
129


[Switch D-E]-4-4F-5
Ac-DWFKAFYEKAVEKFKDAF-NH2
130


 


[A-11 and F-14 switched]




4F-6
Ac-DWFKAFYDKVFEKAKEAF-NH2
131


[Switch D-E]-1-4F-6
Ac-EWFKAFYEKVFDKAKDAF-NH2
132


[Switch D-E]-2-4F-6
Ac-EWFKAFYDKVFDKAKEAF-NH2
133


[Switch D-E]-3-4F-6
Ac-DWFKAFYEKVFDKAKEAF-NH2
134


[Switch D-E]-4-4F-6
Ac-DWFKAFYEKVFEKAKDAF-NH2
135


 


[F-14 and A-17 switched]




4F-7
Ac-DWFKAFYDKVAEKAKEFF-NH2
136


[Switch D-E]-1-4F-7
Ac-EWFKAFYEKVADKAKDFF-NH2
137


[Switch D-E]-2-4F-7
Ac-EWFKAFYDKVADKAKEFF-NH2
138


[Switch D-E]-3-4F-7
Ac-DWFKAFYEKVADKAKEFF-NH2
139


[Switch D-E]-4-4F-7
Ac-DWFKAFYEKVAEKAKDFF-NH2
140


 


[A-17 and F-18 switched]




4F-8
Ac-DWFKAFYDKVAEKFKEFA-NH2
141


[Switch D-E]-1-4F-8
Ac-EWFKAFYEKVADKFKDFA-NH2
142


[Switch D-E]-2-4F-8
Ac-EWFKAFYDKVADKFKEFA-NH2
143


[Switch D-E]-3-4F-8
Ac-DWFKAFYEKVADKFKEFA-NH2
144


[Switch D-E]-4-4F-8
Ac-DWFKAFYEKVAEKFKDFA-NH2
145


 


[W-2 and A-17 switched]




4F-9
Ac-DAFKAFYDKVAEKFKEWF-NH2
146


[Switch D-E]-1-4F-9
Ac-EAFKAFYEKVADKFKDWF-NH2
147


[Switch D-E]-2-4F-9
Ac-EAFKAFYDKVADKFKEWF-NH2
148


[Switch D-E]-3-4F-9
Ac-DAFKAFYEKVADKFKEWF-NH2
149


[Switch D-E]-4-4F-9
Ac-DAFKAFYEKVAEKFKDWF-NH2
150


 


[W-2 and A-11 switched]




4F-10
Ac-DAFKAFYDKVWEKFKEAF-NH2
151


[Switch D-E]-1-4F-10
Ac-EAFKAFYEKVWDKFKDAF-NH2
152


[Switch D-E]-2-4F-10
Ac-EAFKAFYDKVWDKFKEAF-NH2
153


[Switch D-E]-3-4F-10
Ac-DAFKAFYEKVWDKFKEAF-NH2
154


[Switch D-E]-4-4F-10
Ac-DAFKAFYEKVWEKFKDAF-NH2
155


 


[W-2 and Y-7 switched]




4F-11
Ac-DYFKAFWDKVAEKFKEAF-NH2
156


[Switch D-E]-1-4F-11
Ac-EYFKAFWEKVADKFKDAF-NH2
157


[Switch D-E]-2-4F-11
Ac-EYFKAFWDKVADKFKEAF-NH2
158


[Switch D-E]-3-4F-11
Ac-DYFKAFWEKVADKFKEAF-NH2
159


[Switch D-E]-4-4F-11
Ac-DYFKAFWEKVAEKFKDAF-NH2
160


 


[F-3 and A-17 switched]




4F-12
Ac-DWAKAFYDKVAEKFKEFF-NH2
161


[Switch D-E]-1-4F-12
Ac-EWAKAFYEKVADKFKDFF-NH2
162


[Switch D-E]-2-4F-12
Ac-EWAKAFYDKVADKFKEFF-NH2
163


[Switch D-E]-3-4F-12
Ac-DWAKAFYEKVADKFKEFF-NH2
164


[Switch D-E]-4-4F-12
Ac-DWAKAFYEKVAEKFKDFF-NH2
165


 


[F-6 and A-17 switched]




4F-13
Ac-DWFKAAYDKVAEKFKEFF-NH2
166


[Switch D-E]-1-4F-13
Ac-EWFKAAYEKVADKFKDFF-NH2
167


[Switch D-E]-2-4F-13
Ac-EWFKAAYDKVADKFKEFF-NH2
168


[Switch D-E]-3-4F-13
Ac-DWFKAAYEKVADKFKEFF-NH2
169


[Switch D-E]-4-4F-13
Ac-DWFKAAYEKVAEKFKDFF-NH2
170


 


[Y-7 and A-17 switched




4F-14
Ac-DWFKAFADKVAEKFKEYF-NH2
171


[Switch D-E]-1-4F-14
Ac-EWFKAFAEKVADKFKDYF-NH2
172


[Switch D-E]-2-4F-14
Ac-EWFKAFADKVADKFKEYF-NH2
173


[Switch D-E]-3-4F-14
Ac-DWFKAFAEKVADKFKEYF-NH2
174


[Switch D-E]-4-4F
Ac-DWFKAFAEKVAEKFKDYF-NH2
175


 


[V-10 and A-17 switched]




4F-15
Ac-DWFKAFYDKAAEKFKEVF-NH2
176


[Switch D-E]-1-4F-15
Ac-EWFKAFYEKAADKFKDVF-NH2
177


[Switch D-E]-2-4F-15
Ac-EWFKAFYDKAADKFKEVF-NH2
178


[Switch D-E]-3-4F-15
Ac-DWFKAFYEKAADKFKEVF-NH2
179


[Switch D-E]-4-4F-15
Ac-DWFKAFYEKAAEKFKDVF-NH2
180


 


[F3 and Y-7 switched]




4F-16
Ac-DWYKAFFDKVAEKFKEAF-NH2
181


[Switch D-E]-1-4F-16
Ac-EWYKAFFEKVADKFKDAF-NH2
182


[Switch D-E]-2-4F-16
Ac-EWYKAFFDKVADKFKEAF-NH2
183


[Switch D-E]-3-4F-16
Ac-DWYKAFFEKVADKFKEAF-NH2
184


[Switch D-E]-4-4F-16
Ac-DWYKAFFEKVAEKFKDAF-NH2
185


 


[F-3 and V-10 switched]




4F-17
Ac-DWVKAFYDKFAEKFKEAF-NH2
186


[Switch D-E]-1-4F-17
Ac-EWVKAFYEKFADKFKDAF-NH2
187


[Switch D-E]-2-4F-17
Ac-EWVKAFYDKFADKFKEAF-NH2
188


[Switch D-E]-3-4F-17
Ac-DWVKAFYEKFADKFKEAF-NH2
189


[Switch D-E]-4-4F-17
Ac-DWVKAFYEKFAEKFKDAF-NH2
190


 


[Y-7 and F-14 switched]




4F-18
Ac-DWFKAFFDKVAEKYKEAF-NH2
191


[Switch D-E]-1-4F-18
Ac-EWFKAFFEKVADKYKDAF-NH2
192


[Switch D-E]-2-4F-18
Ac-EWFKAFFDKVADKYKEAF-NH2
193


[Switch D-E]-3-4F-18
Ac-DWFKAFFEKVADKYKEAF-NH2
194


[Switch D-E]-3-4F-18
Ac-DWFKAFFEKVADKYKEAF-NH2
195


 


[Y-7 and F-18 switched]




4F-19
Ac-DWFKAFFDKVAEKFKEAY-NH2
196


[Switch D-E]-1-4F-19
Ac-EWFKAFFEKVADKFKDAY-NH2
197


[Switch D-E]-2-4F-19
Ac-EWFKAFFDKVADKFKEAY-NH2
198


[Switch D-E]-3-4F-19
Ac-DWFKAFFEKVADKFKEAY-NH2
199


[Switch D-E]-4-4F-19
Ac-DWFKAFFEKVAEKFKDAY-NH2
200


 


[V-10 and F-18 switched




4F-20
Ac-DWFKAFYDKFAEKFKEAV-NH2
201


[Switch D-E]-1-4F-20
Ac-EWFKAFYEKFADKFKDAV-NH2
202


[Switch D-E]-2-4F-20
Ac-EWFKAFYDKFADKFKEAV-NH2
203


[Switch D-E]-3-4F-20
Ac-DWFKAFYEKFADKFKEAV-NH2
204


[Switch D-E]-4-4F-20
Ac-DWFKAFYEKFAEKFKDAV-NH2
205


 


[W-2 and K13 switched]




4F-21
Ac-DKFKAFYDKVAEKFWEAF-NH2
206


[Switch D-E]-1-4F-21
Ac-EKFKAFYEKVADKFWDAF-NH2
207


[Switch D-E]-2-4F-21
Ac-EKFKAFYDKVADKFWEAF-NH2
208


[Switch D-E]-3-4F-21
Ac-DKFKAFYEKVADKFWEAF-NH2
209


[Switch D-E]-4-4F-21
Ac-DKFKAFYEKVAEKFWDAF-NH2
210


 


[W-3, F-13 and K-2 4F]




4F-22
Ac-DKWKAFYDKVAEKFFEAF-NH2
211


[Switch D-E]-1-4F-22
Ac-EKWKAFYEKVADKFFDAF-NH2
212


[Switch D-E]-2-4F-22
Ac-EKWKAFYDKVADKFFEAF-NH2
213


[Switch D-E]-3-4F-22
Ac-DKWKAFYEKVADKFFEAF-NH2
214


[Switch D-E]-4-4F-22
Ac-DKWKAFYEKVAEKFFDAF-NH2
215


 


[K-2, W10, V-13]




4F-23
Ac-DKFKAFYDKWAEVFKEAF-NH2
216


 


[Switch D-E]-4F analogs




[Switch D-E]-1-4F-23
Ac-EKFKAFYEKWADVFKDAF-NH2
217


[Switch D-E]-2-4F-23
Ac-EKFKAFYDKWADVFKEAF-NH2
218


[Switch D-E]-3-4F-23
Ac-DKFKAFYEKWADVFKEAF-NH2
219


[Switch D-E]-4-4F-23
Ac-DKFKAFYEKWAEVFKDAF-NH2
220


 


[K-2, F-13, W-14 4F]




4F-24
Ac-DKFKAFYDKVAEFWKEAF-NH2
221


 


[Switch D-E]-4F analogs




[Switch D-E]-1-4F-24
Ac-EKFKAFYEKVADFWKDAF-NH2
222


[Switch D-E]-2-4F-24
Ac-EKFKAFYDKVADFWKEAF-NH2
223


[Switch D-E]-3-4F-24
Ac-DKFKAFYEKVADFWKEAF-NH2
224


[Switch D-E]-4-4F-24
Ac-DKFKAFYEKVAEFWKDAF-NH2
225


 


Reverse 4F analogs




Rev-4F
Ac-FAEKFKEAVKDYFAKFWD-NH2
226


[Switch D-E]-1-Rev-4F
Ac-FADKFKDAVKEYFAKFWE-NH2
227


[Switch D-E]-2-Rev-4F
Ac-FADKFKEAVKDYFAKFWE-NH2
228


[Switch D-E]-3-Rev-4F
Ac-FAEKFKDAVKEYFAKFWD-NH2
229


[Switch D-E]-4-Rev-4F
Ac-FAEKFKDAVKDYFAKFWE-NH2
230


 


[A-2 and W-17 switched]




Rev-4F-1
Ac-FWEKFKEAVKDYFAKFAD-NH2
231


[Switch D-E]-1-Rev-4F-1
Ac-FWDKFKDAVKEYFAKFAE-NH2
232


[Switch D-E]-2-Rev-4F-1
Ac-FADKFKEAVKDYFAKFWE-NH2
233


[Switch D-E]-3-Rev-4F-1
Ac-FAEKFKDAVKEYFAKFWD-NH2
234


[Switch D-E]-4-Rev-4F-1
Ac-FAEKFKDAVKDYFAKFWE-NH2
235


 


[Switch A-2 and F-16]




Rev-4F-2
Ac-FFEKFKEAVKDYFAKAWD-NH2
236


[Switch D-E]-1-Rev-4F-2
Ac-FFDKFKDAVKEYFAKAWE-NH2
237


[Switch D-E]-2-Rev-4F-2
Ac-FFDKFKEAVKDYFAKAWE-NH2
238


[Switch D-E]-3-Rev-4F-2
Ac-FFEKFKDAVKEYFAKAWD-NH2
239


[Switch D-E]-4-Rev-4F-2
Ac-FFEKFKDAVKDYFAKAWE-NH2
240


 


[switch F-5 and A-8]




Rev-4F-3
Ac-FAEKAKEFVKDYFAKFWD-NH2
241


[Switch D-E]-1-Rev-4F-3
Ac-FADKAKDFVKEYFAKFWE-NH2
242


[Switch D-E]-2-Rev-4F-3
Ac-FADKAKEFVKDYFAKFWE-NH2
243


[Switch D-E]-3-Rev-4F-3
Ac-FAEKAKDFVKEYFAKFWD-NH2
244


[Switch D-E]-4-Rev-4F-3
Ac-FAEKAKDFVKDYFAKFWE-NH2
245


 


[Switch A-8 and V9]




Rev-4F-4
Ac-FAEKFKEVAKDYFAKFWD-NH2
246


[Switch D-E]-1-Rev-4F-4
Ac-FADKFKDVAKEYFAKFWE-NH2
247


[Switch D-E]-2-Rev-4F-4
Ac-FADKFKEVAKDYFAKFWE-NH2
248


[Switch D-E]-3-Rev-4F-4
Ac-FAEKFKDVAKEYFAKFWD-NH2
249


[Switch D-E]-4-Rev-4F-4
Ac-FAEKFKDVAKDYFAKFWE-NH2
250


 


[Switch V-9 to Y-12]




Rev-4F-5
Ac-FAEKFKEAYKDVFAKFWD-NH2
251


[Switch D-E]-1-Rev-4F-5
Ac-FADKFKDAYKEVFAKFWE-NH2
252


[Switch D-E]-2-Rev-4F-5
Ac-FADKFKEAYKDVFAKFWE-NH2
253


[Switch D-E]-3-Rev-4F-5
Ac-FAEKFKDAYKEVFAKFWD-NH2
254


[Switch D-E]-4-Rev-4F-5
Ac-FAEKFKDAYKDVFAKFWE-NH2
255


 


[Switch Y-12 and F-13]




Rev-4F-6
Ac-FAEKFKEAVKDFYAKFWD-NH2
256


[Switch D-E]-1-Rev-4F-6
Ac-FADKFKDAVKEFYAKFWE-NH2
257


[Switch D-E]-2-Rev-4F-6
Ac-FADKFKEAVKDFYAKFWE-NH2
258


[Switch D-E]-3-Rev-4F-6
Ac-FAEKFKDAVKEFYAKFWD-NH2
259


[Switch D-E]-4-Rev-4F-6
Ac-FAEKFKDAVKDFYAKFWE-NH2
260


 


[Switch K-6 and W-17]




Rev-4F-7
Ac-FAEKFWEAVKDYFAKFKD-NH2
261


[Switch D-E]-1-Rev-4F-7
Ac-FADKFWDAVKEYFAKFKE-NH2
262


[Switch D-E]-2-Rev-4F-7
Ac-FADKFWEAVKDYFAKFKE-NH2
263


[Switch D-E]-3-Rev-4F-7
Ac-FAEKFWDAVKEYFAKFKD-NH2
264


[Switch D-E]-4-Rev-4F-7
Ac-FAEKFWDAVKDYFAKFKE-NH2
265


 


[Switch F-1 and A-2]




Rev-4F-8
Ac-AFEKFKEAVKDYFAKFWD-NH2
266


[Switch D-E]-1-Rev-4F-8
Ac-AFDKFKDAVKEYFAKFWE-NH2
267


[Switch D-E]-2-Rev-4F-8
Ac-AFDKFKEAVKDYFAKFWE-NH2
268


[Switch D-E]-3-Rev-4F-8
Ac-AFEKFKDAVKEYFAKFWD-NH2
269


[Switch D-E]-4-Rev-4F-8
Ac-AFEKFKDAVKDYFAKFWE-NH2
270


 


[F-1 and V-9 are switched]




Rev-F-9
Ac-VAEKFKEAFKDYFAKFWD-NH2
271


[Switch D-E]-1-Rev-4F-9
Ac-VADKFKDAFKEYFAKFWE-NH2
272


[Switch D-E]-2-Rev-4F-9
Ac-VADKFKEAFKDYFAKFWE-NH2
273


[Switch D-E]-3-Rev-4F-9
Ac-VAEKFKDAFKEYFAKFWD-NH2
274


[Switch D-E]-4-Rev-4F-9
Ac-VAEKFKDAFKDYFAKFWE-NH2
275


 


[F-1 and Y-12 are switched]




Rev-4F-10
Ac-YAEKFKEAVKDFFAKFWD-NH2
276


[Switch D-E]-1-Rev-4F-10
Ac-YADKFKDAVKEFFAKFWE-NH2
277


[Switch D-E]-2-Rev-4F-10
Ac-YADKFKEAVKDFFAKFWE-NH2
278


[Switch D-E]-3-Rev-4F-10
Ac-YAEKFKDAVKEFFAKFWD-NH2
279


[Switch D-E]-4-Rev-4F-10
Ac-YAEKFKDAVKDFFAKFWE-NH2
280


 


[F-1 and A-8 are switched]




Rev-4F-11
Ac-AAEKFKEFVKDYFAKFWD-NH2
281


[Switch D-E]-1-Rev-4F-11
Ac-AADKFKDFVKEYFAKFWE-NH2
282


[Switch D-E]-2-Rev-4F-11
Ac-AADKFKEFVKDYFAKFWE-NH2
283


[Switch D-E]-3-Rev-4F-11
Ac-AAEKFKDFVKEYFAKFWD-NH2
284


[Switch D-E]-4-Rev-4F-11
Ac-AAEKFKDFVKDYFAKFWE-NH2
285


 


[A-2 and F-5 are switched]




Rev-4F-12
Ac-FFEKAKEAVKDYFAKFWD-NH2
286


[Switch D-E]-1-Rev-4F-12
Ac-FFDKAKDAVKEYFAKFWE-NH2
287


[Switch D-E]-2-Rev-4F-12
Ac-FFDKAKEAVKDYFAKFWE-NH2
288


[Switch D-E]-3-Rev-4F-12
Ac-FFEKAKDAVKEYFAKFWD-NH2
289


[Switch D-E]-4-Rev-4F-12
Ac-FFEKAKDAVKDYFAKFWE-NH2
290


 


[A-2 and Y12 are switched




Rev-4F-13
Ac-FYEKFKEAVKDAFAKFWD-NH2
291


[Switch D-E]-1-Rev-4F-13
Ac-FYDKFKDAVKEAFAKFWE-NH2
292


[Switch D-E]-2-Rev-4F-13
Ac-FYDKFKEAVKDAFAKFWE-NH2
293


[Switch D-E]-3-Rev-4F-13
Ac-FYEKFKDAVKEAFAKFWD-NH2
294


[Switch D-E]-4-Rev-4F-13
Ac-FYEKFKDAVKDAFAKFWE-NH2
295


 


[A-2 and V-9 are switched]




Rev-4F-14
Ac-FVEKFKEAAKDYFAKFWD-NH2
296


[Switch D-E]-1-Rev-4F-14
Ac-FVDKFKDAAKEYFAKFWE-NH2
297


[Switch D-E]-2-Rev-4F-14
Ac-FVDKFKEAAKDYFAKFWE-NH2
298


[Switch D-E]-3-Rev-4F-14
Ac-FVEKFKDAAKEYFAKFWD-NH2
299


[Switch D-E]-4-Rev-4F-14
Ac-FVEKFKDAAKDYFAKFWE-NH2
300


 


[F-5 and Y-12 are switched]




Rev-4F-15
Ac-FAEKYKEAVKDFFAKFWD-NH2
301


[Switch D-E]-1-Rev-4F-15
Ac-FADKYKDAVKEFFAKFWE-NH2
302


[Switch D-E]-2-Rev-4F-15
Ac-FADKYKEAVKDFFAKFWE-NH2
303


[Switch D-E]-3-Rev-4F-15
Ac-FAEKYKDAVKEFFAKFWD-NH2
304


[Switch D-E]-4-Rev-4F-15
Ac-FAEKYKDAVKDFFAKFWE-NH2
305


 


[F-5 and V-9 are switched]




Rev-4F-16
Ac-FAEKVKEAFKDYFAKFWD-NH2
306


[Switch D-E]-1-Rev-4F-16
Ac-FADKVKDAFKEYFAKFWE-NH2
307


[Switch D-E]-2-Rev-4F-16
Ac-FADKVKEAFKDYFAKFWE-NH2
308


[Switch D-E]-3-Rev-4F-16
Ac-FAEKVKDAFKEYFAKFWD-NH2
309


[Switch D-E]-4-Rev-4F-16
Ac-FAEKVKDAFKDYFAKFWE-NH2
310


 


[A-8 and Y-12 switched]




Rev-4F-17
Ac-FAEKFKEYVKDAFAKFWD-NH2
311


[Switch D-E]-1-Rev-4F-17
Ac-FADKFKDYVKEAFAKFWE-NH2
312


[Switch D-E]-2-Rev-4F-17
Ac-FADKFKEYVKDAFAKFWE-NH2
313


[Switch D-E]-3-Rev-4F-17
Ac-FAEKFKDYVKEAFAKFWD-NH2
314


[Switch D-E]-4-Rev-4F-17
Ac-FAEKFKDYVKDAFAKFWE-NH2
315


 


[V-9 and F-13 are switched]




Rev-4F-18
Ac-FAEKFKEAFKDYVAKFWD-NH2
316


[Switch D-E]-1-Rev-4F-18
Ac-FADKFKDAFKEYVAKFWE-NH2
317


[Switch D-E]-2-Rev-4F-18
Ac-FADKFKEAFKDYVAKFWE-NH2
318


[Switch D-E]-3-Rev-4F-18
Ac-FAEKFKDAFKEYVAKFWD-NH2
319


[Switch D-E]-4-Rev-4F-18
Ac-FAEKFKDAFKDYVAKFWE-NH2
320


 


[V-9 and F-16 switched]




Rev-4F-19
Ac-FAEKFKEAFKDYFAKVWD-NH2
321


[Switch D-E]-1-Rev-4F-19
Ac-FADKFKDAFKEYFAKVWE-NH2
322


[Switch D-E]-2-Rev-4F-19
Ac-FADKFKEAFKDYFAKVWE-NH2
323


[Switch D-E]-3-Rev-4F-19
Ac-FAEKFKDAFKEYFAKVWD-NH2
324


Switch D-E]-4-Rev-4F-19
Ac-FAEKFKDAFKDYFAKVWE-NH2
325


 


[Y-12 and F-16 are switched




Rev-4F-20
Ac-FAEKFKEAVKDFFAKYWD-NH2
326


[Switch D-E]-1-Rev-4F-20
Ac-FADKFKDAVKEFFAKYWE-NH2
327


[Switch D-E]-2-Rev-4F-20
Ac-FADKFKEAVKDFFAKYWE-NH2
328


[Switch D-E]-3-Rev-4F-20
Ac-FAEKFKDAVKEFFAKYWD-NH2
329


[Switch D-E]-4-Rev-4F-20
Ac-FAEKFKDAVKDFFAKYWE-NH2
330


 


[W-1, F-6 and K-17 Rev 4F]




Rev-4F-21
Ac-WAEKFFEAVKDYFAKFKD-NH2
331


[Switch D-E]-1-Rev-4F-7
Ac-WADKFFDAVKEYFAKFKE-NH2
332


[Switch D-E]-2-Rev-4F-7
Ac-WADKFFEAVKDYFAKFKE-NH2
333


[Switch D-E]-3-Rev-4F-7
Ac-WAEKFFDAVKEYFAKFKD-NH2
334


[Switch D-E]-4-Rev-4F-7
Ac-WAEKFFDAVKDYFAKFKE-NH2
335


 


[W-5, F-6 and K-17 Rev-4F]




Rev-4F-22
Ac-FAEKWFEAVKDYFAKFKD-NH2
336


[Switch D-E]-1-Rev-4F-22
Ac-FADKWFDAVKEYFAKFKE-NH2
337


[Switch D-E]-2-Rev-4F-22
Ac-FADKWFEAVKDYFAKFKE-NH2
338


[Switch D-E]-3-Rev-4F-22
Ac-FAEKWFDAVKEYFAKFKD-NH2
339


[Switch D-E]-4-Rev-4F-22
Ac-FAEKWFDAVKDYFAKFKE-NH2
340


 


[V-6, W-9, K-17 Rev-4F]




Rev-4F-23
Ac-FAEKFVEAWKDYFAKFKD-NH2
341


[Switch D-E]-1-Rev-4F-23
Ac-FADKFVDAWKEYFAKFKE-NH2
342


[Switch D-E]-2-Rev-4F-23
Ac-FADKFVEAWKDYFAKFKE-NH2
343


[Switch D-E]-3-Rev-4F-23
Ac-FAEKFVDAWKEYFAKFKD-NH2
344


[Switch D-E]-4-Rev-4F-23
Ac-FAEKFVDAWKDYFAKFKE-NH2
345


 


[Y-2, A-4, W-12, K-17 Rev-4F]




Rev-4F-24 
Ac-FYEKFAEAVKDWFAKFKD-NH2
346


[Switch D-E]-1-Rev-4F-24
Ac-FYDKFADAVKEWFAKFKE-NH2
347


[Switch D-E]-2-Rev-4F-24
Ac-FYDKFAEAVKDWFAKFKE-NH2
348


[Switch D-E]-3-Rev-4F-24
Ac-FYEKFADAVKEWFAKFKD-NH2
349


[Switch D-E]-4-Rev-4F-24
Ac-FYEKFADAVKDWFAKFKE-NH2
350









Based on helical wheel diagrams, it is possible to readily identify biologically active and useful peptides. Thus, for example, the following peptides have been accurately identified as active: 3F1; 3F2; 4F the inverse forms thereof, the reverse (retro) forms thereof and the retro-inverso forms thereof. Thus, in certain embodiments, this invention contemplates active agents comprising a peptide that is 18 amino acids in length and forms a class A amphipathic helix where the peptide has the amino acid composition 2 aspartates, 2 glutamates, 4 lysines, 1 tryptophan, 1 tyrosine, no more than one leucine, no more than 1 valine, no less than 1 and no more than 3 alanines, and with 3 to 6 amino acids from the group: phenylalanine, alpha-naphthalanine, beta-naphthalanine, histidine, and contains either 9 or 10 amino acids on the polar face in a helical wheel representation of the class A amphipathic helix including 4 amino acids with positive charge at neutral pH with two of the positively charged residues residing at the interface between the polar and non-polar faces and with two of the four positively charged residues on the polar face that are contiguous and on the non-polar face two of the amino acid residues from the group: phenylalanine, alpha-naphthalanine, beta-naphthalanine, histidine are also contiguous and if there are 4 or more amino acids from this group on the non-polar face there are also at least 2 residues from this group that are not contiguous.
In certain embodiments, this invention also contemplates certain class Y as well as class A amphipathic helical peptides. Class Y amphipathic helical peptides are known to those of skill in the art (see, e.g., Segrest et al. (1992) J. Lipid Res. 33: 141-166; Oram and Heinecke (2005) Physiol Rev. 85: 1343-1372, and the like). In various embodiments these peptides include, but are not limited to an 18 amino acid peptide that forms a class A amphipathic helix or a class Y amphipathic helix described by Formula XXIV (SEQ ID NO:351):










D X X K Y X X D K X Y D KX K D Y X
XXIV






where the D's are independently Asp or Glu; the Ks are independently Lys or Arg; the Xs are independently Leu, norLeu, Val, Ile, Trp, Phe, Tyr, β-Nal, or α-Nal and all X residues are on the non-polar face (e.g., when viewed in a helical wheel diagram) except for one that can be on the polar face between two K residues; the Y's are independently Ala, His, Ser, Gln, Asn, or Thr non-polar face (e.g., when viewed in a helical wheel diagram) and the Y's are independently one Ala on the polar face, one His, one Ser, one Gln one Asn, or one Thr on the polar face (e.g., when viewed in a helical wheel diagram), where no more than two K are be contiguous (e.g., when viewed in a helical wheel diagram); and where no more than 3 D's are contiguous (e.g., when viewed in a helical wheel diagram) and the fourth D is be separated from the other D's by a Y. Illustrative peptides of this kind which include peptides with histidine, and/or alpha- and/or beta-napthalanine are shown in Table 5. Reverse (retro-), inverse, retro-inverso-, and circularly permuted forms of these peptides are also contemplated.








TABLE 5







Illustrates various class A and/or class Y peptide analogs with His


incorporated into the sequence.











SEQ




ID


Short name
Peptide sequence
NO












[A-5>H]4F
Ac-DWFKHFYDKVAEKFKEAF-NH2
352


 


[A-5>H, D-E switched]4F
Ac-EWFKHFYEKVADKFKDAF-NH2
353


 


[A-5>H, D-1>E]4F
Ac-EWFKHFYDKVAEKFKEAF-NH2
354


 


[A-5>H, D-8>E]4-F
Ac-DWFKHFYEKVAEKFKEAF-NH2
355


 


[A-5>H, E-12>D]4F
Ac-DWFKHFYDKVADKFKEAF-NH2
356


 


[A-5>H, E-16>D]4F
Ac-DWFKHFYDKVAEKFKDAF-NH2
357


 


[F-3>H, A-5>F]-4F
Ac-DWHKFFYDKVAEKFKEAF-NH2
358


 


[F-3>H, A-5>F, D-E switched]-4F
Ac-EWHKFFYEKVADKFKDAF-NH2
359


 


[F-3>H, A-5>F, D-1>E]-4F
Ac-EWHKFFYDKVAEKFKEAF-NH2
360


 


[F-3>H, A-5>F, D-8>E]-4F
Ac-DWHKFFYEKVAEKFKEAF-NH2
361


 


[F-3>H, A-5>F, E-12>D]-4F
Ac-DWHKFFYDKVADKFKEAF-NH2
362


 


[F-3>H, A-5>F, E-16>D]-4F
Ac-DWHKFFYDKVAEKFKDAF-NH2
363


 


[A-5>F, F-6>H]4F
Ac-DWFKFHYDKVAEKFKEAF-NH2
364


 


[A-5>F, F-6>H, D-E switched]4F
Ac-EWFKFHYEKVADKFKDAF-NH2
365


 


[[A-5>F, F-6>H, D-1>E]4F
Ac-EWFKFHYDKVAEKFKEAF-NH2
366


 


[A-5>F, F-6>H, D-8>E]4F
Ac-DWFKFHYEKVAEKFKEAF-NH2
367


 


[A-5>F, F-6>H, E-12>D]4F
Ac-DWFKFHYDKVADKFKEAF-NH2
368


 


[A-5>F, F-6>H, E-16>D]4F
Ac-DWFKFHYDKVAEKFKDAF-NH2
369


 


[A-5>V, V-10>H]4F
Ac-DWFKVFYDKHAEKFKEAF-NH2
370


 


[A-5>V, V-10>H, D-E switched]4F
Ac-EWFKVFYEKHADKFKDAF-NH2
371


 


[A-5>V, V-10>H, D-1>E]4F
Ac-EWFKVFYDKHAEKFKEAF-NH2
372


 


[A-5>V, V-10>H, D-8>E]4F
Ac-DWFKVFYEKHAEKFKEAF-NH2
373


 


[A-5>V, V-10>H, E-12>D]4F
Ac-DWFKVFYDKHADKFKEAF-NH2
374


 


[A-5>V, V-10>H, E16>D]4F
Ac-DWFKVFYDKHAEKFKDAF-NH2
375


 


[[A-17>H]4F
Ac-DWFKAFYDKVAEKFKEHF-NH2
376


 


[A-17>H, D-E switched]4F
Ac-EWFKAFYEKVADKFKDHF-NH2
377


 


[[A-17>H, D-1>E]4F
Ac-EWFKAFYDKVAEKFKEHF-NH2
378


 


[[A-17>H, D-8>E]4F
Ac-DWFKAFYEKVAEKFKEHF-NH2
379


 


[[A-17>H, E-12>D]4F
Ac-DWFKAFYDKVADKFKEHF-NH2
380


 


[[A-17>H, E16>D]4F
Ac-DWFKAFYDKVAEKFKDHF-NH2
381


 


[A-17>F, F-18>H]4F
Ac-DWFKAFYDKVAEKFKEFH-NH2
382


 


[A-17>F, F-18>H, D-E switched]4F
Ac-EWFKAFYEKVADKFKDFH-NH2
383


 


[A-17>F, F-18>H, D-1>E]-4F
Ac-EWFKAFYDKVAEKFKEFH-NH2
384


 


[A-17>F, F-18>H]4F
Ac-DWFKAFYDKVAEKFKEFH-NH2
385


 


[A-17>F, F-18>H, D-8>E]-4F
Ac-DWFKAFYEKVAEKFKEFH-NH2
386


 


[A-17>F, F-18>H, E-12>D]4F
Ac-DWFKAFYDKVAEKFKEFH-NH2
387


 


[A-17>F, F-18>H], E-16>D]-4F
Ac-DWFKAFYDKVAEKFKDFH-NH2
388


 


Rev-4F
Ac-FAEKFKEAVKDYFAKFWD-NH2
389


 


[A-2>H]Rev4F
Ac-FHEKFKEAVKDYFAKFWD-NH2
390


 


Rev-[A-2>H, D>E]-4F
Ac-FHEKFKEAVKEYFAKFWE-NH2
391


 


Rev-[A-2>H, E>D]4F
Ac-FHDKFKDAVKDYFAKFWD-NH2
392


 


[A-2>H, D-E switched]Rev-4F
Ac-FHDKFKDAVKEYFAKFWE-NH2
393


 


[A-2>H, E-3>D]Rev-4F
Ac-FHDKFKEAVKDYFAKFWD-NH2
394


 


[A-2>H, E-7>D]Rev-4F
Ac-FHEKFKDAVKDYFAKFWD-NH2
395


 


[A-2>H, D-11>E]Rev-4F
Ac-FHEKFKEAVKEYFAKFWD-NH2
396


 


[A-2>H, D-18>E]Rev-4F
Ac-FHEKFKEAVKDYFAKFWE-NH2
397


 


[F-1>H, A-2>F]Rev-4F
Ac-HFEKFKEAVKDYFAKFWD-NH2
398


 


[F-1>H, A-2>F, D-E switched]Rev 4F
Ac-HFDKFKDAVKEYFAKFWE-NH2
399


 


[F-1>H, A-2>F, D>E]Rev-4F
Ac-HFEKFKEAVKEYFAKFWE-NH2
400


 


[F-1>H, A-2>F, E-3>D]Rev-4F
Ac-HFDKFKEAVKDYFAKFWD-NH2
401


 


[F-1>H, A-2>F, E-7>D]Rev-4F
Ac-HFEKFKDAVKDYFAKFWD-NH2
402


 


[F-1>H, A-2>F, D-11 >E]Rev-4F
Ac-HFEKFKEAVKEYFAKFWD-NH2
403


 


[F-1>H, A-2>F, D-18>E]Rev-4F
Ac-HFEKFKEAVKDYFAKFWE-NH2
404


 


[A-2>F, F-5>H]Rev D-4F
Ac-FFEKHKEAVKDYFAKFWD-NH2
405


 


[A-2>F, F-5>H, D-E switched]Rev D-4F
Ac-FFDKHKDAVKEYFAKFWE-NH2
406


 


[A-2>F, F-5>H, D>E]Rev D-4F
Ac-FFEKHKEAVKEYFAKFWE-NH2
407


 


[A-2>F, F-5>H, E>D]Rev D-4F
Ac-FFDKHKDAVKDYFAKFWD-NH2
408


 


[A-2>F, F-5>H, E-3>D]Rev D-4F
Ac-FFDKHKEAVKDYFAKFWD-NH2
409


 


[A-2>F, F-5>H, D-11>E]Rev D-4F
Ac-FFEKHKEAVKEYFAKFWD-NH2
410


 


[A-2>F, F-5>H, D-18>E]Rev D-4F
Ac-FFEKHKEAVKDYFAKFWE-NH2
411


 


[A-2>V, V-9>H]Rev D-4F
Ac-FVEKFKEAHKDYFAKFWD-NH2
412


 


[A-2>V, V-9>H, D-E switched]Rev D-4F
Ac-FVDKFKDAHKEYFAKFWE-NH2
413


 


[A-2>V, V-9>H, D>E]Rev D-4F
Ac-FVEKFKEAHKEYFAKFWE-NH2
414


 


[A-2>V, V-9>H, E>D]Rev D-4F
Ac-FVDKFKDAHKDYFAKFWD-NH2
415


 


[A-2>V, V-9>H, E-3>D]Rev D-4F
Ac-FVDKFKEAHKDYFAKFWD-NH2
416


 


[A-2>V, V-9>H, E-7>D]Rev D-4F
Ac-FVEKFKDAHKDYFAKFWD-NH2
417


 


[A-2>V, V-9>H, D-11>E]Rev D-4F
Ac-FVEKFKEAHKEYFAKFWD-NH2
418


 


[A-2>V, V-9>H, D-18>E]Rev D-4F
Ac-FVEKFKEAHKDYFAKFWE-NH2
419


 


[A-8>H]Rev-4F
Ac-FAEKFKEHVKDYFAKFWD-NH2
420


 


[A-8>H, D-E switched]Rev-4F
Ac-FADKFKDHVKEYFAKFWE-NH2
421


 


[A-8>H, D>E]Rev-4F
Ac-FAEKFKEHVKEYFAKFWE-NH2
422


 


[A-8>H, E>D]Rev-4F
Ac-FADKFKDHVKDYFAKFWD-NH2
423


 


[A-8>H, E-3>D]Rev-4F
Ac-FADKFKEHVKDYFAKFWD-NH2
424


 


[A-8>H, E-7>D]Rev-4F
Ac-FAEKFKDHVKDYFAKFWD-NH2
425


 


[A-8>H, D-11>E]Rev-4F
Ac-FAEKFKEHVKEYFAKFWD-NH2
426


 


[A-8>H, D-18>E]Rev-4F
Ac-FAEKFKEHVKDYFAKFWE-NH2
427


 


[A-8>F, F-13>H]Rev-4F
Ac-FAEKFKEFVKDYHAKFWD-NH2
428


 


[A-8>F, F-13>H, D-E switched]Rev-4F
Ac-FADKFKDFVKEYHAKFWE-NH2
429


 


[A-8>F, F-13>H, E-3>D]Rev-4F
Ac-FADKFKEFVKDYHAKFWD-NH2
430


 


[A-8>F, F-13>H, E-7>D]Rev-4F
Ac-FAEKFKDFVKDYHAKFWD-NH2
431


 


[A-8>F, F-13>H, E>D]Rev-4F
Ac-FADKFKDFVKDYHAKFWD-NH2
432


 


[A-8>F, F-13>H, D>E]Rev-4F
Ac-FAEKFKEFVKEYHAKFWE-NH2
433


 


[A-8>F, F-13>H, D-11>E]Rev-4F
Ac-FAEKFKEFVKEYHAKFWD-NH2
434


 


[A-8>F, F-13>H, D-18>E]Rev-4F
Ac-FAEKFKEFVKDYHAKFWE-NH2
435


 


[A-8>F, F16>H]Rev.-4F
Ac-FAEKFKEFVKDYFAKHWD-NH2
436


 


[A-8>F, F16>H, D-E switched]Rev.-4F
Ac-FADKFKDFVKEYFAKHWE-NH2
437


 


[A-8>F, F16>H, D>E]Rev.-4F
Ac-FAEKFKEFVKEYFAKHWE-NH2
438


 


[A-8>F, F16>H, E>D]Rev.-4F
Ac-FADKFKDFVKDYFAKHWD-NH2
439


 


[A-8>F, F16>H, E-3>D]Rev.-4F
Ac-FADKFKEFVKDYFAKHWD-NH2
440


 


[A-8>F, F16>H, E-7>D]Rev.-4F
Ac-FAEKFKDFVKDYFAKHWD-NH2
441


 


[A-8>F, F16>H, D-11>E]Rev.-4F
Ac-FAEKFKEFVKEYFAKHWD-NH2
442


 


[A-8>F, F16>H, D-18>E]Rev.-4F
Ac-FAEKFKEFVKDYFAKHWE-NH2
443


 







Examples of class A 4F and Rev 4F analogs with beta-Nph. Similarly,


alpha-Nph analogs can be designed. Similarly to the above analogs,


His can be incorporated to Nph analogs. D>E analogs, E>D analogs


and D-E switch analogs are additional possibilities similarly to the


above described analogs.









4Nph
Ac-DWNphKANphYDKVAEKNphKEANph-NH2
444


 


[D-E switched]4Nph
Ac-EWNphKANphYEKVADKNphKDANph-NH2
445


 


[D>E]4Nph
Ac-EWNphKANphYEKVAEKNphKEANph-NH2
446


 


[E>D]4Nph
Ac-DWNphKANphYDKVADKNphKDANph-NH2
447


 


[D-1>E]4Nph
Ac-EWNphKANnhYDKVAEKNphKEANph-NH2
448


 


[D-8>E]4Nph
Ac-DWNphKANphYEKVAEKNphKEANph-NH2
449


 


[E-12>D]4Nph
Ac-DWNphKANphYDKVADKNphKEANph-NH2
450


 


[E-16>D]4Nph
Ac-DWNphKANphYDKVAEKNphKDANph-NH2
451


 







As decribed above for 4Nph, a minimum of 7 additional analogs for


each of the analogs given below.









[F-3, 6,>Nph]4F
Ac-DWNphKANphYDKVAEKFKEAF-NH2
452


 


[F-14, 18>Nph]4F
Ac-DWFKAFYDKVAEKNphKEANph-NH2
453


 


[[F-3>Nph]4F
Ac-DWNphKAFYDKVAEKFKEAF-NH2
454


 


[F-6>Nph]4F
Ac-DWFKANphYDKVAEKFKEAF-NH2
455


 


[F-14>Nph]4F
Ac-DWFKAFYDKVAEKNphKEAF-NH2
456


 


[F-18>Nph]4F
Ac-DWFKAFYDKVAEKFKEANph-NH2
457


 







For each of the analog described below, a minimum of 7 additional


analogs are possible as described above by switching D-E,


D>E and E>D and single D or E analogs.









Rev-4Nph
Ac-NphAEKNphKEAVKDYNphAKNphWD-NH2
458


 


[F-3, 6>Nph]Rev 4F
Ac-NphAEKNphKEAVKDYFAKFWD-NH2
459


 


[F-13, 16]Rev-4F
Ac-FAEKFKEAVKDYNphAKNphWD-NH2
460


 


[F-3>Nph]Rev-4F
Ac-NphAEKFKEAVKDYFAKFWD-NH2
461


 


[F-6>Nph]Rev-4F
Ac-FAEKNphKEAVKDYFAKFWD-NH2
462


 


[F-13>Nph]Rev-4F
Ac-FAEKFKEAVKDYNphAKFWD-NH2
463


 


[F-16>Nph]Rev-4F
Ac-FAEKFKEAVKDYFAKNphWD-NH2
464


 







For the analogs described below, additional analogs are possible by 


incorporating His or alpha-Nph and beta-Nph









Rev-[D>E]-4F
Ac-FAEKFKEAVKEYFAKFWE-NH2
465


 


Rev-[E>D]4F
Ac-FADKFKDAVKDYFAKFWD-NH2
466


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
467


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
468


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
469


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
470


 


Rev-[D>E]-4F
Ac-FAEKFKEAVKEYFAKFWE-NH2
471


 


Rev-[E>D]4F
Ac-FADKFKDAVKDYFAKFWD-NH2
472


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
473


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
474


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
475


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
476


 


Rev-[D>E]-4F
Ac-FAEKFKEAVKEYFAKFWE-NH2
477


 


Rev-[E>D]4F
Ac-FADKFKDAVKDYFAKFWD-NH2
478


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
479


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
480


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
481


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
482


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
483


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
484


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
485


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
486


 


Rev-[D>E]-4F
Ac-FAEKFKEAVKEYFAKFWE-NH2
487


 


Rev-[E>D]4F
Ac-FADKFKDAVKDYFAKFWD-NH2
488


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
489


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
490


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
491


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
492


 







For each of the analogs below, additional H and Nph analogs are


possible using the examples described above. Each analog can yield 7


analogs with the changes described in the examples give above.









Rev3F-2
Ac-LFEKFAEAFKDYVAKWKD-NH2
493


 


RevR4-3F-2
Ac-LFERFAEAFKDYVAKWKD-NH2
494


 


RevR10-3F2
Ac-LFEKFAEAFRDYVAKWKD-NH2
495


 


RevR15-3F-2
Ac-LFEKFAEAFKDYVARWKD-NH2
496


 


RevR17-3F-2
Ac-LFEKFAEAFKDYVAKWRD-NH2
497


 


Rev[D>E]3F2
Ac-LFEKFAEAFKEYVAKWKE-NH2
498


 


Rev[E>D]3F-2
Ac-LFDKFADAFKDYVAKWKD-NH2
499


 


Rev-[E3>D]-3F-2
Ac-LFDKFAEAFKDYVAKWKD-NH2
500


 


Rev-[E7>D]-3F-2
Ac-LFEKFADAFKDYVAKWKD-NH2
501


 


Rev[D11>E]3F-2
Ac-LFEKFAEAFKEYVAKWKD-NH2
502


 


Rev-[D18>E]3F-2
Ac-LFEKFAEAFKDYVAKWKE-NH2
503


 


Rev3F-1
Ac-FAEKAWEFVKDYFAKLKD-NH2
504


 


RevR4-3F-1
Ac-FAERAWEFVKDYFAKLKD-NH2
505


 


RevR10-3F-1
Ac-FAEKAWEFVKDYFAKLKD-NH2
506


 


RevR15-3F-1
Ac-FAEKAWEFVKDYFAKLKD-NH2
507


 


RevRl7-3F-1
Ac-FAEKAWEFVKDYFAKLRD-NH2
508


 


Rev[D>E]3F-1
Ac-FAEKAWEFVKEYFAKLKE-NH2
509


 


Rev[E>D}3F-1
Ac-FADKAWDFVKDYFAKLKD-NH2
510


 


Rev[E3>D]-3F-1
Ac-FADKAWEFVKDYFAKLKD-NH2
511


 


Rev[E7>D]3F-1
Ac-FAEKAWDFVKDYFAKLKD-NH2
512


 


Rev-[D11>E]3F-1
Ac-FAEKAWEFVKEYFAKLKD-NH2
513


 


Rev-[D18>E]3F-1
Ac-FAEKAWEFVKDYFAKLKE-NH2
514


 


Rev-5F
Ac-FFEKFKEFVKDYFAKLWD-NH2
515


 


Rev-[D>E]5F
Ac-FFEKFKEFVKEYFAKLWE-NH2
516


 


Rev-[E>D]5F
Ac-FFDKFKDFVKDYFAKLWD-NH2
517


 


Rev-R4-5F
Ac-FFERFKEFVKDYFAKLWD-NH2
518


 


Rev-R6-5F
Ac-FFEKFREFVKDYFAKLWD-NH2
519


 


Rev-R10-5F
Ac-FFEKFKEFVRDYFAKLWD-NH2
520


 


Rev-R15-5F
Ac-FFEKFKEFVKDYFARLWD-NH2
521


 


Rev-[E3>D]-5F
Ac-FFDKFKEFVKDYFAKLWD-NH2
522


 


Rev-[E7>D]5F
Ac-FFEKFKDFVKDYFAKLWD-NH2
523


 


Rev-[D11>E]-5F
Ac-FFEKFKEFVKEYFAKLWD-NH2
524


 


Rev-[D18>E]-5F
Ac-FFEKFKEFVKDYFAKLWE-NH2
525


 


Rev-5F-2
Ac-FLEKFKEFVKDYFAKFWD-NH2
526


 


Rev-[D>E]-5F-2
Ac-FLEKFKEFVKEYFAKFWE-NH2
527


 


Rev-[E>D]-5F-2
Ac-FLDKFKEFVKDYFAKFWD-NH2
528


 


Rev-[E3>D]-5F-2
Ac-FLDKFKEFVKDYFAKFWD-NH2
529


 


Rev-[E7>D]-5F-2
Ac-FLEKFKDFVKDYFAKFWD-NH2
530


 


Rev-[D11>E]-5F-2
Ac-FLEKFKEFVKEYFAKFWD-NH2
531


 


Rev-[D18>E]-5F-2
Ac-FLEKFKEFVKDYFAKFWE-NH2
532


 


Rev-R4-5F-2
Ac-FLERFKEFVKDYFAKFWD-NH2
533


 


Rev-R6-5F-2
Ac-FLEKFREFVKDYFAKFWD-NH2
534


 


RevR10-5F-2
Ac-FLEKFKEFVRDYFAKFWD-NH2
535


 


Rev-R16-5F-2
Ac-FLEKFKEFVKDYFARFWD-NH2
536


 


Rev-6F
Ac-FFEKFKEFFKDYFAKLWD-NH2
537


 


Rev-[D>E]-6F
Ac-FFEKFKEFFKEYFAKLWE-NH2
538


 


Rev-[E>D]-6F
Ac-FFDKFKDFFKDYFAKLWD-NH2
539


 


Rev-R4-6F
Ac-FFERFKEFFKDYFAKLWD-NH2
540


 


Rev-R6-6F
Ac-FFEKFREFFKDYFAKLWD-NH2
541


 


Rev-R10-6F
Ac-FFEKFKEFFRDYFAKLWD-NH2
542


 


Rev-R14-6F
Ac-FFERFKEFFKDYFARLWD-NH2
543


 


Rev-[E3>D]-6F
Ac-FFDKFKEFFKDYFAKLWD-NH2
544


 


Rev-[E7>D]-6F
Ac-FFEKFKDFFKDYFAKLWD-NH2
545


 


Rev-[D11>E]-6F
Ac-FFEKFKEFFKEYFAKLWD-NH2
546


 


Rev-[D18>E]-6F
Ac-FFEKFKEFFKDYFAKLWE-NH2
547


 


Rev-4F
Ac-FAEKFKEAVKDYFAKFWD-NH2
548


 


Rev-[D>E]-4F
Ac-FAEKFKEAVKEYFAKFWE-NH2
549


 


Rev-[E>D]4F
Ac-FADKFKDAVKDYFAKFWD-NH2
550


 


Rev-R4-4F
Ac-FAERFREAVKDYFAKFWD-NH2
551


 


Rev-R6-4F
Ac-FAEKFREAVKDYFAKFWD-NH2
552


 


Rev-R10-4F
Ac-FAEKFKEAVRDYFAKFWD-NH2
553


 


Rev-R14-4F
Ac-FAEKFKEAVKDYFARFWD-NH2
554


 


4F-2
Ac-DKWKAVYDKFAEAFKEFF-NH2
555


 


[D>E]-4F-2
Ac-EKWKAVYEKFAEAFKEFF-NH2
556


 


[E>D]-4F-2
Ac-DKWKAVYDKFADAFKDFF-NH2
557


 


R2-4F-2
Ac-DRWKAVYDKFAEAFKEFF-NH2
558


 


R4-4F-2
Ac-DKWRAVYDKFAEAFKEFF-NH2
559


 


R9-4F-2
Ac-DKWKAVYDRFAEAFKEFF-NH2
560


 


R14-4F-2
Ac-DKWKAVYDKFAEAFREFF-NH2
561


 


Rev4F-2
Ac-FFEKFAEAFKDYVAKWKD-NH2
562


 


Rev-[D>E]-4F-2
Ac-FFEKFAEAFKEYVAKWKE-NH2
563


 


Rev-[E>D]-3F-2
Ac-FFDKFADAFKDYVAKWKD-NH2
564


 


Rev-R4-4F-2
Ac-FFERFAEAFKDYVAKWKD-NH2
565


 


Rev-R10-4F-2
Ac-FFERFAEAFRDYVAKWKD-NH2
566


 


Rev-R15-4F-2
Ac-FFEKFAEAFKDYVARWKD-NH2
567


 


Rev-R17-4F-2
Ac-FFERFAEAFKDYVAKWRD-NH2
568


 


Rev-[E3>D]-4F-2
Ac-FFDKFAEAFKDYVAKWKD-NH2
569


 


Rev-[E7>D]-4F-2
Ac-FFEKFADAFKDYVAKWKD-NH2
570


 


Rev-[D11>E]-4F-2
Ac-FFERFAEAFKEYVAKWKD-NH2
571


 


Rev-[D18>E]-4F-2
Ac-FFERFAEAFKDYVAKWKE-NH2
572


 


Rev-7F
Ac-FFEKFKEFFKDYFAKFWD-NH2
573


 


Rev-[E>D]-7F
Ac-FFDKFKDFFKDYFAKFWD-NH2
574


 


Rev-[D>E]-7F
Ac-FFEKFKEFFKEYFAKFWE-NH2
575


 


Rev-R4-7F
Ac-FFERFKEFFKDYFAKFWD-NH2
576


 


Rev-R6-7F
Ac-FFEKFREFFKDYFAKFWD-NH2
577


 


Rev-R10-7F
Ac-FFEKFKEFFRDYFAKFWD-NH2
578


 


Rev-R14-7F
Ac-FFEKFKEFFKDYFARFWD-NH2
579


 


Rev-[E3>D]-7F
Ac-FFDKFKEFFKDYFAKFWD-NH2
580


 


Rev-[E7>D]7F
Ac-FFEKFKDFFKDYFAKFWD-NH2
581


 


Rev-[D11>E]-7F
Ac-FFEKFKEFFKEYFAKFWD-NH2
582


 


Rev-[D18>E]-7F
Ac-FFEKFKEFFKDYFAKFWE-NH2
583









It is also noted that any of the peptides described herein can comprise non-natural amino acids in addition to or instead of the corresponding natural amino acids identified herein. Such modifications include, but are not limited to acetylation, amidation, formylation, methylation, sulfation, and the like. Illustrative non-natural amino acids include, but are not limited to Ornithine, norleucine, norvaline, N-methylvaline, 6-N-methyllysine, N-methylisoleucine, N-methylglycine, sarcosine, inosine, allo-isoleucine, isodesmolysine, 4-hydroxyproline, 3-hydroxyproline, allo-hydroxylysine, hydroxylisine, N-ethylasparagine, N-ethylglycine, 2,3-diaminopropionic acid, 2,2′-diaminopropionic acid, desmosine, 2,4-diaminobutyric acid, 2-aminopimelic acid, 3-aminoisobutyric acid, 2-aminoisobutyric acid, 2-aminoheptanoic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2-aminobutyric acid, beta-alanine, 3-aminoadipic acid, 2-aminoadipic acid, and the like. In certain embodiments andy one or more of the “natural” amino acids of the peptides described herein, can be substituted with the corresponding non-natural amino acid (e.g., as describe above).
In certain embodiments, this invention contemplates particularly the use of modified lysines. Such modifications include, but are not limited to, biotin modification of epsilon lysines and/or methylation of the epsilon lysines. Illustrative peptide comprising epsilon methylated lysines include, but are not limited to: Ac-D-W—F—K(eCH3)2-A-F—Y-D-K(eCH3)2—V-A-E-K(eCH3)2—F—K(eCH3)2-E-A-F—NH(CH3)2 (SEQ ID NO:635) and: Ac-DWFK(eCH3)2AFYDK(eCH3)2VAEK(eCH3)2FK(eCH3)2EAF-NH(CH3) (SEQ ID NO:636). Other modified amino acids include but are not limited to ornithine analogs and homoaminoalanine analogs (instead of (CH2)4—NH2 for Lys it can be —(CH2)2—NH2 for Haa and —(CH2)3—NH2 for Orn] and the like. It is noted that these modifications are illustrative and not intended to be limiting. Illustrative 4F analogues that possess modified amino acids are shown in Table 6.







TABLE 6







Illustrative 4F analogs that comprise modified amino acids.









SEQ


Peptide
ID NO





εN-Dimethyl-Lys derivative of 4F (εN-Dime):



Ac-D-W-F-K(εN-Dime)-A-F-Y-D-K(εN-Dime)-V-A-E-K(εN-Dime)-F-
586


K(εN-Dime)-E-A-F-NH2



 


Ac-D-W-F-K-(εN-Dime)-A-F-Y-D-K(εN-Dime)-V-A-E-K(εN-Dime)-F-
587


K((εN-Dime)-E-A-F-NH-Me



 


Ac-D-W-F-K-(εN-Dime)-A-F-Y-D-K(εN-Dime)-V-A-E-K(εN-Dime)-F-
588


K(εN-Dime)-E-A-F-N-(Me)2



 


εN-Diethyl-Lys derivatives of 4F (εN-Diet)



Ac-D-W-F-K(εN-Diet)-A-F-Y-D-K(εN-Diet)-V-A-E-K(εN-Diet)-F-
589


K(εN-Diet)-E-A-F-NH2



 


Ac-D-W-F-K(εN-Diet)-A-F-Y-D-K(εN-Diet)-V-A-E-K(εN-Diet)-F-
590


K(εN-Diet)-E-A-F-NH-Et



 


Ac-D-W-F-K(εN-Diet)-A-F-Y-D-K(εN-Diet)-V-A-E-K(εN-Diet)-F-
591


K(εN-Diet)-E-A-F-NH-(Et)2



 


εN-Monomethyl-Lys derivative of 4F (εN-Me)



Ac-D-W-F-K(εN-Me)-A-F-Y-D-K(εN-Me)-V-A-E-K(εN-Me)-F-
592


K(εN-Me)-E-A-F-NH2



 


Ac-D-W-F-K(εN-Me)-A-F-Y-D-K(εN-Me)-V-A-E-K(εN-Me)-F-
593


K(εN-Me)-E-A-F-NH-Me



 


Ac-D-W-F-K(εN-Me)-A-F-Y-D-K(εN-Me)-V-A-E-K(εN-Me)-F-
594


K(εN-Me)-E-A-F-N-(Me)2



 


εN-ethylLys derivative of 4F (εNEt)



Ac-D-W-F-K(εN-Et)-A-F-Y-D-K(εN-Et)-V-A-E-K(εN-Et)-F-
595


K(εN-Et)-E-A-F-NH2



 


Ac-D-W-F-K(εN-Et)-A-F-Y-D-K(εN-Et)-V-A-E-K(εN-Et)-F-
596


K(εN-Et)-E-A-F-NH-Et



 


Ac-D-W-F-K(εN-Et)-A-F-Y-D-K(εN-Et)-V-A-E-K(εN-Et)-F-
597


K(εN-Et)-E-A-F-NH-(Et)2



 


HomoLys analogs of 4F (hK) (—CH2)5—NH2:



Ac-D-W-F-hK-A-F-Y-D-hK-V-A-E-hK-F-hK-E-A-F-NH2
598


 


Ac-D-W-F-hK(εN-Dime)-A-F-Y-D-hK(εN-Dime)-V-A-E-hK(εN-
599


Dime)-F-hK(εN-Dime)-E-A-F-NH2



 


Ac-D-W-F-hK(εN-Dime)-A-F-Y-D-hK(εN-Dime)-V-A-E-hK(εN-
600


Dime)-F-hK(εN-Dime)-E-A-F-N-(Me)2



 


Ac-D-W-F-hK(εN-Dime)-A-F-Y-D-hK(εN-Dime)-V-A-E-hK(εN-
601


Dime)-F-hK(εN-Dime)-E-A-F-NH-Me



 


Ac-D-W-F-hK(εN-Diet)-A-F-Y-D-hK(εN-Diet)-V-A-E-hK(εN-Diet)-F-
602


hK(εN-Diet)-E-A-F-NH-Et



 


Ac-D-W-F-hK(εN-Me)-A-F-Y-D-hK(εN-Me)-V-A-E-hK(εN-Me)-F-
603


hK(εN-Me)-E-A-F-NH2



 


Ac-D-W-F-hK(εN-Me)-A-F-Y-D-hK(εN-Me)-V-A-E-hK(εN-Me)-F-
604


hK(εN-Me)-E-A-F-NH-Me



 


Ac-D-W-F-hK(εN-Me)-A-F-Y-D-hK(εN-Me)-V-A-E-hK(εN-Me)-F-
605


hK(εN-Me)-E-A-F-N-(Me)2



 


Ac-D-W-F-hK(εN-Et)-A-F-Y-D-hK(εN-Et)-V-A-E-hK(εN-Et)-F-
606


hK(εN-Et)-E-A-F-NH2



 


Ac-D-W-F-hK(εN-Et)-A-F-Y-D-hK(εN-Et)-V-A-E-hK(εN-Et)-F-
607


hK(εN-Et)-E-A-F-NH-Et



 


Ac-D-W-F-hK(εN-Et)-A-F-Y-D-hK(εN-Et)-V-A-E-hK(εN-Et)-F-
608


hK(εN-Et)-E-A-F-NH-(Et)2



 


4F analogs in which K is replaced O (O = Ornithine, —(CH2)3—NH2):
609


 


Ac-D-W-F-O-A-F-Y-D-O-V-A-E-O-F-O-E-A-F-NH2
610


 


Ac-D-W-F-O(δN-Dime)-A-F-Y-D-O(δN-Dime)-V-A-E-O(δN-Dime)-
611


F-O(δN-Dime)-E-A-F-NH2



 


Ac-D-W-F-O(δN-Dime)-A-F-Y-D-)(δN-Dime)-V-A-E-O(δN-Dime)-F-
612


O(δN-Dime)-E-A-F-N-(Me)2



 


Ac-D-W-F-O(δN-Dime)-A-F-Y-D-O(δN-Dime)-V-A-E-O(δN-Dime)-
613


F-O(δN-Dime)-E-A-F-NH-Me



 


Ac-D-W-F-O(δN-Diet)-A-F-Y-D-O(δN-Diet)-V-A-E-O(δN-Diet)-F-
614


O(δN-Diet)-E-A-F-NH-Et



 


Ac-D-W-F-O(δN-Me)-A-F-Y-D-O(δN-Me)-V-A-E-O(δN-Me)-F-
615


O(δN-Me)-E-A-F-NH2



 


Ac-D-W-F-O(δN-Me)-A-F-Y-D-O(δN-Me)-V-A-E-O(δN-Me)-F-
616


O(δN-Me)-E-A-F-NH-Me



 


Ac-D-W-F-O(δN-Me)-A-F-Y-D-O(δN-Me)-V-A-E-O(δN-Me)-F-
617


O(δN-Me)-E-A-F-N-(Me)2



 


Ac-D-W-F-O(δN-Et)-A-F-Y-D-O(δN-Et)-V-A-E-O(δN-Et)-F-
618


O(δN-Et)-E-A-F-NH2



 


Ac-D-W-F-O(δN-Et)-A-F-Y-D-O(δN-Et)-V-A-E-O(δN-Et)-F-
619


O(δN-Et)-E-A-F-NH-Et



 


Ac-D-W-F-O(δN-Et)-A-F-Y-D-O(δN-Et)-V-A-E-OdεN-Et)-F-
620


O(δN-Et)-E-A-F-NH-(Et)2









The peptides and modifications shown above are intended to be illustrative and not limiting.
D) Smaller Peptides.
It was also a surprising discovery that certain small peptides consisting of a minimum of three amino acids preferentially (but not necessarily) with one or more of the amino acids being the D-stereoisomer of the amino acid, and possessing hydrophobic domains to permit lipid protein interactions, and hydrophilic domains to permit a degree of water solubility also possess significant anti-inflammatory properties and are useful in treating one or more of the pathologies described herein. The “small peptides” typically range in length from 2 amino acids to about 15 amino acids, more preferably from about 3 amino acids to about 10 or 11 amino acids, and most preferably from about 4 to about 8 or 10 amino acids. In various embodiments the peptides are typically characterized by having hydrophobic terminal amino acids or terminal amino acids rendered hydrophobic by the attachment of one or more hydrophobic “protecting” groups. Various “small peptides” are described in copending applications U.S. Ser. No. 10/649,378, filed Aug. 26, 2003, and in U.S. Ser. No. 10/913,800, filed on Aug. 6, 2004, and in PCT Application PCT/US2004/026288.
In certain embodiments, the peptides can be characterized by Formula XXV, below:

X1—X2−X3n—X4  XXV

where, n is 0 or 1, X1 is a hydrophobic amino acid and/or bears a hydrophobic protecting group, X4 is a hydrophobic amino acid and/or bears a hydrophobic protecting group; and when n is 0 X2 is an acidic or a basic amino acid; when n is 1: X2 and X3 are independently an acidic amino acid, a basic amino acid, an aliphatic amino acid, or an aromatic amino acid such that when X2 is an acidic amino acid; X3 is a basic amino acid, an aliphatic amino acid, or an aromatic amino acid; when X2 is a basic amino acid; X3 is an acidic amino acid, an aliphatic amino acid, or an aromatic amino acid; and when X2 is an aliphatic or aromatic amino acid, X3 is an acidic amino acid, or a basic amino acid.

Longer peptides (e.g., up to 10, 11, or 15 amino acids) are also contemplated within the scope of this invention. Typically where the shorter peptides (e.g., peptides according to Formula XXV) are characterized by an acidic, basic, aliphatic, or aromatic amino acid, the longer peptides are characterized by acidic, basic, aliphatic, or aromatic domains comprising two or more amino acids of that type.
1) Functional Properties of Active Small Peptides.
It was a surprising finding of this invention that a number of physical properties predict the ability of small peptides (e.g., less than 10 amino acids, preferably less than 8 amino acids, more preferably from about 3 to about 5 or 6 amino acids) of this invention to render HDL more anti-inflammatory and to mitigate atherosclerosis and/or other pathologies characterized by an inflammatory response in a mammal. The physical properties include high solubility in ethyl acetate (e.g., greater than about 4 mg/mL), and solubility in aqueous buffer at pH 7.0. Upon contacting phospholipids such as 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in an aqueous environment, the particularly effective small peptides induce or participate in the formation of particles with a diameter of approximately 7.5 nm (±0.1 nm), and/or induce or participate in the formation of stacked bilayers with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing between the bilayers in the stack of approximately 2 nm, and/or also induce or participate in the formation of vesicular structures of approximately 38 nm). In certain preferred embodiments, the small peptides have a molecular weight of less than about 900 Da.
Thus, in certain embodiments, this invention contemplates small peptides that ameliorate one or more symptoms of an indication/pathology described herein, e.g., an inflammatory condition, where the peptide(s): ranges in length from about 3 to about 8 amino acids, preferably from about 3 to about 6, or 7 amino acids, and more preferably from about 3 to about 5 amino acids; are soluble in ethyl acetate at a concentration greater than about 4 mg/mL; are soluble in aqueous buffer at pH 7.0; when contacted with a phospholipid in an aqueous environment, form particles with a diameter of approximately 7.5 nm and/or form stacked bilayers with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing between the bilayers in the stack of approximately 2 nm; have a molecular weight less than about 900 daltons; convert pro-inflammatory HDL to anti-inflammatory HDL or make anti-inflammatory HDL more anti-inflammatory. In certain embodiments the peptides include, but are not limited to peptides having the amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:621801), especially in which Lys-Arg-Asp and Ser are all L amino acids. In certain embodiments, these small peptides protect a phospholipid against oxidation by an oxidizing agent. In certain embodiments the compositions and methods described herein exclude the amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:621), especially in which Lys-Arg-Asp and Ser are all L amino acids.
While these small peptides need not be so limited, in certain embodiments, these small peptides can include the small peptides described below.
2) Tripeptides.
It was discovered that certain tripeptides (3 amino acid peptides) can be synthesized that show desirable properties as described herein (e.g., the ability to convert pro-inflammatory HDL to anti-inflammatory HDL, the ability to decrease LDL-induced monocyte chemotactic activity generated by artery wall cells. In certain embodiments, the peptides are characterized by Formula XXV, wherein N is zero, shown below as Formula XXVI:

X1—X2—X4  XXVI

where the end amino acids (X1 and X4) are hydrophobic either because of a hydrophobic side chain or because the side chain or the C and/or N terminus is blocked with one or more hydrophobic protecting group(s) (e.g., the N-terminus is blocked with Boc-, Fmoc-, nicotinyl-, etc., and the C-terminus blocked with (tBu)-OtBu, etc.). In certain embodiments, the X2 amino acid is either acidic (e.g., aspartic acid, glutamic acid, etc.) or basic (e.g., histidine, arginine, lysine, etc.). The peptide can be all L-amino acids or include one or more or all D-amino acids.

Certain tripeptides of this invention include, but are not limited to the peptides shown in Table 7.







TABLE 7







Examples of certain preferred tripeptides bearing


hydrophobic blocking groups and acidic, basic, or


histidine central amino acids.










X1
X2
X3
X4





Boc-Lys(εBoc)
Arg

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
Arg

Thr(tBu)-OtBu


 


Boc-Trp
Arg

Ile-OtBu


 


Boc-Trp
Arg

Leu-OtBu


 


Boc-Phe
Arg

Ile OtBu


 


Boc-Phe
Arg

Leu-OtBu


 


Boc-Lys(εBoc)
Glu

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
Glu

Thr(tBu)-OtBu


 


Boc-Lys(εBoc)
Asp

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
Asp

Thr(tBu)-OtBu


 


Boc-Lys(εBoc)
Arg

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
Arg

Thr(tBu)-OtBu


 


Boc-Leu
Glu

Ser(tBu)-OtBu


 


Boc-Leu
Glu

Thr(tBu)-OtBu


 


Fmoc-Trp
Arg

Ser(tBu)-OtBu


 


Fmoc-Trp
Asp

Ser(tBu)-OtBu


 


Fmoc-Trp
Glu

Ser(tBu)-OtBu


 


Fmoc-Trp
Arg

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
Glu

Leu-OtBu


 


Fmoc-Leu
Arg

Ser(tBu)-OtBu


 


Fmoc-Leu
Asp

Ser(tBu)-OtBu


 


Fmoc-Leu
Glu

Ser(tBu)-OtBu


 


Fmoc-Leu
Arg

Ser(tBu)-OtBu


 


Fmoc-Leu
Arg

Thr(tBu)-OtBu


 


Boc-Glu
Asp

Tyr(tBu)-OtBu


 


Fmoc-Lys(εFmoc)
Arg

Ser(tBu)-OtBu


 


Fmoc-Trp
Arg

Ile-OtBu


 


Fmoc-Trp
Arg

Leu-OtBu


 


Emoc-Phe
Arg

Ile-OtBu


 


Emoc-Phe
Arg

Leu-OtBu


 


Boc-Trp
Arg

Phe-OtBu


 


Boc-Trp
Arg

Tyr-OtBu


 


Fmoc-Trp
Arg

Phe-OtBu


 


Fmoc-Trp
Arg

Tyr-OtBu


 


Boc-Orn(δBoc)
Arg

Ser(tBu)-OtBu


 


Nicotinyl Lys(εBoc)
Arg

Ser(tBu)-OtBu


 


Nicotinyl Lys(εBoc)
Arg

Thr(tBu)-OtBu


 


Fmoc-Leu
Asp

Thr(tBu)-OtBu


 


Fmoc-Leu
Glu

Thr(tBu)-OtBu


 


Fmoc-Leu
Arg

Thr(tBu)-OtBu


 


Fmoc-norLeu
Arg

Ser(tBu)-OtBu


 


Fmoc-norLeu
Asp

Ser(tBu)-OtBu


 


Fmoc-norLeu
Glu

Ser(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Arg

Ser(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Arg

Thr(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Glu

Ser(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Glu

Thr(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Asp

Ser(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Asp

Thr(tBu)-OtBu


 


Fmoc-Lys(εBoc)
Glu

Leu-OtBu


 


Fmoc-Lys(εBoc)
Arg

Leu-OtBu


 


Fmoc-Lys(εFmoc)
Arg

Thr(tBu)-OtBu


 


Emoc-Lys(εFmoc)
Glu

Ser(tBu)-OtBu


 


Emoc-Lys(εFmoc)
Glu

Thr(tBu)-OtBu


 


Emoc-Lys(εFmoc)
Asp

Ser(tBu)-OtBu


 


Emoc-Lys(εFmoc)
Asp

Thr(tBu)-OtBu


 


Emoc-Lys(εFmoc)
Arg

Ser(tBu)-OtBu


 


Emoc-Lys(εFmoc))
Glu

Leu-OtBu


 


Boc-Lys(εFmoc)
Asp

Ser(tBu)-OtBu


 


Boc-Lys(εFmoc)
Asp

Thr(tBu)-OtBu


 


Boc-Lys(εFmoc)
Arg

Thr(tBu)-OtBu


 


Boc-Lys(εFmoc)
Glu

Leu-OtBu


 


Boc-Orn(δFmoc)
Glu

Ser(tBu)-OtBu


 


Boc-Orn(δFmoc)
Asp

Ser(tBu)-OtBu


 


Boc-Orn(δFmoc)
Asp

Thr(tBu)-OtBu


 


Boc-Orn(δFmoc)
Arg

Thr(tBu)-OtBu


 


Boc-Orn(δFmoc)
Glu

Thr(tBu)-OtBu


 


Fmoc-Trp
Asp

Ile-OtBu


 


Fmoc-Trp
Arg

Ile-OtBu


 


Fmoc-Trp
Glu

Ile-OtBu


 


Fmoc-Trp
Asp

Leu-OtBu


 


Fmoc-Trp
Glu

Leu-OtBu


 


Emoc-Phe
Asp

Ile-OtBu


 


Emoc-Phe
Asp

Leu-OtBu


 


Emoc-Phe
Glu

Leu-OtBu


 


Fmoc-Trp
Arg

Phe-OtBu


 


Fmoc-Trp
Glu

Phe-OtBu


 


Fmoc-Trp
Asp

Phe-OtBu


 


Fmoc-Trp
Asp

Tyr-OtBu


 


Fmoc-Trp
Arg

Tyr-OtBu


 


Fmoc-Trp
Glu

Tyr-OtBu


 


Fmoc-Trp
Arg

Thr(tBu)-OtBu


 


Fmoc-Trp
Asp

Thr(tBu)-OtBu


 


Fmoc-Trp
Glu

Thr(tBu)-OtBu


 


Boc-Phe
Arg

norLeu-OtBu


 


Boc-Phe
Glu

norLeu-OtBu


 


Emoc-Phe
Asp

norLeu-OtBu


 


Boc-Glu
His

Tyr(tBu)-OtBu


 


Boc-Leu
His

Ser(tBu)-OtBu


 


Boc-Leu
His

Thr(tBu)-OtBu


 


Boc-Lys(εBoc)
His

Ser(tBu)-OtBu


 


Boc-Lys(εBoc)
His

Thr(tBu)-OtBu


 


Boc-Lys(εBoc)
His

Leu-OtBu


 


Boc-Lys(εFmoc)
His

Ser(tBu)-OtBu


 


Boc-Lys(εFmoc)
His

Thr(tBu)-OtBu


 


Boc-Lys(εFmoc)
His

Leu-OtBu


 


Boc-Orn(δBoc)
His

Ser(tBu)-OtBu


 


Boc-Orn(δFmoc)
His

Thr(tBu)-OtBu


 


Boc-Phe
His

Ile-OtBu


 


Boc-Phe
His

Leu-OtBu


 


Boc-Phe
His

norLeu-OtBu


 


Boc-Phe
Lys

Leu-OtBu


 


Boc-Trp
His

Ile-OtBu


 


Boc-Trp
His

Leu-OtBu


 


Boc-Trp
His

Phe-OtBu


 


Boc-Trp
His

Tyr-OtBu


 


Boc-Phe
Lys

Leu-OtBu


 


Fmoc-Lys(εFmoc)
His

Ser(tBu)-OtBu


 


Fmoc-Lys(εFmoc)
His

Thr(tBu)-OtBu


 


Fmoc-Lys(εFmoc)
His

Leu-OtBu


 


Fmoc-Leu
His

Ser(tBu)-OtBu


 


Fmoc-Leu
His

Thr(tBu)-OtBu


 


Fmoc-Lys(εBoc)
His

Ser(tBu)-OtBu


 


Fmoc-Lys(εBoc)
His

Thr(tBu)-OtBu


 


Fmoc-Lys(εBoc)
His

Leu-OtBu


 


Fmoc-Lys(εFmoc)
His

Ser(tBu)-OtBu


 


Fmoc-Lys(εFmoc)
His

Thr(tBu)-OtBu


 


Fmoc-norLeu
His

Ser(tBu)-OtBu


 


Emoc-Phe
His

Ile-OtBu


 


Emoc-Phe
His

Leu-OtBu


 


Emoc-Phe
His

norLeu-OtBu


 


Fmoc-Trp
His

Ser(tBu)-OtBu


 


Fmoc-Trp
His

Ile-OtBu


 


Fmoc-Trp
His

Leu-OtBu


 


Fmoc-Trp
His

Phe-OtBu


 


Fmoc-Trp
His

Tyr-OtBu


 


Fmoc-Trp
His

Thr(tBu)-OtBu


 


Nicotinyl Lys(εBoc)
His

Ser(tBu)-OtBu


 


Nicotinyl Lys(εBoc)
His

Thr(tBu)-OtBu









While the peptides of Table 7 are illustrated with particular protecting groups, it is noted that any of these groups may be eliminated and/or substituted with other protecting groups as described herein.
3) Small Peptides with Central Acidic and Basic Amino Acids.
In certain embodiments, the peptides of this invention range from four amino acids to about ten amino acids. The terminal amino acids are typically hydrophobic either because of a hydrophobic side chain or because the terminal amino acids bear one or more hydrophobic protecting groups end amino acids (X1 and X4) are hydrophobic either because of a hydrophobic side chain or because the side chain or the C and/or N terminus is blocked with one or more hydrophobic protecting group(s) (e.g., the N-terminus is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus blocked with (tBu)-OtBu, etc.). Typically, the central portion of the peptide comprises a basic amino acid and an acidic amino acid (e.g., in a 4 mer) or a basic domain and/or an acidic domain in a longer molecule.
These four-mers can be represented by Formula XXV in which X1 and X4 are hydrophobic and/or bear hydrophobic protecting group(s) as described herein and X2 is acidic while X3 is basic or X2 is basic while X3 is acidic. The peptide can be all L-amino acids or include one or more or all D-amino acids.
Certain preferred of this invention include, but are not limited to the peptides shown in Table 8.







TABLE 8







Illustrative examples of small peptides with


central acidic and basic amino acids.















SEQ ID


X1
X2
X3
X4
NO





Boc-Lys(εBoc)
Arg
Asp
Ser(tBu)-OtBu
621


 


Boc-Lys(εBoc)
Arg
Asp
Thr(tBu)-OtBu
622


 


Boc-Trp
Arg
Asp
Ile-OtBu
623


 


Boc-Trp
Arg
Asp
Leu-OtBu
624


 


Boc-Phe
Arg
Asp
Leu-OtBu
625


 


Boc-Phe
Arg
Asp
Ile-OtBu
626


 


Boc-Phe
Arg
Asp
norLeu-OtBu
627


 


Boc-Phe
Arg
Glu
norLeu-OtBu
628


 


Boc-Phe
Arg
Glu
Ile-OtBu
629


 


Boc-Phe
Asp
Arg
Ile-OtBu
630


 


Boc-Phe
Glu
Arg
Ile-OtBu
631


 


Boc-Phe
Asp
Arg
Leu-OtBu
632


 


Boc-Phe
Arg
Glu
Leu-OtBu
633


 


Boc-Phe
Glu
Arg
Leu-OtBu
634


 


Boc-Phe
Asp
Arg
norLeu-OtBu
635


 


Boc-Phe
Glu
Arg
norLeu-OtBu
636


 


Boc-Lys(εBoc)
Glu
Arg
Ser(tBu)-OtBu
637


 


Boc-Lys(εBoc)
Glu
Arg
Thr(tBu)-OtBu
638


 


Boc-Lys(εBoc)
Asp
Arg
Ser(tBu)-OtBu
639


 


Boc-Lys(εBoc)
Asp
Arg
Thr(tBu)-OtBu
640


 


Boc-Lys(εBoc)
Arg
Glu
Ser(tBu)-OtBu
641


 


Boc-Lys(εBoc)
Arg
Glu
Thr(tBu)-OtBu
642


 


Boc-Leu
Glu
Arg
Ser(tBu)-OtBu
643


 


Boc-Leu
Glu
Arg
Thr(tBu)-OtBu
644


 


Fmoc-Trp
Arg
Asp
Ser(tBu)-OtBu
645


 


Fmoc-Trp
Asp
Arg
Ser(tBu)-OtBu
646


 


Fmoc-Trp
Glu
Arg
Ser(tBu)-OtBu
647


 


Fmoc-Trp
Arg
Glu
Ser(tBu)-OtBu
648


 


Boc-Lys(εBoc)
Glu
Arg
Leu-OtBu
649


 


Fmoc-Leu
Arg
Asp
Ser(tBu)-OtBu
650


 


Fmoc-Leu
Asp
Arg
Ser(tBu)-OtBu
651


 


Fmoc-Leu
Glu
Arg
Ser(tBu)-OtBu
652


 


Fmoc-Leu
Arg
Glu
Ser(tBu)-OtBu
653


 


Fmoc-Leu
Arg
Asp
Thr(tBu)-OtBu
654


 


Boc-Glu
Asp
Arg
Tyr(tBu)-OtBu
655


 


Fmoc-Lys(εFmoc)
Arg
Asp
Ser(tBu)-OtBu
656


 


Fmoc-Trp
Arg
Asp
Ile-OtBu
657


 


Fmoc-Trp
Arg
Asp
Leu-OtBu
658


 


Emoc-Phe
Arg
Asp
Ile-OtBu
659


 


Emoc-Phe
Arg
Asp
Leu-OtBu
660


 


Boc-Trp
Arg
Asp
Phe-OtBu
661


 


Boc-Trp
Arg
Asp
Tyr-OtBu
662


 


Fmoc-Trp
Arg
Asp
Phe-OtBu
663


 


Fmoc-Trp
Arg
Asp
Tyr-OtBu
664


 


Boc-Om(δBoc)
Arg
Glu
Ser(tBu)-OtBu
665


 


Nicotinyl Lys(εBoc)
Arg
Asp
Ser(tBu)-OtBu
666


 


Nicotinyl Lys(εBoc)
Arg
Asp
Thr(tBu)-OtBu
667


 


Fmoc-Leu
Asp
Arg
Thr(tBu)-OtBu
668


 


Fmoc-Leu
Glu
Arg
Thr(tBu)-OtBu
669


 


Fmoc-Leu
Arg
Glu
Thr(tBu)-OtBu
670


 


Fmoc-norLeu
Arg
Asp
Ser(tBu)-OtBu
671


 


Fmoc-norLeu
Asp
Arg
Ser(tBu)-OtBu
672


 


Fmoc-norLeu
Glu
Arg
Ser(tBu)-OtBu
673


 


Fmoc-norLeu
Arg
Glu
Ser(tBu)-OtBu
674


 


Fmoc-Lys(εBoc)
Arg
Asp
Ser(tBu)-OtBu
675


 


Fmoc-Lys(εBoc)
Arg
Asp
Thr(tBu)-OtBu
676


 


Fmoc-Lys(εBoc)
Glu
Arg
Ser(tBu)-OtBu
677


 


Fmoc-Lys(εBoc)
Glu
Arg
Thr(tBu)-OtBu
678


 


Fmoc-Lys(εBoc)
Asp
Arg
Ser(tBu)-OtBu
679


 


Fmoc-Lys(εBoc)
Asp
Arg
Thr(tBu)-OtBu
680


 


Fmoc-Lys(εBoc)
Arg
Glu
Ser(tBu)-OtBu
681


 


Fmoc-Lys(εBoc)
Arg
Glu
Thr(tBu)-OtBu
682


 


Fmoc-Lys(εBoc)
Glu
Arg
Leu-OtBu
683


 


Fmoc-Lys(εBoc)
Arg
Glu
Leu-OtBu
684


 


Fmoc-Lys(εFmoc)
Arg
Asp
Thr(tBu)-OtBu
685


 


Emoc- Lys(εFmoc)
Glu
Arg
Ser(tBu)-OtBu
686


 


Emoc- Lys(εFmoc)
Glu
Arg
Thr(tBu)-OtBu
687


 


Emoc- Lys(εFmoc)
Asp
Arg
Ser(tBu)-OtBu
688


 


Emoc- Lys(εFmoc)
Asp
Arg
Thr(tBu)-OtBu
689


 


Emoc- Lys(εFmoc)
Arg
Glu
Ser(tBu)-OtBu
690


 


Emoc- Lys(εFmoc)
Arg
Glu
Thr(tBu)-OtBu
691


 


Emoc- Lys(εFmoc))
Glu
Arg
Leu-OtBu
692


 


Boc-Lys(εFmoc)
Arg
Asp
Ser(tBu)-OtBu
693


 


Boc-Lys(εFmoc)
Arg
Asp
Thr(tBu)-OtBu
694


 


Boc-Lys(εFmoc)
Glu
Arg
Ser(tBu)-OtBu
695


 


Boc-Lys(εFmoc)
Glu
Arg
Thr(tBu)-OtBu
696


 


Boc-Lys(εFmoc)
Asp
Arg
Ser(tBu)-OtBu
697


 


Boc-Lys(εFmoc)
Asp
Arg
Thr(tBu)-OtBu
698


 


Boc-Lys(εFmoc)
Arg
Glu
Ser(tBu)-OtBu
699


 


Boc-Lys(εFmoc)
Arg
Glu
Thr(tBu)-OtBu
700


 


Boc-Lys(εFmoc)
Glu
Arg
Leu-OtBu
701


 


Boc-Orn(δFmoc)
Arg
Glu
Ser(tBu)-OtBu
702


 


Boc-Orn(δFmoc)
Glu
Arg
Ser(tBu)-OtBu
703


 


Boc-Orn(δFmoc)
Arg
Asp
Ser(tBu)-OtBu
704


 


Boc-Orn(δFmoc)
Asp
Arg
Ser(tBu)-OtBu
705


 


Boc-Orn(δFmoc)
Asp
Arg
Thr(tBu)-OtBu
706


 


Boc-Orn(δFmoc)
Arg
Asp
Thr(tBu)-OtBu
707


 


Boc-Orn(δFmoc)
Glu
Arg
Thr(tBu)-OtBu
708


 


Boc-Orn(δFmoc)
Arg
Glu
Thr(tBu)-OtBu
709


 


Fmoc-Trp
Asp
Arg
Ile-OtBu
710


 


Fmoc-Trp
Arg
Glu
Ile-OtBu
711


 


Fmoc-Trp
Glu
Arg
Ile-OtBu
712


 


Fmoc-Trp
Asp
Arg
Leu-OtBu
713


 


Fmoc-Trp
Arg
Glu
Leu-OtBu
714


 


Fmoc-Trp
Glu
Arg
Leu-OtBu
715


 


Emoc-Phe
Asp
Arg
Ile-OtBu
716


 


Emoc-Phe
Arg
Glu
Ile-OtBu
717


 


Emoc-Phe
Glu
Arg
Ile-OtBu
718


 


Emoc-Phe
Asp
Arg
Leu-OtBu
719


 


Emoc-Phe
Arg
Glu
Leu-OtBu
720


 


Emoc-Phe
Glu
Arg
Leu-OtBu
721


 


Fmoc-Trp
Arg
Asp
Phe-OtBu
722


 


Fmoc-Trp
Arg
Glu
Phe-OtBu
723


 


Fmoc-Trp
Glu
Arg
Phe-OtBu
724


 


Fmoc-Trp
Asp
Arg
Tyr-OtBu
725


 


Fmoc-Trp
Arg
Glu
Tyr-OtBu
726


 


Fmoc-Trp
Glu
Arg
Tyr-OtBu
727


 


Fmoc-Trp
Arg
Asp
Thr(tBu)-OtBu
728


 


Fmoc-Trp
Asp
Arg
Thr(tBu)-OtBu
729


 


Fmoc-Trp
Arg
Glu
Thr(tBu)-OtBu
730


 


Fmoc-Trp
Glu
Arg
Thr(tBu)-OtBu
731


 


Emoc-Phe
Arg
Asp
norLeu-OtBu
732


 


Emoc-Phe
Arg
Glu
norLeu-OtBu
733


 


Boc-Phe
Lys
Asp
Leu-OtBu
734


 


Boc-Phe
Asp
Lys
Leu-OtBu
735


 


Boc-Phe
Lys
Glu
Leu-OtBu
736


 


Boc-Phe
Glu
Lys
Leu-OtBu
737


 


Boc-Phe
Lys
Asp
Ile-OtBu
738


 


Boc-Phe
Asp
Lys
Ile-OtBu
739


 


Boc-Phe
Lys
Glu
Ile-OtBu
740


 


Boc-Phe
Glu
Lys
Ile-OtBu
741


 


Boc-Phe
Lys
Asp
norLeu-OtBu
742


 


Boc-Phe
Asp
Lys
norLeu-OtBu
743


 


Boc-Phe
Lys
Glu
norLeu-OtBu
744


 


Boc-Phe
Glu
Lys
norLeu-OtBu
745


 


Boc-Phe
His
Asp
Leu-OtBu
746


 


Boc-Phe
Asp
His
Leu-OtBu
747


 


Boc-Phe
His
Glu
Leu-OtBu
748


 


Boc-Phe
Glu
His
Leu-OtBu
749


 


Boc-Phe
His
Asp
Ile-OtBu
750


 


Boc-Phe
Asp
His
Ile-OtBu
751


 


Boc-Phe
His
Glu
Ile-OtBu
752


 


Boc-Phe
Glu
His
Ile-OtBu
753


 


Boc-Phe
His
Asp
norLeu-OtBu
754


 


Boc-Phe
Asp
His
norLeu-OtBu
755


 


Boc-Phe
His
Glu
norLeu-OtBu
756


 


Boc-Phe
Glu
His
norLeu-OtBu
757


 


Boc-Lys(εBoc)
Lys
Asp
Ser(tBu)-OtBu
758


 


Boc-Lys(εBoc)
Asp
Lys
Ser(tBu)-OtBu
759


 


Boc-Lys(εBoc)
Lys
Glu
Ser(tBu)-OtBu
760


 


Boc-Lys(εBoc)
Glu
Lys
Ser(tBu)-OtBu
761


 


Boc-Lys(εBoc)
His
Asp
Ser(tBu)-OtBu
762


 


Boc-Lys(εBoc)
Asp
His
Ser(tBu)-OtBu
763


 


Boc-Lys(εBoc)
His
Glu
Ser(tBu)-OtBu
764


 


Boc-Lys(εBoc)
Glu
His
Ser(tBu)-OtBu
765









While the peptides of Table 8 are illustrated with particular protecting groups, it is noted that these groups may be substituted with other protecting groups as described herein and/or one or more of the shown protecting group can be eliminated.
4) Small Peptides Having Either an Acidic or Basic Amino Acid in the Center to 2Ether with a Central Aliphatic Amino Acid.
In certain embodiments, the peptides of this invention range from four amino acids to about ten amino acids. The terminal amino acids are typically hydrophobic either because of a hydrophobic side chain or because the terminal amino acids bear one or more hydrophobic protecting groups. End amino acids (X1 and X4) are hydrophobic either because of a hydrophobic side chain or because the side chain or the C and/or N terminus is blocked with one or more hydrophobic protecting group(s) (e.g., the N-terminus is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus blocked with (tBu)-OtBu, etc.). Typically, the central portion of the peptide comprises a basic or acidic amino acid and an aliphatic amino acid (e.g., in a 4 mer) or a basic domain or an acidic domain and an aliphatic domain in a longer molecule.
These four-mers can be represented by Formula XXV in which X1 and X4 are hydrophobic and/or bear hydrophobic protecting group(s) as described herein and X2 is acidic or basic while X3 is aliphatic or X2 is aliphatic while X3 is acidic or basic. The peptide can be all L-amino acids or include one, or more, or all D-amino acids.
Certain preferred peptides of this invention include, but are not limited to the peptides shown in Table 9.







TABLE 9







Examples of certain preferred peptides having


either an acidic or basic amino acid in the center


together with a central aliphatic amino acid.















SEQ ID


X1
X2
X3
X4
NO





Fmoc-Lys(εBoc)
Leu
Arg
Ser(tBu)-OtBu
766


 


Fmoc-Lys(εBoc)
Arg
Leu
Ser(tBu)-OtBu
767


 


Fmoc-Lys(εBoc)
Leu
Arg
Thr(tBu)-OtBu
768


 


Fmoc-Lys(εBoc)
Arg
Leu
Thr(tBu)-OtBu
769


 


Fmoc-Lys(εBoc)
Glu
Leu
Ser(tBu)-OtBu
770


 


Fmoc-Lys(εBoc)
Leu
Glu
Ser(tBu)-OtBu
771


 


Fmoc-Lys(εBoc)
Glu
Leu
Thr(tBu)-OtBu
772


 


Fmoc-Lys(εFmoc)
Leu
Arg
Ser(tBu)-OtBu
773


 


Fmoc-Lys(εFmoc)
Leu
Arg
Thr(tBu)-OtBu
774


 


Fmoc-Lys(εFmoc)
Glu
Leu
Ser(tBu)-OtBu
775


 


Fmoc-Lys(εFmoc)
Glu
Leu
Thr(tBu)-OtBu
776


 


Boc-Lys(Fmoc)
Glu
Ile
Thr(tBu)-OtBu
777


 


Boc-Lys(εFmoc)
Leu
Arg
Ser(tBu)-OtBu
778


 


Boc-Lys(εFmoc)
Leu
Arg
Thr(tBu)-OtBu
779


 


Boc-Lys(εFmoc)
Glu
Leu
Ser(tBu)-OtBu
780


 


Boc-Lys(εFmoc)
Glu
Leu
Thr(tBu)-OtBu
781


 


Boc-Lys(εBoc)
Leu
Arg
Ser(tBu)-OtBu
782


 


Boc-Lys(εBoc)
Arg
Phe
Thr(tBu)-OtBu
783


 


Boc-Lys(εBoc)
Leu
Arg
Thr(tBu)-OtBu
784


 


Boc-Lys(εBoc)
Glu
Ile
Thr(tBu)
785


 


Boc-Lys(εBoc)
Glu
Val
Thr(tBu)
786


 


Boc-Lys(εBoc)
Glu
Ala
Thr(tBu)
787


 


Boc-Lys(εBoc)
Glu
Gly
Thr(tBu)
788


 


Boc--Lys(εBoc)
Glu
Leu
Ser(tBu)-OtBu
789


 


Boc-Lys(εBoc)
Glu
Leu
Thr(tBu)-OtBu
790









While the peptides of Table 9 are illustrated with particular protecting groups, it is noted that these groups may be substituted with other protecting groups as described herein and/or one or more of the shown protecting group can be eliminated.
5) Small Peptides Having Either an Acidic or Basic Amino Acid in the Center Together with a Central Aromatic Amino Acid.
In certain embodiments, the “small” peptides of this invention range from four amino acids to about ten amino acids. The terminal amino acids are typically hydrophobic either because of a hydrophobic side chain or because the terminal amino acids bear one or more hydrophobic protecting groups end amino acids (X1 and X4) are hydrophobic either because of a hydrophobic side chain or because the side chain or the C and/or N terminus is blocked with one or more hydrophobic protecting group(s) (e.g., the N-terminus is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus blocked with (tBu)-OtBu, etc.). Typically, the central portion of the peptide comprises a basic or acidic amino acid and an aromatic amino acid (e.g., in a 4 mer) or a basic domain or an acidic domain and an aromatic domain in a longer molecule.
These four-mers can be represented by Formula XXV in which X1 and X4 are hydrophobic and/or bear hydrophobic protecting group(s) as described herein and X2 is acidic or basic while X3 is aromatic or X2 is aromatic while X3 is acidic or basic. The peptide can be all L-amino acids or include one, or more, or all D-amino acids. Five-mers can be represented by a minor modification of Formula XXV in which X5 is inserted as shown in Table 10 and in which X5 is typically an aromatic amino acid, e.g.,

X1—X2—X3n—X5p—X4  XXVII

where X1, X2, X3, and X4 are as described above, p is 0 or 1 and X5 is typically an aromatic amino acid.

Certain preferred peptides of this invention include, but are not limited to the peptides shown in Table 10.







TABLE 10







Examples of certain preferred peptides having


either an acidic or basic amino acid in the center


together with a central aromatic amino acid.

















SEQ







ID


X1
X2
X3
X5
X4
NO















Fmoc-Lys(εBoc)
Arg
Trp

Tyr(tBu)-OtBu
791


 


Fmoc-Lys(εBoc)
Trp
Arg

Tyr(tBu)-OtBu
792


 


Fmoc-Lys(εBoc)
Arg
Tyr

Trp-OtBu
793


 


Fmoc-Lys(εBoc)
Tyr
Arg

Trp-OtBu
794


 


Fmoc-Lys(εBoc)
Arg
Tyr
Trp
Thr(tBu)-OtBu
795


 


Fmoc-Lys(εBoc)
Arg
Tyr

Thr(tBu)-OtBu
796


 


Fmoc-Lys(εBoc)
Arg
Trp

Thr(tBu)-OtBu
797


 


Fmoc-Lys(εFmoc)
Arg
Trp

Tyr(tBu)-OtBu
798


 


Fmoc-Lys(εFmoc)
Arg
Tyr

Trp-OtBu
799


 


Fmoc-Lys(εFmoc)
Arg
Tyr
Trp
Thr(tBu)-OtBu
800


 


Fmoc-Lys(εFmoc)
Arg
Tyr

Thr(tBu)-OtBu
801


 


Fmoc-Lys(εFmoc)
Arg
Trp

Thr(tBu)-OtBu
802


 


Boc-Lys(εFmoc)
Arg
Trp

Tyr(tBu)-OtBu
803


 


Boc-Lys(εFmoc)
Arg
Tyr

Trp-OtBu
804


 


Boc-Lys(εFmoc)
Arg
Tyr
Trp
Thr(tBu)-OtBu
805


 


Boc-Lys(εFmoc)
Arg
Tyr

Thr(tBu)-OtBu
806


 


Boc-Lys(εFmoc)
Arg
Trp

Thr(tBu)-OtBu
807


 


Boc-Glu
Lys(εFmoc)
Arg

Tyr(tBu)-OtBu
808


 


Boc-Lys(εBoc)
Arg
Trp

Tyr(tBu)-OtBu
809


 


Boc-Lys(εBoc)
Arg
Tyr

Trp-OtBu
810


 


Boc-Lys(εBoc)
Arg
Tyr
Trp
Thr(tBu)-OtBu
811


 


Boc-Lys(εBoc)
Arg
Tyr

Thr(tBu)-OtBu
812


 


Boc-Lys(εBoc)
Arg
Phe

Thr(tBu)-OtBu
813


 


Boc-Lys(εBoc)
Arg
Trp

Thr(tBu)-OtBu
814









While the peptides of Table 10 are illustrated with particular protecting groups, it is noted that these groups may be substituted with other protecting groups as described herein and/or one or more of the shown protecting groups can be eliminated.
6) Small Peptides Having Aromatic Amino Acids or Aromatic Amino Acids Separated by Histidine(s) at the Center.
In certain embodiments, the peptides of this invention are characterized by π electrons that are exposed in the center of the molecule which allow hydration of the particle and that allow the peptide particles to trap pro-inflammatory oxidized lipids such as fatty acid hydroperoxides and phospholipids that contain an oxidation product of arachidonic acid at the sn-2 position.
In certain embodiments, these peptides consist of a minimum of 4 amino acids and a maximum of about 10 amino acids, preferentially (but not necessarily) with one or more of the amino acids being the D-sterioisomer of the amino acid, with the end amino acids being hydrophobic either because of a hydrophobic side chain or because the terminal amino acid(s) bear one or more hydrophobic blocking group(s), (e.g., an N-terminus blocked with Boc-, Fmoc-, Nicotinyl-, and the like, and a C-terminus blocked with (tBu)-OtBu groups and the like). Instead of having an acidic or basic amino acid in the center, these peptides generally have an aromatic amino acid at the center or have aromatic amino acids separated by histidine in the center of the peptide.
Certain preferred peptides of this invention include, but are not limited to the peptides shown in Table 11.







TABLE 11







Examples of peptides having aromatic amino acids


in the center or aromatic amino acids or aromatic


domains separated by one or more histidines.

















SEQ ID


X1
X2
X3
X4
X5
NO





Boc-Lys(εBoc)
Phe
Trp
Phe
Ser(tBu)-OtBu
815


 


Boc-Lys(εBoc)
Phe
Trp
Phe
Thr(tBu)-OtBu
816


 


Boc-Lys(εBoc)
Phe
Tyr
Phe
Ser(tBu)-OtBu
817


 


Boc-Lys(εBoc)
Phe
Tyr
Phe
Thr(tBu)-OtBu
818


 


Boc-Lys(εBoc)
Phe
His
Phe
Ser(tBu)-OtBu
819


 


Boc-Lys(εBoc)
Phe
His
Phe
Thr(tBu)-OtBu
820


 


Boc-Lys(εBoc)
Val
Phe
Phe-
Ser(tBu)-OtBu
821





Tyr




 


Nicotinyl-Lys(εBoc)
Phe
Trp
Phe
Ser(tBu)-OtBu
822


 


Nicotinyl-Lys(εBoc)
Phe
Trp
Phe
Thr(tBu)-OtBu
823


 


Nicotinyl-Lys(εBoc)
Phe
Tyr
Phe
Ser(tBu)-OtBu
824


 


Nicotinyl-Lys(εBoc)
Phe
Tyr
Phe
Thr(tBu)-OtBu
825


 


Nicotinyl-Lys(εBoc)
Phe
His
Phe
Ser(tBu)-OtBu
826


 


Nicotinyl-Lys(εBoc)
Phe
His
Phe
Thr(tBu)-OtBu
827


 


Boc-Leu
Phe
Trp
Phe
Thr(tBu)-OtBu
828


 


Boc-Leu
Phe
Trp
Phe
Ser(tBu)-OtBu
829









While the peptides of Table 11 are illustrated with particular protecting groups, it is noted that these groups may be substituted with other protecting groups as described herein and/or one or more of the shown protecting group can be eliminated.
7) Summary of Tripeptides and Tetrapeptides.
For the sake of clarity, a number of tripeptides and tetrapeptides of this invention are generally summarized below in Table 12.







TABLE 12







General structure of certain peptides of this invention.










X1
X2
X3
X4





hydrophobic side chain
Acidic or
—
hydrophobic side


or hydrophobic
Basic

chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Basic
Acidic
hydrophobic side


or hydrophobic


chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Acidic
Basic
hydrophobic side


or hydrophobic


chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Acidic or 
Aliphatic
hydrophobic side


or hydrophobic
Basic

chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Aliphatic
Acidic or Basic
hydrophobic side


or hydrophobic


chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Acidic or
Aromatic
hydrophobic side


or hydrophobic
Basic

chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Aromatic
Acidic or Basic
hydrophobic side


or hydrophobic


chain or


protecting group(s)


hydrophobic





protecting group(s)


hydrophobic side chain
Aromatic
His Aromatic
hydrophobic side


or hydrophobic


chain or


protecting group(s)


hydrophobic





protecting group(s)









Where longer peptides are desired, X2 and X3 can represent domains (e.g., regions of two or more amino acids of the specified type) rather than individual amino acids. Table 12 is intended to be illustrative and not limiting. Using the teaching provided herein, other suitable peptides can readily be identified.
8) Paired Amino Acids and Dipeptides.
In certain embodiments, this invention pertains to the discovery that certain pairs of amino acids, administered in conjunction with each other or linked to form a dipeptide have one or more of the properties described herein. Thus, without being bound to a particular theory, it is believed that when the pairs of amino acids are administered in conjunction with each other, as described herein, they are capable participating in or inducing the formation of micelles in vivo.
Similar to the other small peptides described herein, it is believed that the pairs of peptides will associate in vivo, and demonstrate physical properties including high solubility in ethyl acetate (e.g., greater than about 4 mg/mL), solubility in aqueous buffer at pH 7.0. Upon contacting phospholipids such as 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in an aqueous environment, it is believed the pairs of amino acids induce or participate in the formation of particles with a diameter of approximately 7.5 nm (±0.1 nm), and/or induce or participate in the formation of stacked bilayers with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing between the bilayers in the stack of approximately 2 nm, and/or also induce or participate in the formation of vesicular structures of approximately 38 nm).
Moreover, it is further believed that the pairs of amino acids can display one or more of the following physiologically relevant properties:

    
    
        1. They convert pro-inflammatory HDL to anti-inflammatory HDL or make anti-inflammatory HDL more anti-inflammatory;
        2. They decrease LDL-induced monocyte chemotactic activity generated by artery wall cells;
        3. They stimulate the formation and cycling of pre-β HDL;
        4. They raise HDL cholesterol; and/or
        5. They increase HDL paraoxonase activity.
    
    


The pairs of amino acids can be administered as separate amino acids (administered sequentially or simultaneously, e.g., in a combined formulation) or they can be covalently coupled directly or through a linker (e.g., a PEG linker, a carbon linker, a branched linker, a straight chain linker, a heterocyclic linker, a linker formed of derivatized lipid, etc.). In certain embodiments, the pairs of amino acids are covalently linked through a peptide bond to form a dipeptide. In various embodiments while the dipeptides will typically comprise two amino acids each bearing an attached protecting group, this invention also contemplates dipeptides wherein only one of the amino acids bears one or more protecting groups.
The pairs of amino acids typically comprise amino acids where each amino acid is attached to at least one protecting group (e.g., a hydrophobic protecting group as described herein). The amino acids can be in the D or the L form. In certain embodiments, where the amino acids comprising the pairs are not attached to each other, each amino acid bears two protecting groups (e.g., such as molecules 1 and 2 in Table 13).







TABLE 13







Illustrative amino acid pairs of this invention.









Amino Acid Pair/dipeptide












1.
Boc-Arg-OtBu*


2.
Boc-Glu-OtBu*


3.
Boc-Phe-Arg-OtBu**


4.
Boc-Glu-Leu-OtBu**


5.
Boc-Arg-Glu-OtBu***





*This would typically be administered in conjunction with a second amino acid.


**In certain embodiments, these dipeptides would be administered in conjunction with each other.


***In certain embodiments, this peptide would be administered either alone or in combination with one of the other peptides described herein..






Suitable pairs of amino acids can readily be identified by providing the pair of protected amino acids and/or a dipeptide and then screening the pair of amino acids/dipeptide for one or more of the physical and/or physiological properties described above. In certain embodiments, this invention excludes pairs of amino acids and/or dipeptides comprising aspartic acid and phenylalanine. In certain embodiments, this invention excludes pairs of amino acids and/or dipeptides in which one amino acid is (−)-N-[(trans-4-isopropylcyclohexane)carbonyl]-D-phenylalanine (nateglinide).
In certain embodiments, the amino acids comprising the pair are independently selected from the group consisting of an acidic amino acid (e.g., aspartic acid, glutamic acid, etc.), a basic amino acid (e.g., lysine, arginine, histidine, etc.), and a non-polar amino acid (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, etc.). In certain embodiments, where the first amino acid is acidic or basic, the second amino acid is non-polar and where the second amino acid is acidic or basic, the first amino acid is non-polar. In certain embodiments, where the first amino acid is acidic, the second amino acid is basic, and vice versa. (see, e.g., Table 14).
Similar combinations can be obtained by administering pairs of dipeptides. Thus, for example in certain embodiments, molecules 3 and 4 in Table 13 would be administered in conjunction with each other.







TABLE 14







Certain generalized amino acid pairs/dipeptides.










First Amino acid
Second Amino acid





1.
Acidic
Basic


2.
Basic
Acidic


3.
Acidic
Non-polar


4.
Non-polar
Acidic


5.
Basic
Non-polar


6.
Non-polar
Basic









It is noted that these amino acid pairs/dipeptides are intended to be illustrative and not limiting. Using the teaching provided herein other suitable amino acid pairs/dipeptides can readily be determined.
In certain embodiments, however, dipeptides and/or amino acid pairs comprising L-Glu-L-Trp, e.g., as described in U.S. Pat. No. 5,807,830 and/or any other peptides disclosed in this patent, are expressly excluded from the methods and/or formulations described herein.
E) Apo-J (G* Peptides).
It was also a discovery of this invention that peptides that mimicking the amphipathic helical domains of apo J are capable of mitigating one or more symptoms of atherosclerosis and/or other pathologies described herein. Apolipoprotein J possesses a wide nonpolar face termed globular protein-like, or G* amphipathic helical domains. The class G amphipathic helix is found in globular proteins, and thus, the name class G. This class of amphipathic helix is characterized by a random distribution of positively charged and negatively charged residues on the polar face with a narrow nonpolar face. Because of the narrow nonpolar face this class does not readily associate with phospholipids. The G* of amphipathic helix possesses similar, but not identical, characteristics to the G amphipathic helix. Similar to the class G amphipathic helix, the G* class peptides possesses a random distribution of positively and negatively charged residues on the polar face. However, in contrast to the class G amphipathic helix which has a narrow nonpolar face, this class has a wide nonpolar face that allows this class to readily bind phospholipid and the class is termed G* to differentiate it from the G class of amphipathic helix.
A number of suitable G* amphipathic peptides are described in copending applications U.S. Ser. No. 10/120,508, filed Apr. 5, 2002, U.S. Ser. No. 10/520,207, filed Apr. 1, 2003, and PCT Application PCT/US03/09988, filed Apr. 1, 2003. In addition, a variety of suitable peptides of this invention that are related to G* amphipathic helical domains of apo J are illustrated in Table 15.







TABLE 15







Certain peptides for use in this invention related


to G* amphipathic helical domains of apo J.










Amino Acid Sequence
SEQ ID NO






LLEQLNEQFNWVSRLANLTQGE
830


 



LLEQLNEQFNWVSRLANL
831


 



NELQEMSNQGSKYVNKEIQNAVNGV
832


 



IQNAVNGVKQIKTLIEKTNEE
833


 



RKTLLSNLEEAKKKKEDALNETRESETKLKEL
834


 



PGVCNETMMALWEECK
835


 



PCLKQTCMKFYARVCR
836


 



ECKPCLKQTCMKFYARVCR
837


 



LVGRQLEEFL
838


 



MNGDRIDSLLEN
839


 



QQTHMLDVMQD
840


 



FSRASSIIDELFQD
841


 



PFLEMIHEAQQAMDI
842


 



PTEFIREGDDD
843


 



RMKDQCDKCREILSV
844


 



PSQAKLRRELDESLQVAERLTRKYNELLKSYQ
845


 



LLEQLNEQFNWVSRLANLTEGE
846


 



DQYYLRVTTVA
847


 



PSGVTEVVVKLFDS
848


 



PKFMETVAEKALQEYRKKHRE
849









The peptides of this invention, however, are not limited to G* variants of apo J. Generally speaking G* domains from essentially any other protein preferably apo proteins are also suitable. The particular suitability of such proteins can readily be determined using assays for protective activity (e.g., protecting LDL from oxidation, and the like), e.g., as illustrated herein in the Examples. Some particularly preferred proteins include G* amphipathic helical domains or variants thereof (e.g., conservative substitutions, and the like) of proteins including, but not limited to apo AI, apo AIV, apo E, apo CII, apo CIII, and the like.
Certain preferred peptides for related to G* amphipathic helical domains related to apoproteins other than apo J are illustrated in Table 16.







TABLE 16







Certain peptides for use in this invention related


to G* amphipathic helical domains related to


apoproteins other than apo J.









SEQ ID


Amino Acid Sequence
NO





WDRVKDLATVYVDVLKDSGRDYVSQF
850


(Related to the 8 to 33 region of apo AI)



 


VATVMWDYFSQLSNNAKEAVEHLQK
851


(Related to the 7 to 31 region of apo AIV)



 


RWELALGRFWDYLRWVQTLSEQVQEEL
852


(Related to the 25 to 51 region of apo E)



 


LSSQVTQELRALMDETMKELKELKAYKSELEEQLT
853


(Related to the 52 to 83 region of apo E)



 


ARLSKELQAAQARLGADMEDVCGRLV
854


(Related to the 91 to 116 region of apo E)



 


VRLASHLRKLRKRLLRDADDLQKRLA
855


(Related to thel3S to 160 region of apo E)



 


PLVEDMQRQWAGLVEKVQA
856


(267 to 285 of apo E.27)



 


MSTYTGIFTDQVLSVLK
857


(Related to the 60 to 76 region of apo CII)



 


LLSFMQGYMKHATKTAKDALSS
858


(Related to the 8 to 29 region of apo CIII)









Additional illustrative G* peptides are shown in Table 17.







TABLE 17







Additional illustrative G* peptides.









SEQ ID


Peptide
NO





Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
859


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Leu-Thr-Glu-Gly-Ser-
860


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Leu-Tyr-His-Leu-Thr-Glu-Gly-Ser-
861


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Val-Tyr-His-Leu-Thr-Glu-Gly-Ser-
862


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-His-Leu-Thr-Glu-Gly-Ser-
863


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Phe-Thr-Glu-Gly-Ser-
864


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Ile-Thr-Glu-Gly-Ser-
865


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Leu-Tyr-His-Val-Thr-Glu-Gly-Ser-
866


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Val-Tyr-His-Tyr-Thr-Glu-Gly-Ser-
867


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-His-Phe-Thr-Glu-Gly-Ser-
868


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-His-Ile-Thr-Glu-Gly-Ser-
869


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-His-Val-Thr-Glu-Gly-Ser-
870


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-His-Tyr-Thr-Glu-Gly-Ser-
871


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Phe-Ile-Trp-His-Leu-Thr-Glu-Gly-Ser-
872


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Leu-Ile-Trp-His-Leu-Thr-Glu-Gly-Ser-
873


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Ile-Ile-Trp-His-Leu-Thr-Glu-Gly-Ser-
874


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Tyr-Ile-Trp-Phe-Leu-Thr-Glu-Gly-Ser-
875


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-Phe-Leu-Thr-Glu-Gly-Ser-
876


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-Leu-Leu-Thr-Glu-Gly-Ser-
877


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Phe-Thr-Glu-Gly-Ser-
878


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Tyr-Thr-Glu-Gly-Ser-
879


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Ile-Thr-Glu-Gly-Ser-
880


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Ser-Glu-Gly-Ser-
881


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Asp-Gly-Ser-
882


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Thr-
883


Ser-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
884


Thr-Glu-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
885


Thr-Asp-Phe-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
886


Thr-Asp-Tyr-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
887


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
888


Thr-Asp-Val-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
889


Thr-Asp-Leu-Lys-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
890


Thr-Asp-Leu-Arg-Ser-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
891


Thr-Asp-Leu-Arg-Thr-Asp-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
892


Thr-Asp-Ile-Lys-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
893


Thr-Asp-Ile-Arg-Ser-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
894


Thr-Asp-Ile-Lys-Ser-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
895


Thr-Asp-Ile-Lys-Ser-Asp-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
896


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Tyr-Ile-Trp-His-Leu-Thr-Glu-Gly-Ser-
897


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
898


Thr-Asp-Ile-Arg-Thr-Asp-Gly-NH2



 


Ac-Arg-Trp-Ile-Phe-His-Leu-Thr-Glu-Gly-Ser-
899


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
900


Thr-Asp-Leu-Lys-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Asp-Gly-Ser-
901


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Asp-Gly-Ser-
902


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-Phe-Leu-Thr-Glu-Gly-Ser-
903


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-Phe-Leu-Thr-Glu-Gly-Ser-
904


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Leu-Thr-Glu-Gly-Ser-
905


Thr-Asp-Phe-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Phe-Tyr-His-Leu-Thr-Glu-Gly-Ser-
906


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Phe-His-Leu-Thr-Glu-Gly-Ser-
907


Thr-Asp-Ile-Arg-Thr-Asp-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
908


Thr-Asp-Ile-Arg-Thr-Asp-Gly-NH2



 


Ac-Arg-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
909


Thr-Asp-Leu-Arg-Thr-Asp-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
910


Thr-Asp-Ile-Lys-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
911


Thr-Asp-Ile-Lys-Thr-Asp-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
912


Thr-Asp-Phe-Lys-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
913


Thr-Asp-Tyr-Lys-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Ile-Tyr-His-Leu-Thr-Glu-Gly-Ser-
914


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
915


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
916


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
917


Thr-Asp-Phe-Arg-Thr-Glu-Gly-NH2



 


Ac-Lys-Trp-Phe-Tyr-His-Phe-Thr-Asp-Gly-Ser-
918


Thr-Asp-Ile-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
919


Thr-Asp-Leu-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
920


Thr-Asp-Phe-Arg-Thr-Glu-Gly-NH2



 


Ac-Arg-Trp-Phe-Tyr-His-Phe-Thr-Glu-Gly-Ser-
921


Thr-Asp-Phe-Arg-Thr-Asp-Gly-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
922


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
923


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Asp-Glu-Phe-Lys-Ser-Leu-
924


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Asp-Phe-Lys-Ser-Leu-
925


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
926


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Val-Asp-Asp-Phe-Lys-Ser-Leu-
927


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
928


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Asp-Asp-Phe-Lys-Ser-Leu-
929


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
930


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Ile-
931


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Val-
932


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Tyr-
933


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
934


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Ile-
935


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Val-
936


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Tyr-
937


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
938


Thr-Thr-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Ile-
939


Ser-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Val-
940


Ser-Thr-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Tyr-
941


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
942


Thr-Thr-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
943


Ser-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
944


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
945


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
946


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
947


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
948


Thr-Ser-Cys-Phe-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
949


Thr-Ser-Cys-Leu-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
950


Thr-Ser-Cys-Ile-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Leu-Lys-Ser-Phe-
951


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
952


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
953


Thr-Ser-Cys-Phe-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
954


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
955


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
956


Thr-Ser-Cys-Phe-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
957


Ser-Ser-Cys-Phe-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
958


Gln-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
959


Gln-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Gln-Phe-
960


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Gln-Leu-
961


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
962


Gln-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Gln-Phe-
963


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Phe-
964


Thr-Ser-Cys-Phe-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
965


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
966


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Arg-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
967


Thr-Ser-Cys-Leu-Glu-Ser-Lys-Ala-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Leu-
968


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
969


Thr-Ser-Cys-Phe-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
970


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
971


Thr-Ser-Cys-Leu-Glu-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Leu-Lys-Ser-Phe-
972


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Arg-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
973


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Ala-Val-Glu-Glu-Phe-Lys-Ser-Phe-
974


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Ala-Val-Glu-Glu-Phe-Lys-Ser-Phe-
975


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Ala-Val-Glu-Glu-Phe-Lys-Ser-Phe-
976


Thr-Ser-Ala-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Ala-Val-Glu-Glu-Phe-Lys-Ser-Phe-
977


Thr-Ser-Ala-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Arg-Ala-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
978


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Arg-Ala-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
979


Thr-Ser-Ala-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Phe-
980


Thr-Ser-Cys-Phe-Glu-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Cys-Tyr-Glu-Glu-Phe-Lys-Ser-Phe-
981


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Asp-Lys-Cys-Trp-Glu-Glu-Phe-Lys-Ser-Phe-
982


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Tyr-
983


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Trp-
984


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Phe-Phe-NH2



 


Ac-Glu-Lys-Cys-Val-Glu-Glu-Phe-Lys-Ser-Trp-
985


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2



 


Ac-Asp-Lys-Cys-Phe-Glu-Glu-Phe-Lys-Ser-Trp-
986


Thr-Ser-Cys-Leu-Asp-Ser-Lys-Ala-Phe-NH2









Other suitable peptides include, but are not limited to the peptides of Table 18.







TABLE 18







Illustrative peptides having an improved hydrophobic phase.











SEQ ID


Name
Sequence
NO





V2W3A5F1017-D-4F
Ac-Asp-Val-Trp-Lys-Ala-Ala-Tyr-Asp-Lys-
987



Phe-Ala-Glu-Lys-Phe-Lys-Glu-Phe-Phe-NH2



 


V2W3F10-D-4F
Ac-Asp-Val-Trp-Lys-Ala-Phe-Tyr-Asp-Lys-
988



Phe-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Phe-NH2



 


W3-D-4F
Ac-Asp-Phe-Trp-Lys-Ala-Phe-Tyr-Asp-Lys-
989



Val-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Phe-NH2









The peptides described here (V2W3A5F10, 17-D-4F; V2W3F10-D-4F; W3-D-4F) may be more potent than the original D-4F.
Still other suitable peptides include, but are not limited to: P1-Dimethyltyrosine-D-Arg-Phe-Lys-P2 (SEQ ID NO:990) and P1-Dimethyltyrosine-Arg-Glu-Leu-P2 (SEQ ID NO:991) where P1 and P2 are protecting groups as described herein. In certain embodiments, these peptides include, but are not limited to BocDimethyltyrosine-D-Arg-Phe-Lys(OtBu) (SEQ ID NO:990) and BocDimethyltyrosine-Arg-Glu-Leu(OtBu) (SEQ ID NO:991).
In certain embodiments, the peptides of this invention include peptides comprising or consisting of the amino acid sequence LAEYHAK (SEQ ID NO:992) comprising at least one D amino acid and/or at least one or two terminal protecting groups. In certain embodiments, this invention includes a peptide that ameliorates one or more symptoms of an inflammatory condition, wherein the peptide: ranges in length from about 3 to about 10 amino acids; comprises an amino acid sequence where the sequence comprises acidic or basic amino acids alternating with aromatic or hydrophobic amino acids; comprises hydrophobic terminal amino acids or terminal amino acids bearing a hydrophobic protecting group. In certain embodiments, the peptide is not the sequence LAEYHAK (SEQ ID NO:992) comprising all L amino acids; where the peptide converts pro-inflammatory HDL to anti-inflammatory HDL and/or makes anti-inflammatory HDL more anti-inflammatory.
It is also noted that the peptides listed in the Tables herein are not fully inclusive. Using the teaching provided herein, other suitable peptides can routinely be produced (e.g., by conservative or semi-conservative substitutions (e.g., D replaced by E), extensions, deletions, and the like). Thus, for example, one embodiment utilizes truncations of any one or more of peptides identified by SEQ ID Nos:830-858.
Longer peptides are also suitable. Such longer peptides may entirely form a class G or G* amphipathic helix, or the G amphipathic helix (helices) can form one or more domains of the peptide. In addition, this invention contemplates multimeric versions of the peptides. Thus, for example, the peptides illustrated in the tables herein can be coupled together (directly or through a linker (e.g., a carbon linker, or one or more amino acids) with one or more intervening amino acids). Suitable linkers include, but are not limited to Proline (-Pro-), Gly4Ser3 (SEQ ID NO:993), and the like. Thus, one illustrative multimeric peptide according to this invention is (D-J336)-P-(D-J336) (i.e. Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-Q-G-E-P-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-Q-G-E-NH2, SEQ ID NO:994).
This invention also contemplates the use of “hybrid” peptides comprising a one or more G or G* amphipathic helical domains and one or more class A amphipathic helices. Suitable class A amphipathic helical peptides are described in PCT publication WO 02/15923. Thus, by way of illustration, one such “hybrid” peptide is (D-J336)-Pro-(4F) (i.e. Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-Q-G-E-P-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2, SEQ ID NO:995), and the like.
Using the teaching provided herein, one of skill can routinely modify the illustrated amphipathic helical peptides to produce other suitable apo J variants and/or amphipathic G and/or A helical peptides of this invention. For example, routine conservative or semi-conservative substitutions (e.g., E for D) can be made of the existing amino acids. The effect of various substitutions on lipid affinity of the resulting peptide can be predicted using the computational method described by Palgunachari et al. (1996) Arteriosclerosis, Thrombosis, & Vascular Biology 16: 328-338. The peptides can be lengthened or shortened as long as the class helix structure(s) are preserved. In addition, substitutions can be made to render the resulting peptide more similar to peptide(s) endogenously produced by the subject species.
While, in preferred embodiments, the peptides of this invention utilize naturally-occurring amino acids or D forms of naturally occurring amino acids, substitutions with non-naturally occurring amino acids (e.g., methionine sulfoxide, methionine methylsulfonium, norleucine, episilon-aminocaproic acid, 4-aminobutanoic acid, tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid, 4-aminobutyric acid, Lys(N(epsilon)-trifluoroacetyl), α-aminoisobutyric acid, and the like) are also contemplated.
New peptides can be designed and/or evaluated using computational methods. Computer programs to identify and classify amphipathic helical domains are well known to those of skill in the art and many have been described by Jones et al. (1992) J. Lipid Res. 33: 287-296). Such programs include, but are not limited to the helical wheel program (WHEEL or WHEEL/SNORKEL), helical net program (HELNET, HELNET/SNORKEL, HELNET/Angle), program for addition of helical wheels (COMBO or COMBO/SNORKEL), program for addition of helical nets (COMNET, COMNET/SNORKEL, COMBO/SELECT, COMBO/NET), consensus wheel program (CONSENSUS, CONSENSUS/SNORKEL), and the like.
F) Blocking Groups and D Residues.
While the various peptides and/or amino acid pairs described herein may be shown with no protecting groups, in certain embodiments (e.g., 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 Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-Q-G-E-NH2 (SEQ ID NO:830 with blocking groups), either or both of these protecting groups can be eliminated and/or substituted with another protecting group as described herein.
Without being bound by a particular theory, it was a discovery of this invention that blockage, particularly of the amino and/or carboxyl termini of the subject peptides of this invention greatly improves oral delivery and significantly increases serum half-life. It was also a surprising discovery, however, that in certain embodiments, particular when used in conjunction with the salicylanilides (e.g., niclosamide) and other delivery agents described herein, any or all of the protecting groups can be omitted and the peptides are still orally administrable. Nevertheless, in certain embodiments the peptides, even when formulated with and/or administered in conjunction with a salicylanilide or other delivery agent as described herein bears one or more protecting groups (e.g., terminal protecting groups).
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.
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 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-1-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), 1-(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), and Trifluoroacetyl (TFA).
Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the peptides of this invention (see, e.g., Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). In one preferred 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 peptides described herein in the examples, rink amide resin was 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.
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, or even every amino acid (e.g., every enantiomeric amino acid) of the peptides described herein is a D-form amino acid.
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.
G) Peptide Mimetics.
In addition to the peptides described herein, it is believed that the salicylanilides (e.g., niclosamide) and other delivery agents described herein are also useful to improve in vivo activity of orally delivered peptide mimetics. 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.
Generally, peptidomimetics are structurally similar to a paradigm polypeptide (e.g., SEQ ID NO:5 shown in Table 1), 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 J Pept 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., European Appln. EP 45665 (1982) 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—)).
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.
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.
H) Small Organic Molecules.
In addition to the peptides described herein, it is believed that the salicylanilides (e.g., niclosamide) and other delivery agents described herein are also useful to improve in vivo activity (apparent activity) of orally delivered small organic molecules, e.g., as described in copending application U.S. Ser. No. 60/600,925, filed Aug. 11, 2004. In various embodiments the small organic molecules are similar to, and in certain cases, mimetics of the tetra- and penta-peptides described in copending application U.S. Ser. No. 10/649,378, filed on Aug. 26, 2003 and U.S. Ser. No. 60/494,449, filed on August 11.
The small organic molecules of this invention typically have molecular weights less than about 900 Daltons. Typically the molecules are highly soluble in ethyl acetate (e.g., at concentrations equal to or greater than 4 mg/mL), and also are soluble in aqueous buffer at pH 7.0.
Contacting phospholipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), with the small organic molecules of this invention in an aqueous environment typically results in the formation of particles with a diameter of approximately 7.5 nm (±0.1 nm). In addition, stacked bilayers are often formed with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing between the bilayers in the stack of approximately 2 nm. Vesicular structures of approximately 38 nm are also often formed. Moreover, when the molecules of this invention are administered to a mammal they render HDL more anti-inflammatory and mitigate one or more symptoms of atherosclerosis and/or other conditions characterized by an inflammatory response.
Thus, in certain embodiments, the small organic molecule is one that ameliorates one or more symptoms of a pathology characterized by an inflammatory response in a mammal (e.g., atherosclerosis), where the small molecule is soluble in ethyl acetate at a concentration greater than 4 mg/mL, is soluble in aqueous buffer at pH 7.0, and, when contacted with a phospholipid in an aqueous environment, forms particles with a diameter of approximately 7.5 nm and forms stacked bilayers with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing between the bilayers in the stack of approximately 2 nm, and has a molecular weight less than 900 daltons.
In certain embodiment, the molecule has the formula:




where P1, P2, P3, and P4 are independently selected hydrophobic protecting groups; R1 and R4 are independently selected amino acid R groups; n, i, x, y, and z are independently zero or 1 such that when n and x are both zero, R1 is a hydrophobic group and when y and i are both zero, R4 is a hydrophobic group; R2 and R3 are acidic or basic groups at pH 7.0 such that when R2 is acidic, R3 is basic and when R2 is basic, R3 is acidic; and R5, when present is selected from the group consisting of an aromatic group, an aliphatic group, a positively charged group, or a negatively charged group. In certain embodiments, R2 or R3 is —(CH2)j-COOH where j=1, 2, 3, or 4 and/or —(CH2)j-NH2 where j=1, 2, 3, 4, or 5, or —(CH2)j-NH—C(═NH)—NH2 where n=1, 2, 3 or 4. In certain embodiments, R2, R3, and R5, when present, are amino acid R groups. Thus, for example, In various embodiments R2 and R3 are independently an aspartic acid R group, a glutamic acid R group, a lysine R group, a histidine R group, or an arginine R group (e.g., as illustrated in Table 1).

In certain embodiments, R1 is selected from the group consisting of a Lys R group, a Trp R group, a Phe R group, a Leu R group, an Orn R group, pr a norLeu R group. In certain embodiments, R4 is selected from the group consisting of a Ser R group, a Thr R group, an Ile R group, a Leu R group, a norLeu R group, a Phe R group, or a Tyr R group.
In various embodiments x is 1, and R5 is an aromatic group (e.g., a Trp R group).
In various embodiments at least one of n, x, y, and i is 1 and P1, P2, P3, and P4 when present, are independently selected from the group consisting of polyethylene glycol (PEG), an acetyl, amide, a 3 to 20 carbon alkyl group, fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic, 9-fluorenone-1-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), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)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), a propyl group, a butyl group, a pentyl group, a hexyl group, and trifluoroacetyl (TFA). In certain embodiments, P1 when present and/or P2 when present are independently selected from the group consisting of Boc-, Fmoc-, and Nicotinyl- and/or P3 when present and/or P4 when present are independently selected from the group consisting of tBu, and OtBu.
While a number of protecting groups (P1, P2, P3, P4) are illustrated above, this list is intended to be illustrative and not limiting. In view of the teachings provided herein, a number of other protecting/blocking groups will also be known to one of skill in the art. Such blocking groups can be selected to minimize digestion (e.g., for oral pharmaceutical delivery), and/or to increase uptake/bioavailability (e.g., through mucosal surfaces in nasal delivery, inhalation therapy, rectal administration), and/or to increase serum/plasma half-life. In certain embodiments, the protecting groups can be provided as an excipient or as a component of an excipient.
In certain embodiments, z is zero and the molecule has the formula:




where P1, P2, P3, P4, R1, R2, R3, R4, n, x, y, and i are as described above.

In certain embodiments, z is zero and the molecule has the formula:




where R1, R2, R3, and R4 are as described above.

In one embodiment, the molecule has the formula:




In certain embodiments, this invention contemplates small molecules having one or more of the physical and/or functional properties described herein and having the formula:




where P1, P2, P3, and P4 are independently selected hydrophobic protecting groups as described above, n, x, and y are independently zero or 1; j, k, and l are independently zero, 1, 2, 3, 4, or 5; and R2 and R3 are acidic or basic groups at pH 7.0 such that when R2 is acidic, R3 is basic and when R2 is basic, R3 is acidic. In certain preferred embodiments, the small molecule is soluble in water; and the small molecule has a molecular weight less than about 900 Daltons. In certain embodiments, n, x, y, j, and l are 1; and k is 4.

In certain embodiments, P1 and/or P2 are aromatic protecting groups. In certain embodiments, R2 and R3 are amino acid R groups, e.g., as described above. In various embodiments least one of n, x, and y, is 1 and P1, P2, P3 and P4 when present, are independently protecting groups, e.g., as described above. In certain embodiments the protecting groups, when present, are independently selected from the group consisting of polyethylene glycol (PEG), an acetyl, amide, 3 to 20 carbon alkyl groups, Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic, 9-fluorenone-1-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), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)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), a propyl group, a butyl group, a pentyl group, a hexyl group, and trifluoroacetyl (TFA). In certain embodiments, P1 when present and/or P2 when present are independently selected from the group consisting of Boc-, Fmoc-, and Nicotinyl- and/or P3 when present and/or P4 when present are independently selected from the group consisting of tBu, and OtBu.
IV. Pharmaceutical Formulations.
A) Pharmaceutical Formulations.
In order to carry out the methods of the invention, one or more therapeutic peptides, mimetics, or small organic molecules described herein are administered in conjunction with a salicylanilide (e.g., niclosamide or niclosamide analogue) or one of the other delivery agents described herein to a mammal, e.g., to an individual diagnosed as having one or more symptoms of atherosclerosis, or as being at risk for atherosclerosis and or the various other pathologies described herein.
In various embodiments the “active agent(s)”, therapeutic peptides, mimetics, or small organic molecules described herein, are formulated in combination with one or more of the salicylanilides (e.g., niclosamide or niclosamide analogue) or one of the other delivery agents described herein. In certain embodiments one or more active agent(s) are combined with one or more salicylanilides (e.g., niclosamide or niclosamide analogs) to form an adduct. The active agent(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.
Similarly, the delivery agent(s) can also be formulated as salts, esters, amides, and the like.
Methods of formulating such derivatives are known to those of skill in the art. For example, the disulfide salts of a number of delivery agents are described in PCT Publication WO 00/059863 which is incorporated herein by reference. Similarly, acid salts of therapeutic peptides, mimetics, and small organic molecules can be 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.
Preparation of Esters Typically Involves Functionalization of Hydroxyl and/or carboxyl groups which 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 active agents 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). 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.
In addition to administration in conjunction with or formulation with one or more delivery agents, the active agents of this invention can also 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.
Other physiologically acceptable compounds, particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to binders, diluent/fillers, disentegrants, lubricants, susupending agents, and the like.
In certain embodiments, to manufacture an oral dosage form (e.g., a tablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrator (e.g. calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone etc.), a binder (e.g. alpha-starch, gum arabic, microcrystalline cellulose, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), for instance, are added to the active component or components (e.g., active peptide and salicylanilide) and the resulting composition is compressed. Where necessary, the compressed product is coated, e.g., known methods for masking the taste or for enteric dissolution or sustained release. Suitable coating materials include, but are not limited to ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).
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).
In certain embodiments the excipients are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. For various oral dosage form excipients such as tablets and capsules sterility is not required. The USP/NF standard is usually sufficient.
In therapeutic applications, the compositions of this invention are administered, e.g., orally administered, to a patient suffering from one or more symptoms of the 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 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 active agent(s) can vary widely, and will be selected primarily based on activity of the active ingredient(s), 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 11.0 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.
In certain embodiments, the active agents of this invention are administered orally (e.g., via a tablet, capsule, caplet, gel cap, etc.). It was a surprising discovery that therapeutic peptides can be orally administered and achieve therapeutically effective levels, particularly when administered with a salicylanilide (e.g., niclosamide or a niclosamide analogue) or one of the other delivery agents described herein. It was particularly surprising that when so administered, the therapeutic peptide can be an L-form peptide and need not bear protecting groups. The combination of therapeutic peptide with a salicylanilide or other delivery agent is not limited to unprotected L-form peptides. To the contrary, the use salicylanilides and/or other delivery agent(s) with L-form peptides bearing one or more protecting groups, D-form peptides, and D-form peptides bearing one or more protecting groups is also contemplated.
In certain embodiments the active agents of this invention are administered as an injectable in accordance with standard methods well known to those of skill in the art. In other preferred embodiments, the agents, can 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.
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, polyethylenes, 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.
Other formulations for topical drug delivery include, but are not limited to, ointments and creams. Ointments are semisolid preparations that 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.
As indicated above, various buccal, and sublingual formulations are also contemplated.
The use of salicylanilide or other delivery agents as described herein need not be limited to oral delivery. In certain embodiments the use of such delivery agents is also contemplated in formulations intended for transdermal delivery, injectable delivery, surgical implantation, nasal delivery, rectal delivery, and the like.
In another embodiment, one or more components of the formulation (e.g., delivery agent and/or active agent) can be provided as a “concentrate”, e.g., in a storage container (e.g., in a premature volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water. In certain embodiments the salicylanilide or other delivery agent and the therapeutic agent are provided separately. Thus for example, salicylanilide or other delivery agent is provided as a solution that is administered immediately or some time prior to administration of the therapeutic agent (e.g., therapeutic peptide), or the salicylanilide or other delivery agent is provided as a solution used while swallowing the active agent(s) formulated as a capsule, tablet, gel cap, etc.
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.
In certain embodiments, the active agents and/or salicylanilide or other delivery agent(s) of this invention 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.
Without being bound by a particular theory, it was discovered of this invention that administration (e.g., oral administration) 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 active agents of this invention confer a number of advantages: They protect the active agents from digestion or hydrolysis, they improve uptake, and they improve HDL/LDL ratios.
The lipids can be formed into liposomes that encapsulate the active agents of this invention and/or they can be complexed/admixed with the active agents and/or they can be covalently coupled to the active agents. 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).
Preferred 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 preferred fatty acids are illustrated in Table 19.







TABLE 19







Preferred fatty acids in the sn-1 and/or sn-2 position of the


preferred phospholipids for administration of active agents


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. Particularly preferred phospholipids have phosphorylcholine at the sn-3 position.
V. Additional Pharmacologically Active Agents.
A) Combined Active Agents
In various embodiments, the use of combinations of two or more active agents described 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.
By way of illustration, it is noted that D-4F and L-4Frapidly associates with pre-beta HDL and HDL and then are rapidly cleared from the circulation (it is essentially non-detectable 6 hours after an oral dose), while D-[113-122]apoJ slowly associates with pre-beta HDL and to a lesser extent with HDL but remains associated with these HDL fractions for at least 36 hours. FREL associates with HDL and only HDL but remains detectable in HDL for much longer than D-4F (i.e., it is detectable in HDL 48 hours after a single oral dose in mice). In certain embodiments this invention thus contemplates combinations of, for example, these three peptides to reduce the amount to reduce production expense, and/or to optimize dosage regimen, therapeutic profile, and the like. In certain embodiments combinations of the active agents described herein can be simply coadministered and/or added together to form a single pharmaceutical formulation. In certain embodiments the various active agent(s) can be complexed together (e.g., via hydrogen bonding) to form active agent complexes that are more effective than the parent agents.
B) Use with Additional Pharmacologically Active Materials.
Additional pharmacologically active materials (i.e., drugs) can be delivered in conjunction with one or more of the active agents 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 blockers and thiazide diuretic combinations, statins, aspirin, ace inhibitors, ace receptor inhibitors (ARBs), and the like.
It was discovered that, adding a low dosage active agent (e.g., of D-4F) (1 μg/ml) to the drinking water of apoE null mice for 24 hours did not significantly improve HDL function (see, e.g., related application U.S. Ser. No. 10/423,830, filed on Apr. 25, 2003, which is incorporated herein by reference). In addition, adding 0.05 mg/ml of atorvastatin or pravastatin alone to the drinking water of the apoE null mice for 24 hours did not improve HDL function. However, when D-4F1 μg/ml was added to the drinking water together with 0.05 mg/ml of atorvastatin or pravastatin there was a significant improvement in HDL function). Indeed the pro-inflammatory apoE null HDL became as anti-inflammatory as 350 μg/ml of normal human HDL (h, HDL see, e.g., related application U.S. Ser. No. 10/423,830).
Thus, doses of D-4F alone, or statins alone, which by themselves had no effect on HDL function when given together acted synergistically. When D-4F and a statin were given together to apo E null mice, their pro-inflammatory HDL at 50 μg/ml of HDL-cholesterol became as effective as normal human HDL at 350 μg/ml of HDL-cholesterol in preventing the inflammatory response induced by the action of HPODE oxidizing PAPC in cocultures of human artery wall cells.
Thus, in certain embodiments this invention provides methods for enhancing the activity of statins. The methods generally involve administering one or more of the active agents described herein, as described herein in conjunction with one or more statins. The active agents achieve synergistic action between the statin and the agent(s) to ameliorate one or more symptoms of atherosclerosis. In this context 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 pravastatin (Pravachol/Bristol-Myers Squibb), simvastatin (Zocor/Merck), lovastatin (Mevacor/Merck), and the like.
In various embodiments the active agent(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 (SectralTM), atenolol (TenorminTM), betaxolol (KerloneTM), bisoprolol (ZebetaTM), metoprolol (LopressorTM), and the like. Suitable non-selective blockers (block beta 1 and beta 2 equally) include, but are not limited to carteolol (CartrolTM), nadolol (CorgardTM), penbutolol (LevatolTM), pindolol (ViskenTM), propranolol (InderalTM), timolol (BlockadrenTM), labetalol (NormodyneTM, TrandateTM), 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., CapotenTM by Squibb), benazepril (e.g., LotensinTM by Novartis), enalapril (e.g., VasotecTM by Merck), fosinopril (e.g., MonoprilTM by Bristol-Myers), lisinopril (e.g., PrinivilTM by Merck or ZestrilTM by Astra-Zeneca), quinapril (e.g., AccuprilTM by Parke-Davis), ramipril (e.g., AltaceTM by Hoechst Marion Roussel, King Pharmaceuticals), imidapril, perindopril erbumine (e.g., AceonTM by Rhone-Polenc Rorer), trandolapril (e.g., MavikTM by Knoll Pharmaceutical), and the like. Suitable ARBS (Ace Receptor Blockers) include but are not limited to losartan (e.g., CozaarTM by Merck), irbesartan (e.g., AvaproTM by Sanofi), candesartan (e.g., AtacandTM by Astra Merck), valsartan (e.g., DiovanTM by Novartis), and the like.
In various embodiments, one or more agents described herein are administered with one or more of the drugs identified below.
Thus, in certain embodiments one or more active agents 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, 1-[[[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-δ12,14-prostaglandin J2, Fenofibrate, Fmoc-Leu-OH, GW1929, GW7647, 8(S)-Hydroxy-(5Z,9E,11Z,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.
In certain embodiments one or more of the active agents 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), Gö 6976, H-7. dihydrochloride, 1-O-Hexadecyl-2-O-methyl-rac-glycerol, Hexadecyl-phosphocholine (C16:0); 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-erythro-Sphingosine (isolated), D-erythro-Sphingosine (synthetic), Sphingosine, N,N-dimethyl, D-erythro-Sphingosine, Dihydro-, D-erythro-Sphingosine, N,N-Dimethyl-, D-erythro-Sphingosine chloride, N,N,N-Trimethyl-, Staurosporine, Bisindolylmaleimide I, G-6203, and the like).
In certain embodiments, one or more of the active agents are administered in conjunction with ApoAI, Apo A-I derivatives and/or agonists (e.g., 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. Pat. Nos. 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®; DuraclonTM (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. Pat. No. 5,639,726, and the like), and/or low molecular weight heparin, and/or AGE inhibitorsibreakers (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 B1 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 VIIa 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.
C) Administration.
Typically the active agent(s) described hereinwill be administered (typically in conjunction with a salicylanilide (e.g., niclosamide or niclosamide analogue) or other delivery agent as described herein) 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.
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).
The methods involve the administration of a single active agent of this invention or the administration of two or more different active agents, typically in conjunction with a salicylanilide (e.g., niclosamide or niclosamide analogue) or other delivery agent as described herein. The active agents 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).
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.
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.
VI. Kits for the Treatment of One or More Indications.
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 active agents described herein. The active agent(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 excipients.
In various embodiments the kits typically additionally comprise a salicylanilide or other delivery agent described herein. The salicylanilide or other delivery agent can be formulated as a compound formulation with one or more of the active agents described herein. Alternatively, the salicylanilide or other delivery agent can be provided separately, e.g., in a separate container.
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.
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 active agent(s) of this invention 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 may also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
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.
VII. Indications.
The active agents (e.g., peptides, small organic molecules, amino acid pairs, etc.) 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. In particular, the active agents (e.g., peptides, small organic molecules, amino acid pairs, etc.) described herein are effective for mitigating one or more symptoms of atherosclerosis. Without being bound to a particular theory, it is believed that the peptides bind the “seeding molecules” required for the formation of pro-inflammatory oxidized phospholipids such as Ox-PAPC, POVPC, PGPC, and PEIPC.
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 active agents described herein appear to be efficacious.
A number of pathologies for which the active agents described herein appear to be a palliative and/or a preventative are shown in Table 20.







TABLE 20





Summary of conditions in which the active agents (e.g., D-4F)


have been shown to be or 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)



  Sjögren's syndrome



  Scleroderma



  polymyositis.



AIDS inflammatory response



infections:



 bacterial



 fungal



 viral



 parasitic



 influenza



  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



osteoarthritis



inflammatory bowel disease



allergic rhinitis



cachexia



diabetes



Alzheimer's disease



implanted prosthesis



biofilm formation



Crohns' disease



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










It is noted that the conditions listed in Table 20 are intended to be illustrative and not limiting.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Niclosamide Enhances Uptake/Bioavailability of Orally Administered Peptides
We previously reported that the amino acid sequence D-W—F—K-A-F—Y-D-K—V-A-E-KF—K-E-A-F (SEQ-ID-NO:5) bearing at least one protecting group (see, e.g., U.S. Pat. No. 6,933,279) when synthesized from all L-amino acids (L-4F) and administered orally to mice was rapidly degraded and did not significantly alter the protective capacity of HDL to inhibit LDL-induced monocyte chemotactic activity in cultures of human artery wall cells (Navab et al. (2002) Circulation 105: 290-292).
It was a surprising finding of this invention that administering L-4F with niclosamide orally to mice resulted in significant improvement in the ability of HDL from these mice to inhibit LDL-induced monocyte chemotactic activity. In contrast orally administering either agent alone was ineffective or significantly less effective.
As shown in FIG. 8, the combination of oral Niclosamide and L-4F was potent in a mouse model of atherosclerosis. 11-month-old female apoE null mice were fasted during the day. At night the mice were provided chow containing or not containing additions. In the first experiment the mice were given chow alone (C) or chow supplemented with 8.0 micrograms of Niclosamide (2′,5-Dichloro-4′-nitrosalicylanilide; Niclosamide, Sigma catalog number N-3510 Page 1711 2006-2007 catalog Empirical Formula (Hill Notation): C13H8C12N2O4 Formula Weight: 327.12, CAS Number: 50-65-7 Batch 105K0666 EC 200-056-8) per gram of chow (D) or chow supplemented with 2.0 micrograms of L-4F (free base) per gram of chow (E), or chow supplemented with 8.0 micrograms of Niclosamide together with 2.0 micrograms of L-4F (free base) per gram of chow (F). The mice were only given one gram of chow per mouse (n=8 mice per group) so that they would consume all of the chow. In the morning after the chow was consumed the mice were bled and their plasma was sucrose cryopreserved and fractionated by FPLC and the HDL-containing fractions were tested for their ability to inhibit monocyte chemotactic activity induced by a standard control human LDL (A) in cultures of human aortic endothelial cells. The mouse HDL (C-J) was also compared to a standard human HDL (B) that was added at the same concentrations as the mouse HDL. The resulting monocyte chemotactic activity was normalized to the standard control LDL added alone (A). The results are plotted as the HDL-inflammatory index, which is the result of dividing the monocyte chemotactic activity measured for each condition by the monocyte chemotactic activity obtained by the standard control LDL added alone, which was normalized to 1.0 as described previously (Navab et al. (2004) J Lipid Res, 45: 993-1007).
A second experiment was performed as described for the first experiment with 8 mice in each group except that the additions to the chow were different. Chow alone in the second experiment (G) was compared to chow supplemented with 100 micrograms of Niclosamide per gram of chow (H), or supplemented with 10 micrograms of L-4F (free base) per gram of mouse chow (I), or supplemented with 10 micrograms of L-4F (free base) together with 100 micrograms of Niclosamide per gram of chow (J). As in the first experiment the mice were only given one gram of chow per mouse so that they would consume all of the chow. In the morning this second group of mice were bled and their HDL tested in the human artery wall cell culture together with the HDL from the first experiment.
The data indicate that addition of either 2 (E) or 10 (I) micrograms of L-4F to the chow slightly but significantly improved the HDL-inflammatory index and the difference between these two doses in the absence of Niclosamide was not significant confirming our previous report (Navab et al. (2002) Circulation, 105: 290-292). As shown in FIG. 1 (D) and (H), administering Niclosamide by itself was ineffective. Surprisingly the oral combination of Niclosamide with L-4F in each case resulted in dramatic statistically significant improvement in the HDL-inflammatory index. The use of 10 micrograms of L-4F together with 100 micrograms of Niclosamide (J) was significantly better than 2 micrograms of L-4F together with 8 micrograms of Niclosamide (F).
As shown in FIG. 9, administration of Niclosamide as an oral bolus by gastric gavage (stomach tube) immediately followed by administration of L-4F as an oral bolus by stomach tube rendered apoE null mouse HDL anti-inflammatory. Ten mg of Niclosamide was placed in a glass-glass homogenizer with mortar and round bottom pestle (Kontes Dounce Tissue grinder, K885300-0015 available from Fisher, VWR) and 200 μL of ethanol was added. The Niclosamide ethanol mixture was homogenized using 2-3 strokes and distilled water was added and the mixture further homogenized using 5-10 strokes and the volume was adjusted to 10 mL with distilled water. Serial dilutions of this mixture were made using distilled water to give the micrograms of Niclosamide shown on the x-axis, which were contained in 100 μL. L-4F (free base) was diluted with water to give 10 μg per 100 μL of water. One hundred microliters of the Niclosamide solution was given by stomach tube to each mouse in each group of twelve-month-old non-fasting female apoE null mice (n=4 per group) and immediately followed by 100 μL containing 10 μg of L-4F (free base) in water. The mice were fasted and after 7 hours they were bled and their plasma was sucrose cryopreserved. The plasma was fractionated by FPLC and the HDL-containing fractions were tested for their ability to inhibit the induction of monocyte chemotactic activity by a standard control human LDL, which was added to cultures of human aortic endothelial cells. The standard control human LDL was also added by itself or with a standard control human HDL. The values obtained by the standard control human LDL alone were normalized to 1.0. The values obtained after the addition of the standard control HDL or the mouse HDL were compared to the values obtained by the standard control LDL alone to give the HDL Inflammatory Index.
FIG. 10 shows that Administration of Niclosamide as an oral bolus by stomach tube immediately followed by administration of L-4F as an oral bolus by stomach tube significantly reduced the ability of apoE null mouse LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells. The LDL fractions from the mice described in FIG. 9 were tested for their ability to induce monocyte chemotactic activity in cultures of human aortic endothelial cells and compared to a standard control human LDL whose values were normalized to 1.0 for the LDL-inflammatory index.
FIG. 11 shows that oral administration of niclosamide (5.0 mg/kg body weight) immediately followed by oral administration of L-4F (0.5 mg/kg/body weight) renders monkey HDL anti-inflammatory. One hundred mg of niclosamide was placed in a glass-glass homogenizer with mortar and round bottom pestle (Kontes Dounce Tissue grinder, K885300-0015 available from Fisher, VWR) and 200 μL of ethanol was added. The Niclosamide ethanol mixture was homogenized using 2-3 strokes and distilled water was added and the mixture further homogenized using 5-10 strokes and the volume was adjusted to 10 mL with distilled water. The niclosamide mixture was again mixed immediately before the dose was removed as the Niclosamide tends to settle out. Each of 4 monkeys (2 Female and 2 Male) were given 5.0 mg/kg body weight of Niclosamide contained in 2.5 mL of the mixture by stomach tube. L-4F (free base) was added to 10 mL of distilled water in the glass-glass homogenizer and homogenized using 5-10 strokes. Immediately after administration of the Niclosamide mixture each monkey was given 0.5 mg/kg body weight of L-4F (free base) contained in 2.5 mL water by stomach tube. Blood was obtained 5 hours later and the plasma was separated by FPLC and the lipoproteins tested as described in FIG. 8 for the HDL-inflammatory index and FIG. 10 for the LDL-inflammatory index. The data shown are the Mean±S.D. for the HDL Inflammatory Index for monkey HDL before and 5 hours after treatment (the data for the standard control human LDL alone and the standard control human LDL plus the standard control human HDL are not shown in the figure).
Oral administration of niclosamide (5.0 mg/kg body weight) immediately followed by oral administration of L-4F (0.5 mg/kg/body weight) significantly reduced the ability of monkey LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells (see, e.g., FIG. 12). The LDL fractions from the monkey plasma described in FIG. 11 were tested as described in FIG. 10.
Niclosamide is relatively insoluble in aqueous solutions even when added in ethanol and homogenized. It was a surprising finding of this invention that L-4F solubilized niclosamide in aqueous solution as shown in FIG. 13. Niclosamide at 10 mg per mL was added to water, or to water containing 1.0 mg/mL L-4F (free base) and was homogenized in a glass-glass homogenizer. The solutions were stored at 4° C. for ten days and photographed (see FIG. 13).
The solutions of Niclosamide with or without L-4F shown above in FIG. 13 were serially diluted and given by gastric gavage (stomach tube) to fasting seven month old female apoE null mice in a volume of 100 μL per mouse (n=8 per group). Blood was collected 6 hrs following treatment while the mice were still fasting and the plasma was separated by FPLC and the HDL fractions were tested as described in FIG. 8 and the data are shown in FIG. 14.
The micrograms of L-4F and/or niclosamides are shown on the X-axis. Six hours after administration the mice were bled and the ability of mouse HDL (m) or human HDL (h) to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells was determined and plotted as the HDL-inflammatory index as described for FIG. 8.
As shown in FIG. 15, administration of the L-4F together with the solubilized niclosamide resulted in a significant reduction in the ability of mouse LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells.
The data in FIGS. 14 and 15 demonstrate the remarkable, novel, and unexpected findings that the peptide L-4F solubilizes niclosamide and results in a therapeutic combination that renders HDL anti-inflammatory and significantly reduces the inflammatory properties of LDL in a mouse model of atherosclerosis.
It was also a surprising finding of this invention that administration of Niclosamide in mouse chow greatly enhanced the ability of L-4F to render HDL anti-inflammatory and to decrease the ability of LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells even when the L-4F was administered in the drinking water (see, e.g., FIGS. 16 and 17).
L-4F was previously thought to be ineffective in rendering HDL anti-inflammatory and ineffective in reducing the ability of LDL to induce monocyte chemotactic activity in cultures of human aortic endothelial cells if the peptide was given orally (see, e.g., Navab et al. (2002) Circulation, 105: 290-292). The data in FIGS. 8-17 demonstrate the surprising and unexpected finding that if L-4F is given orally with niclosamide it is highly effective in rendering HDL anti-inflammatory and highly effective in reducing the inflammatory properties of LDL. This invention also demonstrates the surprising and unexpected finding that L-4F solubilizes niclosamide.
Example 2
Salicylanilides Combined with L-4F Enhance Formation of Pre-Beta HDL
Niclosamide plus L-4F causes the formation of pre-β HDL in apoE null mice after oral administration (see, e.g., FIG. 18). D-4F (free base) was dissolved in 0.1% Tween20 in ammonium bicarbonate buffer (ABCT) pH 7.0. L-4F (free base) plus niclosamide were dissolved in ABCT in a ratio of 1:10 (L-4F:Niclosamide; wt:wt). ABCT alone or ABCT containing the micrograms of L-4F or D-4F with or without the micrograms of niclosamide shown in FIG. 18 on the X-axis were administered in 100 μL by stomach tube to 8 month old female apoE null mice that were fasted overnight (n=8 per group). Thirty to forty minutes later the mice were bled and the percent of apolipoprotein A-I contained in pre-β-1 HDL was determined in triplicate 2-dimensional gels by scanning. The data shown are the Mean±S.D.
It was also a surprising discovery that oral co-administration of niclosamide and L-4F improved the inflammatory properties of apoE null mouse HDL (as measured in a cell-based assay) to a degree similar to that seen when niclosamide was administered with D-4F (see, e.g., FIG. 19).
Similar results were obtained when the inflammatory properties of HDL were measured by a cell-free assay (see, e.g., FIG. 20).
It was also a surprising discovery that when niclosamide and L-4F were co-administered orally to apoE null mice the increase in paraoxonase activity was similar to that seen when niclosamide was co-administered with D-4F (see, e.g., FIG. 21).
Oral co-administration of niclosamide with either D-4F or L-4F enhanced the ability of both peptides to improve HDL inflammatory properties in apoE null mice. In the absence of niclosamide, however, D-4F was able to render apoE null mouse HDL anti-inflammatory to a degree comparable to normal human HDL while L-4F was only able to achieve this degree of efficacy when co-administered with niclosamide (see, e.g., FIG. 22).
As shown in FIG. 23 the inflammatory properties of LDL from apoE null mice were reduced by the co-administration orally of niclosamide and L-4F.
It was a surprising discovery of this invention that some of the salicylanilides described in FIGS. 24-26 were even more potent than niclosamide in rendering apoE null mouse HDL anti-inflammatory when administered orally together with either D-4F or L-4F. As shown in FIG. 24 neither niclosamide nor the new salicylanilides were anti-inflammatory when administered without the peptides.
As shown in FIG. 25 the new salicylanilides (BP-1001 and BP-1012) were also more potent in reducing the inflammatory properties of LDL than niclosamide when co-administered with D-4F or L-4F.
As shown in FIG. 26, other salicylanilides were similar to niclosamide (BP-124) in bioactivity while still others were more potent.
Example 3
Niclosamide Increases L-4F Absorption in Apoe Null Mice
L-4F absorption was determined with and without niclosamide (BP-124) using 14C-L-4F. Fasted female apoE null mice 6-months of age (n=4 per group) were administered by stomach tube L-4F (21,000 dpm containing 10 micrograms of L-4F per mouse) with or without 100 micrograms of niclosamide in 200 μL 0.1% Tween20 in ammonium bicarbonate at pH 7.0. Fasting was continued and the mice were bled at the time points shown on the X-axis in FIG. 27 and the dpm per mL plasma determined. The area under the curve (AUC) in FIG. 27 for the mice receiving L-4F+niclosamide was 4.4 times greater than the AUC for the mice receiving L-4F without niclosamide.
The data indicate that one of the mechanisms by which niclosamide enhances the in vivo bioactivity of L-4F is by increasing the absorption of L-4F.
The foregoing data (Examples 1, 2, and 3) show that the combination of niclosamide or other salicylanilides with L-4F, and presumably other therapeutic peptides, appears to have great potential for oral therapy. Based on these data it is believed that the use of niclosamide or other salicylanilides with other peptides or proteins will make new oral therapeutics possible.
The data in FIG. 27 indicate that without niclosamide administration of 14C-L-4F by stomach tube resulted in low plasma levels that lasted no more than 5 minutes. In contrast, when 14C-L-4F was administered with niclosamide a Cmax of approximately 150 nanograms/mL was achieved which persisted for more than an hour and at a lower level for up to four hours.
The data in FIG. 28 demonstrate that the 14C-L-4F used in FIG. 28 was biologically active when given with niclosamide. Fasted apoE null mice 5-months of age (n=4 per group) were administered by stomach tube 14C-L-4F (21,000 dpm containing 10 μg of L-4F per mouse) with or without 100 μg of niclosamide (Niclos.) in 200 μL. Fasting was continued and the mice were bled 5 hours later and the HDL inflammatory index determined in cultures of human aortic endothelial cells as described in FIG. 8. Briefly, To determine the HDL-inflammatory index lipoproteins were added to human aortic endothelial cell cultures as described previously (Navab et al. (2005) Circulation Research 97: 524-532). A normal control human LDL was added to each well in triplicate at a final concentration of 100 μg/mL of LDL-cholesterol. A normal human HDL was added to three wells containing human LDL at a final concentration of 50 μg/mL HDL-cholesterol as a positive control. HDL from the mice at a final concentration of 50 μg/mL HDL-cholesterol was added in triplicate to other wells containing human LDL. After 8 hours of culture the supernatants were removed and monocyte chemotactic activity was determined as previously described (Navab et al. (2001) J. Lipid Res., 42: 1308-1317; Danciger et al. (2004) J. Immunol. Meth., 288: 123-124). The values obtained from wells containing the human LDL but no HDL were normalized to 1.0. The values obtained from wells containing the human LDL with either human or mouse HDL were divided by the values obtained from wells with human LDL without added HDL to give the HDL-inflammatory index as previously described (Ansell et al. (2003) Circulation 108: 2751-2756). The data in FIG. 28 demonstrate that the 14C-L-4F used in the experiments described in FIG. 27 was biologically active.
Example 4
Niclosamide Plus L-4F Administered Orally (but not L-4F Alone) Reduces Lesions in Mouse Models of Atherosclerosis
In another experiment, seventeen week old female apoE null mice were divided into three groups: Group I received niclosamide 250 μg/mouse/day in rodent chow. Group II received L-4F at 25 μg/mouse/day in rodent chow. Group III received niclosamide at 250 μg/mouse/day together with L-4F 25 μg/mouse/day in rodent chow. All three groups received pravastatin 50 μg/mouse/day in drinking water. After 14 weeks the mice were sacrificed and aortic sinus lesion area was determined. As shown in FIGS. 29-31 oral administration of L-4F together with niclosamide but not without niclosamide significantly inhibits atherosclerosis in apoE Null mice.
In still another experiments, nine and half months-old female apoE null mice were divided into four groups: Group I was sacrificed to establish base line lesion area (Time Zero). Group II received niclosamide at 2 mg/mouse/day in rodent chow. Group III received L-4F at 200 μg/mouse/day in rodent chow. Group IV received niclosamide (Niclos.) at 2 mg/mouse/day together with L-4F 200 μg/mouse/day in rodent chow. Groups II-IV received pravastatin 50 μg/mouse/day in drinking water. After 26 weeks the mice were sacrificed and aortic sinus lesion area was determined. The data in FIGS. 32-34 demonstrate that the combination of L-4F plus niclosamide caused lesion regression in old apoE null mice. In contrast, neither niclosamide nor L-4F without niclosamide significantly reduced lesions.
L-4F forms a class A amphipathic helix. The sequence comprising residues 113-122 in apolipoprotein J (apoJ) comprises a potential G* helix. Administration of this peptide synthesized from all D-amino acids, D-[113-122]apoJ, dramatically improved HDL inflammatory properties and reduced atherosclerosis in apoE null mice (Navab et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25: 1932-1937).
To determine whether niclosamide could improve activity of the L-form of apoJ, ten month old apoE null mice (n=4 per group) were administered by stomach tube 2 mg of niclosamide or 200 μg of L-[113-122]apoJ or 2 mg of niclosamide together with 100 or 200 μg of L-[113-122]apoJ or were administered 2 mg of niclosamide together with 100 or 200 μg of L-4F. Eight hours later the mice were bled and the HDL inflammatory index was determined in cultures of human aortic endothelial cells as described in FIG. 8. As shown in FIG. 35 oral administration of the same peptide but synthesized from all L-amino acids and administered with niclosamide rendered apoE null mouse HDL anti-inflammatory to the same degree as normal human HDL, but when the peptide was administered orally without niclosamide this was not the case.
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

1. A method of mitigating one or more symptoms of atherosclerosis in a mammal, said method comprising:
orally administering to said mammal an effective amount of a “D” or “L” peptide and niclosamide, wherein said peptide ranges in length from about 18 to about 50 amino acids and comprises an amino acid sequence selected from the group consisting of peptide D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (4F, SEQ ID NO:5), F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D (retro 4F, SEQ ID NO: 104), D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F (6F, SEQ ID NO:7), and F-F-E-K-F-K-E-F-F-K-D-Y-F-A-K-L-W-D (retro 6F, SEQ ID NO:537).

2. The method of claim 1, wherein said niclosamide is administered before said peptide.

3. The method of claim 1, wherein said niclosamide is administered simultaneously with peptide.

4. The method of claim 1, wherein said niclosamide and said peptide are administered as a single formulation.

5. The method of claim 1, wherein said niclosamide and said peptide are combined to form an adduct prior to administration.

6. The method of claim 1, wherein all the amino acids of said peptide are “D” amino acids.

7. The method of claim 1, wherein all the amino acids of said peptide are “L” amino acids.

8. The method of claim 1, wherein said peptide comprises a protecting group at the amino or carboxyl terminus.

9. The method of claim 8, wherein said protecting group is a protecting group selected from the group consisting of acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-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), 1-(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), and Trifluoroacetyl (TFA).

10. The method of claim 8, wherein said peptide comprises a first protecting attached to the amino terminus and a second protecting group attached to the carboxyl terminus.

11. The method of claim 10, wherein said first protecting group is a protecting group selected from the group consisting of acetyl, propionyl, and a 3 to 20 carbon alkyl.

12. The method of claim 10, wherein said second protecting group is an amide.

13. The method of claim 1, wherein said niclosamide and said peptide are administered as a unit dosage formulation.

14. The method of claim 1, wherein said niclosamide and said peptide are administered as a unit dosage formulation formulated so that the niclosamide is released or solubilized before the peptide.

15. The method of claim 1, wherein said peptide has a formula selected from the group consisting of Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (4F, SEQ ID NO:5), Ac-F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D-NH2 (retro 4F, SEQ ID NO: 104), Ac-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2 (6F, SEQ ID NO:7), and Ac-F-F-E-K-F-K-E-F-F-K-D-Y-F-A-K-L-W-D-NH2 (retro 6F, SEQ ID NO:537).

16. The method of claim 1, wherein said peptide consists of all “L” amino acids and the amino acid sequence of said peptide consists of the sequence D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F (SEQ ID NO:7).

17. The method of claim 16, wherein said peptide has the formula: Ac-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH2 (6F, SEQ ID NO:7).

18. The method of claim 1, wherein said peptide consists of all “L” amino acids and the amino acid sequence of said peptide consists of the sequence F-F-E-K-F-K-E-F-F-K-D-Y-F-A-K-L-W-D (SEQ ID NO:537).

19. The method of claim 18, wherein said peptide has the formula Ac-F-F-E-K-F-K-E-F-F-K-D-Y-F-A-K-L-W-D-NH2(SEQ ID NO:537).

20. The method of claim 1, wherein said peptide consists of all “L” amino acids and the amino acid sequence of said peptide consists of the sequence D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO:5).

21. The method of claim 1, wherein said peptide has the formula Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO:5).

22. The method of claim 1, wherein said peptide consists of all “L” amino acids and the amino acid sequence of said peptide consists of the sequence F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D (SEQ ID NO: 104).

23. The method of claim 22, wherein said peptide has the formula Ac-F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D-NH2 (SEQ ID NO: 104).