Melanocortin receptor-specific cyclic peptides containing reverse amide bonds.

Abstract

Xaa 1 R1, R2, R3, R4, R7, R8, R9, R 10 The present invention relates to melanocortin receptor-specific cyclic peptides of the formula (I), wherein t, x, and y are as defined herein, compositions and formulations comprising peptides of said formula, and methods of preventing, ameliorating, or treating melanocortin receptor-mediated diseases, symptoms, conditions, and syndromes using melanocortin receptor-specific cyclic peptides of formula (I). [Formula 1] JPEG2023512067000033.jpg68170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 969,315, entitled "Reverse Amide-Linked Melanocortin Receptor-Specific Cyclic Peptides," filed February 3, 2020, the specification and claims of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Field of the invention (technical field): The present invention relates to head-to-tail reverse amide linked melanocortin receptor-specific cyclic peptides, including cyclic peptides that are agonists, partial agonists, antagonists, or mixed agonist-antagonists at melanocortin receptors, and the use of melanocortin receptor-specific reverse amide linked cyclic peptides in the treatment of melanocortin receptor-mediated diseases, indications, conditions, and syndromes.

[0003] Description of related art: Peptides are cyclized through the side chains of two amino acid residues, one proximal to the N-terminus and the other proximal to the C-terminus of the peptide sequence, typically through a disulfide bond (e.g., through the side chains of two Cys residues) or through an amide bond (e.g., through the side chains of two residues, one having a side chain containing a carboxyl group and one having a side chain containing an amine). Head-to-tail cyclized peptides are also known, such as peptides where an amide is formed by coupling an N-terminal group (e.g., amine) and a C-terminal group (e.g., carboxy), thereby forming an amide-linked cyclic peptide.

[0004] A family of melanocortin receptor types and subtypes has been identified, including melanocortin receptor 1 (MC1r), which is expressed in normal human melanocytes, melanoma cells, macrophages, and other cells; melanocortin receptor 2 (MC2r) for ACTH (adrenocorticotropin), which is expressed in cells of the adrenal gland; melanocortin receptor 3 and melanocortin receptor 4 (MC3r and MC4r), which are expressed in cells in the hypothalamus, midbrain, brainstem, and peripheral tissues; and melanocortin receptor 5 (MC5r), which is expressed in a widespread distribution in peripheral tissues. MC1r is thought to be involved in mediating inflammation, hair and skin pigmentation, and other functions; MC2r is thought to mediate steroid production; MC3r is thought to be involved in energy homeostasis, feeding behavior, mediating inflammation, and other functions; MC4r is thought to be involved in feeding behavior, energy homeostasis, sexual function, and other functions; and MC5r is thought to be involved in regulating the exocrine system and other functions.

[0005] Melanocortin receptor-specific compounds, including agonist and antagonist peptides, are known. For example, MC4r agonist peptides are believed to be useful in treating obesity or obesity-induced weight loss, and various forms of sexual dysfunction, including male erectile dysfunction and female sexual dysfunction. MC4r antagonist peptides are believed to have potential utility in treating conditions that result in weight gain, such as cachexia and other wasting syndromes and conditions.

[0006] Peptide analogs of the endogenous agonist alpha-melanocortin stimulating hormone (α-MSH) are known. These include both linear and cyclic peptides. Peptides specific for cyclic melanocortin receptors are typically cyclized via a side chain such as an amide or cysteine ​​bond, acylated at the N-terminus, and amidated at the C-terminus (endogenous α-MSH is acylated at the N-terminus and amidated at the C-terminus). However, as disclosed in U.S. Pat. No. 6,579,968, α-MSH analogs may also have a C-terminal carboxyl group. Summary of the Invention [Problem to be solved by the invention]

[0007] Despite the intense scientific and pharmaceutical interest in melanocortin receptor-specific peptides, evidenced by numerous articles in the scientific literature and numerous patent applications and issued patents, the only melanocortin receptor-specific peptide drugs approved in the United States are bremelanotide, sold under the trade name VYLEESI®, indicated for hypoactive sexual desire disorder in premenopausal women; afamelanotide, sold under the trade name SCENESSE®, indicated for the prevention of phototoxicity in adult patients with erythropoietic protoporphyria; and setomelanotide, sold under the trade name INCIVREE™, indicated for the treatment of obesity resulting from deficiencies of proopiomelanocortin (POMC), proprotein convertase subtilisin / kexin type 1 (PCSK1), or leptin receptor (LEPR). There remains a significant and substantial need for melanocortin receptor-specific peptides for pharmaceutical applications, particularly for use in the treatment of inflammation-related diseases, symptoms, conditions, and syndromes. It is against this background that the present invention was constructed. [Means for solving the problem]

[0008] Brief description of the invention In one aspect, the present invention provides a peptide of formula I: [ka] (In the formula: Xaa 1 is -R5-R6; R1 is substituted or unsubstituted indole, phenyl, or naphthyl; R2 is -(CH2) u - and; R3 is H or a C1-C9 linear or branched aliphatic chain, optionally containing one or more C=C double bonds; R4 is -H or -CH3; R5 is optionally present and, when present, is 1 to 3 L- or D-isomer amino acids, or combinations thereof, wherein any backbone nitrogen atom is optionally methylated; R6 is H or C1 to C 17 acyl groups, including optionally substituted linear or branched alkyl, cycloalkyl, alkylcycloalkyl, aryl, aralkyl, or heteroaryl; R7 is -H, -CH3 or -CH2-, and when it is -CH2-, it can be combined with R8 to form the general structure [ka] forming a ring of; R8 is -H, and when R8 forms a ring with R7, that is, R8 is -(CH2)3, -N(R 12a )(R 12b ), -NH(CH2) z -N(R 12a )(R 12b ), -C(=O)-N(R 12a )(R 12b ), -O-(R 12a ), -S-(=O)2-CH3, -S-(=O)-CH3, substituted or unsubstituted phenyl, -O-CH2-phenyl, where phenyl is substituted or unsubstituted. [ka] and; R9 is substituted or unsubstituted phenyl or naphthyl; R 10 teeth, -N(R 12a )(R 12b ), -NH(CH2) z -N(R12a )(R 12b ), -NH-C(=NH)-N(R 12a )(R 12b ), -NH-C(=O)-N(R 12a )(R 12b ), -O(R 12a ), -C1~C 17 linear, branched or cyclic alkyl chains, -S(=O)2-CH3, -S(=O)-CH3, -C(=O)-O(R 12a ), R 11 is -O-CH2-phenyl, where phenyl is substituted or unsubstituted [ka] and; R 12a and R 12b are each independently, and independently in each occurrence, H or a C1-C4 linear, branched or cyclic alkyl chain; y is 0 or 1, when it is 0 the bracket group is absent, and when it is 1 the bracket group is present; t is, independently in each occurrence, 1 to 4; x is 1 to 5; u is 1 to 8; and z is 1 to 3. including all enantiomers, stereoisomers or diastereoisomers thereof, or pharmaceutically acceptable salts of any of the foregoing.

[0009] In one embodiment, R9 is unsubstituted naphthyl. In another embodiment, any substituted phenyl or naphthyl present in the cyclic peptide of Formula (I) is substituted, independently in each occurrence, with between 1 and 3 ring substituents, where the substituents are the same or different and each independently, halo, (C1-C 10 ) Alkyl-halo, (C1-C10 ) alkyl, (C1-C 10 ) alkoxy, (C1-C 10 ) alkylthio, aryl, (C1-C 10 ) alkylaryl, aryloxy, nitro, nitrile, sulfonamido, amino, monosubstituted amino, disubstituted amino, hydroxy, carbamoyl, carboxy, carbamoyl, alkoxy-carbonyl, or aryloxy-carbonyl.

[0010] In one embodiment of the cyclic peptide of Formula (I), R5 comprises at least one L- or D-isomer amino acid. In another embodiment, R5 is a single L- or D-isomer amino acid having an aliphatic side chain, including where the aliphatic side chain is -(CH2)3-CH3. In another embodiment, R5 is a single L- or D-isomer amino acid having a side chain containing at least one nitrogen atom, including where R5 is an L- or D-isomer of Arg, Lys, Orn, Dab, Dap, or Cit.

[0011] The cyclic peptide of formula (I) has the formula: [ka] The cyclic peptides include:

[0012] In the cyclic peptide of formula (I), R7 and R8 are each a group: [ka] may be included together.

[0013] In the cyclic peptide of formula (I), R8 is C(=O)-N(R 12a )(R 12b )(wherein, R 12a and R 12b is H).

[0014] In the cyclic peptide of formula (I), R8 may be an imidazole ring.

[0015] In the cyclic peptides of formula (I), R5 may be absent, and in such cases, optionally, R6 is a C4-C 17 It may also be an acyl group.

[0016] In another aspect, the present invention provides a compound of formula (II): [ka] (In the formula, Z is H or an N-terminal group; Xaa 1 is optionally present and, if present, is 1 to 3 amino acids, wherein any backbone nitrogen atom is optionally methylated; Xaa 2 is Xaa 7 an L- or D-isomer of an amino acid having a side chain containing an amine group that forms an amide with the carboxyl group of Xaa 3 is an L- or D-isomer amino acid of Pro, optionally substituted with hydroxyl, halogen, sulfonamide, alkyl, O-alkyl, aryl, alkyl-aryl, alkyl-O-aryl, alkyl-O-alkyl-aryl, -O-alkyl-aryl, or -O-aryl, or Xaa 3 is an L- or D-isomer amino acid having at least one primary amine, secondary amine, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, ether, sulfide, or carboxyl-containing side chain; Xaa 4 is an L- or D-isomer amino acid having a substituted or unsubstituted aryl-containing side chain; Xaa 5 is an L- or D-isomer amino acid having a side chain containing at least one primary amine, secondary amine, guanidine, urea, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or ether; 6 If Xaa is not present, 7having a C-terminal carboxyl group that forms an amide bond with the amine group of Xaa 6 is optionally present and, if present, is an L- or D-isomer amino acid having a side chain containing at least one aryl or heteroaryl, optionally substituted with one or more ring substituents, where one or more, if present, are the same or different and independently hydroxyl, halogen, sulfonamide, alkyl, -O-alkyl, aryl, or -O-aryl; 7 having a C-terminal carboxyl group that forms an amide bond with the amine of Xaa 7 is an amino acid selected from glycine, β-alanine, γ-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, and 8-aminocaprylic acid) or a pharmaceutically acceptable salt thereof.

[0017] In the cyclic peptide of formula (II), Z is a C1-C alkyl group, including linear or branched alkyl, cycloalkyl, alkylcycloalkyl, aryl, or aralkyl. 17 The N-terminal group may be selected from the group consisting of acyl groups.

[0018] In the cyclic peptide of formula (II), Xaa 1 may be a single amino acid residue selected from the group consisting of Gly or Ala, Nle, Leu, Ile or Val, and the L- or D-isomers thereof. Alternatively, in the cyclic peptide of formula (II), Xaa 1 may be a single amino acid having a side chain containing at least one primary amine, guanidine, or urea group. Alternatively, in the cyclic peptide of formula (II), Xaa 1 may be the L- or D-isomer of Arg, Lys, Orn, Dab, Dap or Cit.

[0019] In the cyclic peptide of formula (II), Xaa 3may be D-Phe or Phe, optionally substituted with 1 to 3 ring substituents. The ring substituents may be the same or different and each independently include halo, (C1 to C 10 ) Alkyl-halo, (C1-C 10 ) alkyl, (C1-C 10 ) alkoxy, (C1-C 10 ) alkylthio, aryl, (C1-C 10 ) alkylaryl, aryloxy, nitro, nitrile, sulfonamido, amino, monosubstituted amino, disubstituted amino, hydroxy, carboxy, or alkoxy-carbonyl. Alternatively, in the cyclic peptide of formula (II), Xaa 3 may be D-Nal1 or D-Nal2.

[0020] In the cyclic peptide of formula (II), Xaa 5 may be the L- or D-isomer of Arg, Lys, Orn, Dab or Dap.

[0021] In the cyclic peptide of formula (II), Xaa 6 may be the L- or D-isomer of Trp, Na11, or Na12.

[0022] In one embodiment of the cyclic peptide of formula (II), Z is a C1-C7 linear alkyl acyl group; Xaa 1 is the L- or D-isomer of Nle or Arg; Xaa 2 is the L- or D-isomer of Dab, Dap, Orn, or Lys, where the side chain amine group is 7 forms an amide bond with the carboxyl of Xaa 3 is the L- or D-isomer of His, Hyp(Bzl), Met(O2), or Asn; Xaa 4 is a substituted or unsubstituted L- or D-isomer of Phe, Na1 or Na12; Xaa 5is the L- or D-isomer of Arg; and Xaa 6 is the L- or D-isomer of Trp, Na1, or Na12, in which the C-terminal carboxyl group is 7 It forms an amide bond with the amine of

[0023] The cyclic peptides of formula (II) further include embodiments as described above, wherein at least one backbone nitrogen atom thereof comprises a methyl group.

[0024] In one aspect, a cyclic peptide template is provided that may be utilized in the generation of receptor-specific peptides for biological receptors.

[0025] In another aspect, there are provided peptide-based pharmaceutical compositions specific for melanocortin receptors for use in the treatment of melanocortin receptor-mediated diseases, indications, conditions and syndromes.

[0026] In another aspect, peptide-based melanocortin receptor-specific pharmaceuticals are provided, wherein the peptides are selective and are agonists at MC1r and antagonists at MC4r.

[0027] In another aspect, peptide-based melanocortin receptor-specific pharmaceuticals are provided, wherein the peptides are selective and are agonists at MC1r and partial agonists at MC4r.

[0028] In another aspect, peptide-based melanocortin receptor-specific pharmaceuticals are provided, wherein the peptides are selective and agonists of MC4r.

[0029] In another embodiment, a sub-nanomolar EC 50 Receptor-specific peptides are provided that are functionally active at one or more melanocortin receptors.

[0030] In another embodiment, an EC60 of less than 1 nM50 Melanocortin receptor-specific peptides are provided that are agonists or partial agonists at one or more of MC1r, MC3r, and MC5r.

[0031] Other aspects and novel features of the present invention, and further scope of applicability, will be set forth in part in the detailed description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. Aspects of the present invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description of the Invention 1.0 Definition. Before proceeding with the description of the present invention, certain terms are defined as described herein.

[0033] In the sequences given in the peptides disclosed herein, the amino acid residues have their ordinary meanings as set forth in Chapter 2400 of the Manual of Patent Examining Procedure, 9th Ed. Thus, for example, "Ala" is alanine, "Asn" is asparagine, "Asp" is aspartic acid, "Arg" is arginine, "Cys" is cysteine, "Gly" is glycine, "Gln" is glutamine, "Glu" is glutamic acid, "His" is histidine, "Ile" is isoleucine, "Leu" is leucine, "Lys" is lysine, "Met" is methionine, "Phe" is phenylalanine, "Pro" is proline, "Ser" is serine, "Thr" is threonine, "Trp" is tryptophan, "Tyr" is tyrosine, and "Val" is valine. It should be understood that D-isomers are designated by the three-letter code or "D-" before the amino acid name, e.g., D-Phe is D-phenylalanine. Amino acid residues not encompassed by the foregoing include, but are not limited to, those having the following side chains, with the understanding that such amino acid residues may be either the L- or D-isomer:

[0034] [Table 1]

[0035] [Table 2]

[0036] [Table 3]

[0037] [Table 4]

[0038] The amino acid residues further include, but are not limited to, the following, and it is understood that such amino acid residues may be either the L- or D-isomer.

[0039] [Table 5]

[0040] The term "alpha amino acid" refers to the general structure (represented in its non-ionized form): [ka] where R is any side chain group or hydrogen, including but not limited to the amino acid residues or side chain groups set forth in the preceding tables and paragraphs.

[0041] The terms "L- or D-isomer amino acid" or "L- or D-isomer amino acids" include any isomeric form of any amino acid residue as defined herein, and specifically include any alpha, beta, gamma, or delta amino acid, including, but not limited to, amino acids directly encoded by DNA, post-translationally modified amino acids, amino acids expressed by biological means other than directly by DNA, proteinogenic or non-proteinogenic amino acids, or any synthetic or artificial amino acid.

[0042] Amino acids, including L- or D-isomer amino acids, are linked together by "amide bonds" or amide linkages to form covalent peptide bonds that link the backbone carboxylic acid group of one amino acid with the backbone amino group of another amino acid, thereby forming a peptide bond (-C(=O)-NH-) or backbone amide bond.

[0043] , The term "acyl" includes the group R(C=O)-, where R is an organic group such as alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl. When reference is made herein to a substituted acyl group, it means that the organic group (R) is substituted. Non-limiting examples of acyl groups include CH3-C(=O)-, referred to herein as an acetyl group or "Ac"; CH3-(CH2)4-C(=O)-, referred to herein as hexanoyl or "Hex"; CH3-(CH2)4-C(=O)-, referred to herein as heptanoyl or "Hept"; and various cyclyl groups such as phenylpropanoyl and cyclopentylacetyl.

[0044] A peptide or aliphatic moiety is "acylated" when an aliphatic or substituted aliphatic group, or an aromatic substituted aromatic group, is attached through a carbonyl {-(C=O)-} group to form an acyl group. Peptides are most commonly acylated at the N-terminus.

[0045] The term "alkane" includes linear or branched saturated hydrocarbons. Examples of linear alkane groups include methane, ethane, propane, etc. Examples of branched or substituted alkane groups include methylbutane or dimethylbutane, methylpentane, dimethylpentane, or trimethylpentane, etc. In general, any alkyl group may be a substituent of an alkane.

[0046] The term "alkene" includes unsaturated hydrocarbons with one or more double carbon-carbon bonds. Examples of such alkene groups include ethylene, propene, and the like.

[0047] The term "alkenyl" includes linear monovalent hydrocarbon radicals of two to six carbon atoms or branched monovalent hydrocarbon radicals of three to six carbon atoms having at least one double bond; examples thereof include ethenyl, 2-propenyl, and the like.

[0048] The "alkyl" groups specified herein include alkyl radicals of the designated length that are either straight- or branched-chain saturated aliphatic hydrocarbon groups. 1~10 Alkyl means an alkyl having 1 to 10 carbon atoms. Non-limiting examples of such alkyl radicals include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl, and the like.

[0049] The term "alkyne" includes a linear monovalent hydrocarbon radical of two to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbon atoms having at least one triple bond; examples thereof include ethyne, propyne, butyne, and the like.

[0050] The term "aryl" includes a monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 12 ring atoms, optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cycloalkyl, alkoxy, alkythio, halo, nitro, acyl, cyano, amino, monosubstituted amino, disubstituted amino, hydroxy, carboxy, or alkoxy-carbonyl. Examples of aryl groups include phenyl, biphenyl, naphthalene, naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like. Similarly, the term "naphthyl" includes 1-naphthyl and 2-naphthyl, and "naphthalene" includes 1-naphthalene and 2-naphthalene.

[0051] The term "aralkyl" refers to the radical -R a R b (In the formula, R a is an alkylene (divalent alkyl) group, and R b is an aryl group as defined above. Examples of aralkyl groups include benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the like.

[0052] The term "aliphatic" includes compounds having hydrocarbon chains such as, for example, alkyls, aryls, heteroaryls, alkanes, alkenes, alkynes, and derivatives thereof.

[0053] As used herein, the term "amide" refers to a trivalent nitrogen bonded to a carbonyl group, i.e., -C(=O)-NH2 (i.e., primary amide), -C(=O)-NHR c and -C(=O)-NR c R d (In the formula, R c and R d where each independently represents hydrogen or an organic group. When reference is made to a substituted amide group, it is understood that the reference is to a substituted amide group. c and R d ) is substituted. Examples of amide include methylamide, ethylamide, and propylamide.

[0054] "Amine" refers to the amino group (-NH2), -NHR a and -NR a R b (In the formula, R a and R b each independently represents hydrogen or an organic group). When reference is made to a substituted amine group, it is a and R b ) is substituted.

[0055] "Nitrile" is a carboxylic acid derivative and includes compounds having a (-CN) group attached to an organic group.

[0056] The term "halogen" is intended to include the halogen atoms fluorine, chlorine, bromine, and iodine, as well as groups containing one or more halogen atoms, such as, for example, --CF.sub.3.

[0057] The term "composition," such as in the case of a pharmaceutical composition, is intended to encompass a product comprising an active ingredient and an inactive ingredient that forms a carrier, as well as any product that results directly or indirectly from the combination, complexation, or aggregation of any two or more of the ingredients, or from the dissociation of one or more of the ingredients, or from any other type of reaction or interaction of one or more of the ingredients. Thus, a pharmaceutical composition encompasses any composition made by mixing an active ingredient with one or more pharmaceutically acceptable carriers.

[0058] A melanocortin receptor "agonist" refers to an endogenous substance, drug substance, or compound, including certain peptide compounds disclosed herein, that can interact with a melanocortin receptor and initiate a pharmacological response, including activation of the receptor, including initiating signal transduction characteristic of the melanocortin receptor, such as adenyl cyclase activation. A melanocortin receptor agonist may be an agonist at one or more of MC1r, MC2r, MC3r, MC4r, and MC5r.

[0059] A melanocortin receptor "antagonist" refers to an endogenous substance, drug substance, or compound, including certain peptide compounds disclosed herein, that blocks or attenuates the action of an agonist at a melanocortin receptor. A melanocortin receptor antagonist may be an antagonist at one or more of MC1r, MC2r, MC3r, MC4r, and MC5r. Certain compounds, including certain peptide compounds disclosed herein, may be agonists at one or more melanocortin receptors and antagonists at one or more other melanocortin receptors.

[0060] "α-MSH" means the peptide Ac-Ser-Tyr-Ser-Met-Glu-His-Pro-Arg-Glu-Lys-Pro-Val-NH and analogs and homologs thereof, including but not limited to NDP-α-MSH.

[0061] "NDP-α-MSH" means the peptide Ac-Tyr-Ser-Tyr-Ser-Nle-Glu-His-DlE-Pro-Arg-Glu-Lys-Pro-Val-NH and analogs and homologs thereof.

[0062] "EC 50 " means the molar concentration of an agonist, including partial agonists, that produced 50% of the maximum possible response for that agonist. By way of example, a test compound that, at a concentration of 72 nM, produces 50% of the maximum possible response for that compound, as determined in a cAMP assay in an MC4r cell expression system, has an EC 50 Unless otherwise specified, EC 50 The molar concentrations relevant to the determination of are in nanomoles (nM) per liter.

[0063] "Ki (nM)" refers to the equilibrium inhibitor dissociation constant, which represents the molar concentration of a competitor compound that binds to half of the receptor's binding sites at equilibrium in the absence of a competitor. Generally, the value of Ki is inversely related to the affinity of a compound for a receptor, and the lower the Ki, the higher the affinity. Ki is calculated according to the formula of Cheng and Prusoff (Cheng Y., Prusoff WH, Biochem.Pharmacol.22:3099-3108,1973):

number

[0064] "Inhibition" refers to the percent attenuation or reduction in receptor binding in a competitive inhibition assay compared to a known standard. Thus, "inhibition at 1 μM (NDP-α-MSH)" refers to the percent reduction in NDP-α-MSH binding upon addition of a determined amount of test compound, e.g., 1 μM test compound, under assay conditions such as those described below. For example, a test compound that does not inhibit NDP-α-MSH binding has 0% inhibition, and a test compound that completely inhibits NDP-α-MSH binding has 100% inhibition. Typically, for competitive inhibition studies, e.g., as described below, a test compound is added to the receptor, e.g., 1 μM. 125 Detectable label assays using labeled NDP-α-MSH or lanthanide chelate fluorescence assays using, for example, Eu-NDP-α-MSH are used. However, other methods for testing competitive inhibition, including the use of different labeling or tagging systems, are known, and generally, any method known in the art for testing competitive inhibition may be used in the present invention. Therefore, it may be recognized that "inhibition" is one measure for determining whether a test compound attenuates the binding of α-MSH to the melanocortin receptor.

[0065] "Binding affinity" means the ability of a compound or agent to bind to its biological target, expressed herein as Ki (nM).

[0066] "E max " refers to the maximum functional activity achievable by a compound in a cell line expressing a particular melanocortin receptor, such as maximal stimulation of adenylyl cyclase. The maximal stimulation achieved by NDP-α-MSH is 100% E max A compound capable of stimulating half of the maximal activity of NDP-α-MSH is designated as 50% E max Under the assay conditions described herein, an E of 70% or greater is designated. max Compounds of the invention having an E of between 10% and 70% may be classified as agonists. max Compounds having an E of less than 10% may be classified as partial agonists. maxmay be classified as inactive.

[0067] Generally, "functional activity," such as in the case of melanocortin receptors, is a measure of receptor signaling or a measure of changes in receptor-associated signaling upon receptor activation by a compound. Melanocortin receptors initiate signaling through the activation of heterotrimeric G proteins. In one embodiment, melanocortin receptors signal through Gα, which catalyzes the production of cAMP by adenylyl cyclase. Thus, measuring stimulation of adenylyl cyclase, such as measuring maximal stimulation of adenylyl cyclase, is one measure of functional activity and is the primary measure exemplified herein. However, it should be understood that alternative measures of functional activity may be utilized in the practice of the present invention and are specifically contemplated and within the scope of the present invention. Thus, in one example, intracellular free calcium may be measured using specific fluorescent molecules that bind calcium, such as Fura2, as described by or using the methods disclosed in Mountjoy KGet et al., Melanocortin receptor-medicated mobilization of intracellular free calcium in HEK293 cells. Physiol Genomics 5:11-19, 2001, or Newman et al., Activation of the melanocortin-4 receptor mobilizes intracellular free calcium in immortalized hypothalamic neurons. J Surg Res:132:201-207, 2006.Fluo-4 is a more commonly used alternative calcium-binding dye (Nohr et al., The orphan G protein-coupled receptor GPR139 is activated by the peptides: adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW. Neurochem Int 102:105-113, 2017). Additionally, commercially available HTRF assays, such as Fluo-4, can measure Ca2+. +Activation can also be measured by measuring the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-biphosphate, within the same pathway, upstream of the release event (Liu et al., Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophosphate-1 and intracellular calcium in a 1536-well plate format. Curr Chem Genomics 1:70-77, 2008). Yet another measure of functional activity is receptor internalization resulting from activation of a regulatory pathway, such as that described in Nickoll's SA et al., Functional selectivity of melanocortin 4 receptor peptide and nonpeptide agonists: evidence for ligand-specific conformational states. J Pharm Exper Therapeutics 313:1281-1288, 2005. Yet another measure of functional activity is the exchange and rate of nucleotides associated with G protein receptor activation, such as the exchange of GDP (guanosine diphosphate) for GTP (guanosine triphosphatase) on G protein α subunits, which are guanosine 5'-(γ-[ 35The activity of Gα may be measured by several means, including radiometric assays using [S]thio)-triphosphate. A relatively new assay platform has been devised to measure the activity / association of 14 different Gα species belonging to the Gi, Gq, Gs, and Gi2 / i3 subfamilies as they relate to receptors using a BRET (bioluminescence resonance energy transfer)-based biosensor to measure the disengagement of Gα and Gγ subunits upon ligand binding (Zhao et al., Biased signaling of protease-activated receptors. Front Endocrinol 5:67, 2014; and van der Westhuizen et al., Quantification of ligand bias for clinically relevant β2-adrenergic receptor ligands: Implications for drug taxonomy. Molecular Pharm 85:492-509, 2014). To measure the activation of G-coupled proteins, various gene-based assays have been developed, such as those disclosed in Chen W. et al., A colorimetric assay from measuring activation of Gs- and Gq-coupled signaling pathways. Anal Biochem 226:349-354, 1995; Kent TC et al., Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors. Biomol Screening 5:437-446, 2005; or Kotarsky K. et al., Improved receptor gene assays used to identify ligands acting on orphan seven-transmembrane receptors. Pharmacology & Toxicology 93:249-258, 2003.As disclosed in Hruby VJ et al., "Cyclic lactam α-melanocortin analogues of Ac-Nle4-cyclo[Asp5,D-Phe7,Lys10] α-melanocyte-stimulating hormone-(4-10)-NH2 with bulky aromatic amino acids at position 7 shows high antagonist potency and selectivity at specific melanocortin receptors," J Med Chem 38:3454-3461, 1995, the colorimetric assay of Chen et al. has been adapted for use in measuring melanocortin receptor activation. In general, functional activity may be measured by any method, including methods that measure G-coupled receptor activation and / or signaling, including further methods that may be later developed or reported. Each of the foregoing articles, and the methods disclosed therein, is incorporated herein by reference as if fully set forth.

[0068] The terms "treat," "treating," and "treatment," as used herein, contemplate the action of reducing the severity of a particular disease or disorder while the patient is suffering from the disease or disorder.

[0069] As used herein, the term "pharmacologically effective amount" (including "therapeutically effective amount") means an amount of a peptide according to the present invention that is sufficient to induce a desired therapeutic or biological effect.

[0070] As used herein, the term "therapeutically effective amount" means an amount of a compound, including a peptide of the present invention, that will elicit a biological or medical response in a mammal being treated by a physician or other clinician.

[0071] As used herein, the term "prophylactically effective" or "prophylactic" refers to an amount of a compound, including a peptide of the present invention, that will prevent or inhibit or alleviate suffering in a mammal having a medical condition that a physician or other clinician seeks to prevent, inhibit, or alleviate before the patient begins to suffer from a particular disease or disorder.

[0072] 2.0 Clinical Indications and Usefulness The compositions and methods disclosed herein can be used in both medical and animal husbandry or veterinary applications. The term "patient" is intended to refer to a human and is used as such throughout the specification and in the claims. The primary application of the peptides disclosed herein, or of the formulas disclosed herein, involves human patients, although the peptides disclosed herein, or of the formulas disclosed herein, may also be applied to laboratory, farm, zoo, wildlife, pet, sport, or other animals. Clinical applications and specific utilities include the following:

[0073] 2.1 Inflammatory and fibrotic diseases and conditions The peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists or partial agonists, or any combination thereof, may be used in the treatment of inflammatory diseases and conditions in patients. There are several inflammatory diseases and conditions that may be so treated. In one embodiment, the inflammatory condition is caused by diseases including, but not limited to, forms of arthritis, including osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudogout, juvenile idiopathic arthritis, Still's disease and ankylosing spondylitis, and arthritis secondary to other diseases such as arthritis secondary to lupus erythematosus, Henoch-Schönlein purpura, psoriatic arthritis, reactive arthritis, hemochromatosis, hepatitis, Wegener's granulomatosis, vasculitis syndromes, Lyme disease, familial Mediterranean fever, hyperglobulin Demia with relapsing fever, TNF receptor-associated periodic syndromes, and inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. In another embodiment, the inflammatory condition results from a disease including forms of inflammatory bowel disease such as Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, fecal diversion colitis, Behcet's syndrome, infectious colitis, and atypical colitis. In another embodiment, the inflammatory condition results from an autoimmune disease, including, but not limited to, systemic syndromes such as systemic lupus erythematosus, Sjogren's syndrome, scleroderma, rheumatoid arthritis, and polymyositis, or syndromes affecting only a localized body system such as the endocrine system (type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, etc.), dermatological system (pemphigus vulgaris), hematological system (autoimmune hemolytic anemia), or nervous system (multiple sclerosis).Autoimmune diseases therefore include, in addition to the common syndromes discussed above, diseases and conditions such as acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, gestational pemphigoid, Good Pasteur's disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, Kawasaki disease, lupus erythematosus, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, primary biliary cirrhosis, Reiter's syndrome, Sjögren's syndrome, Takayasu's arteritis, temporal arteritis, autoimmune hemolytic anemia, and Wegener's granulomatosis.

[0074] In another aspect, the inflammatory condition is caused by or associated with chronic obstructive pulmonary disease (COPD), also known as chronic obstructive airway disease, including, but not limited to, diseases characterized by pathological airflow restriction in the airways that is not fully reversible, such as chronic bronchitis, emphysema, pneumoconiosis, lung neoplasms, and other lung disorders. Other inflammatory conditions include diseases and disorders of the upper or lower respiratory tract, such as allergic asthma, non-allergic asthma, allergic rhinitis, vasomotor rhinitis, allergic conjunctivitis, and non-allergic conjunctivitis, as well as airway diseases associated with external toxins or substances, such as various forms of pneumoconiosis (coal workers' pneumoconiosis, asbestosis, silicosis, bauxite fibrosis, beryllium lung disease, or siderosis), assinosis, or hypersensitivity interstitial pneumonia (farmer's lung or bird breeder's disease). Other lung diseases involving inflammatory conditions include acute respiratory distress syndrome. The peptides and compositions of the present invention are particularly useful in treating conditions in which glucocorticoids are either ineffective or inadequate to produce the desired pharmacological response, such as COPD, asthma in smokers, and other conditions characterized, in whole or in part, by eosinophil accumulation in the lung, neutrophil infiltration and activation, alveolar macrophage recruitment and activation, epithelial cell expression of IL-8, or increased expression of TNF-α. In airway or lung disorders, in one embodiment, the peptides of the present invention are delivered systemically; in another embodiment, the peptides of the present invention are delivered locally, such as by inhalation administration.

[0075] In yet another aspect, the inflammatory condition is caused by or associated with some form of transplant-related pathology or syndrome, such as graft-versus-host disease, hyperacute rejection, acute rejection, or chronic rejection. Graft-versus-host disease is a common complication of allogeneic bone marrow transplantation, but can also occur in other transplants, particularly in the case of T cells present in the graft either as contaminants or intentionally introduced. Hyperacute, acute, or chronic rejection can occur in transplants of bodily organs such as kidney, liver, pancreas, spleen, uterus, heart, or lung, as well as bone, cornea, face, hand, penis, or skin. In one embodiment, a pharmaceutical composition comprising one or more peptides of the present invention is administered prophylactically, such as immediately before, during, or after transplantation of a body fluid, organ, or part, to limit or prevent a transplant-related pathology or syndrome. In another embodiment, the transplanted body fluid, organ, or part is perfused with a solution of a pharmaceutical composition comprising one or more peptides of the present invention. In yet another embodiment, one or more of the peptides of the invention are administered in conjunction with, in combination with, or sequentially with one or more other agents against transplant rejection, such as calcineurin inhibitors including cyclosporine or tacrolimus, mTOR inhibitors including sirolimus or everolimus, antiproliferative agents including azathioprine or mycophenolic acid, corticosteroids including prednisolone or hydrocortisone, antibodies such as monoclonal anti-IL-2Rα receptor antibodies, basiliximab or daclizumab, or polyclonal anti-T cell antibodies such as antithymocyte globulin or antilymphocyte globulin.

[0076] In yet another aspect, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists or partial agonists, or any combination thereof, may be used in the treatment of fibrotic and sclerosing diseases, signs, conditions, and syndromes in patients. There are several fibrotic and sclerosing diseases, signs, conditions, and syndromes that may be so treated. Many fibrotic and sclerosing diseases, signs, conditions, and syndromes include an inflammatory component, and therefore many may also be classified as inflammatory diseases or conditions, as listed above. In addition to including an inflammatory component, fibrotic and sclerosing diseases and conditions may also be idiopathic, toxic, genetic, and / or pharmacologically induced disorders. Generally, fibrotic disorders are characterized by excessive production of extracellular matrix, primarily type I collagen, which can lead to impaired organ function. Without wishing to be bound by theory, it is believed that agonism of MC1r results in the suppression of transforming growth factor-β1-induced collagen synthesis by human skin fibroblasts, thereby providing therapeutic and / or prophylactic benefit against fibrotic and sclerosing diseases, signs, conditions, and syndromes. Exemplary fibrotic and sclerosing diseases and conditions that may be so treated include, but are not limited to, localized sclerosis, systemic sclerosis, cutaneous graft-versus-host disease, idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, cyclosporine-induced nephropathy, cirrhosis of the liver, hypertrophic scars, keloids, and the like.

[0077] In yet another embodiment, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, particularly MC1r and MC5r agonists, may be used in the treatment of fibrotic diseases, conditions, and syndromes in patients. Such fibrotic processes can be secondary to chronic inflammation, the development of fibrosis being a common consequence of chronic inflammation. Diseases in which fibrosis contributes to mortality and morbidity are widespread, including pulmonary fibrosis, liver fibrosis and cirrhosis, chronic kidney disease, myocardial infarction, and systemic autoimmune diseases such as systemic sclerosis. Fibrosis can also occur in ocular diseases, particularly those characterized by chronic inflammation. It is believed that the peptides and compositions of the present invention can inhibit the formation of fibrosis and possess regenerative properties that reduce or ameliorate the effects of fibrosis.

[0078] In yet another aspect, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, may be used in the treatment of diseases associated with increased cytokine expression in patients and related diseases, symptoms, conditions, and syndromes. Expression of various cytokines is increased during inflammatory processes, including those secondary to circulatory shock, ischemia, reperfusion injury, and the like. TNF-α is a pleiotropic cytokine produced primarily by macrophages, but also by other types of cells. Other cytokines that are increased during inflammatory processes, including those secondary to circulatory shock, ischemia, reperfusion injury, and the like, include IL-1 and IL-6. While cytokines such as TNF-α often have beneficial effects, significantly increased levels, for example, secondary to circulatory shock, ischemia, reperfusion injury, and the like, can have pathological effects. In one aspect, reperfusion of hypoxic or ischemic tissue, for example, secondary to circulatory shock, results in an inflammatory response, including increased cytokine expression.

[0079] In one embodiment, the present invention is directed to methods of using one or more of the peptides of the present invention to reduce the production and expression of pro-inflammatory cytokines, including reducing the production and expression of pro-inflammatory cytokines secondary to circulatory shock, ischemia, reperfusion injury, etc. The reduction in the production and expression of pro-inflammatory cytokines, including but not limited to one or more of TNF-α, IL-1, and IL-6, preferably occurs within a short period of time following administration of a composition comprising one or more of the peptides of the present invention.

[0080] In related embodiments, the present invention is directed to methods of using one or more of the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, to increase the production and expression of anti-inflammatory cytokines, including but not limited to IL-10, within a short period of time following administration of a composition comprising one or more of the peptides of the present invention.

[0081] 2.2 Skin signs In yet another embodiment, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r agonists, partial agonists, or antagonists, or combinations thereof, may be used in the treatment of dermatological and cosmetic diseases, symptoms, conditions, and syndromes. In one embodiment, the peptides and compositions of the present invention are MC1r agonists that stimulate melanocytes and related cells, increasing the level of melanin in the skin. Increasing the level of melanin in the skin provides protection against ultraviolet radiation (UVR) and sunlight, for example, protection against cutaneous phototoxicity and photosensitivity caused by UVR, the sun, and light.

[0082] In yet another aspect, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, may be utilized in the prophylactic and / or therapeutic treatment of skin diseases, signs, conditions and syndromes, such as acne vulgaris (commonly referred to as acne), atopic dermatitis (commonly referred to as atopic eczema or eczema), polymorphous light eruption, psoriasis, rosacea, seborrheic dermatitis, vitiligo, porphyria, porphyria cutanea tarda, erythropoietic protoporphyria, solar urticaria, urticaria pigmentosa or xeroderma pigmentosum. In another aspect, the peptides, compositions, and methods of the present invention may be used to prevent, limit, or treat photosensitive or photoresponsive viral infections, such as herpes simplex virus (commonly referred to as cold sores and genital herpes, depending on the site of infection), human papilloma virus, and varicella zoster virus. In another aspect, the peptides, compositions, and methods of the present invention may be used to prevent, limit, or treat skin cancer, including precancerous conditions, actinic keratosis, basal cell carcinoma, melanoma, or squamous cell carcinoma. In another aspect, the peptides, compositions, and methods of the present invention may be used to prevent or limit the adverse effects of various treatments, including phototherapy, such as photodynamic therapy. In yet another aspect, the peptides, compositions, and methods of the present invention may be used to induce sunburn, reduce hair graying, or for similar related purposes associated with increased melanin production.

[0083] 2.3 Cancer. Certain cancers, such as mesothelioma, have been reported to be highly sensitive to the growth-promoting effects of cytokines and growth factors and may be treatable using peptides selective for MC1r. Canania, A., et al., "Autocrine inhibitory influences of α-melanocyte-stimulating hormone in malignant pleural mesothelioma," J. Leukoc. Biol. 75:253-259 (2004). Cancers that may be so treated include pleural mesothelioma, which is known to express mRHA and receptor proteins in MC1r, as well as other tumors that express MC1r, including, but not limited to, adenocarcinomas such as lung adenocarcinoma.

[0084] 2.4 Eye Diseases and Symptoms There are several ocular diseases, signs, conditions, and syndromes characterized by inflammation, including, but not limited to, increased cytokine production, that may be treated with the peptides and compositions of the present invention, including, but not limited to, peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof. One example is dry eye disease, also known as dry eye syndrome or keratoconjunctivitis sicca, an ocular disease that affects approximately 10-20% of the population. This disease affects an increasingly larger percentage of the population with age, and the majority of these patients are women. Furthermore, ocular irritation or symptoms and / or signs of dry eye disease are sometimes experienced under certain circumstances, such as prolonged visual challenges (e.g., working on a computer), dry environments, or the use of medications that cause dry eyes. In individuals with dry eye, the protective layer of tears that normally protects the ocular surface is impaired as a result of insufficient or unhealthy production of one or more tear components. This can cause exposure of the ocular surface, ultimately promoting the drying and damage of surface cells.The signs and symptoms of dry eye include, but are not limited to, keratitis, conjunctival and corneal staining, redness, blurred vision, decreased tear film breakup time, decreased tear production, tear volume and tear flow, increased conjunctival redness, excessive debris in the tear film, dry eyes, eye grittiness, eye stinging, intraocular foreign body sensation, excessive tearing, photophobia, stinging eye pain, refractive error, eye sensitivity and eye irritation.Patients may experience one or more of these symptoms.

[0085] There are many variables that may affect a patient's signs or symptoms of dry eye, including circulating hormone levels, various autoimmune diseases (e.g., Sjögren's syndrome and systemic lupus erythematosus), eye surgery including PRK or LASIK, numerous medications, environmental conditions, visual tasks such as computer use, eye strain, contact lens wear, and mechanical influences such as corneal sensitivity, partial eyelid closure, surface irregularities (e.g., pterygium), and eyelid irregularities (e.g., ptosis, entropion / ectropion, pinguecula). Dehydrating environments, such as low-humidity environments, sitting in a car with a defroster, or living in arid climates, can cause or worsen dry eye symptoms. Furthermore, visual tasks can worsen symptoms. Tasks that can significantly affect symptoms include watching television or using a computer for extended periods of time when blink rate is reduced.

[0086] Uveitis is an ocular disease involving inflammation of the middle layer, or uvea, of the eye, and may be understood to include any inflammatory process involving the interior of the eye. Uveitis includes anterior, intermediate, posterior, and panuveitic forms, including inflammation of the iris and anterior chamber (most uveitis cases are located in the anterior segment). This condition may occur as a single episode that can subside with appropriate treatment, or it may become recurrent or chronic. Symptoms include red eyes, injected conjunctiva, pain, and decreased vision. Signs include dilated ciliary vessels, the presence of cells and redness in the anterior chamber, and corneal deposits on the posterior surface of the cornea. Intermediate uveitis involves inflammation and the presence of inflammatory cells in the vitreous cavity, while posterior uveitis involves inflammation of the retina and choroid. Uveitis can be secondary to any of several diseases and disorders, including acute posterior multifocal patchy pigment epitheliopathy, ankylosing spondylitis, Behçet's disease, shotcretal chorioretinopathy, brucellosis, herpes simplex, herpes zoster, inflammatory bowel disease, juvenile rheumatoid arthritis, Kawasaki disease, leptospirosis, Lyme disease, multiple sclerosis, psoriatic arthritis, Reiter's syndrome, sarcoidosis, syphilis, systemic lupus erythematosus, toxocariasis, toxoplasmosis, tuberculosis, Vogt-Koyanagi-Harada syndrome, Whipple's disease, or polyarteritis nodosa.

[0087] Other intraocular inflammatory conditions that one or more of the peptides of the invention may be used to treat include, but are not limited to, corneal ulcers, corneal erosions, corneal abrasions, corneal degeneration, corneal perforations, corneal scars, epithelial defects, keratoconjunctivitis, idiopathic uveitis, corneal transplants, age-related macular degeneration, diabetic eye, blepharitis, glaucoma, ocular hypertension, postoperative eye pain and inflammation, posterior segment neovascularization, proliferative vitreoretinopathy, cytomegalovirus, and cytomegalovirus. These include: Irvine retinitis, endophthalmitis, choroidal neovascular membrane, vascular occlusive disease, allergic eye disease, tumors, retinitis pigmentosa, eye infection, scleritis, ptosis, miosis, eye pain, mydriasis, neuralgia, cicatricial ocular surface disease, eye infection, inflammatory eye disease, ocular surface disease, corneal disease, retinal disease, ocular manifestations of systemic disease, inherited eye conditions, eye tumors, increased intraocular pressure, herpes infection, pterygium, wound sustained to ocular surface, eye pain and inflammation after photorefractive keratectomy, thermal or chemical burns to the cornea, scleral wounds, keratoconus, or conjunctival wounds.

[0088] In one embodiment, the present invention is directed to a method of using one or more of the peptides of the present invention for the treatment of any of the aforementioned ocular diseases, signs, conditions, and syndromes. Such treatment may include treatment with eye drops, ointments, gels, rinses, implants, plugs, or other means and methods for delivering one or more of the peptides of the present invention to the ocular surface, or alternatively, treatment by intravitreal injection or similar means that results in delivery to the vitreous humor, or alternatively, treatment by systemic administration, including oral, subcutaneous, or intravenous injection, to a patient who responds thereto.

[0089] 2.5 Ischemia and related symptoms In yet another embodiment, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, may be used in the treatment of ischemia and related diseases, symptoms, conditions, and syndromes. Ischemia includes any reduction or cessation in blood supply to any bodily organ, tissue, cell, or part, particularly when the reduction or cessation causes or increases the likelihood of causing ischemic damage to the bodily organ, tissue, cell, or part. An "ischemic episode" refers to any transient or sustained period of ischemia. Ischemia can result from any narrowing or occlusion of the vasculature or from circulatory shock, such as hemorrhagic shock or hypovolemic shock. Reduced or absent blood flow results in reduced or absent oxygen to the affected part of the body and can also result in an increase in inflammatory disease mediators, such as various cytokines and other substances. During certain surgical procedures, such as cardiac surgery and organ transplants, blood flow is temporarily stopped and then resumed (reperfusion), resulting in ischemia-reperfusion injury. During a heart attack, blood supply to the heart is stopped, resulting in ischemia that can further develop into infarction. Current treatments for alleviating heart attacks involve reperfusion of the ischemic area of ​​the heart, for example, by using thrombolytic drugs or coronary angioplasty.

[0090] The peptides and compositions of the present invention have particular utility in preventing damage resulting from renal ischemia, including lung damage secondary to renal ischemia, preventing or limiting ischemic cardiac damage secondary to myocardial infarction, and preventing or limiting ischemic brain damage secondary to cardiovascular injury, including, but not limited to, myocardial infarction, stroke, etc. Neuroprotection is provided by administration of the compositions of the present invention to patients with cerebral ischemia or stroke, particularly those who are concomitantly hypotensive. The peptides and compositions of the present invention have further particular utility in preventing or limiting ischemic organ damage in organ transplantation, including heart, kidney, liver, lung, pancreas, or small intestine transplantation. In one aspect, the pharmaceutical compositions of the present invention may be used for perfusion of transplanted organs, where perfusion may be before, during, or after organ transplantation.

[0091] In one embodiment, the present invention is directed to a method of using one or more of the peptides of the present invention to protect the heart, brain, or other organs of a patient against damage caused by ischemia, wherein the protective effect against ischemia occurs immediately or within a short period of time after administration of a composition comprising one or more of the peptides of the present invention.

[0092] Ischemia can result from any of a variety of diseases or conditions, and in one embodiment, the invention is directed to a method of using one or more peptides of the invention to protect a patient's organs against damage due to ischemia caused by a disease or condition. Such diseases or conditions may include, for example, but are not limited to, atherosclerotic diseases such as atheroma, as well as thrombosis, embolism from blood vessels from the heart or any organ, vasospasm, hypotension due to cardiac disease, hypotension due to systemic disease including infection or allergic reaction, or hypotension due to administration, ingestion, or other exposure to one or more toxic compounds or drugs. Ischemia can also be secondary ischemia, and in another embodiment, the invention is directed to a method of using one or more peptides of the invention to protect a patient's organs against damage due to secondary ischemia. Such secondary ischemia may be secondary to diseases or conditions such as diabetes, hyperlipidemia, hyperlipoproteinemia, dyslipidemia, Buerger's disease, also known as thromboangiitis obliterans, Takayasu's arteritis, temporal arteritis, Kawasaki disease, also known as lymph node syndrome, mucocutaneous node disease, infantile polyarteritis, cardiovascular syphilis, and various connective tissue diseases and disorders.

[0093] In yet another embodiment, peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, may be used in the treatment of ischemia-reperfusion injury and related diseases, symptoms, conditions, and syndromes. While restoration of blood flow after ischemia is essential to preserve functional tissue, reperfusion itself is known to be harmful to tissue. Both ischemia and reperfusion are known to be important contributors to tissue necrosis. Several mechanisms appear to play a causative role in the generation of tissue damage associated with ischemia-reperfusion injury. Certain peptides and compositions of the present invention have particular utility in preventing or limiting the severity of renal reperfusion injury, including lung injury secondary to renal reperfusion, including myocardial infarction, stroke, etc.; preventing or limiting reperfusion cardiac injury secondary to myocardial infarction; and preventing or limiting reperfusion brain injury secondary to cardiovascular injury. The present invention has further particular application in preventing or limiting reperfusion organ injury in organ transplantation, including heart, kidney, liver, lung, pancreas or small intestine transplantation. In one aspect, the pharmaceutical compositions of the present invention may be used in the perfusion of a transplanted organ, wherein perfusion may be before, during or after transplantation of the organ.

[0094] In one embodiment, the present invention is directed to a method of using one or more of the peptides of the present invention to protect a patient's heart, brain, or other organ against damage caused by ischemia-reperfusion injury, including damage caused by or during reperfusion. The protective effect against ischemia-reperfusion injury occurs immediately or within a short period of time after administration of a composition comprising one or more of the peptides of the present invention.

[0095] In yet another aspect, peptides and compositions of the present invention, including but not limited to peptides that are MC1r, MC3r, MC4r, and / or MC5r agonists, partial agonists, antagonists, or any combination thereof, may be used in the treatment of circulatory shock and related diseases, symptoms, conditions, and syndromes in patients. The present invention provides peptides for use, compositions, and methods for treating or preventing shock, including hemorrhagic shock, in patients, comprising administering a composition comprising one or more of the peptides of the present invention to a patient diagnosed with blood loss. Blood loss may, but need not, be measured as a percentage of the subject's blood volume, such as, for example, blood loss of more than about 15% of the total blood volume, or blood loss of more than 20%, 25%, 30%, 35%, 40%, or 50% of the subject's total volume. Alternatively, blood loss may, but need not, be measured in terms of a drop in blood volume by any amount sufficient to cause hemorrhagic shock in a particular subject, such as, for example, a drop of about 750 mL, 1000 mL, about 1500 mL, or about 2000 mL or more in a human subject. Blood loss may also be measured in terms of a drop in systolic blood pressure, such as, for example, a drop in systolic blood pressure of about 20 mmHg, about 30 mmHg, about 40 mmHg, about 50 mmHg, about 60 mmHg, about 70 mmHg, about 80 mmHg, about 90 mmHg, or about 100 mmHg or more than about 100 mmHg below the subject's normal systolic blood pressure. In certain embodiments, the subject is undergoing or has undergone a medical procedure, such as, but not limited to, surgery, a blood transfusion, or childbirth. In other specific embodiments, the subject has suffered trauma, for example, but not limited to, resulting from a car accident, an industrial injury, or a gunshot wound.

[0096] In further embodiments of the present invention, the compositions and methods are used to treat cardiogenic shock, hypovolemic shock, and vasodilatory shock, each of which may be any stage of shock. In one specific embodiment of the present invention, the method is used to treat cardiogenic shock. Cardiogenic shock is generally low blood flow or perfusion caused by cardiac dysfunction, when the heart does not eject enough blood. Causes may include, but are not limited to, any condition that interferes with ventricular filling or emptying, such as embolism, ischemia, regurgitation, and valvular dysfunction. In another specific embodiment of the present invention, the method is used to treat vasodilatory shock. Vasodilatory shock is caused by severe venous or arteriolar dilation, resulting in insufficient blood flow. Several known causes contribute to vasodilatory shock, including, but not limited to, cerebral trauma, drug or toxin toxicity, anaphylaxis, liver failure, bacteremia, and sepsis. In another further specific embodiment of the present invention, the method is used to treat shock caused by sepsis or bacteremia. In a further specific embodiment, the compositions and methods are used to treat septic shock or bacteremic shock, which are referred to as stage I, II, or III shock. In yet another embodiment, the compositions and methods of the present invention are used to treat hypovolemic shock. Hypovolemic shock is generally a decrease in intravascular volume, which can be relative or absolute. Hemorrhage from conditions such as, but not limited to, ulcers, gastrointestinal injuries, trauma, accidents, surgery, and aneurysms can cause hypovolemic shock; however, other fluid losses can also cause hypovolemic shock. For example, loss of renal fluid, loss of intravascular fluid, loss of water or other ascites fluid can contribute to hypovolemic shock. In a specific embodiment of the present invention, compositions and methods comprising administration of one or more peptides of the present invention are used to treat hypovolemic shock. In a further specific embodiment, the compositions and methods are used to treat hypovolemic shock in stage I, stage II, or stage III.

[0097] In one embodiment, the present invention is directed to a method of using one or more peptides of the present invention to protect the heart, brain, or other organs of a patient against damage caused by circulatory shock, wherein the protective effect against circulatory shock occurs immediately or within a short period of time, preferably within at least about 40 minutes after administration of a composition comprising one or more peptides of the present invention.

[0098] 2.6 Signs of MC4r responsiveness In yet another aspect, the peptides and compositions of the present invention, including but not limited to peptides that are MC4r agonists or partial agonists or MC3r agonists, partial agonists, antagonists, or any combination thereof, are useful for treating diseases, disorders and / or conditions that are responsive to modulation of MC4r function, more particularly activation of MC4r, i.e., that would benefit from agonism (including full or partial agonism) at MC4r, or for treating diseases, disorders and / or conditions that are responsive to modulation of MC3r function, more particularly activation of MC3r, i.e., that would benefit from agonism (including full or partial agonism) at MC3r. The compounds may be used in the treatment of diseases, disorders and / or conditions that result in or are responsive to modulation of both MC4r and MC3r function, including those related to energy homeostasis and metabolism (such as diabetes, especially type 2 diabetes; dyslipidemia; fatty liver; gout; hypercholesterolemia; hypertriglyceridemia; hyperuricemia; impaired glucose tolerance; impaired fasting glucose; insulin resistance syndrome; and metabolic syndrome), food intake (such as excessive appetite; binge eating; bulimia; and hyperphagia), and / or energy balance and weight-related diseases, disorders and / or conditions, more particularly those characterized by excess body weight and / or excessive food intake. In one aspect, the compounds of the present invention are used to treat conditions associated with various expression or receptor genetic disorders, such as proopiomelanocortin deficiency due to mutations in the POMC gene (POMC heterozygous deficiency obesity), Prader-Willi syndrome, obesity due to MC4r deficiency, leptin receptor deficiency obesity, leptin deficiency obesity, congenital leptin deficiency, Bardet-Biedl syndrome, Alström syndrome, and various other diseases, conditions, genetic defects, metabolic disorders, and syndromes.

[0099] Such peptides are believed to be useful, inter alia, for the treatment of weight-related diseases, disorders and / or conditions characterized by excess weight, including obesity and overweight (by promoting weight loss, maintaining weight loss, and / or preventing weight gain, including drug-induced weight gain or weight gain secondary to smoking cessation), as well as for the treatment of diseases, disorders and / or conditions associated with obesity and / or overweight, such as insulin resistance; impaired glucose tolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (including hyperlipidemia); hypertension; heart disorders (e.g., coronary heart disease, myocardial infarction); cardiovascular disorders; non-alcoholic fatty liver disease (including non-alcoholic steatohepatitis); joint disorders (including secondary osteoarthritis); gastroesophageal reflux; sleep apnea; atherosclerosis; stroke; macro- and microvascular disease; fatty liver (e.g., of the liver); gallstones; and gallbladder disorders.

[0100] MC4r is part of the leptin-melanocortin pathway, or proopiomelanocortin (POMC)-MC4r pathway. Members of this pathway include a wide variety of proteins, including α-MSH, POMC, leptin, and the leptin receptor. Certain diseases, conditions, and syndromes result from mutations and alterations, including genetic defects, associated with or in one or more members of the POMC-MC4r pathway. The compounds of the present invention may be useful in treating diseases, conditions, and syndromes resulting from mutations and alterations, including genetic defects, associated with or in one or more members of the POMC-MC4r pathway, as described below.

[0101] The hypothalamic POMC-MC4r pathway is part of a regulatory system that regulates eating behavior, appetite, and body weight. Several diseases, conditions, and syndromes associated with disruption of the hypothalamic POMC-MC4r pathway, thought to be due to genetic defects or disruptions, including defects or disruptions in genes within the POMC-MC4r pathway, have been described. For example, Prader-Willi syndrome is manifested by significant hyperphagia and severe obesity and may include other features and symptoms such as learning disabilities, abnormal neurological function, hypogonadism, short stature, and developmental and cognitive delays. The compounds of the present invention may be useful in treating Prader-Willi syndrome and other diseases, conditions, and syndromes involving defects or disruptions in genes within the POMC-MC4r pathway, as described below.

[0102] Therefore, the compounds of the present invention may be used to treat obesity and hyperphagia associated with POMC deficiency caused by homozygous or compound heterozygous loss of function mutations in the POMC gene located at position 23.3 on chromosome 2, and treatment is indicated. Mutations in the POMC gene that result in a complete deficiency of the POMC polypeptide or significantly reduce its production result in no or reduced production of α-MSH. This lack of endogenous α-MSH results in significantly reduced MC4r activity, resulting in hyperphagia and obesity. The compounds of the present invention may also be used as MC4r agonist replacement therapy in patients with little or no endogenous α-MSH.

[0103] For various diseases, conditions, or syndromes associated with disruption of the hypothalamic POMC-MC4r pathway, various genetic and genotyping tests may be used as part of the diagnosis of a prospective patient and the suitability of such patient for use with the compounds of the present invention. For example, but not limited to, in the case of Prader-Willi syndrome, genetic tests such as DNA-based methylation testing can be used to confirm the loss of an active gene within a specific portion of chromosome 15, the 15q11-q13 region, specifically a deletion of at least the 15q11-q13 region of paternal chromosome 15. Similarly, POMC deficiency may be diagnosed by loss-of-function mutations in the POMC gene. Therefore, treatment with the compounds of the present invention may involve various diagnostic and genetic tests to confirm the presence of loss-of-function mutations or other mutations in the POMC-MC4r pathway, including, but not limited to, loss-of-function mutations in Prader-Willi syndrome affecting the 15q11-q13 region, loss-of-function mutations in the POMC gene, leptin gene, leptin receptor gene, and various other genes in the POMC-MC4r pathway.

[0104] In yet another aspect, peptides and compositions of the present invention, including but not limited to peptides that are MC4r agonists or partial agonists, may be used in the treatment of sexual dysfunction, including both male erectile dysfunction and female sexual dysfunction. Female sexual dysfunction includes, but is not limited to, hypoactive sexual desire disorder. In one specific embodiment, peptides, compositions, and methods of the present invention are used in male patients to enhance erectile function, including but not limited to enhancing erectile function to allow for vaginal intercourse. In another specific embodiment, peptides, compositions, and methods of the present invention are used to treat female sexual dysfunction, including but not limited to increasing arousal success rate, desire success rate, and levels of arousal and desire. For female sexual dysfunction, including hypoactive sexual desire disorder, endpoints may, but need not, be determined by any of several validated instruments, including, but not limited to, the Female Sexual Distress Scale, Female Sexual Encounter Profile, Female Sexual Function Index, and Global Assessment Questionnaire. Patients treated for female sexual dysfunction may be premenopausal or postmenopausal women.

[0105] In yet another embodiment, the peptides and compositions of the present invention, including but not limited to peptides that are MC4r agonists or partial agonists, may be used to inhibit alcohol consumption, reduce alcohol consumption, treat or prevent alcohol dependence, treat or prevent alcohol abuse, or treat or prevent alcohol-related disorders. In another related embodiment, one or more of the present peptides may be used to inhibit consumption of drugs of abuse, reduce consumption of drugs of abuse, treat or prevent drug abuse, or treat or prevent drug abuse-related disorders. Drugs of abuse are typically controlled substances. These include controlled naturally occurring drugs such as heroin, morphine, opium, cocaine, and marijuana, as well as synthetically produced drugs such as Vicodin®, Lortab®, Lorcet®, Percocet®, Percodan®, Tylox®, hydrocodone, OxyContin®, methadone, tramadol, various methamphetamines, and other tranquilizers, stimulants, or sedatives known to be abused, as well as drugs with no established pharmaceutical utility such as ecstasy, LSD, or PCP.

[0106] In yet another embodiment, the peptides and compositions of the present invention, including MC4r antagonist or inverse agonist peptides, which may be agonists, partial agonists, antagonists, or inverse agonists at one or more of MC1r, MC2r, MC3r, and MC5r, may be used in the treatment of various body weight disorders, including cachexia, sarcopenia, and wasting syndromes or diseases, as well as in the treatment of inflammatory and immune disorders. Body weight disorders include one or more "wasting" disorders (e.g., wasting syndromes, cachexia, sarcopenia) that cause unwanted, unhealthy weight loss or a decrease in somatic cell mass. In elderly individuals and cancer and AIDS patients, wasting disorders can result in unwanted weight loss, including both the obese and non-obese compartments. Wasting disorders can be the result of inadequate food intake and / or metabolic changes associated with disease and / or the aging process. Cancer and AIDS patients, as well as patients undergoing extensive surgery or with chronic infections, immune disorders, hyperthyroidism, Crohn's disease, psychogenic disorders, chronic heart failure, or other severe trauma, often suffer from wasting diseases. Wasting diseases, sometimes referred to as cachexia, are generally recognized as metabolic disorders, sometimes as eating disorders. Cachexia may additionally be characterized by hypermetabolism and hypercatabolism. Sarcopenia, another disorder that may affect aging individuals, is typically characterized by a loss of muscle mass. Such end-stage wasting diseases can occur in individuals with either cachexia or sarcopenia.

[0107] 2.7 Nuclear Medicine and Drug Delivery Applications In yet another embodiment, the peptides and compositions of the present invention, including but not limited to peptides that are MC1r agonists, partial agonists, or antagonists, may be used in targeted imaging and cytotoxic therapy for specific cancers, such as melanoma and other indications, in patients in need thereof. The peptides, compositions, and methods of the present invention may be used, for example, by diagnostic imaging using a radionuclide in combination with the peptides of the present invention, for imaging melanoma and other cancers or diseases or conditions characterized in part by relatively high expression of MC1r. For diagnostic imaging, the peptides of the present invention are typically conjugated to a radionuclide by the use of a linker, such as a cross-linking agent, that couples the peptide of the present invention to the radionuclide. The radionuclide is preferably a gamma emitter that may be imaged using a gamma detector or camera, such as single-photon emission computed tomography, or a positron emitter that may be imaged using positron emission tomography. Gamma emitters that may be used in this way include, inter alia: 99m Tc, 111 In, 123 I and 67 Positron emitters that may be used include: 11 C. 13 N, 15 O and 18 Contains F.

[0108] In a related aspect, the peptides, compositions, and methods of the invention may be used for the cytotoxic therapy of melanoma, other cancers, or diseases or conditions characterized, in part, by relatively high expression of MC1r, for example, by using chemotherapeutic agents, including toxins or radiotherapeutic agents, in combination with the peptides of the invention. Chemotherapeutic agents include any antineoplastic or chemical agent, such as, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Non-limiting examples of alkylating agents include cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; examples of antimetabolites include azathioprine and mercaptopurine; examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone; examples of plant alkaloids include vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine, and taxanes such as paclitaxel and docetaxel; examples of topoisomerase inhibitors include camptothecins such as irinotecan and topotecan, and type II topoisomerase inhibitors such as amsacrine, etoposide, etoposide phosphate, and teniposide. However, any agent suitable for use in targeted cytotoxic therapy may be used in this manner. Non-limiting examples of radiotherapeutic agents that may be so used include, among others: 131 I, 125 I, 211 At, 186 Re, 188 Re, 90 Y, 153 Sm, 212 Bi and 32 Examples include P.

[0109] Diagnostic imaging agents or cytotoxic therapeutic agents may be used, for example, instead of non-radioactive isotopes, particularly 11 C. 13 N, 15They may be incorporated into the peptides of the invention, such as by using O; directly linked to the peptides of the invention, such as by halogenation or other direct conjugation methods; or indirectly linked to the peptides of the invention, such as by conjugation with a linker or chelating unit. Linker units are well known in the art and include, but are not limited to, chemically linked conjugates comprising at least one disulfide bond, thioether bond, or covalent bond between free reactive groups. Representative cross-linking and conjugation reagents are disclosed in, inter alia, U.S. Pat. Nos. 7,169,603, 7,820,164, and 5,443,816, and U.S. Patent Application Publication No. 2009 / 0297444, which are incorporated herein by reference.

[0110] 3.0 Combination Therapies for Specific Indications The peptides, compositions, and methods of the present invention may be used in the treatment of any of the aforementioned diseases, symptoms, conditions, or syndromes, or any disease, symptom, condition, or syndrome mediated or responsive to MC1r, by administration in combination with one or more other pharmaceutically active compounds. Such combined administration may be via a single dosage form containing both the peptide of the present invention and another pharmaceutically active compound, such as a single dosage form including a tablet, capsule, spray, inhalation powder, injectable solution, etc. Alternatively, combined administration may be via administration of two different dosage forms, one dosage form containing the peptide of the present invention and the other dosage form containing another pharmaceutically active compound. In this case, the dosage forms may be the same or different. The term "co-administered" indicates that each of at least two compounds in a combination therapy is administered during a time frame in which their respective periods of biological activity or effect overlap. Thus, the term includes sequential and simultaneous administration of compounds when one compound is one or more peptides of the present invention. When two or more compounds are co-administered, the routes of administration of the two or more compounds need not be the same. While not intending to limit the combination therapy, the following are examples of particular combination therapies that may be used.

[0111] 3.1 Combination therapy with anti-inflammatory agents In treating inflammation-related diseases, symptoms, conditions, and syndromes, the peptides of the present invention may be used in combination therapy, for example, by co-administration with one or more anti-inflammatory agents. One class of anti-inflammatory agents is the glucocorticoids, including but not limited to cortisone, including cortisone acetate, hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, prednisone, fludrocortisone acetate, deoxycorticosterone acetate, and aldosterone. Other anti-inflammatory agents that may be used in combination therapy, e.g., with co-administration, include aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen and naproxin), TNF-α inhibitors (such as tenidap and rapamycin or derivatives thereof) or TNF-α antagonists (e.g., infliximab, OR1384), cyclooxygenase inhibitors (i.e., cyclooxygenase-1 and / or cyclooxygenase-2 inhibitors such as naproxen® or Celebrex®), CTLA4-Ig agonists / antagonists, CD40 ligand antagonists, IMPDH inhibitors, e.g., mycophenolate (CellCept®), integrin antagonists, alpha4beta7 integrin antagonists, and the like. antagonists, cell adhesion inhibitors, interferon gamma antagonists, ICAM-1, prostaglandin synthesis inhibitors, budesonide, clofazimine, p38 mitogen-activated protein kinase inhibitors, protein tyrosine kinase (PTK) inhibitors, IKK inhibitors, therapeutic agents for the treatment of irritable bowel syndrome (e.g., Zelmac® and Maxi-K® openers disclosed in U.S. Pat. No. 6,184,231), or other NF-κB inhibitors, such as corticosteroids, calphostin, CSAIDs, 4-substituted imidazo[1,2-α]quinoxalines (as disclosed in U.S. Pat. No. 4,200,750); interleukin-10, salicylic acid, nitric oxide, and other immunosuppressants; and nuclear transport inhibitors such as deoxyspergualin (DSG).

[0112] 3.2 Combination therapy with phosphodiesterase inhibitors In certain applications and indications, it is desirable to increase the production and maintenance of levels of cyclic adenosine 3',5'-monophosphate (cAMP), a nucleotide messenger associated with inflammatory cell activity. The peptides of the present invention can be co-administered with compounds or substances that increase intracellular levels of cAMP and inhibit the degradation of cAMP. cAMP is hydrolyzed to an inactive form by phosphodiesterases (PDEs); compounds or substances that inhibit PDEs can thereby maintain and / or increase available cAMP. A class of compounds known as PDE inhibitors has been extensively tested for use in the treatment of inflammatory diseases such as asthma, COPD, and acute respiratory distress syndrome. Inhibitors of PDE types 1, 2, 3, 4, 7, 8, 10, or 11 are preferred; in one embodiment, this includes cAMP-PDE inhibitors, i.e., selective PDE type 4 inhibitors, or inhibitors that have selectivity for one particular type of PDE4 isoenzyme, such as rolipram, cilomilast, ibudilast, and piclamilast.

[0113] 3.3 Combination therapy in ocular indications For ophthalmic indications, eye drop dosage forms may contain, in addition to one or more peptides of the invention, one or more active ingredients, such as, for example, an artificial tear component, a topical corticosteroid, a nonsteroidal anti-inflammatory drug, or a calcineurin inhibitor such as cyclosporine A ophthalmic emulsion (Restasis® - Allergan). Co-administration may also include administration of one or more additional compounds given separately from the peptides of the invention, such as separate administration of an eye drop form containing an artificial tear component, a topical corticosteroid, a nonsteroidal anti-inflammatory drug, a calcineurin inhibitor such as cyclosporine A, or a combination of any of the foregoing.

[0114] Eye drop combinations may also be used, particularly including solutions containing two or more active pharmaceutical ingredients. In one embodiment, nonsteroidal anti-inflammatory drugs (NSAIDs) are used in combination with the peptides of the present invention. NSAIDs suitable for use in eye drop combinations include, but are not limited to, propionic acid compounds such as naproxen, flurbiprofen, oxaprozin, ibuprofen, ketoprofen, and fenoprofen; ketorolac tromethamine; acetic acid derivatives such as sulindac, indomethacin, and etodolac; phenylacetic acid derivatives such as diclofenac, bromfenac, and suprofen; arylacetic acid prodrugs such as nepafenac and amfenac. prodrugs); salicylates such as aspirin, salsalate, diflunisal, and choline magnesium trisalicylate; para-aminophenol derivatives such as acetaminophen; naphthyl alkanones such as nabumetone; enolic acid derivatives such as piroxicam and meloxicam; fenamic acids such as mefenamic acid, meclofenamic acid, and flufenamic acid; pyrroleacetic acids such as tolmetin; and pyrazolones such as phenylbutazone; and drugs that inhibit cyclooxygenase (COX)-1 and / or -2 enzymes, including COX-2 selective inhibitors such as celecoxib, valdecoxib, parecoxib, etoricoxib, and luaricoxib, their esters, and pharmaceutically acceptable salts thereof. The eye drops may further comprise other active ingredients, including, but not limited to, vasoconstrictors, anti-allergens, anti-infectives, steroids, anesthetics, anti-inflammatory drugs, analgesics, dry eye treatments (e.g., secretagogues, mucomimetics, polymers, lipids, antioxidants), and the like, or may be administered in conjunction with (simultaneously or sequentially with) a pharmaceutical composition containing other active ingredients, including, but not limited to, vasoconstrictors, anti-allergens, anti-infectives, steroids, anesthetics, anti-inflammatory drugs, analgesics, dry eye treatments (e.g., secretagogues, mucomimetics, polymers, lipids, antioxidants), and the like.

[0115] 3.4 Concomitant therapy in shock-related conditions Furthermore, the methods of treating or preventing circulatory shock of the present invention involve co-administering one or more substances to a subject in addition to one or more peptides of the present invention. For example, one or more peptides of the present invention may be co-administered with androstentriol, androstenediol or derivatives thereof, various vasopressin agonists, or other pharmaceutically active substances, such as catecholamines or other alpha-, alpha-, beta-, or beta-adrenergic agonists, including, but not limited to, epinephrine, norepinephrine, dopamine, isoproterenol, vasopressin, and dobutamine. Alternatively, one or more peptides of the present invention may be co-administered with a body fluid or other substance capable of alleviating, attenuating, preventing, or eliminating symptoms in a subject suffering from, experiencing, or at risk of suffering from hypovolemic shock, vasodilatory shock, or cardiogenic shock. The type of bodily fluid that may be co-administered with one or more peptides of the present invention should be specific to the surrounding environment of a particular subject suffering from, exhibiting symptoms of, or at risk of suffering from shock. For example, bodily fluids that may be co-administered with one or more peptides of the present invention include, but are not limited to, saline solutions such as sodium chloride and sodium bicarbonate, as well as whole blood, synthetic blood substitutes, plasma, serum, serum albumin, and colloid solutions. Colloid solutions include, but are not limited to, solutions containing hetastarch, albumin, or plasma. In one specific embodiment of the present invention, bodily fluids such as one or more of saline solutions, colloid solutions, whole blood, synthetic blood substitutes, plasma, or serum are co-administered with one or more peptides of the present invention to a patient suffering from or exhibiting symptoms of hypovolemic shock, such as hemorrhagic shock.

[0116] 3.5 Combination Therapy for Obesity and Related Metabolic Syndromes One or more peptides of the present invention may be combined with one or more other pharmacologically active agents useful in the treatment of various weight- and eating-related disorders, such as obesity and / or overweight, particularly other anti-obesity agents that affect energy expenditure, glycolysis, gluconeogenesis, glycogenolysis, lipolysis, lipogenesis, fat absorption, fat storage, fat excretion, hunger and / or satiety and / or appetite mechanisms, appetite / motivation, food intake, or gastrointestinal motility. Agents that reduce energy intake include a variety of pharmacological agents, sometimes referred to as anorectic agents, that are used, in part, as adjuncts to behavioral therapy in weight loss programs.

[0117] Generally, the total dosage of the obesity control agents or drugs described below, when used in combination with one or more peptides of the present invention, can range from 0.01 to 3,000 mg / day, preferably about 0.1 to 50 mg / day, and more preferably about 0.1 to 10 mg / day, in single or 2 to 4 divided doses. However, the exact dosage will be determined by the attending clinician and will depend on factors such as the potency of the compound administered, the age, weight, condition and response of the patient.

[0118] One or more peptides of the present invention may be combined with one or more other pharmacologically active agents that are useful in the treatment of diabetes, such as other antidiabetic agents.

[0119] One or more peptides of the present invention may additionally or alternatively be further combined with one or more other pharmacologically active agents useful in the treatment of diseases, disorders and / or conditions associated with obesity and / or overweight, such as insulin resistance; impaired glucose tolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (including hyperlipidemia); hypertension; cardiac disorders (e.g., coronary heart disease, myocardial infarction); cardiovascular disorders; non-alcoholic fatty liver disease (including non-alcoholic steatohepatitis); joint disorders (including secondary osteoarthritis); gastroesophageal reflux; sleep apnea; atherosclerosis; stroke; macro- and microvascular disease; steatosis (e.g., of the liver); gallstones; and gallbladder disorders.

[0120] According to a further aspect of the invention, a pharmacologically effective amount of a peptide according to the invention, or a pharmaceutically acceptable salt thereof, optionally in association with a pharmaceutically acceptable diluent or carrier, is administered, -insulin and insulin analogues; - insulin secretagogues, such as sulfonylureas (e.g., glipizide) and prandial glucose regulators (sometimes called "short-acting secretagogues"), for example meglitinides (e.g., repaglinide and nateglinide); -Medications that improve incretin action, such as dipeptidyl peptidase IV (DPP-4) inhibitors (e.g., vildagliptin, saxagliptin, and sitagliptin) and glucagon-like peptide-1 (GLP-1) agonists (e.g., exenatide); - insulin sensitizers, such as peroxisome proliferator-activated receptor gamma (PPARγ) agonists, for example, thiazolidinediones (e.g., pioglitazone and rosiglitazone), and agents with any combination of PPAR alpha, gamma, and delta activity; - drugs that regulate hepatic glucose balance, such as biguanides (e.g., metformin), fructose 1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, glycogen synthase kinase inhibitors, and glucokinase activators; -Medications designed to reduce / delay the absorption of glucose from the intestine, such as alpha-glucosidase inhibitors (e.g., miglitol and acarbose); - drugs that antagonize the action of glucagon or reduce its secretion, such as amylin analogues (e.g., pramlintide); -Medications that inhibit glucose reabsorption by the kidney, such as sodium-dependent glucose transporter 2 (SGLT-2) inhibitors (e.g., dapagliflozin); - Medications designed to treat complications of long-term hyperglycemia, such as aldose reductase inhibitors (e.g., epalrestat and ranirestat); and medications used to treat complications associated with microangiopathy; - anti-dyslipidemic agents, such as HMG-CoA reductase inhibitors (statins, e.g., rosuvastatin) and other cholesterol-lowering drugs; PPARα agonists (fibrates, e.g., gemfibrozil and fenofibrate); bile acid sequestrants (e.g., cholestyramine); cholesterol absorption inhibitors (e.g., plant sterols (i.e., phytosterols), synthesis inhibitors); cholesteryl ester transfer protein (CETP) inhibitors; inhibitors of the ileal bile acid transport system (IBAT inhibitors); bile acid-binding resins; nicotinic acid (niacin) and its analogs; antioxidants, e.g., probucol; and omega-3 fatty acids; - antihypertensive drugs, adrenergic receptor antagonists, such as beta-blockers (e.g., atenolol), alpha-blockers (e.g., doxazosin), and mixed alpha / beta-blockers (e.g., labetalol); adrenergic receptor agonists, such as alpha-2 agonists (e.g., clonidine); angiotensin-converting enzyme (ACE) inhibitors (e.g., lisinopril), calcium channel blockers, such as dihydropridine (e.g., nifedipine), phenylalkylamines (e.g., verapamil), and benzothiazepines (e.g., diltiazem); angiotensin II receptor antagonists (e.g., candesartan); aldosterone receptor antagonists (e.g., eplerenone); centrally acting adrenergic drugs, such as central alpha-agonists (e.g., clonidine); and diuretics (e.g., furosemide); - hemostatic modulators, such as antithrombotic agents, for example, fibrinolytic activators; thrombin antagonists; factor VIIa inhibitors; anticoagulants, for example, vitamin K antagonists (e.g., warfarin), heparin and its low molecular weight analogues, factor Xa inhibitors, and direct thrombin inhibitors (e.g., argatroban); antiplatelet agents, for example, cyclooxygenase inhibitors (e.g., aspirin), adenosine diphosphate (ADP) receptor inhibitors (e.g., clopidogrel), phosphodiesterase inhibitors (e.g., cilostazol), glycoprotein IIB / IIA inhibitors (e.g., tirofiban), and adenosine reuptake inhibitors (e.g., dipyridamole); antiobesity drugs, such as appetite suppressants (e.g., ephedrine), noradrenergic agents (e.g., phentermine) and serotonergic agents (e.g., sibutramine), pancreatic lipase inhibitors (e.g., orlistat), microsomal transfer protein (MTP) modulators, diacylglycerol acyltransferase (DGAT) inhibitors, and cannabinoid (CB1) receptor antagonists (e.g., rimonabant); Feeding behavior regulators, such as orexin receptor modulators and melanin-concentrating hormone (MCH) modulators; -glucagon-like peptide-1 (GLP-1) receptor modulators; -Neuropeptide Y (NPY) / NPY receptor modulators; -pyruvate dehydrogenase kinase (PDK) modulators; -Serotonin receptor modulators; -Leptin / leptin receptor modulators; - a ghrelin / ghrelin receptor modulator; or monoamine transport modulators, such as selective serotonin reuptake inhibitors (SSRIs) (e.g., fluoxetine), noradrenaline reuptake inhibitors (NARIs), noradrenaline-serotonin reuptake inhibitors (SNRIs), triple monoamine reuptake blockers (e.g., tesofensine), and monoamine oxidase inhibitors (MAOIs) (e.g., toloxatone and amiflamine), or a pharmaceutically acceptable salt, solvate, solvate of such a salt, or prodrug thereof, optionally together with a pharmaceutically acceptable carrier, to a mammal, such as a human, in need of such therapeutic treatment.

[0121] According to a further aspect of the present invention there is provided a combination therapy comprising the simultaneous, sequential or separate administration of a very low calorie diet (VLCD) or a low calorie diet (LCD) together with the administration of a pharmacologically effective amount of a compound according to the present invention, or a pharmaceutically acceptable salt thereof, optionally in association with a pharmaceutically acceptable carrier.

[0122] According to a further aspect of the present invention, one or more peptides of the present invention, preferably peptides that are MC4r agonists, may be administered in conjunction with a GLP-1 receptor agonist, as disclosed in WO 2016 / 168388 "Therapies for Obesity, Diabetes and Related Indications", which is incorporated herein by reference. Thus, the present invention provides, on a dosage basis: a peptide of the invention that is an MC4r agonist, in an amount sufficient to induce at least minimal weight loss when administered as monotherapy without a GLP-1 receptor agonist; and A GLP-1 receptor agonist at a dose sufficient to induce glycemic control but not weight loss when administered as monotherapy without an MC4r agonist for subcutaneous administration in the treatment of obesity or for inducing weight loss, which preferably has a synergistic anti-obesity effect.

[0123] In a related aspect, the present invention provides a method of treating a patient with obesity, diabetes, or metabolic syndrome, comprising administering to the patient (a) a peptide of the invention that is an MC4r agonist, in an amount sufficient to induce at least minimal weight loss when administered as monotherapy without a GLP-1 receptor agonist, and (b) a GLP-1 receptor agonist in an amount sufficient to induce glycemic control, but not weight loss, when administered as monotherapy without an MC4r agonist. Preferably, the pulmonary method induces a synergistic effect on the treatment of obesity.

[0124] In another aspect, the present invention provides a method for reducing side effects associated with a therapeutic agent in the treatment of obesity, diabetes, or metabolic syndrome in a patient, comprising: administration of a peptide of the invention that is an MC4r agonist in an amount where the amount of the MC4r agonist peptide administered, when administered as monotherapy without a GLP-1 receptor agonist, is insufficient to initiate a desired pharmacological response in the treatment of at least one condition from the group consisting of obesity, diabetes, and metabolic syndrome in a patient when administered as monotherapy; and Administration of a GLP-1 receptor agonist in an amount that, when administered as monotherapy without an MC4r agonist, is insufficient to initiate a desired pharmacological response in the treatment of at least one condition from the group consisting of obesity, diabetes, and metabolic syndrome in a patient when administered as monotherapy. Including, wherein the amount of the MC4r agonist and the amount of the GLP-1 receptor agonist treat at least one condition from the group consisting of obesity, diabetes, and metabolic syndrome in a patient; Thereby, methods are provided which are both effective in initiating a desired pharmacological response that reduces the side effects of treating at least one of obesity, diabetes, or metabolic syndrome in a patient.

[0125] 3.6 Combination therapy for sexual dysfunction It is also possible and contemplated that the cyclic peptides of the present invention may be used in combination with other drugs or agents, for example, for the treatment of sexual dysfunction. These other drugs and agents may include agents that induce erectile activity, including phosphodiesterase-5 (PDE-5) inhibitors, testosterone, prostaglandins, etc. In a preferred embodiment of the present invention, the cyclic peptides of the present invention are used in combination with a therapeutically effective amount of a cyclic GMP-specific phosphodiesterase inhibitor or an alpha-adrenergic receptor antagonist. The teachings and disclosures of U.S. Patent No. 7,235,625, entitled "Multiple Agent Therapy for Sexual Dysfunction," are incorporated herein by reference as if fully set forth.

[0126] Thus, the present invention provides a method for treating sexual dysfunction, comprising administering a therapeutically effective amount of a cyclic peptide of the present invention in combination with a therapeutically effective amount of a second sexual dysfunction medication to a patient suffering from or at risk of suffering from sexual dysfunction. The cyclic peptide of the present invention may be administered simultaneously with, before, or after the administration of the therapeutically effective amount of the second sexual dysfunction medication. Preferably, the peptide of the present invention is administered within one hour, preferably within less than 30 minutes, of the administration of the therapeutically effective amount of the second sexual dysfunction medication. However, in certain forms of combination therapy, for example, in combination with a therapeutically effective amount of a hormone or hormone-related sexual dysfunction medication, the hormone or hormone-related sexual dysfunction medication may be administered on an independent schedule such that there is no set or specific temporal relationship between the administration of the peptide of the present invention and the administration of the hormone or hormone-related sexual dysfunction medication. Thus, for example, the hormone or hormone-related sexual dysfunction medication may be administered together with the administration of the peptide of the present invention, in daily or other doses, or using a patch or other continuous administration schedule, as desired or required by the patient.

[0127] Therefore, the present invention provides a method for treating sexual dysfunction, comprising administering to a patient suffering from or at risk of suffering from sexual dysfunction a therapeutically effective amount of a cyclic peptide of the present invention in combination with another compound useful in treating sexual dysfunction. In a preferred embodiment of the combination therapy, the sexual dysfunction is female sexual dysfunction. In another preferred embodiment of the combination therapy, the sexual dysfunction is erectile dysfunction.

[0128] The present invention also provides a pharmaceutical composition comprising a cyclic peptide of the present invention and a second compound useful for treating sexual dysfunction. In an embodiment of the composition, the additional compound useful for treating sexual dysfunction is preferably selected from the group consisting of, but not limited to, phosphodiesterase inhibitors, cyclic GMP-specific phosphodiesterase inhibitors, prostaglandins, apomorphine, oxytocin modulators, α-adrenergic antagonists, androgens, selective androgen receptor modulators (SARMs), buproprion, vasoactive intestinal peptide (VIP), neutral endopeptidase inhibitors (NEP), and neuropeptide Y receptor antagonists (NPY).

[0129] In an embodiment of the methods and compositions, the second sexual dysfunction medication is testosterone.

[0130] In another embodiment of the combination therapy, the second sexual dysfunction drug is a type V phosphodiesterase (PDE-5) inhibitor. For example, the PDE-5 inhibitor may be Viagra®, the brand name for sildenafil, Levitra®, the brand name for vardenafil monohydrochloride, or Cialis®, the brand name for tadalafil. Other PDE-5 inhibitors are disclosed in U.S. Patent No. 7,235,625, entitled "Multiple Agent Therapy for Sexual Dysfunction," issued June 22, 2007, which is incorporated herein by reference.

[0131] In another embodiment of the composition, the compound useful for treating sexual dysfunction is an estrogen agonist / antagonist.In one embodiment, the estrogen agonist / antagonist is (-)-cis-6-phenyl-5-[-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-2-ol (also known as lasofoxifene) or its optical or geometric isomer; pharmaceutically acceptable salt, N-oxide, ester, quaternary ammonium salt; or prodrug thereof.More preferably, the estrogen agonist / antagonist is in the form of D-tartrate.

[0132] In yet another embodiment of the above composition, the estrogen agonist / antagonist is selected from the group consisting of tamoxifen, 4-hydroxytamoxifen, raloxifene, droloxifene, toremifene, centchroman, idoxifene, 6-(4-hydroxy-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-naphthalen-2-ol, {4-[2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy]-phenyl}-[6-hydroxy-2-(4-hydroxy-phenyl)-benzo[b]thiophen-3-yl]-methanone, EM-652, EM-800, GW5368, GW7604, TSE-424, and optical or geometric isomers thereof; and pharmaceutically acceptable salts, N-oxides, esters, quaternary ammonium salts, and prodrugs thereof.

[0133] In yet another embodiment, the cyclic peptides of the present invention may be used in conjunction with any known mechanical aid or device.

[0134] 4.0 Methods of Administration and Use Methods of administration and use will vary depending on the characteristics of the particular peptide disclosed herein or of the formulas disclosed herein, the disease, symptom, condition or syndrome being treated, and other factors known to those of skill in the art. Generally, any method of administration and use known in the art or later developed may be used with peptides disclosed herein or of the formulas disclosed herein. Without limiting the foregoing, the following methods of administration and use have particular use for the indicated indications.

[0135] 4.1 Subcutaneous injection use In one aspect, a composition comprising one or more peptides of the present invention is formulated for subcutaneous injection, and the subcutaneous injections are administered at specific intervals, such as once or more weekly or daily. In another aspect, the composition is formulated as an injectable sustained-release formulation. In one embodiment, the peptides of the present invention are formulated with polyethylene glycol, such as polyethylene glycol 3350, and optionally one or more additional excipients and preservatives, including, but not limited to, excipients such as salts, polysorbate 80, sodium hydroxide, or hydrochloric acid for pH adjustment. In another embodiment, the peptides of the present invention are formulated with poly(orthoesters), which may be autocatalytic poly(orthoesters) with varying percentages of lactic acid in the polymer backbone, and optionally one or more additional excipients. In one embodiment, poly(D,L-lactide-co-glycolide) polymers (PLGA polymers), preferably PLGA polymers with hydrophilic end groups, such as PLGA RG502H from Boehringer Ingelheim, Inc. (Ingelheim, Germany), are used. Such formulations may be made, for example, by combining a peptide of the present invention in a suitable solvent, such as methanol, with a solution of PLGA in methylene chloride and adding a continuous phase solution of polyvinyl alcohol thereto under suitable mixing conditions in a reactor. Generally, any of several injectable biodegradable polymers, preferably also adhesive polymers, may be used in sustained-release injectable formulations. The teachings of U.S. Patent Nos. 4,938,763, 6,432,438, and 6,673,767, and the biodegradable polymers and formulation methods disclosed therein, are incorporated herein by reference. Injections may be formulated so that they are required weekly, monthly, or on other periodic basis, depending on the concentration and amount of peptide, the biodegradation rate of the polymer, and other factors known to those skilled in the art.

[0136] 4.2 Inhalation use In one embodiment, compositions comprising one or more peptides of the invention are formulated for administration to the respiratory tract, e.g., in the form of an aerosol or solution for a nebulizer, or as a finely divided powder for insufflation or inhalation (e.g., locally to the lungs and / or respiratory tract), alone or in combination with one or more inert carriers or additional active pharmaceutical ingredients, as well as in the form of a solution, suspension, aerosol, or dry powder formulation. See generally, Cryan, S.-A., "Carrier-based strategies for targeting protein and peptide drugs to the lungs," The AAPS Journal 7:E20-41 (2005). In general, the peptides of the present invention may be prepared using any of the following U.S. patents or patent applications, each of which is incorporated herein by reference: U.S. Patent Application Publication No. 20090241949, "Dry powder inhalation system"; U.S. Patent Application Publication No. 20080066741, "Methods and systems of delivering medication via inhalation"; U.S. Patent Application Publication No. 20070298116, "Amorphous, spray-dried powders having a reduced moisture content and a high long-term stability"; U.S. Patent Application Publication No. 20070140976, "Aqueous inhalation pharmaceutical composition"; U.S. Patent Application Publication No. 20060054166, "Inhalation nebulizer"; U.S. Patent Application Publication No. 20050211244, "Dry powder preparations"; U.S. Patent Application Publication No. 20050123509, "Modulating charge density to produce improvements in the characteristics of spray-dried proteins”; U.S. Patent Application Publication No. 20040241232, “Dry powder medicament formulations”;U.S. Patent No. 7,582,284, "Particulate materials"; U.S. Patent No. 7,481,212, "Increased dosage metered dose inhaler"; U.S. Patent No. 7,387,794, "Preparation of powder agglomerate"; U.S. Patent No. 7,258,873, "Preservation of bioactive materials by spray drying"; U.S. Patent No. 7,186,401, "Dry powder for inhalation"; U.S. Patent No. 7,143,764, "Inhalation device"; U.S. Patent No. 7,022,311, "Powdery inhalational preparations and process for producing the same"; U.S. Patent No. 6,962,151, "Inhalation nebulizer"; U.S. Patent No. 6,907,880, "Inhalation device"; U.S. Patent No. 6,881,398, "Therapeutic dry powder preparation"; U.S. Patent No. 6,698,425, "Powder inhaler”; U.S. Patent No. 6,655,380, “Inhalation device”; U.S. Patent No. 6,645,466, “Dry powder for inhalation”; U.S. Patent No. 6,632,456, “Compositions for inhalation”; U.S. Patent No. 6,610,272, “Medicinal aerosol formulation”; U.S. Patent No. 6,596,261, “Method of administering a medicinal aerosol formulation”; U.S. Patent No. 6,585,957, “Medicinal aerosol formulation”; U.S. Patent No. 6,582,729, “Powered pharmaceutical formulations having improved dispersibility”;U.S. Patent No. 6,572,893, "Systems and processes for spray drying hydrophobic drugs with hydrophilic excipients"; U.S. Patent No. 6,551,578, "Modulated release particles for aerosol delivery"; U.S. Patent No. 6,520,179, "Inhalation device"; U.S. Patent No. 6,518,239, "Dry powder compositions having improved dispersivity"; U.S. Patent No. 6,503,481, "Compositions for aerosolization and inhalation"; U.S. Patent No. 6,358,530, "Powdered pharmaceutical formulations having improved dispersibility"; U.S. Patent No. 6,325,061, "Inhalation device"; U.S. Patent No. 6,257,232, "Inhalation device"; U.S. Patent No. 6,187,344, "Powdered pharmaceutical formulations having improved dispersibility"; U.S. Patent No. 6,116,237, "Methods of dry powder No. 5,934,272, "Device and method of creating aerosolized mist of respiratory drug"; and U.S. Patent No. 5,558,085, "Intrapulmonary delivery of peptide drugs";

[0137] The composition may be a dry powder composition for localized delivery to the lungs by inhalation. The composition may contain a powder mixture for inhalation of the peptide of the present invention and a suitable powder base, diluent, or carrier material, such as lactose, glucose, dextran, mannitol, or another sugar or starch. The composition may be used in any of a variety of dry powder devices, such as a reservoir dry powder inhaler, a multi-dose dry powder inhaler, or a metered-dose inhaler. The composition may contain additional excipients, such as alcohol, surfactants, lubricants, antioxidants, or stabilizers. Suitable propellants include hydrocarbon, chlorofluorocarbon, and hydrofluoroalkane propellants, or mixtures of any such propellants.

[0138] Additionally, inhalation solutions may be formulated with a liquefied propellant for aerosol delivery, such as with a pressurized metered dose inhaler. In yet another formulation, the solution may be in the form of an nebulized aqueous suspension or solution, with or without suitable pH or osmolality adjustment, as either a single-dose or multi-dose device.

[0139] 4.3 Nasal Delivery Formulations or compositions suitable for nasal administration in which the carrier is a solid include coarse powders, e.g., having a particle size in the range of 20 to 500 microns, administered in the manner in which snuff is administered (i.e., by rapid inhalation through the nasal passages from a container of the powder held up to the nose). Suitable powder compositions include, by way of example, powder formulations of the active ingredient thoroughly mixed with lactose or other inert powders acceptable for intrabronchial administration. Powder compositions may be administered via an aerosol dispenser or may be enclosed in breakable capsules which may be punctured by the patient and inserted into a device that expels the powder in a steady stream suitable for inhalation. Alternatively, suitable formulations may comprise aqueous or oily solutions of the active ingredient or may comprise a liquid carrier, such as a nasal spray or nasal drops.

[0140] 4.4 Buccal and mucosal delivery Pharmaceutical compositions may further comprise, for example, one or more of water, a buffer (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, a carbohydrate (e.g., glucose, mannose, sucrose, or dextran), mannitol, a protein, an adjuvant, a polypeptide or amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, and / or a preservative. Additionally, one or more other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein.

[0141] 4.5 Oral Delivery In one embodiment, peptides of the invention, including MC1r agonists, are orally administered and delivered substantially intact to the lumen of all or part of the intestinal tract, including, in certain embodiments, the patient's colon, for the treatment of inflammatory bowel disease, colitis, or other melanocortin receptor-mediated or -responsive diseases, symptoms, conditions, and syndromes of the gastrointestinal tract. Delayed-release polymer formulations comprising peptides of the invention, including, but not limited to, pH-dependent release polymers, may also be used. The teachings and disclosures of International Publication No. WO 2019 / 183472, filed under International Application No. PCT / US2019 / 023575 and entitled "Melanocortin Receptor-Specific Formulations and Methods for Gastrointestinal Tract-Specific Delivery," are incorporated herein by reference as if fully set forth.

[0142] For systemic administration, compositions comprising one or more peptides disclosed herein or of formulas disclosed herein may be orally administered in a separate dosage form, such as a tablet or capsule. In a preferred embodiment, the separate dosage form comprises an enteric coating and, optionally, one or more agents intended to increase uptake, reduce protease degradation, increase cell permeability, etc. Any of a variety of delivery techniques may be used in oral delivery of the peptides of the invention, including, but not limited to, liposomal compositions, mucoadhesive or gastroretentive delivery systems, absorption enhancers, multifunctional drug delivery systems, co-administration of permeation enhancers and / or protease inhibitors, covalent conjugation with various chemical or biological adjuvants (e.g., to enhance cell permeability), enteric coatings, various nanoparticles, etc.

[0143] 5.0 How to Make In general, peptides disclosed herein or of the formulas disclosed herein may be synthesized by any means known in the art, including solid phase synthesis, and purified according to methods known in the art. Any of several well-known methods using a variety of resins and reagents may be used to prepare peptides disclosed herein or of the formulas disclosed herein.

[0144] Solid phase peptide synthesis methods are well known and practiced in the art. In such methods, synthesis of the peptides of the invention can be carried out by sequentially incorporating the desired amino acid residues one at a time into a growing peptide chain according to the general principles of solid phase methods.

[0145] In the chemical synthesis of peptides, the reactive side groups of the various amino acid residues are protected with suitable protecting groups that prevent chemical reactions from occurring at that site until the protecting group is removed. Furthermore, it is common for the alpha amino group of an amino acid residue or fragment to be protected while the entity reacts at a carboxyl group, followed by selective removal of the alpha amino protecting group, allowing subsequent reactions to occur at that site. Specific protecting groups have been disclosed and are known for solid-phase and solution-phase synthesis.

[0146] The alpha amino group may be protected by a suitable protecting group, including urethane-type protecting groups, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, e.g., p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz), and aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl (Alloc). Fmoc is particularly suitable for alpha amino protection.

[0147] The guanidino group may be protected by a suitable protecting group such as nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromansulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), and Boc. Pbf and Pmc are preferred protecting groups for Arg. Other reactive groups, including amine and carboxylic acid groups, may be similarly protected, e.g., 1-tert-butyl ester (OtBu) for Glu, Boc for Trp, trityl (Trt) for His, etc.

[0148] The linear peptide precursors of the peptides of the invention described herein were prepared using solid-phase synthesis on an automated peptide synthesizer, using the programming module as provided by the manufacturer and following the protocols described in the manufacturer's instructions.

[0149] The linker from the side chain amine group to the C-terminal carboxyl group of the linear peptide is a straight chain alkyl amino acid with no side chains other than hydrogen, such as glycine, β-alanine, γ-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, etc.

[0150] In one embodiment utilizing solid phase synthesis, synthesis is initiated from the C-terminus of the peptide, a linear alkyl amino acid is coupled to a suitable resin, thereby forming the starting resin, and then a protected alpha amino acid is coupled to the linear alkyl amino acid. For example, a preloaded trityl group may be utilized, to which Fmoc-8-aminocaprylic acid (ChemImpex, catalog number 04945) Fmoc-7-aminoheptanoic acid (ChemImpex, catalog number 07157) Fmoc-6-aminohexanoic acid (ChemImpext, catalog number 02490) Fmoc-5-aminovaleric acid (ChemImpex, catalog number 04797) Fmoc-γ-aminobutyric acid (ChemImpex, catalog number 02692) Fmoc-β-alanine (ChemImpex, catalog number 02374) Fmoc-glycine (ChemImpex, catalog number 02416) An Fmoc-protected linear alkyl amino acid, such as methylamino acid (Fmoc-protected methylamino acid), is attached to the peptide. However, other resins, such as Merrifield resin, Wang resin, bromobenzyl resin, 2-chlorotrityl resin, or other resins, may be used to generate peptide acids. In general, cyclic peptides disclosed herein or of the formulas disclosed herein may be readily synthesized by known conventional methods for forming peptide bonds between amino acids. Such conventional methods include, for example, any solution-phase method that allows for the condensation of the free alpha amino group of an amino acid residue bearing a protected carboxyl group and other reactive group with the free primary carboxyl group of another amino acid residue bearing a protected amino group or other reactive group. Any of several well-known methods using a variety of resins and reagents may be used to prepare peptides disclosed herein or of the formulas disclosed herein.

[0151] For Peptide No. 16 of the present invention, the synthesis began with manually preloaded 2-chlorotrityl chloride resin (ChemImpex, Catalog No. 03498, 0.9 g, 1.0 mmol) with Fmoc-5-aminovaleric acid (Fmoc-5-Ava-OH, ChemImpex, Catalog No. 04797, 1.4 g, 4.0 mmol). The resulting Fmoc-5-Ava-2Cl trityl resin (approximately 1.0 mmol) was loaded onto a peptide synthesizer. Next, the Fmoc-protected amino acids Trp(Boc), Arg(Pbf), d-Phe(4-F), His(Trt), Dab(Boc), and Nle were coupled individually and sequentially.

[0152] After Fmoc deprotection from Fmoc-Nle, the resulting N-terminal amine group is acylated, such as by using acetic anhydride and pyridine in DMF for a suitable period of time, to yield the peptide resin: Ac-Nle-Dab(Boc)-His(Trt)-D-Phe(4-F)-Arg(Pbf)-Trp(Boc)-NH(CH2)4COO-Resin was obtained.

[0153] The peptide resin was mixed with 30 mL of cleavage solution containing TFA / TIS / HO (95:2.5:2.5, v / v / v) for 20 min to further cleave the orthogonal protecting groups. This was followed by repeated mixing with another 30 mL of freshly prepared cleavage solution for 20 min. The combined filtrates were stored at room temperature for 2 h and concentrated using a purging N stream. The cleaved linear peptide was precipitated from cold ether. The solid / oil residue was dissolved in 50% t-butanol / water and lyophilized to give the crude linear peptide (approximately 1.0 mmol): Ac-Nle-Dab-His-D-Phe(4-F)-Arg-Trp-NH-(CH2)4-COOH was obtained.

[0154] The crude linear peptide (approximately 1.0 mmol) was dissolved in a mixture of 7.5 mL of DMF and 7.5 mL of DCM and cooled in an ice-water bath. To the cooled solution, EDC (0.288 g, 1.5 mmol) and HOAt (0.45 mL of a 0.6 M solution in DMF, 0.75 mmol) were added, followed by triethylamine (TEA) (0.4 mL, 3.0 mmol) until the pH reached 8-9. The reaction mixture was stirred while warming to room temperature and overnight at room temperature. LC / MS analysis indicated complete cyclization. 5 mL of 1N HCl was added to the reaction mixture, adjusting the pH to 3-4, and stirred for 1 hour. The reaction mixture was filtered through a 0.45 μ syringe filter and directly loaded onto a preparative HPLC column. Pure fractions were pooled and lyophilized to give 73 mg of the cyclic peptide (6% yield): [ka]

[0155] Other peptides of the invention may be made by similar means.

[0156] Generally, each deprotection step may involve repeated cycles as necessary, for example, using piperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole (HOBT), N,N-dimethylformamide (DMF), etc., followed by washing cycles with DMF or methyl tert-butyl ether (MBTE), etc.

[0157] Each coupling step may involve the use of a desired protected amino acid, such as FMOC-AA-OH, and coupling reagents including, among others, dichloromethane (DCM), HOBT, N,N-diisopropylethylamine (DIPEA), DMF, or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). After coupling, a wash cycle, such as with DMF or MBTE, may be used.

[0158] While the synthetic peptide is coupled to the resin in solution, the N-terminus may be modified, for example, by acetylation. In one embodiment, after removing the N-terminal protecting group, the resin-bound peptide is reacted with acetic anhydride in dichloromethane in the presence of an organic base such as diisopropylethylamine. Other methods of N-terminal acetylation, including liquid-phase acetylation, are known in the art and may be used.

[0159] The resulting resin-bound peptide may be cleaved from the resin by any means known in the art, such as by mixing the resin-bound peptide with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water, e.g., TFA / TIS / HO (95:2.5:2.5, v / v / v), at a suitable temperature, e.g., room temperature, for a suitable period of time, e.g., 20 minutes. If desired, one or more additional cycles of mixing the resin-bound peptide with a mixture of TFA / TIS / HO may be performed after filtration. The combined filtrate may be stored, e.g., at room temperature for 2 hours, and then concentrated by purging with a stream of N. The cleaved linear peptide may then be precipitated from cold ether, and the resulting residue may then be dissolved in 50% t-butanol / water and lyophilized to yield the linear peptide.

[0160] The resulting crude linear peptide may then be cyclized in solution by conventional reaction procedures for cyclization via amide bond condensation. The linear peptide is first dissolved in a suitable solvent such as DMF, tetrahydrofuran (THF), DCM, or 1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include, for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), HBTU, benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), or N,N'-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole (DCCI / HOBt). Coupling is usually initiated by the use of a suitable base such as DIPEA, sym-collidine or N-methylmorpholine (NMM).

[0161] After solution cyclization, the resulting mixture may be concentrated by known means and then partially purified, for example, by trituration with methyl tert-butyl ether (MTBE). The resulting fractions may then be pooled and lyophilized.

[0162] Typically, orthogonal protecting groups may be used as needed. For example, peptides of the present invention have multiple amino acids with amino group-containing side chains. Any of a variety of protecting groups may be used, including orthogonal protecting groups cleavable under different reaction conditions, used for allyl-alloy protecting schemes for certain amino acids and other amino acids with amino group-containing side chains. Thus, for example, amino acids with amine group-containing side chains may have different orthogonal protecting groups, e.g., Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Pbf)-OH, Fmoc-Dab(Pbf)-OH. Other protecting groups may be used as well; for example, but not limited to, Mtt (4-methyltrityl) or Mtt / OPp (4-methyltrityl / 2-phenylisopropyl) may be used for the side chain of His, and orthogonal protecting groups may be used at other positions that are not cleavable using conditions suitable for cleavage of Mtt or Mtt / OPp.

[0163] The reactive groups in the peptide can be selectively modified either during solid-phase synthesis or after removal from the resin. For example, the peptide can be modified to obtain an N-terminal modification such as acetylation while still on the resin, or can be removed from the resin by using a cleavage reagent and then modified. Similarly, methods for modifying the side chains of amino acids are well known to those skilled in the art of peptide synthesis. The choice of modifying the reactive groups present on the peptide will be determined, in part, by the characteristics desired in the peptide.

[0164] Although the synthesis is described primarily with reference to solid phase Fmoc chemistry, it should be understood that other chemistries and synthetic methods may be used to make the cyclic peptides of the invention, including, but not limited to, methods using Boc chemistry, solution chemistry, and other chemistries and synthetic methods.

[0165] 6.0 Formulation The formulation of the composition comprising one or more cyclic peptides disclosed herein or of the formula disclosed herein may be modified according to the desired route of administration.Thus, the formulation may be suitable for subcutaneous injection, delayed-release subcutaneous injection, intravenous injection, nasal spray application, inhalation application, oral administration, including but not limited to oral release for the treatment of gastrointestinal disorders, buccal or other mucosal application, other transdermal application, etc.In general, the formulation may be used for any administration form of the peptide of the present invention.

[0166] 6.1 Salt Forms of Cyclic Peptides The cyclic peptides disclosed herein or of the formulas disclosed herein may be in the form of any pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid, including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese salts, manganese, potassium, sodium, zinc, etc. Ammonium, calcium, lithium, magnesium, potassium, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0167] When a cyclic peptide disclosed herein, or of a formula disclosed herein, is basic, acid addition salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carboxylic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, malonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, TFA, and the like. Acid addition salts of peptides disclosed herein or of formulas disclosed herein are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, TFA, citric acid, tartaric acid, maleic acid, succinic acid, or methanesulfonic acid.

[0168] Acetate, ammonium acetate, and TFA salt forms are particularly useful. When a peptide disclosed herein or of a formula disclosed herein contains an acidic moiety, suitable pharmaceutically acceptable salts may include alkali metal salts, such as sodium or potassium salts, or alkaline earth metal salts, such as calcium or magnesium salts. It should also be understood that certain peptides of Formulas (I)-(V) can exist in solvated and unsolvated forms, including solvates of the free peptide or a salt of the compound. The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol. The term "hydrate" is used when the solvent is water. It should be understood that all polymorphs, including mixtures of different polymorphs, are included within the scope of the claimed peptides.

[0169] 6.2 Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising a cyclic peptide disclosed herein or of a formula disclosed herein and a pharmaceutically acceptable carrier.The carrier may be a liquid formulation, preferably a buffer, an isotonic solution, or an aqueous solution.Pharmaceutically acceptable carriers also include excipients such as diluents and carriers, and additives such as stabilizers, preservatives, solubilizers, and buffers, as described below.

[0170] Cyclic peptide compositions disclosed herein or of a formula disclosed herein may be formulated or compounded into pharmaceutical compositions comprising at least one cyclic peptide disclosed herein or of a formula disclosed herein, together with one or more pharmaceutically acceptable carriers, including excipients such as diluents, carriers, and additives such as stabilizers, preservatives, solubilizers, and buffers, as may be desired. Formulation excipients may include polyvinylpyrrolidone, gelatin, hydroxypropyl cellulose, acacia, polyethylene glycol, mannitol, sodium chloride, and sodium citrate. For injection or other liquid administration formulations, water containing at least one or more buffer components is preferred, and stabilizers, preservatives, and solubilizers may also be used. For solid administration formulations, any of a variety of thickeners, fillers, extenders, and additive carriers, such as starches, sugars, cellulose derivatives, fatty acids, and the like, may be used. For topical administration formulations, any of a variety of creams, ointments, gels, lotions, and the like may be used. In most pharmaceutical formulations, inactive ingredients will comprise a larger portion of the formulation by weight or volume. It is also contemplated that pharmaceutical formulations may be formulated in dosages that provide for extended delivery of the peptides disclosed herein or of the formulas disclosed herein, by using any of a variety of measured-release, delayed-release, or sustained-release formulations and additives.

[0171] Generally, the actual amount of a cyclic peptide disclosed herein or of a formula disclosed herein administered to a patient will vary over a fairly wide range, depending on the mode of administration, the formulation used, and the response desired.

[0172] In practice, the cyclic peptides disclosed herein or of the formulas disclosed herein can be combined as an active ingredient in a mixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, such as oral, parenteral (including intravenous), urethral, ​​vaginal, nasal, buccal, or sublingual. In preparing compositions for oral dosage forms, any of the following carriers may be used: conventional pharmaceutical media, such as water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents, for oral liquid preparations such as suspensions, elixirs, and solutions; or starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like, for oral solid preparations such as powders, hard and soft capsules, and tablets.

[0173] Tablets and capsules represent an advantageous oral dosage unit form due to their ease of administration. If necessary, tablets may be coated by standard aqueous or non-aqueous techniques. Due to the amount of active peptide in such therapeutically useful compositions, an effective dosage will be obtained. In another advantageous dosage unit form, sublingual structures such as sheets, wafers, tablets, etc. may be used.

[0174] Tablets, pills, capsules, etc. may further contain binders such as povidone, gum tragacanth, acacia, corn starch, or gelatin; diluents; fillers such as microcrystalline cellulose; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, or alginic acid; preservatives; coloring agents; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil. Various other materials may be used as coating agents or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor.

[0175] When formulated for oral delivery, the peptide may be formulated and prepared so that it is enclosed in an enteric protective agent, more preferably so that it is not released until the tablet or capsule has passed through the stomach and, optionally, further through a portion of the small intestine. In the context of this application, the term enteric coating or material will be understood to refer to a coating or material that will pass through the stomach essentially intact but will disintegrate and release the active ingredient after passing through the stomach. Materials that may be used include cellulose acetate phthalate, hydroxypropylmethyl-ethylcellulose succinate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, and methacrylic acid-methyl methacrylate copolymer. The enteric coating used may be selected to promote dissolution of the dosage form primarily outside the stomach and so that the enteric coating dissolves at a pH of at least about 5.5, more preferably from about 6.0 to about 8.0.

[0176] Upon dissolution of the enteric coating, any of a variety of permeation enhancers may be used to increase intestinal uptake. In one aspect, the permeation enhancer enhances either the paracellular or transcellular transport system. Representative, non-limiting examples of such permeation enhancers include calcium chelators, bile salts (such as sodium cholate), and fatty acids. In some embodiments, a peptide or polypeptide that acts as a substrate for intestinal proteases is further added.

[0177] Cyclic peptides may also be administered parenterally. Solutions or suspensions of these active peptides may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Furthermore, dispersions may be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. These preparations may optionally contain preservatives to prevent the growth of microorganisms.

[0178] Pharmaceutical dosage forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the dosage form must be sterile and must be fluid to the extent that it can be administered by syringe.The dosage form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols such as glycerol, propylene glycol or liquid polyethylene glycol, suitable mixtures thereof, and vegetable oils.

[0179] The cyclic peptides disclosed herein may be therapeutically applied via nasal administration. The peptides may be present in aqueous solutions, such as solutions containing saline, citrate, or other common excipients or preservatives, as well as in absorption or permeation enhancers, cell permeation enhancers, mucoadhesive polymers, and various carrier systems. The peptides may also be present in dry or powder formulations. The cyclic peptides disclosed herein or of the formulas disclosed herein may be formulated with any of a variety of agents that enhance the effective nasal absorption of drugs, including peptide drugs. These agents may enhance nasal absorption without unacceptable damage to the mucosa. In particular, U.S. Patent Nos. 5,693,608, 5,977,070, and 5,908,825 teach several pharmaceutical compositions that may be used, including absorption enhancers; the teachings of each of the foregoing, as well as all references and patents cited therein, are incorporated by reference.

[0180] When present in aqueous solution, the cyclic peptide may be suitably buffered using saline, acetate, phosphate, citrate, acetate, or other buffers at any physiologically acceptable pH, generally about pH 4 to about pH 7. Additionally, combinations of buffers, such as phosphate-buffered saline, saline, and acetate buffer, may be used. In the case of saline, 0.9% saline may be used. In the case of acetate, phosphate, citrate, etc., 50 mM solutions may be used. In addition to the buffer, a suitable preservative may be used to prevent or limit the growth of bacteria and other microorganisms. One such preservative that may be used is 0.05% benzalkonium chloride.

[0181] In an alternative embodiment, the cyclic peptides disclosed herein or of the formulas disclosed herein may be administered directly to the lung. Pulmonary administration may be accomplished using a metered-dose inhaler, a device that, when actuated by the patient during inspiration, allows for self-administration of a metered bolus of the peptides disclosed herein or of the formulas disclosed herein. In one aspect of this embodiment, the cyclic peptide may be present in a dry, particulate form, e.g., particles between about 0.5 and 6.0 μm, such that the particles have sufficient mass to settle on the lung surface without being exhaled, but are small enough not to deposit on the surface of the airways before reaching the lung. Any of a variety of different techniques may be used to create dry powder particulates, including, but not limited to, micromilling, spray drying, and freeze-drying after rapid-freezing aerosolization. In the case of particulates, the peptide may be deposited deep into the lung, thereby resulting in rapid and efficient absorption into the bloodstream. Furthermore, with such approaches, permeation enhancers are sometimes not required, such as in the case of transdermal, nasal, or oral mucosal delivery routes. Any of a variety of inhalers can be used, including propellant-based aerosols, nebulizers, single-dose dry powder inhalers, and multi-dose dry powder inhalers. Common devices currently in use include metered-dose inhalers, which are used to deliver medications for the treatment of asthma, chronic obstructive pulmonary disease, and the like. Preferred devices include dry powder inhalers designed to produce a fine cloud or aerosol with a particle size consistently less than about 6.0 μm.

[0182] Microparticle size, including average size distribution, may be controlled using the fabrication method. In micromilling, the size of the milling head, rotor speed, processing time, etc., control the microparticle size. In spray drying, the nozzle size, flow rate, dryer heat, etc., control the microparticle size. In fabrication using quick-freeze aerosol followed by freeze-drying, the nozzle size, flow rate, concentration of the aerosolized solution, etc., control the microparticle size. These parameters and others may be used to control the microparticle size.

[0183] Cyclic peptides disclosed herein or of a formula disclosed herein may be therapeutically administered using injections of sustained-release formulations. In one embodiment, cyclic peptides disclosed herein or of a formula disclosed herein are formulated for deep intramuscular injection, for example, in the gluteus or deltoid muscles, with polyethylene glycol, such as polyethylene glycol 3350, and optionally one or more additional excipients and preservatives, including, but not limited to, excipients such as salt, polysorbate 80, sodium hydroxide, or hydrochloric acid for pH adjustment. In another embodiment, cyclic peptides disclosed herein or of a formula disclosed herein are formulated with poly(orthoesters), which may be autocatalytic poly(orthoesters) with varying percentages of lactic acid in the polymer backbone, and optionally one or more additional excipients. In one embodiment, poly(D,L-lactide-co-glycolide) polymers are used. In general, in one aspect, any of several injectable biodegradable polymers, preferably also adhesive polymers, may be used in sustained-release injectable formulations. Alternatively, other sustained release formulations may be used, including formulations that allow for subcutaneous injection, and other formulations may include one or more of nano / microspheres (such as compositions comprising PLGA polymers), liposomes, emulsions (such as water-in-oil emulsions), gels, insoluble salts, or suspensions in oil. Formulations may be made so that injections are required on a daily, weekly, monthly, or other periodic basis, depending on the concentration and amount of cyclic peptide, the sustained release rate of the material used, and other factors known to those skilled in the art.

[0184] 6.3 Route of Administration When a composition comprising one or more peptides disclosed herein or of a formula disclosed herein is administered by injection, the injection may be intravenous, subcutaneous, intramuscular, intraperitoneal, or other means known in the art. The peptides disclosed herein or of a formula disclosed herein may be formulated by any means known in the art, including, but not limited to, tablets, capsules, caplets, suspensions, powders, lyophilized formulations, suppositories, ocular drops, skin patches, orally soluble formulations, sprays, aerosols, and the like, and may be mixed with and formulated with buffers, binders, excipients, stabilizers, antioxidants, and other agents known in the art. Generally, any route of administration may be used in which the peptides of the present invention are introduced through the epidermal layer of cells. Thus, administration routes may include mucosal, buccal, oral, transdermal, inhalation, nasal, urethral, ​​intravaginal, and the like.

[0185] 6.4 Therapeutically Effective Dose Generally, the actual amount of cyclic peptides disclosed herein or of the formulas disclosed herein administered to a patient will vary widely depending on the mode of administration, the formulation used, and the desired response. Therapeutic dosages are administered by any of the above means or any other means known in the art in an amount sufficient to produce the desired therapeutic effect. The cyclic peptides disclosed herein or of the formulas disclosed herein are generally highly active. For example, cyclic peptides can be administered at about 0.001, about 0.01, about 0.1, about 0.5, or about 1 μg / kg body weight, depending on the specific peptide selected, the desired therapeutic response, the route of administration, the formulation, and other factors known to those skilled in the art.

[0186] 7.0 Tests and Assays Used in Evaluating Peptides Peptides specific for the melanocortin receptors disclosed herein or of the formulas disclosed herein may be tested in a variety of assay systems and animal models to determine binding, functional status and efficacy.

[0187] 7.1 Assays for agonist activity performed at CEREP To assess the agonist activity of compounds at melanocortin receptors, their effects on cAMP production were determined by measuring them using the HTRF detection method at CEREP (Eurofins CEREP SA, Celle-Levescault, France). Cells were suspended in HBSS buffer (Invitrogen) supplemented with 20 mM HEPES (pH 7.4) and 500 μM IBMX, then distributed into microplates and incubated in the presence of HBSS (basal control), test compound, or reference agonist. Incubation time, temperature, cell number, reference agonist, and cell line information are included in Table 1 below. For stimulation control measurements, separate assay wells contained the reference compound. After incubation, cells were lysed, and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) were added. After 60 min at room temperature, fluorescence input was measured using a microplate reader (Envision, Perkin Elmer) at an excitation wavelength of 337 nm and emission wavelengths of 620 and 665 nm. The cAMP concentration was determined by dividing the signal measured at 665 nm by the signal measured at 620 nm (ratio).

[0188] Results are expressed as a percentage of the control response to a 1 μM reference. A standard reference agonist was tested at several concentrations in each experiment and its EC 50 A concentration-response curve is constructed from which values ​​are calculated.

[0189] [Table 6]

[0190] 7.2 Assays for antagonist activity performed at CEREP This assay was used to evaluate the antagonist activity of compounds at melanocortin receptors, determined by measuring their effect on cAMP production using HTRF detection methods.

[0191] The desired cells bearing melanocortin receptors were suspended in HBSS buffer (Invitrogen) supplemented with 20 mM Hepes (pH 7.4) and 500 μM IBMX and then distributed onto a microplate in the presence of HBSS (basal control), test compound, or reference antagonist. A concentration of agonist was then added to stimulate cAMP production. For basal control measurements, separate assay wells contained no reference agonist. Information on incubation time, temperature, cell number, reference agonist, and cell line is included in Table 2 below.

[0192] After incubation, cells are lysed and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 minutes at room temperature, fluorescence input is measured using a microplate reader (Envision, Perkin Elmer) at an excitation wavelength of 337 nm and emission wavelengths of 620 and 665 nm. The cAMP concentration is determined by dividing the signal measured at 665 nm by the signal measured at 620 nm (ratio). Results are expressed as percent inhibition of the control response to a reference agonist. A standard reference antagonist is tested at several concentrations in each experiment, and its IC 50 A concentration-response curve is constructed from which values ​​are calculated.

[0193] [Table 7]

[0194] 7.3 Alternative Assays for Agonist Activity Intracellular cAMP accumulation was tested as a measure of the ability of peptides to elicit functional responses in either HEK-293 cells (mouse) expressing recombinant MC3r, MC4r, or B16-F10, or in the HBL (human) cell line expressing native MC1r. Confluent cells were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Hank's balanced salt solution containing 10 mM Hepes (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin, and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. Cells were cultured at 0.5 × 10 5 Cells were distributed into 96-well plates at a density of 10 cells / well and preincubated for 10 minutes. Cells were exposed to peptides dissolved in DMSO (1% final DMSO concentration) at concentrations ranging from 0.05 to 5000 nM in a total assay volume of 200 μL for 15 minutes at 37°C. NDP-α-MSH was used as a reference agonist. cAMP levels were determined using the HTRF® cAMP cell-based assay system from Cisbio Bioassays, using cryptate-labeled anti-cAMP and D2-labeled cAMP, and plates were read at 665 and 620 nM on a Perkin-Elmer Victor plate reader. Data analysis was performed by nonlinear regression analysis using Graph-Pad Prism® software. The maximum efficacy of the test peptides was compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

[0195] 7.4 High and Low Density hMC4r Functional Assays The HEK293 cell line transfected with human MC4r (Palatin Technologies, USA, licensed from the University of Michigan) was used. Human MC4r was introduced into HEK293 cells using the T-REx™ System (Invitrogen). The T-REx™ System employs a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon. Using the T-REx™ System, expression of the gene of interest, the human MC4r gene, is repressed in the absence of tetracycline or doxycycline and induced in the presence of tetracycline or doxycycline (see the T-REx™ System Manual published by Invitrogen).

[0196] HEK293-T-REx-MC4r cells were cultured in DMEM (Gibco 11965) supplemented with L-glutamine (Gibco 25030), 10% fetal bovine serum (FBS), 200 μg / mL Zeocin (Invitrogen 46-0072), and 6 mg / mL blasticidin (Invitrogen 46-1120) at 37°C, 5% CO2, and 95% humidity. A T-150 flask of cells at 75% confluence was incubated with two concentrations of doxycycline (0.1 ng / mL to generate low-density hMC4r lines and 10 ng / mL to generate high-density hMC4r lines) for 16–18 hours at 37°C, 5% CO2, to induce MC4r expression. On the day of the assay, cells were washed with PBS (Gibco 14190), collected using cell dissociation buffer (Gibco 13150-016), centrifuged, and resuspended in Hank's balanced salt solution (+Ca, +Mg) (Gibco 14025), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.4 (Sigma H0887), 1 mM L-glutamine (Gibco 25030), 1 mg / mL bovine serum albumin (BSA) (Sigma A3311), and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX).

[0197] The cells were then plated in 198 μL (approximately 5 × 10 4 Cells were distributed at 100 μL total assay volume, 10 cells / well and incubated for 10 minutes at 37°C. -5 ~10 -13 The cells were exposed to peptides dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 1 M, and NDP-α-MSH was used as a reference agonist. The reaction was stopped by adding 15 μL of lysis buffer / well, and the plate was shaken for 30 min at room temperature.

[0198] cAMP levels were determined using the HTRF® cAMP cell-based assay system from Cisbio Bioassays, using cryptate-labeled anti-cAMP and d2-labeled cAMP, and plates were read at 665 and 620 nM on a Perkin-Elmer Victor plate reader. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism® software. The maximum efficacy of the test peptides was compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

[0199] Agonist stimulation of MC4r activates adenylate cyclase, an enzyme that catalyzes the formation of 3',5'-cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Therefore, agonist stimulation of MC4r increases cAMP levels. cAMP levels were measured using the cAMP dynamic 2HTRF kit (CisBio catalog number 62AM4PEC; see the instructions published by CisBio). cAMP levels were normalized to plate controls (1% DMSO for 0% and 400 nM NDP-α-MSH for 100%) and a calibration curve ranging from 712 nM to 0.04 nM cAMP (as described in the CisBio HTRF kit). Plates were incubated on a shaker at room temperature for 1 hour and read at 665 and 620 nm on a Perkin-Elmer Victor plate reader. Fluorescence ratios were then calculated as described in the CisBio HTRF kit, and GraphPad Prism software was used to plot the change in percent fluorescence versus cAMP concentration using a variable slope dose-response curve. EC values ​​were calculated based on the calculated cAMP concentrations. 50 and E max The value was determined.

[0200] 8.0 Example Peptide Structures In one aspect, cyclic peptides are provided having a core sequence derived from the sequence His-Phe-Arg-Trp within the cyclic moiety, or modifications thereof, cyclized via the side chains of the amino acids immediately adjacent to the N-terminal His (or derivatives, modifications, or substitutions of His) and the C-terminal group of the peptide. The cyclic peptides are cyclic pentapeptides having at least five amino acids within the cyclic moiety, and optionally cyclic hexapeptides, heptapeptides, or larger cyclic peptides having one or more additional amino acid residues outside the N-terminal cyclic moiety.

[0201] In MC4r antagonists, which may also contain MC1r, MC3r, or MC54 agonists, or combinations thereof, the core sequence derived from His-Phe-Arg-Trp in some embodiments will contain D-Phe at the Phe position rather than L-Phe, Na1, or Na12 substitutions at the Phe position, such as D-Nal1 or D-Nal2, or alternatively, may contain a substituted Phe at the Phe position, such as substituted D-Phe or substituted L-Phe. Various amino acids may be used for the remaining amino acids in the core sequence. In general, the His position may be substituted or unsubstituted Pro, or an amino acid having a side chain containing at least one primary amine, secondary amine, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, alcohol, ether, sulfide, sulfone, sulfoxide, carbamoyl, or carboxyl. Arg positions may be substituted or unsubstituted Pro or may be an amino acid having a side chain containing at least one primary amine, secondary amine, guanidine, urea, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or ether. Trp positions may be an amino acid having a side chain containing at least one substituted or unsubstituted aryl or heteroaryl, or may alternatively be omitted.

[0202] The peptides encompassed within formulas (I), (II), (III), (IV), and (V) possess one or more asymmetric arrangements, such as asymmetric centers or axes, such that the peptides encompassed within such formulas may exist in different stereoisomeric forms. For both specifically described peptides and generically described peptides, including those encompassed within formulas (I), (II), (III), (IV), and (V), all isomeric forms at all chiral or other isomeric centers, including enantiomers and diastereomers, are intended to be encompassed herein. The peptides of the present invention each contain multiple chiral centers and may be used as racemic mixtures or enantiomerically enriched mixtures, in addition to using the peptides of the present invention in enantiopure formulations. Typically, the peptides of the present invention will be synthesized by the use of chirally pure reagents, such as specific L- or D-isomer amino acids, using reagents, conditions, and methods such that enantiomeric purity is maintained, although it is possible and contemplated that racemic mixtures may be produced. Such racemic mixtures may optionally be separated using well-known techniques, and the individual enantiomers may be used alone. Under certain conditions, such as temperature, solvent, and pH, at which the peptide exists in tautomeric forms, each tautomeric form is considered to be included in the present invention, regardless of whether it exists in equilibrium or predominantly in one form. Optically active forms, therefore, of the peptides of Formulas (I)-(V), as single enantiomers, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or resolution of the racemates.

[0203] Although the peptides disclosed herein are particular stereoisomeric forms of the peptides of Formulas (I)-(V), the present invention should not be construed as being limited to the stereoisomeric forms encompassed by the peptides disclosed herein.

[0204] The present invention further contemplates prodrugs of the present peptides, which upon administration undergo chemical conversion by metabolic processes before becoming pharmacologically active peptides. Generally, such prodrugs will be functional derivatives of the present peptides that are readily convertible in vivo to the peptides of formulas (I)-(V). A prodrug is any covalent compound that releases the active parent peptide drug of formulas (I)-(V) in vivo. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Typical examples of prodrugs have biologically labile protecting groups on functional groups, for example, by esterification of a hydroxyl, carboxyl, or amino function. Thus, for example, and without limitation, prodrugs include peptides of Formula (I), (II), or (III) in which an ester prodrug form is used, such as, for example, a lower alkyl ester of the R group of Formula (I), (II), or (III) (e.g., R is -OH), where the lower alkyl ester may contain 1 to 8 carbons in the alkyl radical or an aralkyl ester (having 6 to 12 carbons in the aralkyl radical). Generally, prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated in vivo to generate the active parent peptide drug of Formula (I).

[0205] The present invention also includes peptides that correspond to those set forth in formula (I) except for the fact that one or more atoms depicted in formula (I) are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into the peptides of the present invention include: 2 H, 3 H, 13 C. 14 C. 15 N, 18 O and 17Included within the scope of the present invention are isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 0. Peptides disclosed herein or of the formulas disclosed herein, and pharmaceutically acceptable salts or solvates of said peptides, that contain the above isotopes and / or other isotopes of other atoms, are included within the scope of the present invention. Certain isotopically labeled peptides, e.g. 3 H and 14 Peptides incorporating radioisotopes such as C may have use in a variety of assays, such as drug and / or substrate tissue distribution assays. Substitution of one or more hydrogen atoms with heavier isotopes, such as deuterium (H), may in some cases provide pharmacological advantages, including increased metabolic stability. Isotopically labeled peptides of formula (I) can generally be prepared by replacing an isotopically labeled reagent with a non-isotopically labeled reagent.

[0206] 9.0 Working Examples The present invention is further illustrated by the following non-limiting examples: The peptides of the following structures were synthesized by the general method described above, and the EC 50 Values ​​were determined as indicated. ECs marked with "*" 50 Values ​​were determined by CEREP. "%" indicates EC 50 E in the case of value max The percentages (percentage of the maximum response obtained with the positive control) are shown. EC of "NC" 50 The value is EC 50 Indicates that the value was greater than 10,000 nM and therefore could not be calculated.

[0207] [Table 8]

[0208] [Table 9]

[0209] [Table 10]

[0210] [Table 11]

[0211] [Table 12]

[0212] [Table 13]

[0213] [Table 14]

[0214] Although the present invention has been described in detail with particular reference to these preferred embodiments, other embodiments may achieve the same results. Variations and modifications of the present invention will be apparent to those skilled in the art, and it is intended to cover all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above are incorporated herein by reference.

Claims

[Claim 1] A peptide selected from the group consisting of the following compounds No. 1 to 19, or a pharmaceutically acceptable salt thereof. 【Chemistry 1-1】 【Chemistry 1-2】 【Chemistry 1-3】 【Chemistry 1-4】 【Chemistry 1-5】 【Chemistry 1-6】 【Chemistry 1-7】

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