KR20070032054A - Cyclic peptides for treatment of cachexia - Google Patents

Abstract

A highly selective melatocortin-4 receptor antagonist cyclic peptide having the following formula and methods for treating weight disorders including cassia, sarcopenia and wasting syndrome, and methods for treating inflammatory and immune diseases.

Wherein R ₁ , R ₂ , R ₃ , R _4a , R _4b , R ₅ , R ₆ , R ₇ , x, y and z are as defined in the specification.

Description

CYCLIC PEPTIDES FOR TREATMENT OF CACHEXIA}

Related application cross-reference

This application is a priority claim application based on US Provisional Patent Application No. 60 / 585,971, filed Jul. 6, 2004, entitled "Capeccia Therapeutic Cyclic Peptides," wherein the specification and provisional claims of the provisional patent application are Reference is incorporated.

Technical Fields:

The present invention is a highly specific antagonist for the melanocortin-4 receptor (MC4-R), but also a variety of body weights including cachexia, sarcopenia and wasting syndrome or wasting disease. Cyclic peptides that can be used for the treatment of related diseases and for the treatment of inflammatory and immune diseases.

Description of related technology:

The following discussion refers to some publications and recent publications of the author (s), and since the publication date is recent, some documents are not considered mutually prior art in relation to the present application. The discussion of these publications in the main text is intended to be a complete description of the background art and does not recognize such publications as prior art for patent evaluation purposes.

Melanocortin Receptor . -1 receptor (MC1-R) expressed in normal human melanocytes and melanoma cells, melanocortin-2 receptor (AC2-R) for ACTH (adrenocorticotropin) expressed in adrenal cells, hypothalamus Melanocortin-3 and Melanocortin-4 receptors (MC3-R and MC4-R) mainly expressed in cells of the brain, midbrain and brain stem, and Melanocortin-5 receptors widely distributed in peripheral tissues (MC5-R ), A group of melanocortin receptor types and subtypes have been identified.

Intensive research has been conducted to determine the structure of the melanocortin receptor, including the nucleic acid sequence encoding the receptor and the amino acid sequence constituting the receptor. MC4-R is believed to be expressed mainly in the brain as a G protein-coupled 7-transmembrane receptor. Inactivation of this receptor by gene targeting in mice has been reported to cause adult obesity syndrome associated with overeating, hyperinsulinemia and hyperglycemia. Hyperglycemia (Huszar D., Lynch CA, Fairchild-Huntress V., et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice Cell 88:. 131-141 (1997)). MC4-R is a molecular target in the therapeutic intervention of energy homeostasis.

In general, compounds specific for MC4-R, and secondary compounds specific for MC3-R and MC5-R, are useful for controlling energy homeostasis in mammals, including their use as drugs to mitigate food intake and weight gain. It is believed to be. MC4-R antagonists are believed to be useful for weight gain and are useful for treating, for example, cachexia, sarcopenia and wasting syndrome or wasting disease and anorexia. In contrast, MC4-R agonists are believed to be effective in treating obesity by reducing food intake and weight gain. In addition, antagonist compounds specific for MC3-R and MC4-R are believed to be useful in regulating blood pressure, heart rate and other neurophysiological variables.

Cajuncia and other wasting diseases. Weight-related disorders include one or more "consumable" disorders (eg, wasting syndrome, cacacia, sarcopenia) that cause undesirable and infrequent weight loss or somatic weight loss. In elderly and cancer patients and in AIDS patients, wasting disease can result in undesirable weight loss, such as reducing both fat and fat free components. Consumable diseases may be due to improper intake of food and / or metabolic changes associated with the disease and / or aging process. Cancer patients, AIDS patients, and patients with severe surgical procedures or those with chronic infections, immunological disorders, hyperthyroidism, Crohn's disease, psychogenic disease, chronic heart failure, or other serious trauma often suffer from this wasting disease. Consumable disease is sometimes referred to as cajuncia and is generally recognized as a metabolic disease and sometimes an eye related disease. Cacexia may also be characterized by the Great Jindae and Ewha Daedaejin. Although cathesia and wasting disease are often used interchangeably to refer to wasting symptoms, there is at least one area that distinguishes cacacia from wasting symptoms, namely, in terms of loss of fat-free weight, especially somatic body weight. (Roubenoff R. The pathophysiology of wasting in the elderly.J. Nutr. 129 (1S Suppl.): 256S-259S (1999)). Another of these diseases, sarcopenia, which affects the elderly, is generally characterized by a loss of muscle weight. The above terminally wasting disease may occur in individuals suffering from Cajuncia or sarcopenia.

Melanocortin antagonist peptide . Antagonist peptides are based on a modification of His-Phe-Arg-Trp (SEQ ID NO: 1), an alpha-melanin cell stimulating hormone (α-MSH) core sequence, and is generally a Phe position, most often 1- or 2- D-amino acids in D-amino acids having a naphthyl ring or phenyl ring (which may be a substituted ring if necessary). Thus, U. S. Patent No. 5,731, 408 is a nonspecific antagonist for the melanocortin receptors MC3-R and MC4-R, and contains cyclic lactam heptapeptides containing D-Phe (4-I) or D-Nal 2 instead of Phe residues. It is starting. Of particular note among those described in US Pat. No. 5,731,408 are peptides commonly called SHU 91 19 (Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -NH ₂ ). SHU91 19 has been widely used in various studies as a reference for non-specific melanocortin antagonists. U. S. Patent No. 6,054, 556 describes related cyclic lactam heptapeptides, which are antagonists for the melanocortin receptors MC1-R, MC3-R, MC4-R and MC5-R. These peptides all contain substituted D-Phe or D-Nal instead of Phe residues as needed. The peptides disclosed in US Pat. Nos. 5,731,408 and 6,054,556 all have C-terminal NH ₂ groups commonly found in melanocortin-specific peptides.

Other patent documents also disclose the use of melanocortin antagonists in the treatment of carcassia and other weight related diseases. See, eg, US Pat. No. 6,716,810; 6,699,873; 6,693,165; 6,613,874; 6,476,187; 6,284,729; 6,100,048; And 5,908,609. However, none of these patent documents discloses the peptide of the present invention. U.S. Patent 6,693,165 discloses cyclic heptapeptides and hexapeptides which are alleged to be selective MC4-R antagonists. These peptides all include D-amino acids containing 1- or 2-naphthyl, 3-benzothienyl or phenyl optionally substituted in place of the Phe residue in the His-Phe-Arg-Trp (SEQ ID NO: 1) core sequence do. However, the peptides described in US Pat. No. 6,693,165 optionally omit His in the His-Phe-Arg-Trp (SEQ ID NO: 1) sequence and are limited to Lys or His when the His position is present. Each of the peptides disclosed in US Pat. No. 6,693,165 and the general formula disclosed in this document have a C terminal NH ₂ group.

U.S. Patent Application 2003/0113263, "Methods and Reagents for Using Mammalian Melanocortin Receptor Antagonists to Treat Cachexia," describes the use of MC4-R antagonists to treat animals with Cactusia, including the use of MC4-R antagonists. Disclosed are methods for characterizing compounds useful for the treatment of animals with carcassia, including, in particular, SHU91 19. U.S. Patent Application 2003/0105024, "Methods and Reagents for Discovering and Using Mammalian Melanocortin Receptor Agonists and Antagonists to Modulate Feeding Behavior in Animals" discloses SHU91 19 as an MC receptor antagonist experimentally used to stimulate food intake activity. have. U.S. Patent 6,476,187, "Methods and Reagents for Discovering and Using Mammalian Melanocortin Receptor Agonists and Antagonists to Modulate Feeding Behavior in Animals", also discloses SHU91 19 as an MC receptor antagonist experimentally used to stimulate food intake activity. U.S. Patent Application Publication 2003/0032791, "Novel Melanocortin-4 Receptor Sequences and Screening Assays to Identify Compounds Useful in Regulating Animal Appetite and Metabolic Rate," describes the experimental use of SHU 91 19 in various assays. U.S. Patent Application 2002/0016291, "Cyclic Peptides as Potent and Selective Melanocortin-4 Receptor Antagonists," discloses SHU 91 19 as an antagonist at MC3 and MC4 receptors. In 1977, SHU91 19 was initiated to promote food intake activity. Fan W., Boston BA, Kesterson RA., Hruby VJ, Cone RD Role of Melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385: 165-168 (1997); See also Rossi M., Kim MS, Morgan DG, et al. A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo. Endocrinology 139: 4428-31 (1998); Wisse BE, Frayo RS, Schwartz MW, Cummings DE Reversal of cancer anorexia by blockade of central Melanocortin Receptor in rats. Endocrinology 142: 3292-3301 (2001); Marks DL, Ling N., Cone RD Role of the central Melanocortin system in Cachexia. Cancer Research 61: 1432-1438 (2001).

There is an urgent need for ligands that have high specificity for isolated melanocortin receptors, particularly MC4-R, and ligands that are antagonists, or inverse agonists of MC4-R, if necessary. In order to reduce unwanted pharmacological responses, ligands are required to be highly specific for target MC receptors such as MC4-R. Thus, a binding affinity such as at least 10 times higher for MC4-R is required compared to other MC receptors. Highly affinity peptides of melanocortin receptors can be used as antagonists or inverse agonists to develop various physiological responses associated with melanocortin receptors. For example, antagonists or inverse agonists of MC4-R can be used to treat eating disorders, wasting diseases, and cassia. In addition, the melanocortin receptor has an effect on the activity of various cytokines, it is possible to control the cytokine activity by using a high affinity peptide ligand of the melanocortin receptor. Thus, such peptide ligands may be used to treat inflammation and other immune diseases.

Summary of the Invention

The present invention provides cyclic peptides having the formula

Wherein R ₁ is H,

R ₃ is 4-imidazolyl or 3-indolyl;

R _4a and R _4b are each any ring substituents, and if one or both are present, they are the same or the same as each other and independently represent a hydroxy, halogen, alkyl, or aryl group, which can be attached directly or via an ether bond;

R ₅ is —NH ₂ or —NH (C═NH) NH ₂ ;

R ₆ is 1- or 2-naphthyl or 3-indolyl, optionally having one or two ring substituents, and when one or both ring substituents are present, they are the same or different from one another and are independently hydroxy, halogen, alkyl, Or aryl groups, which can be attached directly or via an ether bond;

;R ₇ is -OH or

;

R ₈ is H, NH ₂ , lower aliphatic C ₁ to C ₄ straight or branched alkyl chains, C ₁ to C ₄ aralkyl, or C ₁ to C ₄ omega amino derivatives;

R ₉ is H, lower aliphatic C ₁ to C ₄ straight or branched alkyl chain, C ₁ to C ₄ aralkyl, or C ₁ to C ₄ omega amino derivatives;

R ₁₀ is aliphatic L- or D-amino acid, N-acylated L- or D-amino acid or straight or branched C ₁ to C ₁₇ alkyl, aryl, heteroaryl, alkene, alkenyl, or aralkyl chain;

R ₁₁ and R ₁₂ are each independently H or C ₁ to C ₄ straight or branched alkyl chains, provided that both R ₉ and R ₁₀ are not H;

x is 1 to 4, y is 1 to 5, provided that x + y is 2 to 7;

z is 2 to 5.

In one embodiment, the cyclic peptides of the invention have formula II:

Wherein R ₃ and R ₆ are as defined for the peptide of formula (I) above. Representative cyclic peptides of formula (II) include:

Ac- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH;

Ac- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH; or

Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH.

In another embodiment, the cyclic peptides of the invention have the formula (III):

Wherein R ₃ and R ₆ are as defined in the peptide structure of formula (I) above. Representative cyclic peptides of formula (III) include:

Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH;

Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH; or

Ac-Nle- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH.

In another embodiment, the cyclic peptides of the invention have the formula (IV):

Wherein R ₃ , R ₆ , R ₁₁ and R ₁₂ are as defined for the peptide structure of formula (I). Representative cyclic peptides of formula (IV) include:

Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃ ;

Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂ ; or

Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ .

In another embodiment, the cyclic peptide has the formula (V):

Wherein R ₃ , R ₅ , R ₆ , R ₁₁ , R ₁₂ and z are as defined for the peptide structure of formula (I). Representative cyclic peptides of formula (V) include:

H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃ ;

H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ ;

H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂ ;

H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₂ -CH ₃ ;

H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₃ ; or

H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -N (CH ₃ ) ₂ .

Another embodiment of the present invention provides a pharmaceutical formulation comprising a cyclic peptide of formula (I) to (V) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Yet another embodiment of the present invention provides a method of treating Cajuncia. This method comprises administering a pharmaceutically sufficient amount of the pharmaceutical formulation provided above. Another embodiment of the invention provides a method of treating inflammation and immune related diseases. This method comprises administering a pharmaceutically sufficient amount of the pharmaceutical formulation provided above.

In another embodiment, the present invention provides cyclic hexapeptides having C-terminal hydroxyl or N-alkyl groups, wherein the N-alkyl groups have one or two C ₁ to C ₄ straight or branched alkyl chains. The hexapeptide contains a core sequence of His-D-Nal 2-XY or Trp-D-Nal 2-XY, where X is an L-amino acid selected from Arg, Lys, Orn, Harg and Hlys and Y is Nal L- or D-amino acid selected from 1, Nal 2 and Trp, wherein any aromatic ring in the core sequence optionally includes one or two substituents, and when one or both rings are present, they are the same or different and independently Selected from hydroxyl, halogen, alkyl, or aryl groups attached directly or through an ether bond. In one embodiment, the cyclic hexapeptide has an N-terminal Ac or NH ₂ group. This cyclic hexapeptide can be cyclized by forming an amide bond between the amino group of the side chain of the amino acid at position 1 or the amino group of the N-terminal group of the amino acid at position 1 and the side chain carboxyl group of the amino acid residue at position 6. Alternatively, this cyclic hexapeptide may be cyclized by the formation of an amide bond between the side chain carboxyl of the amino acid residue at position 1 and the side chain amino group of the amino acid at position 6. In another example, the cyclic peptide may be used to form covalent bonds including amide, disulfide, thioether, Schiff base, reduced Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. May be cyclized. In a preferred embodiment of the cyclic hexapeptide, the core sequence is at positions 2-5 and is His-D-Nal 2-X-Nal 2 and cyclized via amino acids at positions 1 and 6. In another preferred embodiment of the cyclic hexapeptide, the core sequence is at positions 2-5 and is Trp-D-Nal 2-X-Nal 2 and cyclized via amino acids at positions 1 and 6. For cyclic hexapeptides according to another preferred embodiment, the core sequence is at positions 2 to 5 and is His-D-Nal 2-X-Trp and is cyclized via amino acids at positions 1 and 6. The position is determined by conventional methods of counting the position of amino acid residues from the N-terminus to the C-terminus.

One object of the present invention is to provide a medicament for the treatment of melanocortin receptor-specific cassistia based on peptides, wherein the peptides are highly selective MC4-R antagonists or inverse agonists.

Another object of the present invention is to provide a medicament for the treatment of melanocortin receptor-specific caschesia based on peptides, wherein the peptide has a C-terminal hydroxyl.

Yet another object of the present invention is to provide a medicament for the treatment of melanocortin receptor-specific caschesia based on a peptide, wherein the peptide has a C-terminal N-alkyl group.

It is another object of the present invention to provide peptides which are highly specific for the melanocortin receptor MC4-R and which are antagonists or inverse agonists.

It is still another object of the present invention to provide a medicament for the treatment of melanocortin receptor-specific inflammation and other immune related diseases based on peptides.

It is another object of the present invention to provide a melanocortin receptor-specific therapeutic medicament based on peptides, which is used for treatment by nasal administration.

According to another embodiment of the present invention, the C-terminal hydroxyl cyclic peptide, which is a highly specific MC4-R antagonist or inverse agonist, suitable for use as a medicament specific for treating eating-related diseases and effective in small amounts. Is provided.

Another embodiment of the present invention provides a C-terminal N-alkyl cyclic peptide, which is a highly specific MC4-R antagonist or inverse agonist suitable for use as a specific medicament for the treatment of eating-related diseases and which is effective in small amounts. do.

Another aspect of the invention provides highly specific MC4-R cyclic peptide antagonists or inverse agonists that are effective in a significant dosage range.

Another aspect of the present invention provides highly specific MC4-R cyclic peptide antagonists or inverse agonists used to treat eating disorders, which are highly potent in small amounts, and therefore, with conventional intravenous, subcutaneous or intramuscular injections. Administration via the same route as well as oral delivery, nasal delivery system and mucosal delivery system is not limited to these routes of administration.

Other objects, advantages, new features and additional applications of the present invention are set forth in the following detailed description together with the accompanying drawings, and those skilled in the art may clearly understand the following description or practice the present invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

Definition Certain terms used throughout this specification and claims are defined as follows.

The terms "bind", "binding", "complex", "complexing" generally refer to all types of physicochemical bonds, throughout the specification and claims, Reaction, compounding, pulling, and chelating.

In the present invention, "peptide" means a) natural, b) produced by chemical synthesis, c) produced by recombinant DNA technology, d) produced by biochemical or enzymatic fragmentation of larger molecules Or e) produced by a combination of the methods of a) to d), or f) produced by any other means for producing a peptide.

By using chemical synthesis, which is the preferred means of production, it is possible to introduce various non-naturally occurring amino acids into the chain or to modify the N- or C-terminus, thereby improving stability and formulation and making them resistant to protease degradation. You can enjoy such advantages.

As used herein, the term "peptide" encompasses any structure comprising two or more amino acids, including chemical modifications and amino acid derivatives. The amino acids forming all or part of a peptide may be natural amino acids, stereoisomers or variants of these amino acids, nonprotein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, structures or structures designed to mimic amino acids, and the like. Accordingly, the term "peptide" includes both pseudopeptides and peptidomimetics, including structures having a non-peptide backbone. The term "peptide" is also a dimer or multimer of a peptide. A “manufactured” peptide refers to a peptide that is made by chemical synthesis, recombinant DNA technology, biochemical or enzymatic fragmentation of larger molecules, combinations thereof, or other methods.

In the present invention, including the specification and claims, the term "amino acid side chain moidty" includes all of the side chains of "amino acid" as defined herein. Thus the term also includes side chain moieties present in natural amino acids. The term also encompasses the side chain portion of a propagated natural amino acid, such as glycosylated amino acid. The term also encompasses stereoisomers such as natural protein amino acids, nonprotein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derived amino acids, structures or structures designed to mimic amino acids, and side chain portions in the variants. For example, side chain portions of all amino acids disclosed herein are included. The term "derivative" of an amino acid side chain portion is encompassed by the definition of an amino acid side chain portion.

A "derivative" of an amino acid side chain portion includes any modification or variation of the amino acid side chain portion, including modifications of the natural amino acid side chain portion. Examples of derivatives of amino acid side chain moieties include straight or branched chain, cyclic or bicyclic, substituted or unsubstituted, saturated or unsaturated alkyl, aryl or aralkyl moieties.

As used herein in the embodiments of the present invention, "amino acids" and as used herein and in the claims, encompass both known natural protein amino acids, generally referred to as three letter abbreviations and one letter abbreviations. See, eg, Synthetic Peptides: A User's Guide , GA Grant, editor, WH Freeman & Co., New York (1992). The disclosure of this document is incorporated herein by reference in its entirety, including its text and the tables set forth on pages 11-24. As mentioned above, the term "amino acid" refers to stereoisomers and variants, such as natural protein amino acids, nonprotein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derived amino acids, structures or structures designed to mimic amino acids. Include all of them. Modified and specific amino acids are generally described in Synthetic Peptides: A User's Guide ; Hruby VJ, Al-obeidi F., Kazmierski W., Biochem. J. 268: 249-262 (1990); And Toniolo C., Int. J. Peptide Protein Res. 35: 287-300 (1990); The disclosures of these documents are all incorporated by reference herein. In addition, the following abbreviations have the meanings given:

7'-Amino-heptanoyl-NH _2- (CH ₂ ) ₆ CO-

Harg-Homo Arginine

Hlys-Homo Lysine

Nal 1-3- (1-naphthyl) alanine

Nal 2-3- (2-naphthyl) alanine

In the list of peptides according to the invention, conventional amino acid residues have the usual meaning as described in the Manual of Patent Examining Procedure , chapter 24008 of the eighth edition. Thus, "Nle" is norleucine, "Asp" is aspartic acid, "His" is histidine, "D-Phe" is D-phenylalanine, "Arg" is arginine, "Trp" is tryptophan, "Lys" is lysine, etc. Point to.

The term "alkene" includes unsaturated hydrocarbons containing one or more carbon-carbon double bonds. Examples of such alkenes include ethylene, propene and the like.

"Alkenyl" means a linear monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched monovalent hydrocarbon radical of 3 to 6 carbon atoms containing at least one double bond; Examples thereof include ethenyl and 2-propenyl.

"Alkyl" groups specified herein include linear or branched alkyl radicals of the indicated length. Examples of such alkyl radicals include methyl, ethyl, propyl, isopropyl, butyl, secondary-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl and the like.

"Alkynyl" includes linear linear hydrocarbon radicals of 2 to 6 carbon atoms or branched monovalent hydrocarbon radicals of 3 to 6 carbon atoms containing at least one triple bond; Examples thereof include ethynyl, propinyl, butynyl and the like.

The term "aryl" includes monovalent or divalent aromatic hydrocarbon radicals of 6 to 12 ring atoms, and such radicals include alkyl, haloalkyl, cycloalkyl, alkoxy, alkylthio, halo, nitro, acyl, cyano, amino And may be independently optionally substituted with one or more substituents selected from mono-substituted amino, disubstituted amino, hydroxy, carboxy or alkoxy-carbonyl. Examples of the aryl group include phenyl, biphenyl, naphthyl, 1-naphthyl and 2-naphthyl, derivatives thereof and the like.

The term "aralkyl" includes the radical -R ^a R ^b where R ^a is an alkylene (divalent alkyl) group and R ^b is an aryl group as defined above. Examples of the aralkyl group include benzyl, phenylethyl, 3- (3-chlorophenyl) -2-methylpentyl and the like.

The term "aliphatic" includes compounds having hydrocarbon chains such as, for example, alkanes, alkenes, alkynes and derivatives thereof.

The term "acyl" includes an RCO- group, where R is an organic group. An example of an acetyl group is CH ₃ CO- abbreviated as “Ac” in the text.

When an alkyl or substituted alkyl group as defined above is bonded through one or more carbonyl {-(C = O)-} groups, the peptide or aliphatic moiety is said to be "acylated".

"Omega amino derivatives" include aliphatic moieties having terminal amino groups. Examples of omega amino derivatives include aminoheptanoyl such as the amino acid side chain portion of lysine and ornithine and 7'-amino-heptanoyl.

"Heteroaryl" includes mono- and bicyclic aromatic rings containing 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur. 5- or 6-membered heteroaryls are monocyclic heteroaromatic rings; Examples thereof include thiazole, oxazole, thiophene, furan, pyrrole, imidazole, isoxazole, pyrazole, triazole, thiadiazole, tetrazole, oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine and the like. Can be mentioned. Bicyclic heteroaromatic rings include benzothiadiazole, indole, benzothiophene, benzofuran, benzimidazole, benzisoxazole, benzothiazole, quinoline, benzotriazole, benzoxazole, isoquinoline, purine, furypyridine and Thieropyridine, including but not limited to.

"Amide" includes compounds having trivalent nitrogen (-CCNH ₂ ) attached to a carbonyl group, such as methylamide, ethylamide, propylamide, and the like.

"Imide" includes compounds containing an imido group (-CO.NH.CO-).

"Amine" includes compounds containing an amino group (-NH ₂ ).

"Nitrile" includes compounds containing (-CN) groups bonded to organic groups as carboxylic acid derivatives.

The term "halogen" encompasses halogen atoms fluorine, chlorine, bromine and iodine and groups containing one or more halogen atoms, such as -CF ₃ .

As in pharmaceutical compositions, "composition" refers to the active ingredient (s) and the inert ingredient (s) constituting the carrier, as well as to the combination, complexation or aggregation of two or more ingredients, directly or indirectly, or one or more It is intended to encompass any product comprising any product resulting from the dissociation of components or by other reactions or interactions of one or more components. Accordingly, the pharmaceutical composition of the present invention encompasses any composition made by mixing the cyclic peptide and the pharmaceutically acceptable carrier of the present invention.

Single amino acids, including stereoisomers and variants, including natural protein amino acids, nonprotein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derived amino acids, structures or structures designed to mimic amino acids, are sometimes In the present invention, it is referred to as "residue".

Melanocortin Receptor By "agonist" is meant a natural substance or a manufactured drug substance or composition that interacts with the melanocortin receptor and initiates a pharmacological response characteristic to the melanocortin receptor. Melanocortin Receptor The term "antagonist" means a natural substance or a manufactured drug substance or composition that counteracts a melanocortin receptor-related response usually caused by a melanocortin receptor agonist. Melanocortin Receptor "Inverse agonist" refers to a drug or compound that stabilizes the inactive arrangement of the Melanocortin receptor to inhibit basal activity.

"Eating disorders" refers to diseases associated with underweight, cacacia, anorexia, or bulimia, whatever the cause for a person.

"Cachexia" generally refers to an unhealthy and malnourished condition. This symptom is often associated with or caused by malignant cancer, cystic fibrosis or AIDS, and is characterized by loss of appetite, weight loss, especially lean body mass and muscle wasting.

"Anorexia" refers to a simple loss of appetite caused by medical, physiological or mental factors. Anorexia is often closely linked to and encourages cathesia, which is observed in patients with advanced cancer and other symptoms.

Cyclic Peptides of the Invention

According to one embodiment of the invention, the core sequence His-D-Nal 2-Arg-Nal 2, Trp-D-Nal 2-Arg-Nal 2, His-, including peptides having one or more substituted ring groups in the core sequence Cyclic peptides are provided comprising D-Nal 2-Arg-Trp or homologs or analogs thereof. In each of the foregoing materials, Arg may be substituted with Lys. Another embodiment of the invention is the core sequence His-D-Nal 2-Arg-Nal 2, Trp-D-Nal 2-Arg-Nal 2, His-D-Nal 2-Arg-Trp, or homologs thereof Or a cyclic peptide comprising an analog, wherein Arg may be replaced with Lys, wherein the peptide is deaminoated, ie without the -NH ₂ group at the C-terminus. In a preferred embodiment, the deaminoated α-MSH cyclic peptides of the invention have a -OH group at the C-terminus and are thus in the free acid form of the cyclic peptide. In another preferred embodiment, the peptide may have a substituted amide, in particular an N-alkyl group at the C-terminus.

Another aspect of the invention provides particular cyclic peptides that are highly specific for the melanocortin receptor, preferably MC4-R, or both MC4-R and MC3-R. Most preferably this cyclic peptide binds to MC4-R with high affinity and has a Ki value of at least 100 nM, preferably at least 10 nM and most preferably from about 0.01 nM to about 2 nM. In some embodiments the cyclic peptide is a functional inverse agonist with respect to such a receptor or receptors. However, the peptides of the present invention need not be inverse agonists. Such peptides can be preferably used to treat eating related diseases, and include, by way of non-limiting example, partial weight gain in mammals such as rodents, dogs, and humans.

Peptides of the invention are cyclic peptides. Cyclic peptides can be obtained by inducing covalent bond formation between the N-terminal (if provided) amino group and the C-terminal (if provided) carboxyl group of the peptide. Cyclic peptides can also be obtained by forming a covalent bond between the terminal reactive group and the reactive amino acid side chain moiety, or between two reactive amino acid side chain moieties. Cyclic peptides can also be obtained by forming disulfide covalent bonds between two sulfhydryl group containing amino acid side chain moieties or between the terminal sulfhydryl group and the sulfhydryl group of the other amino acid side chain moiety. Peptides having lanthionine, cystathionine or pentionine covalent bonds can also be obtained, such as cyclic bonds formed from cysteine, homocysteine or penicylamine amino acid residues. Galande AK, Spatola AF Lett. , Which discloses a method for synthesizing such bonds . Pept. Sci. 8: 247 (2001) is incorporated herein by reference. Thus, a cyclic peptide may be obtained by forming a thioether covalent bond between two reactive amino acid side chain moieties or between a terminal reactive group and a reactive amino acid side chain moiety. Those skilled in the art will recognize that the means for making a given peptide a cyclic peptide may be determined depending on the reactive groups present on the peptide and the desired properties of the peptide.

The cyclic peptides disclosed in some embodiments of the present invention have the preferred feature of being highly selective for MC4-R in part. For example, in the case of SHU91 19, the ratio of Ki value for MC4-R and Ki value for MC3-R was less than about 1: 6 under the analytical conditions used in the present invention, and the ratio of the ratio of MC4-R to MC5-R The case was less than about 1: 3 and the ratio of MC4-R to MC1-R was less than about 1: 7. Other researchers (eg, Schioth HB et al. Peptides 18: 1009-1013 (1997)) infer that SHU91 19 is nonselective, although reporting different values. Thus SHU9119 does not show very high selectivity for MC4-R. In contrast, certain peptides of the invention are much more selective. Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH, a cyclic peptide of Example 16, has a Ki value for MC4-R versus Ki for MC3-R under the same assay conditions. The ratio of values is about 1: 110, the ratio of MC4-R to MC5-R is about 1: 187, and the ratio of MC4-R to MC1-R is about 1: 12,095. Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ , the cyclic peptide of Example 19, has a Ki-value for MC4-R versus MC3-R under the same assay conditions. The ratio of Ki to R is about 1:54, the ratio of MC4-R to MC5-R is about 1: 101 and the ratio of MC4-R to MC1-R is about 1: 10,531. Thus, at pharmaceutically reasonable dosage levels, the cyclic peptides of the invention can be seen as highly selective for MC4-R.

The cyclic peptides disclosed through some embodiments of the present invention are not agonists for any MC receptor and additionally have the property of being inactive or antagonist for all MC receptors other than MC4-R. All cyclic peptides of the invention are functional antagonists for MC4-R. Certain peptides of the invention are partial agonists or antagonists for MC1-R: they are peptides His-D-Nal 2-Arg-Nal 2, His-D-Nal 2-Arg-Trp, or Trp-D-Nal 2 Peptides having an -Arg-Nal 2 core sequence and a C-terminal N-alkyl or hydroxyl. However, these peptides are functional antagonists for MC3-R and MC4-R.

Peptide Synthesis

The cyclic peptides disclosed in some embodiments of the present invention can be readily synthesized by all conventional methods known for forming peptide bonds between amino acids. Examples of such conventional methods include, for example, solid phase processes that cause condensation between the free alpha amino acid of an amino acid residue having a carboxyl group or other protected reactive group and the free primary carboxyl group of another amino acid residue thereof having an amino group or other protected reactive group thereof. Can be mentioned. In a preferred commercial method, the cyclic peptide of the present invention can be synthesized by solid phase synthesis and purified according to a known method. Several well known methods using various resins and reagents can be used to prepare the cyclic peptides of the present invention.

Synthesis of cyclic peptides involves adding each amino acid in a desired sequence one at a time to another amino acid residue one by one or by synthesizing a peptide fragment having a desired amino acid sequence according to the conventional method and then condensing the desired peptide. How to make it. The resulting peptide is then cyclized to make the cyclic peptide of the present invention.

Solid phase peptide synthesis methods are well known and practiced in the art. In this method, the synthesis of the peptides of the present invention can be carried out by sequentially integrating the desired amino acid residues one at a time into the growing peptide chain, according to the general principles of the solid phase method. This method is Merrifield RB, Solid phase synthesis (Nobel lecture). Angew. Chem. 24: 799-810 (1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology , Vol. 2, Gross E. and Meienhofer J., Eds. It is described in various documents, including Academic Press 1-284 (1980).

In chemical synthesis of peptides, the reactive side chains of various amino acid residues are protected with appropriate protecting groups which prevent the chemical reaction from occurring at that position until the protecting group is removed. It is also commonly used to protect the alpha amino group of an amino acid residue or fragment while the whole is reacted with a carboxyl group and then selectively remove the alpha amino protecting group to cause subsequent reactions at that position. Certain protecting groups used in solid phase and solution phase synthesis are well known.

Alpha amino groups can be protected by suitable protecting groups, examples of which include urethane type protecting groups such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyls such as p-chlorobenzyloxycarbonyl, p-nitrobenzyl Oxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); Aliphatic urethane type protecting groups such as t-butoxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl and allyloxycarbonyl. Fmoc is preferred for alpha amino protection.

Guanidino groups such as nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromansulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and It may be protected by a suitable protecting group such as Boc. Pmc is a preferred protecting group for Arg.

Peptides described in the present invention were prepared using a Symphony Multiplex Peptide Synthesizer (Rainin Instrument Company) automated peptide synthesizer, using a programming module provided by the manufacturer according to the protocol set forth in the manufacturer's manual.

Solid phase synthesis was initiated from the C-terminus of the peptide by coupling the protected alpha amino acid to the appropriate resin. These starting materials attach alpha amino-protected amino acids to p-benzyloxybenzyl alcohol (Wang) resins or 2-chlorotrityl chloride resins by ester linkages, or p-[(R, S) -α Benz by amide linkage between Fmoc-Linkers such as [1- (9H-Fluoro-en-9-yl) -methoxyformamido] -2,4-dimethyloxybenzyl J-phenoxyacetic acid (Rink Link) It is prepared by adhering to hydrylamine (BHA) resin or by other known means. Fmoc-Linker-BHA resin supports are commercially available and generally used well. These resins are used repeatedly as necessary to add amino acids sequentially. The alpha amino Fmoc protecting group is removed under basic conditions. Piperidine, piperazine, diethylamine or morpholine (20-40% v / v) in N, N-dimethylformamide (DMF) can be used for this purpose.

After removal of the alpha amino protecting group, the protected amino acids are sequentially coupled in the desired order to obtain the intermediate, protected peptide-resin. Activators used to couple amino acids in solid phase synthesis of these peptides are well known in the art. After peptide synthesis, orthogonally protected side chain protecting groups, if necessary, may be removed by known methods for further peptide derivatization.

Reactive groups in the peptide can be selectively modified during solid phase synthesis or after removal from the resin. For example, peptides may be modified to be N-terminally modified by acetylation on the resin or removed from the resin using a degradation reagent and then modified. N-terminal modification methods such as acetylation, or C-terminal modification methods such as the introduction of N-acetyl groups are well known in the art. Likewise, methods for modifying the side chains of amino acids are also well known to those skilled in the art of peptide synthesis. The choice of modification to the reactive group present on the peptide is determined in part by taking into account the properties required for that peptide.

In one embodiment the peptide may be cyclized prior to separation from the peptide resin. For cyclization through the reactive side chain moiety, the desired side chain is deprotected and the peptide and cyclic coupling agent suspended in the appropriate solvent are added. Suitable solvents include, for example, DMF, dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling agents include, for example, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2- (1H-benzotria) Zol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (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 using a suitable base such as N, N-diisopropylethylamine (DIPEA), sym-colidine or N-methylmorpholine (NMM).

After the peptide synthesized by the solid phase synthesis method is separated from the solid phase, the peptide is separated into various methods such as C _18. Purification by reverse phase high performance liquid chromatography (RP-HPLC) can be carried out using a suitable column such as a column. Other separation or purification methods can also be used, for example based on the size or charge of the peptide. Once purified, the peptides can be characterized by various methods such as high performance liquid chromatography (HPLC), amino acid analysis, mass spectrophotometry, and the like.

Peptides of the invention having a substituted amino derivative C-terminus, typically an N-alkyl group, can be prepared by initiating solid phase synthesis from the C-terminus of the peptide by coupling the protected alpha amino acid to an appropriate resin. Such methods for preparing substituted amide derivatives in the solid phase are well known in the art. For example, Barn DR, Morphy JR, Rees DC Synthesis of an array of amides by aluminum chloride assisted cleavage of resin-bound esters. tetrahedron Lett . 37, 3213-3216 (1996); DeGrado WF Kaiser ET Solid-phase synthesis of protected peptides on a polymer bound oxime: Preparation of segments comprising the sequences of a cytotoxic 26-peptide analogue. J. Org . Chem . 47: 3258-3261 (1982). Such starting materials can be prepared by attaching alpha amino-protected amino acids to p-benzyloxybenzyl alcohol (Wang) resins by ester linking using known means. The peptide chain is grown to the desired amino acid sequence to cyclize the peptide and treat the peptide-resin with a solution of the appropriate amine and aluminum chloride (eg versus methyl amine, dimethyl amine, ethylamine, etc.) in dichloromethane. The resulting peptide amide derivative is released from the resin in solution. The resin is filtered off and the peptide amide derivative is recovered by solvent concentration, followed by ether precipitation. The crude peptide is dried and the remaining amino acid side chain protecting groups are separated using trifluoroacetic acid (TFA) in the presence of water and 1,2-ethanedithiol (EDT). Cold ether is added to precipitate the final product and then collected by filtration. Final purification by RP-HPLC using a C ₁₈ column.

Formulation and Uses

The cyclic peptides described herein can be used for both medical and animal husbandry or veterinary use. Generally, this product is used in humans, but can also be used in other mammals. "Patient" refers to a mammalian subject and is used in this specification and claims in that sense. Although the primary use of the present invention relates to human patients, the present invention may also be applied to laboratories, farms, zoos, wild animals, pets, sports or other animals.

In general, the cyclic peptides of the present invention can be synthesized by solid phase synthesis according to known methods and then purified. Several known methods using various resins and reagents can be used to prepare the cyclic peptides of the invention.

Cyclic Salt Forms of Peptides . The cyclic peptides of the invention can be made in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to salts made of pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric iron, ferrous, lithium, magnesium, manganese salts, divalent manganous, potassium, sodium, zinc and the like. Especially preferred are the ammonium, calcium, lithium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable non-toxic organic bases include primary, secondary and tertiary amines, substituted amines such as naturally occurring substituted amines, cyclic amines, basic ion exchange resins such as arginine, betaine , Caffeine, choline, N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperi Dean, Glucamine, Glucosamine, Histidine, Hydrabamine, Isopropylamine, Lysine, Methylglucamine, Morpholine, Piperazine, Piperidine, Polyamine resin, Procaine, Purine, Theobromine, Triethylamine, Trimethylamine, Tri Propylamine, tromethamine, and the like.

When the cyclic peptide of the present invention is a base, acid addition salts can be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. These acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carboxylic acid, citric acid, ethanesulfonic acid, formic acid, formaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isetionic acid, lactic acid, maleic acid, Malic acid, mandelic acid, methanesulfonic acid, malonic acid, mucic acid, nitric acid, palmic acid, pantothenic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid and the like. Acid addition salts of the peptides of the invention are prepared from peptides and excess acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, succinic acid or methanesulfonic acid in a suitable solvent. do. Acetate forms are particularly useful. When the peptide of an embodiment of the present invention comprises an acid 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.

Pharmaceutical composition . Another embodiment of the invention provides a pharmaceutical composition comprising a cyclic peptide of the invention and a pharmaceutically acceptable carrier. The carrier may be a liquid formulation and is preferably a buffered, isotonic aqueous solution. Pharmaceutically acceptable carriers may also include excipients such as diluents, carriers and the like and additives such as stabilizers, preservatives, solubilizers, buffers and the like, as described below.

The cyclic peptide composition of some embodiments of the invention comprises at least one cyclic peptide of the invention, including excipients such as excipients such as diluents, carriers and the like and additives such as stabilizers, preservatives, solubilizers, buffers and the like. It may be formulated or prepared as a pharmaceutical composition containing together with the above pharmaceutically acceptable carrier. Formulation excipients include polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride and sodium citrate. In preparations for injection or other liquid administration, one or more buffering ingredients comprising water are preferred, and stabilizers, preservatives and solubilizers can also be used. In the solid dosage form, various thickening agents, fillers, bulking agents and carrier additives can be used, such as starch, sugars, fatty acids and the like. For formulations for topical administration, various creams, ointments, gels, lotions and the like can be used. For most pharmaceutical formulations, the inactive ingredient occupies more formulation than the active ingredient in weight or dose. In the case of pharmaceutical formulations, various measured release, sustained release or timed release formulations and additives can be used to allow delivery of the peptides of the invention for a period of time.

In general, the actual amount of cyclic peptide administered to a patient can vary greatly depending on the mode of administration, the agent used and the response desired.

In practical use, the cyclic peptides of the present invention can be combined as active ingredients by mixing with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take the form suitable for formulation according to the route of administration desired, such as oral, parenteral (including intravenous), urinary tract, vaginal, nasal, buccal, sublingual and the like. In preparing compositions for oral dosage forms, oral liquid preparations such as suspensions, elixirs and solutions include conventional pharmaceutical media such as water, glycols, oils, alcohols, flavors, preservatives, colorants and the like; For oral solid preparations such as powders, hard and soft capsules and tablets, starches, sugars, microcrystalline celluloses, diluents, granulating agents, lubricants, binders, disintegrating agents and the like can be used.

Tablets and capsules are preferred examples of unit dosage forms for oral administration because of ease of administration. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. The amount of active peptide in such therapeutically useful compositions is such that an effective dosage will be obtained. In another advantageous unit dosage form, sublingual structures such as sheets, wafers, tablets, and the like can be used. The active peptide may also be administered through the nose, for example liquid drops or sprays.

Tablets, pills, capsules and the like also binders such as gum tragacanth, acacia, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, or alginic acid; Lubricants such as magnesium stearate; And sweeteners such as sucrose, lactose or saccharin. If the unit dosage form is a capsule, it may contain a liquid carrier such as fatty oil, in addition to the substances of the aforementioned type.

Various other materials can be used as coatings or made as physical unit dosage forms. For example, tablets may be coated with shellac, sugar or both. Syrups or elixirs may contain, in addition to the active ingredient, flavors and pigments such as sucrose as sweetener, methylparaben and propylparaben as preservatives, cherry or orange flavors.

Cyclic peptides can also be administered parenterally. Solutions or suspensions of these active peptides can be made in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Such formulations may also contain preservatives to prevent microbial growth, if desired.

Pharmaceutical formulations suitable for injection may contain sterile aqueous solutions or dispersants and sterile powders for the instant preparation of sterile injectable solutions or dispersions. In all such cases, the form must be sterile and must be fluid flowing enough to be injected through a syringe. Such formulations must be stable during manufacture and storage and must be safely preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols such as glycerol, propylene glycol or liquid polyethylene glycols, suitable mixtures thereof, vegetable oils and the like.

The cyclic peptides disclosed herein can be applied therapeutically through the nose. By "nasal administration" is meant any form of administration of the cyclic peptide of the invention into the nose. The peptide may be in the form of an aqueous solution, such as an aqueous solution, such as saline, citrate or other commonly used excipients or solutions containing preservatives. Such peptides may also be in the form of dry or powdered preparations.

In another embodiment, the cyclic peptide may be administered directly into the lung. Administration into the lung is performed by a metered dose inhaler, a device that allows self administration of a metered bolus of the peptides of the invention when the patient is operated while breathing.

According to another embodiment of the present invention, the cyclic peptides of the present invention may be formulated with various agents that enhance the efficient nasal absorption of drugs, including these peptide drugs. Such formulations increase nasal absorption without unacceptable damage to the mucosa. In particular, US Patent No. 5,693,608, 5,977,070 and 5,908,825 disclose various pharmaceutical compositions that can be used, including absorption accelerators, and the contents of all references cited so far, including the aforementioned references and all patent documents, are incorporated herein by reference.

In aqueous solution, certain cyclic peptides of the present invention may be appropriately buffered by saline, acetate, phosphate, citrate, acetate or other buffer means, which may be used at physiologically acceptable pH, generally between about pH 4 and about pH 7. Range. Combinations of buffers may also be used, such as phosphate buffered saline, saline and acetate buffers, and the like. For saline solution, 0.9% saline solution can be used. In the case of acetates, phosphates, citrate, acetates and the like, 50 mM solutions can be used. In addition to buffers, suitable preservatives may be used to prevent the growth of bacteria and other microorganisms. One such preservative that can be used is 0.05% benzalkonium chloride.

It is also possible and practice to use the cyclic peptides of the invention in the form of dry particles. In a preferred embodiment, such particles may be about 0.5 to 6.0 μm, and these particles have a sufficient weight to settle to the lung surface, so that they may not be spit out, but are small enough in size to reach the airways before reaching the lungs. Is not deposited on its surface. Various techniques can be used to make dry powder particulates, which include, but are not limited to, micromilling, spray drying, and lyophilization followed by a quick freeze aerosol. Using microparticles, the peptide can be deposited deep into the lungs for rapid and effective absorption into the bloodstream. In addition, this approach does not require penetration enhancers, as is the case with transdermal, oral or oral mucosal delivery routes. Various types of inhalers can be used, including propelled aerosols, nebulizers, single dose dry powder inhalers, and multiple dose dry powder inhalers. Currently widely used devices include metered dose inhalers used to deliver medications for the treatment of asthma and chronic obstructive pulmonary disease. Preferred devices are dry powder inhalers designed to form a cloud or aerosol of fine powder with a particle size of always less than about 6.0 μm.

The size, including the average size distribution of the microparticles, can be adjusted according to the manufacturing method means. In the micromilling method, the particle size is controlled by the size of the milling head, the rotor speed, the processing time and the like. In spray drying, the nozzle size, flow rate, and heat of the dryer controls the size of the particles. When prepared by rapid freezing aerosol and lyophilization means, the nozzle size, flow rate, concentration of aerosol solution, etc., control the size of the particulates. These and other variables can be used to control the particle size.

The cyclic peptides of the present invention are administered therapeutically by deep intramuscular injection of a formulation for injection over time into an injection means, typically a muscle such as the parietal muscle or deltoid muscle. In one embodiment, the cyclic peptides of the invention are formulated with polyethylene glycol, such as polyethylene glycol 3350, and optionally one or more additional excipients and preservatives, examples of which include, but are not limited to, salts, polysorbates 80, Or hydrochloric acid or sodium hydroxide for pH adjustment. In another embodiment the cyclic peptides of the invention are formulated with poly (ortho esters), which may be autocatalyzed poly (ortho esters) having varying percentages of lactic acid in the polymer backbone and, if desired, one or more additional excipients. Can be. In one embodiment a poly (D, L-lactide-co-glycolide) polymer (PLGA polymer) is used, preferably a PLGA polymer with hydrophilic end groups, such as Boehringer Ingelheim, Inc. It is preferable to use PLGA RG502H commercially available from (Ingelheim, Germany) This preparation combines the cyclic peptide of the invention with a solution of PLGA in methylene chloride in a suitable solvent such as methanol, which is then subjected to poly under appropriate mixing conditions in the reactor. It is prepared by adding to a continuous phase solution of vinyl alcohol In general, various injectable and biodegradable polymers, preferably adhesive polymers, can be used in the preparation for sustained release injecting, as described in U.S. Patent Nos. 4,948,763, 6,432,438 and 6,673,767. The disclosure and the biodegradable polymers and formulations described herein are incorporated herein by reference. And sheep, the formulation according to the biodegradation rate, and other factors known to those skilled in the art of polymer per week, it is possible to scan according to a month or other periodic reference.

Route of administration . When administered by injection, this method of injection may be by intravenous, subcutaneous, intramuscular, intraperitoneal injection or other known means. The peptides of the present invention may be made into non-limiting formulations such as tablets, capsules, caplets, suspensions, sprays, lyophilized formulations, suppositories, eye drops, dermal patches, oral soluble preparations, sprays, aerosols, and the like, It may be mixed with binders, excipients, stabilizers, antioxidants and other materials known in the art. In general, administration routes may be employed which introduce the peptides of the invention through the epidermal layer of cells. Therefore, administration routes such as mucosa, buccal administration, oral administration, transdermal administration, inhalation administration, nasal administration, urinary tract administration and vaginal administration can be used.

Therapeutically effective amount . In general, the actual dosage of the cyclic peptides of the present invention to the patient can vary greatly depending on the mode of administration, the agent used and the desired response. Dosages for treatment are sufficient to achieve the desired therapeutic effect according to any of the methods described or known administration methods. Thus, a therapeutically effective amount may be used to treat a disease or condition associated with cathesia, including therapeutically alleviating an eating related disease in a patient, or preventing or delaying the onset or recurrence of an eating disease, or symptoms associated with immune and cancer. It is an amount sufficient to manage eating-related diseases in patients with the disease.

In general, the cyclic peptides of the present invention are very active. For example, such cyclic peptides may be about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, or depending on the particular peptide selected, the desired therapeutic response, the route of administration, the agent, and other variables known to those skilled in the art. It may be administered in an amount of 500 μg / kg body weight.

Inflammatory and immune-mediated diseases . The peptides of the present invention can also be used to treat inflammatory and immune mediated diseases. See, eg, Catania A. et al . , Trends Endocrinol. Metab. 11: 304-308 (2000); Gantz I. and Fong ™, Am. J. Physiol. Endocrinol. Metab . 284: E468-E474 (2003); And Catania A., Gatti S., Colombo G., Lipton JM, Pharmacol. Rev. 56: 1-29 (2004); These documents are referenced in the present invention.

Combination therapy

Cyclic peptides according to some embodiments of the present invention may also be used in combination with other drugs or agents, particularly for the treatment of cathesia. These other drugs and agents may include agents that induce weight gain, such as corticosteroids and progestational agents. In a preferred embodiment of the invention, the cyclic peptides of the invention are used in combination with a therapeutically effective amount of a second weight increasing pharmaceutical agent.

According to another embodiment of the invention, there is provided a method of treating Cajuncia. The method comprises administering to a patient at risk of or having Cacexia, a therapeutically effective amount of the cyclic peptides disclosed herein, together with a therapeutically effective amount of other compounds useful for the treatment of Cacexia.

It is therefore an object of the present invention to provide a pharmaceutical composition comprising 1) a cyclic peptide of one embodiment of the present invention and 2) a second compound useful for the treatment of carcassia.

In one embodiment, the second compound useful for the treatment of carcassia is preferably an ADP-ribose polymerase inhibitor, an ADP-ribose-transferase inhibitor, a NADase inhibitor, nicotinamide benzamide, theophylline, thymine and analogs thereof; Omega-3-fatty acids such as alpha-linolenic acid, stearic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid or mixtures thereof; Branched chain amino acids valine, leucine, isoleucine or mixtures thereof, tryptophan and 5-hydroxytryptophan concentrations decreased or were not reduced; Antioxidant selected from beta carotene, vitamin C, vitamin E, selenium or mixtures thereof; L-glutamine, vitamin A, vitamin C, vitamin E, and selenium; Azaftig; Quinine derivatives including 3,5,6-trimethyl-2- (3-pyridyl) methyl-1,4-benzoquinone hydrochloride; Interleukin 2; Benzaldehyde; 4,6-O-benzylidene-D-glucose; Friedelan-3-one; Hydrazine sulfate; Methoxyprogesterone acetate; Beta 2-adrenoceptor agonists; Corticosteroids such as dexamethasone; Vitor ^TM ; Pro-Stat ^™ ; Megestrol acetate (Megace ^™ ); Dronabinol (Marinol ^™ ); Magesterol acetate (Megace ^™ ); Thhalidomid ^™ ; Fluoxymesterone (Halotestin ^™ ); Pentoxifylline (Trental ^™ ); Ciproheptadine (Periactin ^™ ); Metoclopramide (Reglan ^™ ); Total parenteral nutrition; Or other MC4-R antagonists. In another embodiment, the second compound useful for treating Cathesia is somatotropin (Secrostim ^™ ), an injectable form of human growth hormone.

According to yet another embodiment of the present invention there is provided a kit for treating cathesia. The kit comprises a first pharmaceutical composition containing a cyclic peptide according to one embodiment of the invention, a second pharmaceutical composition containing a second compound useful for the treatment of carcassia, and a container for the first composition and the second composition It includes.

The present invention is explained in more detail by way of the non-limiting examples described.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present invention with reference to the detailed description of the principles of the invention. These drawings are presented solely for the purpose of illustrating one or more preferred embodiments of the invention and should not be construed as limiting the invention to the scope of the drawings. In the drawing:

1 is a graph of 24 hours food intake in male rats comparing the effect of IV administration of 1 mg / kg of the compound of Example 12 with the same volume of vehicle.

FIG. 2 is a graph showing 24 hour weight change in the animals of FIG. 1.

Example 1 [I ¹²⁵ Competitive Suppression Analysis with] -NDP-α-MSH

0.4 nM, 0.2 nM, 0.4 nM or 0.1 nM [I ¹²⁵ ] -NDP-αMSH (New England Nuclear, respectively) in 50 mM HEPES buffer at pH 7.2 containing 1 mM MgCl ₂ , 2 mM CaCl ₂ , and 5 mM KCl, respectively. Competitive inhibitory binding assay using membrane prepared with HEK-293 cells transfected with hMC3-R, hMC4-R or hMC5-R gene construct, and B-16 mouse melanoma cells (containing MC1-R) Do this. Assay tubes contain a test peptide of the present invention at a specific concentration, usually 1 μM, to determine the efficacy of [I ¹²⁵ ] -NDP-α-MSH to bind its receptor. In assays with 1 μM NDP-α-MSH, nonspecific binding is measured by complete inhibition of [I ¹²⁵ ] -NDP-αMSH.

The assay mixture was incubated for 90 minutes at room temperature, then filtered and washed three times with ice cold buffer. The filter was dried and the residual radioactivity bound to the membrane was counted with a gamma counter. The difference in radioactivity (cpm) bound to the cell membrane in the absence and presence of 1 μM NDP-α-MSH is defined as 100% specific binding. The cpm obtained in the presence of the test peptide is normalized with respect to 100% specific binding to determine the percentage inhibition of [I ¹²⁵ ] -NDP-α-MSH binding. Each analysis is performed three times. Ki (nM) of certain peptides of the invention are measured using similar assay protocols by testing the peptides in a broader dosage range.

Example 2 EC in Functional Activity Assay ₅₀  General method for measuring

Determination of peptides at the melanocortin receptor by measuring the accumulation of intracellular cAMP in HEK-293 cells expressing hMC3-R, hMC4-R or hMC5-R, and B-16 mouse melanoma cells expressing MC1-R. Perform a functional assessment. Earle's balanced salt containing 10 mM HEPES (pH 7.5), 5 mM MgCl ₂ , 1 mM glutamine, 0.1% albumin and 0.6 mM 3-isobutyl-1-methyl-xanthine, phosphodiesterase inhibitor Cells suspended in solution are plated in 96 well plates at a density of 0.5 × 10 ⁵ cells per well. Cells are incubated with the test peptide in the presence or absence of α-MSH for 1 hour at 37 ° C. CAMP concentration in cell lysate is measured using the EIA kit (Amersham). Data analysis and EC ₅₀ values are measured using nonlinear regression analysis by Prism Graph-Pad software.

Example  3 Function status

Agonist / antagonist states for MC1-R, MC4-R and MC5-R of certain peptides of the invention are measured. Antagonistic activity is determined by measuring inhibition of α-MSH-induced or NDP-α-MSH-induced cAMP after exposure of the peptides as described above.

Agent Analysis. Evaluation of molecules that elicit a functional response in HEK-293 cells expressing hMC4-R to agonist activity by measuring the accumulation of intracellular cAMP after treatment. Confluent HEK-293 cells overexpressing MC4-R are detached with reactive small cell suspension buffer. Oral balanced salt containing cells with 10 mM HEPES (pH 7.5), 1 mM MgCl ₂ , 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine, phosphodiesterase inhibitor Suspension in solution. Cells are plated in 96 well plates at a density of 0.5 × 10 ⁵ cells per well and then preincubated for 30 minutes. Cells are then challenged with test peptides dissolved in dimethylsulfoxide (DMSO) at a concentration range of 0.05-5000 nM in 200 μl total assay volume for 1 hour at 37 ° C. The concentration of DMSO is always kept at 1% in the assay mixture. NDP-α-MSH is used as a reference agent. At the end of the incubation period cells are disrupted by adding 50 μl lysis buffer from the cAMP EIA kit (Amersham). Suck the cells several times with a pipette and repeat them to rupture the cells completely. After appropriate dilution using the EIA kit (Amersham) method, the cAMP concentration in the cell lysate is measured. Data analysis and EC ₅₀ Values are measured using nonlinear regression by Prism Graph-Pad software. Peptides with a response rate of at least 0.7 compared to NDP-α-MSH at 5000 nM concentration are classified as complete agents. Peptides with a ratio of 0.1 to 0.7 are classified as partial agents. Peptides with a reaction rate of less than 0.1 are evaluated for antagonistic activity.

Analysis of neutral antagonists . For MC4-R membranes peptides that bind with high affinity but have low efficiency (EC ₅₀ > 1000 nM) and low response rates (<0.1) are analyzed for their ability to antagonize the stimulatory effects of the agent NDP-α-MSH. This study is performed on HEK-293 cells expressing hMC4-R. The degree of antagonism is measured by incubating the cells with peptides in the presence of NDP-α-MSH agonists and measuring a decrease in intracellular cAMP concentration. Antagonist screening of peptides against a single concentration of NDP-α-MSH (0.1 nM) is performed for a peptide concentration range of 0.5-5000 nM. If the peptide exhibits strong antagonism that induces pA ₂ values from Schild's analysis, further studies are conducted.

Experimental details are similar to agonist activity and the foregoing. In summary, cells are preincubated for 30 minutes with test peptides at concentration ranges from 0.5 nM to 5000 nM. The cells are then stimulated with 1 nM concentration of NDP-α-MSH for 1 hour. For shield analysis, studies are carried out over the full range of agents (0.005-5000 nM) using at least three peptide concentrates separated in log units. After appropriate dilution the cAMP concentration in the cell lysate is measured. Nonlinear regression analysis is performed using Prism Graph-Pad software to obtain shield analysis and EC ₅₀ values. pA ₂ values are derived from the shield plot.

Inverse agonist analysis. Peptides with low EC ₅₀ values (EC ₅₀ > 1000 nM) or low reaction rates (<0.1) have the ability to act as inverse agonists, ie in HEK-293 cells expressing the hMC4-R receptor Investigate the ability to reduce basal or continuous concentrations of cAMP. The experimental protocol is basically as described above. The cells are exposed to the test peptide over a concentration range of 0.05 nM to 5000 nM for 1 hour at 37 ° C. Aguti-related protein (Aguti) Biologically active fragments of Aguti protein, such as AgRP (83-132) (Ser-Ser-Arg-Arg-Cys-Val-Arg-Leu- His-Glu-Ser -Cys-Leu-Gly-Gln-Gln-Val-Pro-Cys-Cys-Asp-Pro-Cys-Ala-Thr-Cys-Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr -Cys-Arg-Lys-Leu-Gly-Thr-Ala-Met-Asn-Pro-Cys-Ser-Arg-Thr (SEQ ID NO: 2)) is used as reference inverse agonist. Data Analysis W and EC ₅₀ values are determined by nonlinear regression analysis using Prism Graph-Pad software.

Example 4 ICV Food Absorption and Weight Change

Food intake and body weight changes are assessed against selected peptides. Rats with intrinsic endothelial cannula (ICV rats) were developed from Hilltop Lab Animals, Inc. From Scottadale, Pa. Animals are kept in cages floating in conventional plexiglasses and maintained using a 12 hour on / off photoperiod. Eat water and powder (LabDiet, 5P00 Prolab RMH 3000) or pellet (Harlan Tekland 2018 18% Protein Rodent Diet). One week before treatment, 24 hours of food intake and body weight change are recorded and used as the baseline of the group upon vehicle treatment. Rats are dosed ICV with vehicle or selected cyclic peptides (0.3-3 nmol). Changes in body weight and food intake are measured for 24 hours after dosing. Changes in body weight and food intake over 48 and 72 hours after dosing are also recorded to determine the reversal of changes in body weight and food intake effects to baseline levels.

Example 5 IV and IP food intake and weight changes

Evaluate food intake and body weight changes for the selected peptides. Male Sprague-Dawley rats were purchased from Taconic (Germantown, NY). Animals are each housed in a conventional suspended plexiglass cage and maintained using a 12 hour on / off photoperiod. Eat water and powder (LabDiet, 5P00 Prolab RMH 3000) or pellet (Harlan Tekland 2018 18% Protein Rodent Diet). One week before treatment, 24 hours of food intake and body weight change are recorded and used as the baseline of the group upon vehicle treatment. Rats are dosed IV or IP with vehicle or selected cyclic peptides (0.5-3 mg / kg). Changes in body weight and food intake are measured for 24 hours after dosing. Changes in body weight and food intake over 48 and 72 hours after dosing are also recorded to determine the reversal of changes in body weight and food intake effects to baseline levels.

Example 6 Behavior satiety sequence

Feeding male Sprague-Dawley rats is limited to 20 g of powder per day. Food is given at the same time during the light cycle and saline or test peptides are administered 2 hours before food. Provide 20 g of feed in a pre-weighed bowl and observe rat behavior for 1 hour. Behavioral observations are divided into three areas: food intake, activities (hair whipping, drinking and wheezing / searching activities), and rest (activity reduction and sleep). Record the length of time each action takes. Measure food intake after the observation period.

Example 7 Conditioned taste avoidance

Male Sprague-Dawley rats were given a limited time to drink 30 minutes a day during the ritual and allowed to eat pelleted chow. LiCl administration in laboratory animals conditioned the aversion to saccharin's new and good taste (Seeley RJ, Blake K., Rushing PA et al. The role of CNS glucagons-like peptide-1 (7-36) amide receptors in mediating the visceral illness effects of lithium chloride.J. Neurosci. 20: 1616-1621 (2000). To familiarize animals, injections of LiCl or test peptide are administered immediately after initial delivery of 0.1% saccharin solution. After 2 days, saccharin solution is given again and fluid intake is measured. Reduced drinking of saccharin solution suggests a conditional taste aversion.

Example 8 Lipopolysaccharide-Induced Cajuncia Model

Rats with intrinsic endothelial cannula (ICV rats) were developed from Hilltop Lab Animals, Inc. From Scottadale, Pa. Animals are kept in cages floating in conventional plexiglasses and maintained using a 12 hour on / off photoperiod. Eat water and powder (LabDiet, 5P00 Prolab RMH 3000) or pellet (Harlan Tekland 2018 18% Protein Rodent Diet). Lipopolysaccharide (LPS) (E. Coli 055: B5, Sigma Chemical Co.) is dissolved in normal saline and injected intraperitoneally. For the first LPS injection, 6-7 weeks old male animals are used. In the same replicate, female animals use 5 weeks of age. The basal feeding status was monitored for these animals for 2 days, and then Lp. Saline injection and monitoring for 12 hours each. Certain peptides of the invention are administered and 50 μg / kg LPS is administered after 3 hours. Administration of the second 100 μg / kg LPS is performed 60 hours after the first dose in the second experiment. Food is not provided until the LPS administration after the peptide administration. After LPS administration, the feeding amount is measured for 24 hours every 6 hours and then for 48 hours every 12 hours.

In the sham group, baseline meals are measured every 6 hours in two age and sex-matched groups after simulated ICV injection and intraperitoneal saline injection. 24 hours later, the selected peptide is administered and LPS is intraperitoneally administered 3 hours later. Feeds are measured every 6 hours for 24 hours and every 12 hours for the next 48 hours. Differences in the feeding curves between the two groups are expressed as body weight normalized intake and basal feeding percentages versus saline and post-treatment ICV injections.

Example 9 Tumor-induced Cacetia Model

Lewis lung carcinoma (LLC) cells and Englebreth-Holm Swarm sarcoma (EHS) tumors were treated as primary cultures in DMEM containing 10% fetal bovine serum or in vivo, respectively, as recommended by the supplier. Keep it. LLC tumor cells are harvested during the logarithmic growth phase of the culture, washed with Hanks balanced saline solution and the cells injected subcutaneously above the flanks of the animals. EHS sarcoma tissue is dissected from the donor animal and a lump of about 3 mm tissue is subcutaneously implanted over the posterior flank. In vain treated animals, donor animal muscle tissue is transplanted in similar amounts. In all cases, the timing of the appearance of tumor masses was recognized and all animals were touched within 4 days (LLC) or 8 days (EHS) of the start of the experiment. The animal is sacrificed and the tumor is extracted from the surrounding tissue and weighed. Visual inspection of all organs did not detect the presence of observable metastases. For serum leptin measurements using the radioimmunoassay kit, arterial blood is drawn when the animal is sacrificed.

The animals are kept in cages, each floating in a conventional plexiglass, and maintained using a 12 hour on / off photoperiod. The effect of the administration of certain peptides of the invention on hypophagia and weight loss animals due to the presence of a growing tumor is tested. In the initial experiment, the daily food intake and body weight are measured until the food intake of the tumor-bearing animal is 75-80% of the baseline for three consecutive days. On average this occurs 12 days after implantation or 4 days after the development of a palpable tumor. ICV injections of selected peptides are then monitored to assess food intake changes in animals.

In the second experiment, the selected peptides are tested for their ability to prevent the onset of cassia and maintain normal food intake and growth. Test daily for tumor touch in the animal. All animals developed tumors up to 14 days after transplantation and none until 12 days. The selected peptide is then administered or vanished every 48 hours until the animal is killed. For comparison, the sham-tumor transplant group was included in the trial to administer the peptide as well.

In all experiments, differences in feeding, activity, and water consumption curves are analyzed in two ways, and ANOVA is measured repeatedly over time and treatment as measurement variables. The final tumor and body weight are analyzed by the Student's t-test if the two groups are involved, or one-way ANOVA with the post hoc analysis if the three groups are involved. For repeated measures of ANOVA, data sets are analyzed for statistical significance using PRISM software package (GRaphPad) or EXCEL (Microsoft) using Student's t-test.

Example 10 Weight Measurement and Nuclear Magnetic Resonance Analysis

The weight of the peptides of the invention is measured using a Waters MicroMass ZQ device in positive mode. Weighing values are expressed in the form of mass weight plus circles (M + 1 or M + H) in comparison with the calculated values.

Proton NMR data are obtained using a Bruker 300 MHz spectrophotometer. This spectrum is obtained after the peptide is dissolved in a suitable deuterated solvent such as chloroform, DMSO or methanol.

Example 11 Ac-Nle- Cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH

Peptide Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH was synthesized by conventional peptide synthesis. The weight of this peptide was determined to be 1189. Competitive inhibition test and Ki (nM) of peptides were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist with an EC ₅₀ (nm) of 57 for MC1-R, and an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 7.95.

Example 12 Ac-Nle- Cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH

Peptide Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH was synthesized by conventional peptide synthesis. The weight of this peptide was determined to be 1200. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist with an EC ₅₀ (nm) of 5 for MC1-R, an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.12.

FIG. 1 is a diagram showing the cumulative increase in food intake when rats were administered with the compound of Example 12 compared to when vehicle alone was administered. Rats were administered 1 mg / kg of the peptide of Example 12 and food intake was measured at selected time points for 24 hours. In summary, male Sprague-Dawley rats (300-350 g) were individually housed in 12 h light / dark cycles in shoebox cages. Food intake and body weight were monitored 24 hours prior to study start. Rats were randomized irrespective of body weight and then administered IV with the compound of Example 12 or the same volume of vehicle just prior to extinction. A pre-weighed amount of food was provided and then the food intake was measured at 2, 4, 20 and 24 hours. In FIG. 1, "*" indicates probability p <0.05 and "**" indicates probability p <0.01. FIG. 2 shows cumulative changes in body weight at 24 hours of the animal of FIG. 1.

Example 13 Ac-Nle- Cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH

Peptide Ac-Nle- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1249. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist for MC1-R and an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 7.59.

Example 14 Ac- Cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH

Peptide Ac- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1076. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In the cAMP assay to measure agonist / antagonist state, the peptides for the MC1-R for MC3-R for the EC ₅₀ (nm) is 4420 of agonists, MC3-R and MC4-R EC ₅₀ (nm) is It was determined to be an antagonist of 1.0. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 6.73.

Example 15 Ac- Cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH

Peptide Ac- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1088. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist with an EC ₅₀ (nm) of 2153 for MC1-R, and an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.0.

Example 16 Ac- Cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH

Peptide Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1137. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist with an EC ₅₀ (nm)> 1000 for MC1-R, and an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.45.

Example 17 Ac- Cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃

Peptide Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃ was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1163. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be a partial agonist with an EC ₅₀ (nm)> 1000 for MC1-R, and an antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.76, and the pA ₂ (M) value for MC3-R was 8.04.

Example 18 Ac- Cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂

Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂ was synthesized according to a conventional peptide synthesis method. The weight of this peptide was determined to be 1163. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, this peptide was determined to be inactive for MC1-R and antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.69, and the pA ₂ (M) value for MC3-R was 7.29.

Example 19 Ac- Cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃

Peptide Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ was synthesized according to conventional peptide synthesis. The weight of this peptide was determined to be 1149. Competitive inhibition test and Ki (nM) of this peptide were measured according to the method of Example 1. The functional state of this peptide was measured according to the method of Examples 2 and 3.

In cAMP analysis to determine agonist / antagonist status, at 1 μm concentration the peptide was determined to be inactive for MC1-R and antagonist for MC3-R and MC4-R. In the test for functional antagonism as in Example 3, the pA ₂ (M) value for MC4-R was determined to be 8.9, and the pA ₂ (M) value for MC3-R was 8.07.

Example 20-25 Additional peptides

The following peptides were synthesized according to conventional peptide synthesis methods:

20.H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃

21.H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃

22.H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂

23.H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₂ -CH ₃

24.H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₃

25.H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -N (CH ₃ ) ₂

Competitive inhibition test and Ki (nM) of the peptides of Examples 20-25 were measured according to the method of Example 1. The functional state of the peptides of Examples 20-25 was measured according to the method of Examples 2 and 3.

Example 26 ICV Feed Research

A series of ICV feeding studies was performed on rats. All animals were fed with saline solution ICV on the first day and fed food in pre-weighted containers 2 and 21 hours after ICV-injection. On day 2, animals were randomized based on food consumption at 21 hours and animals that had eaten or spilled some food were excluded. Animals were administered vehicle (salt), positive control (1 nmol of SHU 91 19) or peptide of the invention (0.3 or 3 nmol). The food weights were recorded again 2, 4, 21 and 24 hours after the ICV injection. In most cases, different tests are performed several times for groups of 8 to 12 per group; The minimum value is shown below. Measurements are expressed as percentage increase (decrease) in total food intake. "ND" indicates no test was performed at that dose level.

The above-described examples can repeatedly achieve similar results even with appropriate substitution of the reactants and / or the conditions of implementation generally or specifically described herein.

Although the present invention has been described with reference to certain preferred embodiments, the same results can be obtained in other embodiments. Various modifications and variations of the present invention will be apparent to those skilled in the art, and all such modifications and equivalents are all within the scope of the present invention. All references, patent applications, patent documents, and publications mentioned above are hereby incorporated by reference in their entirety.

<110> PALATIN TECHNOLOGIES, INC. <120> CYCLIC PEPTIDES FOR TREATMENT OF CACHEXIA <130> KP-CH-077287 <150> US 60 / 585,971 <151> 2004-07-06 <150> US 11 / 174,845 <151> 2005-07-05 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Core alpha-melanocyte stimulating hormone messenger sequence <400> 1 His Phe Arg Trp   One <210> 2 <211> 50 <212> PRT <213> Homo sapiens <400> 2 Ser Ser Arg Arg Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln   1 5 10 15 Val Pro Cys Cys Asp Pro Cys Ala Thr Cys Tyr Cys Arg Phe Phe Asn              20 25 30 Ala Phe Cys Tyr Cys Arg Lys Leu Gly Thr Ala Met Asn Pro Cys Ser          35 40 45 Arg Thr      50  

Claims (20)

Cyclic peptides having the formula:

Wherein R ₁ is H,

R ₃ is 4-imidazolyl or 3-indolyl; R _4a and R _4b are each any ring substituents, and if one or both are present, they are the same or the same as each other and independently represent a hydroxy, halogen, alkyl, or aryl group, which can be attached directly or via an ether bond; R ₅ is —NH ₂ or —NH (C═NH) NH ₂ ; R ₆ is 1- or 2-naphthyl or 3-indolyl, optionally having one or two ring substituents, and when one or both ring substituents are present, they are the same or different from one another and are independently hydroxy, halogen, alkyl, Or aryl groups, which can be attached directly or via an ether bond; R ₇ is -OH or

; R ₈ is H, NH ₂ , lower aliphatic C ₁ to C ₄ straight or branched alkyl chains, C ₁ to C ₄ aralkyl, or C ₁ to C ₄ omega amino derivatives; R ₉ is H, lower aliphatic C ₁ to C ₄ straight or branched alkyl chain, C ₁ to C ₄ aralkyl, or C ₁ to C ₄ omega amino derivatives; R ₁₀ is aliphatic L- or D-amino acid, N-acylated L- or D-amino acid or straight or branched C ₁ to C ₁₇ alkyl, aryl, heteroaryl, alkene, alkenyl, or aralkyl chain; R ₁₁ and R ₁₂ are each independently H or C ₁ to C ₄ straight or branched alkyl chains, provided that both R ₉ and R ₁₀ are not H; x is 1 to 4, y is 1 to 5, provided that x + y is 2 to 7; z is 2 to 5. The cyclic peptide of claim 1 having the formula:

Wherein R ₃ and R ₆ are as defined in claim 1. The cyclic peptide of claim 2, wherein the cyclic peptide is: Ac- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH; Ac- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH; or Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH. The cyclic peptide of claim 1 having the formula:

Wherein R ₃ and R ₆ are as defined in claim 1. The cyclic peptide of claim 4, wherein the cyclic peptide is as follows: Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Trp-Lys) -OH; Ac-Nle- cyclo (-Asp-His-D-Nal 2-Arg-Nal 2-Lys) -OH; or Ac-Nle- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -OH. The cyclic peptide of claim 1 having the formula:

Wherein R ₃ , R ₆ , R ₁₁ and R ₁₂ are as defined in claim 1. The cyclic peptide of claim 6, wherein the cyclic peptide is: Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃ ; Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂ ; or Ac- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ . The cyclic peptide of claim 1 having the formula:

Wherein R ₃ , R ₅ , R ₆ , R ₁₁ , R ₁₂ and z are as defined in claim 1. The cyclic peptide of claim 8, wherein the cyclic peptide is: H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₂ -CH ₃ ; H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -NH-CH ₃ ; H- cyclo (-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys) -N (CH ₃ ) ₂ ; H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₂ -CH ₃ ; H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -NH-CH ₃ ; or H- cyclo (-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys) -N (CH ₃ ) ₂ . A pharmaceutical formulation comprising the cyclic peptide of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A method of treating cathesia comprising administering to a mammal a pharmaceutically sufficient amount of the pharmaceutical formulation of claim 10. A method of treating inflammation and immune-mediated disease, comprising administering to a mammal a pharmaceutically sufficient amount of the pharmaceutical formulation of claim 10. A cyclic hexapeptide having a C-terminal hydroxyl or N-alkyl group, wherein the N-alkyl group has one or two C ₁ to C ₄ straight or branched alkyl chains and the hexapeptide is His-D-Nal 2 Contains a core sequence of -XY or Trp-D-Nal 2-XY, where X is an L-amino acid selected from Arg, Lys, Orn, Harg and Hlys and Y is L selected from Nal 1, Nal 2 and Trp Or D-amino acids, wherein any aromatic ring in the core sequence optionally comprises one or two substituents, and when one or both imaginary ring substituents are present, they are the same or different and independently direct or ether bonds And a cyclic hexapeptide selected from hydroxyl, halogen, alkyl, or aryl groups attached through. The cyclic hexapeptide of claim 13 having a N-terminal Ac group. The hexapeptide according to claim 13, wherein the hexapeptide is cyclized by forming an amide bond between the amino group of the side chain of the amino acid at position 1 or the amino group of the N-terminal group of the amino acid at position 1 and the side chain carboxyl group of the amino acid residue at position 6 Cyclic hexapeptide. The cyclic hexapeptide of claim 13, wherein the hexapeptide is cyclized by the formation of an amide bond between the side chain carboxyl group at the amino acid residue at position 1 and the side chain amino group at the amino acid at position 6. The hexapeptide of claim 13, wherein the hexapeptide is used to form a covalent bond comprising an amide, disulfide, thioether, schiff base, reduced Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. Cyclic hexapeptide that is cyclized by 14. The cyclic hexapeptide of claim 13, wherein the core sequence is at positions 2-5 and is His-D-Nal 2-X-Nal 2 and cyclized through amino acids at positions 1 and 6. The cyclic hexapeptide of claim 13, wherein the core sequence is at positions 2-5 and is Trp-D-Nal 2-X-Nal 2, and is cyclized via amino acids at positions 1 and 6. The cyclic hexapeptide of claim 13, wherein the core sequence is at positions 2 to 5 and is His-D-Nal 2-X-Trp and is cyclized via amino acids at positions 1 and 6.

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