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Title:
POLYETHYLENE GLYCOL LINKED MC4R OR MC3R AGONIST PEPTIDES
Document Type and Number:
WIPO Patent Application WO/2006/073772
Kind Code:
A1
Abstract:
The invention provides MC4R or MC3R agonist peptides coupled to at least one polyethylene glycol molecule or derivative thereof, resulting in a biologically active peptide with an extended half-life and a slower clearance when compared to that of unPEGylated peptide. These PEGylated MC4R or MC3R agonist peptides and compositions are useful in treating obesity, diabetes, sexual dysfunction, cachexia, sarcopenia, dyslipidemia, to increase weight loss, or to increase muscle mass.

Inventors:
Flora, David Benjamin (5096 North 300 East, Greenfield, Indiana, 46140, US)
Mayer, John Philip (5839 North Washington Boulevard, Indianapolis, Indiana, 46220, US)
Yan, Liang Zeng (12420 Springbrooke Run, Carmel, Indiana, 46033, US)
Application Number:
PCT/US2005/045868
Publication Date:
July 13, 2006
Filing Date:
December 16, 2005
Export Citation:
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Assignee:
ELI LILLY AND COMPANY (Lilly Corporate Center, Indianapolis, Indiana, 46285, US)
Flora, David Benjamin (5096 North 300 East, Greenfield, Indiana, 46140, US)
Mayer, John Philip (5839 North Washington Boulevard, Indianapolis, Indiana, 46220, US)
Yan, Liang Zeng (12420 Springbrooke Run, Carmel, Indiana, 46033, US)
International Classes:
A61K47/48; A61P1/18; A61P3/04; A61P15/10; A61P21/00; C07K7/56; C07K14/68
Attorney, Agent or Firm:
Davis, Paula K. (ELI LILLY AND COMPANY, P.O. Box 6288 Indianapolis, Indiana, 46206-6288, US)
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Claims:
We Claim:
1. A PEGylated MC4R or MC3R agonist peptide of the formula: and pharmaceutically acceptable salts thereof, wherein W is GIu or Asp; R1 is C(O)CH3, C(O)(CH2)14CH3, C(O)(CH2)14NHC(NH)NH2, AcTyrβ hArg, gluconoylTyrArg, Acdiaminobutyryl, Acdiaminopropionyl, Npropionyl, Nbutyryl, Nvaleryl, NmethylTyrArg, NglutarylTyrArg, NsuccinylTyrArg, R6SO2NHC(O)CH2CH2C(O), R6SO2NHC(O)CH2CH2C(O)Arg, R6SO2NHCH2CH2CH2C(O), C3C7 cycloalkylcarbonyl, phenylsulfonyl, C8C14 bicyclic arylsulfonyl, phenyl(CH2)qC(O), C8C14 bicyclic aryl(CH2)qC(O), , wherein R2 is H, NHC(O)CH3, NHC(O)(CH2)14CH3, NHTyrC(O)CH3, R6SO2NH, AcCyaNH, HO(C6Hs)CH2CH2C(O)NH, or CH3(C6Hs)C(O)CH2CH2C(O)NH; R3 is C1C4 straight or branched alkyl, NH2CH2(CH2)q, HOCH2, (CH3)2CHNH(CH2)4, R6(CH2)q, R6SO2NH, Ser, He, q is O, 1, 2, or 3; R6 is a phenyl or C8C14 bicyclic aryl; m is 1 or 2; p is 1 or 2; R4 is H, C1C4 straight or branched alkyl, phenyl, benzyl, or (C6Hs)CH2OCH2; X is H, Cl, F, Br, methyl, or methoxy; and R5 is NH2, OH, glycinol, NH2ProSer, NH2ProLys, HOSer, HOProSer, HOLys, Ser alcohol, SerPro alcohol, LysPro alcohol, HOCH2CH2OCH2CH2NH, NH2PheArg, NH2GIu, NH2CH2RCH2NH, RHN, or RO where R is a C1C4 straight or branched alkyl; and wherein one PEG molecule is covalently bonded to the carboxylic acid moiety of GIu or Asp at position W.
2. A PEGylated MC4R or MC3R agonist peptide comprising one PEG molecule covalently bonded to a carboxylic acid moiety of a GIu or Asp residue in an unPEGylated MC4R or MC3R agonist peptide selected from the group consisting of Compound Numbers 1100.
3. A method of agonizing an MC4 or MC3 receptor in a subject in need of such agonism, said method comprising the step of administering to the subject an effective amount of a PEGylated MC4R or MC3R agonist peptide of Claim 1 or 2.
4. The method of Claim 3, wherein the subject is treated for obesity.
5. The method of Claim 3, wherein the subject is treated for diabetes.
6. The method of Claim ,3, wherein the subject is treated for sexual dysfunction.
7. The method of Claim 3, wherein the subject is treated for cachexia.
8. The method of Claim 3, wherein the subject is treated for sarcopenia.
9. The method of Claim 3, wherein the subject is treated for dyslipidemia.
10. The method of Claim 3, wherein the subject is treated to increase weight loss.
11. The method of Claim 3, wherein the subject is treated to increase muscle mass.
12. Use of a PEGylated MC4R or MC3R agonist peptide of Claim 1 or 2 in the manufacture of a medicament for the treatment of obesity, diabetes, sexual dysfunction, cachexia, sarcopenia, dyslipidemia, to increase weight loss, or to increase muscle mass.
13. The use of Claim 12, wherein the medicament is used to treat obesity.
14. The use of Claim 12, wherein the medicament is used to treat diabetes.
15. The use of Claim 12, wherein the medicament is used to treat sexual dysfunction.
16. The use of Claim 12, wherein the medicament is used to treat cachexia.
17. The use of Claim 12, wherein the medicament is used to treat sarcopenia.
18. The use of Claim 12, wherein the medicament is used to treat dyslipidemia.
19. The use of Claim 12, wherein the medicament is used to increase weight loss.
20. The use of Claim 12, wherein the medicament is used to increase muscle mass.
Description:
POLYETHYLENE GLYCOL LINKED MC4R OR MC3R AGONIST PEPTIDES

The present invention relates to melanocortin 4 receptor (MC4R) agonist peptides or melanocortin 3 receptor (MC3R) agonist peptides covalently bonded to a molecule of polyethylene glycol or a derivative thereof, and related compositions and methods useful in treating conditions or disorders benefited by inducing weight loss, reducing obesity, or increasing muscle mass.

MacNeil et al provide an excellent overview of the melanocortin receptors and their function relating to body weight regulation (EMr. J. Pharm. 440(2-3): 141-57, 2002). Both MC3R and MC4R are expressed in the brain. MC4R is expressed throughout the brain, whereas MC3R is expressed predominantly in the hypothalamus, leading to an abundance of MC3R there (Roselli-Rehfuss et al, PNAS USA 90:8856-60, 1993). Both MC3R and MC4R are involved in regulating energy metabolism. Analysis of Mc3r-/- mice suggests that MC3R is complementary to the MC4R's role in regulating body weight. The mice, although not significantly overweight, exhibit increased adiposity, with an increased feeding efficiency (Butler et al, Endocrinol 141:3518-21, 2000; Chen et al, Nat. Genet. 26:97-102, 2000). Kim et al suggest that MC4R regulates food intake and energy expenditure, whereas MC3R regulates feeding efficiency and partitioning of nutrients into fat {Diabetes 49:177-82, 2000; /. Clin. Invest. 105(7): 1005-11, 2000). Furthermore, both receptors regulate insulin activity (Fan et al, Endocrinol. 141(9):3072-79, 2000; Obici et al, J. Clin. Invest. 108:1079-85, 2001).

Additionally, MC3R is expressed in peripheral tissues such as heart, gut, stomach, pancreas, placenta, testis, ovary, muscle, and kidney (Gantz et al, J. Biol. Chem. 268:8246-50, 1993; Chhajlani, Biochem. MoI Biol Int. 38:73-80, 1996). Gamma-melanocortin stimulating hormone (γ-MSH), a compound that reduces blood pressure and heart rate when administered by intracerebroventricular administration, preferentially activates MC3R. This suggests that the MC3R may play a role in regulating cardiovascular functions (Versteeg et al, Eur. J. Pharmacol. 360:1-14, 1998).

The usefulness of peptides and proteins as therapeutic drugs is often limited by

enzymatic instability and the short plasma half-life of peptide- and protein-based drug molecules. Enzymatic degradation contributes partially to short plasma half-lives. Additionally, the relatively small size of peptides allows rapid in vivo clearance from the circulation. Consequently, many MC4R and MC3R agonist peptides have half -lives between fifteen minutes and two hours. Approaches are needed to decrease MC4R or MC3R agonist peptide clearance and increase half -life, thereby improving its usefulness as a therapeutic.

The PEGylated MC4R or MC3R agonist peptides of the present invention are useful because they retain all or a portion of biological activity of the corresponding unPEGylated MC4R or MC3R agonist peptide, yet they have enhanced half-life and/or reduced clearance when compared to that of the unPEGylated MC4R or MC3R agonist peptide. Such PEGylated MC4R or MC3R agonist peptides may be used therapeutically to treat subjects with disorders including, but not limited to, obesity, diabetes, sexual dysfunction, cachexia, sarcopenia and other frailty disorders, and dyslipidemia and its pathological sequelae, with a particular advantage being that the PEGylated MC4R or MC3R agonist peptides of the invention require fewer doses during a 24-hour period compared to the corresponding unPEGylated peptides.

The invention described herein provides MC4R or MC3R agonist peptides covalently bonded to a molecule of polyethylene glycol (PEG), or a derivative thereof, wherein PEG is covalently bonded to a carboxylic acid moiety of a GIu or Asp residue in the peptide, resulting in PEGylated MC4R or MC3R agonist peptides with an elimination half -life of at least one hour, preferably at least 3, 5, 7, 10, 15, or 20 hours and most preferably at least 24 hours. The PEGylated MC4R or MC3R agonist peptides of the present invention preferably have a clearance value of 15 mL/min/kg or less, more preferably 10, 7, 5, 3, or 2 mL/min/kg or less, and most preferably less than 1, 0.5, or 0.1 mL/min/kg.

One embodiment of the present invention is directed to PEGylated MC4R or MC3R agonist peptides represented by the following Structural Formula I:

and pharmaceutically acceptable salts thereof, wherein W is GIu or Asp;

R 1 is -C(O)CH 3 , -C(O)(CH 2 ) 1-4 CH 3 , -C(O)(CH 2 ) 1-4 NHC(NH)NH 2 , Ac-Tyr-β- hArg-, gluconoyl-Tyr-Arg-, Ac-diaminobutyryl-, Ac-diaminopropionyl-, N-propionyl-, N-butyryl-, N-valeryl-, N-methyl-Tyr-Arg-, N-glutaryl-Tyr-Arg-, N-succinyl-Tyr-Arg-, R 6 -SO 2 NHC(O)CH 2 CH 2 C(O)-, R 6 -SO 2 NHC(O)CH 2 CH 2 C(O)Arg-, R 6 -SO 2 NHCH 2 CH 2 CH 2 C(O)-, C 3 -C 7 cycloalkylcarbonyl, phenylsulfonyl, C 8 -C 14 bicyclic arylsulfonyl, phenyl-(CH 2 ) q C(O)-, C 8 -C 14 bicyclic aryl-(CH 2 ) q C(O)-,

, wherein

R 2 is -H, -NHC(O)CH 3 , -NHC(O)(CH 2 ) 1-4 CH 3 , -NH-TyrC(O)CH 3 , R 6 SO 2 NH-, Ac-Cya-NH-,

- A -

HO-(C 6 Hs)-CH 2 CH 2 C(O)NH-, or CH 3 -(C 6 Hs)-C(O)CH 2 CH 2 C(O)NH-;

R 3 is C 1 -C 4 straight or branched alkyl, NH 2 -CH 2 -(CH 2 ) q -, HO-CH 2 -, (CH 3 ) 2 CHNH(CH 2 ) 4 -, R 6 (CH 2 ) q -, R 6 SO 2 NH-, Ser, De,

q is O, 1, 2, or 3;

R 6 is a phenyl or C 8 -C 14 bicyclic aryl; m is 1 or 2; p is 1 or 2; R 4 is H, C 1 -C 4 straight or branched alkyl, phenyl, benzyl, or

(C 6 Hs)-CH 2 -O-CH 2 -; X is H, Cl, F, Br, methyl, or methoxy; and R 5 is -NH 2 , -OH, glycinol, NH 2 -Pro-Ser-, NH 2 -Pro-Lys-, HO-Ser-,

HO-Pro-Ser-, HO-Lys-, -Ser alcohol, -Ser-Pro alcohol, -Lys-Pro alcohol, HOCH 2 CH 2 -O-CH 2 CH 2 NH-, NH 2 -Phe-Arg-, NH 2 -GIu-,

NH 2 CH 2 RCH 2 NH-, RHN-, or RO- where R is a C 1 -C 4 straight or branched alkyl; and wherein one PEG molecule is covalently bonded to the carboxylic acid moiety of GIu or Asp at position W.

A preferred embodiment of the present invention is directed to PEGylated MC4R or MC3R agonist peptides, comprising one PEG molecule covalently bonded to a carboxylic acid moiety of a GIu or Asp residue in an unPEGylated MC4R or MC3R agonist peptide selected from the group consisting of Compound Numbers 1-100 of Table 1, below.

Table 1. UnPEGylated MC4R or MC3R Agonist Peptides

The polyethylene glycol polymers used in the invention ("PEG") preferably have molecular weights between 500 and 100,000 Daltons, more preferably between 2,000 and 40,000 Daltons, and most preferably between 5,000 and 20,000 Daltons. The polyethylene glycol polymer may be a linear or branched molecule, and it may be a polyethylene glycol derivative as described in the art.

The present invention also encompasses a method of agonizing the MC4 or MC3 receptor in a subject in need of such agonism, said method comprising the step of administering to the subject an effective amount of a PEGylated MC4R or MC3R agonist peptide described herein. Subjects in need of MC4 or MC3 receptor agonism may include those with obesity, diabetes, sexual dysfunction, cachexia, sarcopenia and other frailty disorders, dyslipidemia and its pathological sequelae, or those with a need to increase weight loss or increase muscle mass.

The present invention describes modifications to MC4R or MC3R agonist peptides resulting in extended elimination half-life and/or reduced clearance. Polyethylene glycol (PEG) or PEG derivative may be covalently bonded to a carboxylic acid moiety of a GIu or Asp residue of the peptide, resulting in a PEGylated MC4R or MC3R agonist peptide. The terms "PEGylated" or "PEGylation," when referring to an MC4R or MC3R agonist peptide of the present invention, refer to an MC4R or MC3R agonist peptide that is chemically modified by covalent attachment of a molecule of polyethylene glycol or a derivative thereof. Furthermore, it is intended that the terms "polyethylene glycol" or

"PEG" refer to polyethylene glycol or a derivative thereof as are known in the art {see, e.g., U.S. Patent Nos: 5,446,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659; 6,515,100; 6,514,491; and the present application). In PEGylated MC4R or MC3R agonist peptides of the present invention, PEG is covalently bonded to a carboxylic acid moiety of a GIu or Asp residue of the MC4R or MC3R agonist peptide. Optionally, the PEG molecules may be attached to the MC4R or MC3R agonist peptide via a linker or spacer molecule (see example spacer molecules described in U.S. Patent No. 6,268,343).

"*" means that both the D- and L- isomers are possible. "Ac" refers to acetyl (i.e. , -C(O)CH 3 ).

"Orn" refers to ornithine.

"hCys" refers to homocysteine.

"hArg" refers to homoarginine.

"Lys(ipr)" refers to lysine(N-isopropyl). "Cit" refers to citrulline.

"nLeu" refers to norleucine.

"Me" refers to methyl.

"OMe" refers to methoxy.

"Cya" refers to cysteic acid. "Dap" refers to diaminopropionyl.

"Dab" refers to diaminobutyryl.

"βArg" refers to beta-arginine.

"β-hArg" refers to beta-homoarginine.

"Cya" refers to cysteic acid. "Bom" refers to benzyloxymethyl.

Modified amino acids are indicated by parentheses around the amino acid and the modification thereto (e.g., (4-Cl-D-Phe) is a 4-chloro modification on the D-isomer of phenylalanine). With respect to moieties depicted in Structural Formula I, the single letter designations are as defined and do not refer to single letter amino acids corresponding to those letters (e.g. , W and X).

The letter "D" preceding the above-mentioned 3-letter abbreviations, e.g., "D-Phe," means the D-form of the amino acid. When the single letter abbreviation is used

for an amino acid, a "d" will precede the letter to designate the D-form of the amino acid (e.g. dF = D-Phe). Unless otherwise indicated herein, absence of a "D" or "L" designation indicates that the abbreviation refers to both D- and L- forms.

An "amino alcohol" is an amino acid that has been modified by reducing the carbonyl group of the C-terminus to a methylene group. Amino alcohols are denoted by the general nomenclature "Xaa alcohol," wherein Xaa is the specific amino acid from which the carbonyl group has been removed. To illustrate, "Ser alcohol" has the structure H 2 N-CH(CH 2 OH)-CH 2 OH as opposed to the Ser amino acid structure of H 2 N-CH(CH 2 OH)-COOH. For the purposes of the present invention, an in vitro MC4 or MC3 receptor potency assay is used to determine whether a PEGylated peptide of the present invention will exhibit biological activity. "In vitro potency" as used herein, is the measure of the ability of a peptide to activate the MC4 or MC3 receptor in a cell-based assay. In vitro potency is expressed as the "EC 50 " which is the effective concentration of compound that results in 50% activity in a single dose-response experiment. For the purposes of the present invention, in vitro potency is determined using an assay that employs HEK-293 cells that stably express the human MC4 or MC3 receptor. See Examples 1 and 2. Relative in vitro potency values may be established by running NDP-αMSH or the respective unPEGylated MC4R or MC3R agonist peptide as a control and assigning the control a reference value of 100%.

The term "plasma half-life" refers to the time in which half of the relevant molecules circulate in the plasma prior to being cleared. An alternatively used term is "elimination half-life." The term "extended" or "longer" used in the context of plasma half-life or elimination half -life indicates there is a statistically significant increase in the half-life of a PEGylated MC4R or MC3R agonist peptide relative to that of the reference molecule (e.g., the unPEGylated peptide) as determined under comparable conditions. Preferably a PEGylated MC4R or MC3R agonist peptide of the present invention has an elimination half-life of at least one hour, more preferably at least 3, 5, 7, 10, 15, 20 hours and most preferably at least 24 hours. Those of skill in the art appreciate that half -life is a derived parameter that changes as a function of both clearance and volume of distribution. "Clearance" is the measure of the body's ability to eliminate a drug. As clearance decreases due, for example, to modifications to a drug, half -life would be expected to

increase. However, this reciprocal relationship is exact only when there is no change in the volume of distribution. A useful approximate relationship between the terminal log- linear half-life (t y 2 ), clearance (C), and volume of distribution (V) is given by the equation: t γ 2 ~ 0.693 (V/C). Clearance does not indicate how much drug is being removed but, rather, the volume of biological fluid such as blood or plasma that would have to be completely freed of drug to account for the elimination. Clearance is expressed as a volume per unit of time. The PEGylated MC4R or MC3R agonist peptides of the present invention preferably have a clearance value of 15 mL/min/kg or less, more preferably 10, 7, 5, 3, or 2 mL/min/kg or less, and most preferably less than 1, 0.5, or 0.1 mL/min/kg.

A peptide for use in the invention is modified by covalently bonding to a carboxylic acid moiety of a GIu or Asp residue of the peptide. A wide variety of methods have been described in the art to produce peptides and proteins covalently bonded to PEG, and the specific method used for the present invention is not intended to be limiting (see, e.g., Roberts, M. et al. Advanced Drug Delivery Reviews, 54:459-476, 2002). PEGylation of proteins may overcome many of the pharmacological and toxicological/immunological problems associated with using peptides or proteins as therapeutics. However, for any individual peptide, it is uncertain whether the PEGylated form of the peptide will have significant loss in bioactivity as compared to the unPEGylated form of the peptide.

The bioactivity of PEGylated proteins can be affected by factors such as: i) the size of the PEG molecule; ii) the particular sites of attachment; iii) the degree of modification; iv) adverse coupling conditions; v) whether a linker is used for attachment or whether the polymer is directly attached; vi) generation of harmful co-products; vii) damage inflicted by the activated polymer; or viii) retention of charge. Depending on the coupling reaction used, polymer modification of cytokines, in particular, has resulted in dramatic reductions in bioactivity. [Francis, G.E. et ah, PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimization of coupling techniques, Intl. J. Hem. 68:1-18, 1998]. Although some PEGylated MC4R or MC3R agonist peptides of the invention may have biological activity lower than that of unPEGylated MC4R or MC3R agonist peptide as measured in a particular assay, this

activity decrease is compensated by the compound's extended half-life and/or lower clearance value and may even be a favorable characteristic for an MC4R or MC3R agonist peptide with an extended elimination half-life.

In its typical form, PEG is a linear polymer with terminal hydroxyl groups and has the formula HO-CH 2 CH 2 -(CH 2 CH 2 O)ZX-CH 2 CH 2 -OH, where n is from about 8 to about 4000. The terminal hydrogen may be substituted with a protective group such as an alkyl or alkanol group. Preferably, PEG has at least one hydroxy group; more preferably, it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with the peptide. There are many forms of PEG useful for the present invention. Numerous derivatives of PEG exist in the art and are suitable for use in the invention. {See, e.g., U.S. Patent Nos: 5,446,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659; 6,515,100 and 6,514,491 and Zalipsky, S. Bioconjugate Chem. 6: 150-165, 1995). The PEG molecule covalently attached to MC4R or MC3R agonist peptides in the present invention is not intended to be limited to a particular type. PEG's molecular weight is preferably from 500-100,000 Daltons, more preferably from 2,000-40,000 Daltons, and most preferably from 5,000-20,000 Daltons. PEG may be linear or branched, and PEGylated MC4R or MC3R agonist peptides of the invention may have one or more PEG molecules attached to the peptide. It is most preferably that there be one PEG molecule per PEGylated MC4R or MC3R agonist peptide molecule; however, when there are more than one PEG molecules per peptide molecule, it is preferred that there be no more than two. It is further contemplated that both ends of the PEG molecule may be homo- or hetero-functionalized for crosslinking two or more MC4R or MC3R agonist peptides together.

The present invention provides MC4R or MC3R agonist peptides with one or more PEG molecules covalently attached thereto. A side-product may also be produced, wherein an acidic moiety in the peptide, such as a GIu or Asp residue, is also PEGylated.

A method for preparing the PEGylated MC4R agonist peptides of the present invention is covalently bonding the PEG to a carboxylic acid moiety of a GIu or Asp residue. This reaction occurs according to the following scheme, shown with an example MC4R agonist peptide:

It is contemplated that use of a PEGylated MC4R agonist peptide of the present invention includes use in the manufacture of a medicament for the treatment of obesity, diabetes, sexual dysfunction, cachexia, sarcopenia and other frailty disorders, dyslipidemia and its pathological sequelae, to increase weight loss, or to increase muscle mass.

PEGylation of an MC4R or MC3R agonist peptide may be combined with other modifications known in the art to increase peptide half -life and thereby increase the half- life of the compound even further than PEGylation alone or other modification methods alone.

As used herein, the terms "MC4R or MC3R agonist peptide," "MC4R agonist peptide," and "MC3R agonist peptide" also include pharmaceutically acceptable salts of the compounds described herein. An MC4R or MC3R agonist peptide of this invention can possess a sufficiently acidic group, a sufficiently basic group, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. See, e.g., Patent Application No. PCT/US04/16625 for

example salts of the MC4R or MC3R agonist peptides of the present invention.

The PEGylated MC4R or MC3R agonist peptides of the present invention are particularly suited for parenteral administration. Administration of the PEGylated peptide may be accomplished by, but is not limited to, delivery via pump, depot, suppository, pessary, transdermal patch or other topical administration (such as buccal, sublingual, spray, ointment, creme, or gel) using, for example, subcutaneous, intramuscular, intraperitoneal, intravenous, intracerebral, or intraarterial administration.

The PEGylated MC4R or MC3R agonist peptides can be administered to the subject in conjunction with an acceptable pharmaceutical carrier, diluent or excipient as part of a pharmaceutical composition for treatment as discussed above. The pharmaceutical composition can be a solution or, if administered parenterally, a suspension of the PEGylated peptide or a suspension of the PEGylated peptide complexed with a divalent metal cation such as zinc. Suitable pharmaceutical carriers may contain inert ingredients that do not interact with the peptide or peptide derivative. Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA). Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer' s-lactate and the like. Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol.

A "therapeutically effective amount" means that amount of a compound, or salt thereof, that will elicit the biological or medical response of a tissue, system, or mammal and/or is capable of treating the conditions described herein, or that is capable of agonizing the MC3 and/or MC4 receptors. An "effective amount" of the peptide administered to a subject will also depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The recipient patient's physician should determine the therapeutic dose administered in light of the relevant circumstances. A therapeutically effective amount can be administered prophylactically to a patient thought to be susceptible to development of a disease or condition. Such amount, when administered prophylactically to a patient, can also be effective to prevent or lessen

the severity of the mediated condition. The dosage regimen utilizing the compounds of the present invention is selected by one of ordinary skill in the medical or veterinary arts, in view of a variety of factors, including, without limitation, the route of administration, the prior medical history of the recipient, the pathological condition or symptom being treated, the severity of the condition/symptom being treated, and the age and sex of the recipient patient. However, it will be understood that the therapeutic dose administered will be determined by the attending physician in the light of the relevant circumstances.

Generally, an effective minimum daily dose of a compound of the present invention will exceed about 0.01 mg. Typically, an effective maximum daily dose will not exceed about 1000 mg. More preferably, an effective minimum daily dose will be between about 0.05 mg and 50 mg, more preferably between 0.1 mg and 10 mg. Most preferably, an effective minimum daily dose of an MC4R or MC3R agonist peptide in the present invention will exceed about 2 μg/kg and will not exceed about 20 μg/kg. The exact dose may be determined, in accordance with the standard practice in the medical arts of "dose titrating" the recipient; that is, initially administering a low dose of the compound, and gradually increasing the dose until the desired therapeutic effect is observed. The desired dose may be presented in a single dose or as divided doses administered at appropriate intervals.

A "subject" is a mammal, preferably a human, but can also be an animal, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

The peptides used to generate the PEGylated MC4R or MC3R agonist peptides of the present invention can be prepared by using standard methods of solution phase or solid-phase peptide synthesis techniques. See, e.g., Patent Application No. PCT/US04/16625 for peptide syntheses. Peptide synthesizers are commercially available from, for example, Applied Biosystems in Foster City CA. Reagents for solid phase synthesis are commercially available, for example, from Midwest Biotech (Fishers, IN). Solid phase peptide synthesizers can be used according to manufacturers' instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, decoupling, and capping of unreacted amino acids.

The invention is illustrated by the following examples that are not intended to be limiting in any way.

EXAMPLES

Example 1: Construction of MC Receptor Expression Plasmids Construction of human MCl expression plasmid:

Human MCl cDNA is cloned by PCR using human genomic DNA (Clontech Cat. # 6550-1) as a template. A forward hMCl gene-specific primer containing initiation codon (ATG) and EcoRI site and a reverse hMCl gene specific primer containing a stop codon and Xbal site are used in the PCR. The full-length hMCl cDNA generated by PCR is cloned into ρUC18/SmaI plasmid (Pharmacia Cat. # 27-5266-01), and the correct hMCl cDNA is confirmed by DNA sequencing. The sequenced pUC18hMCl is digested with EcoRI and Xbal, and the hMCl cDNA fragment is then subcloned into pcDNA3.1 (Invitrogen Cat. # V790-20) to generate expression plasmid pCDNA3-hMCl. Construction of human MC3 expression plasmid:

Human MC3 cDNA is cloned by PCR using human genomic DNA (Clontech Cat. # 6550-1) as a template. A forward hMC3 gene-specific primer containing initiation codon (ATG) and EcoRI site and a reverse hMC3 gene specific primer containing a stop codon and Xbal site are used in the PCR. The full-length hMC3 cDNA generated by PCR is cloned into pUC18/SmaI plasmid (Pharmacia Cat# 27-5266-01), and the correct hMC3 cDNA is confirmed by DNA sequencing. The sequenced pUC18hMC3 is digested with EcoRI and Xbal, and the hMC3 cDNA fragment is then subcloned into pcDNA3.1 (Invitrogen Cat. # V790-20) to generate expression plasmid pCDNA3-hMC3. Construction of human MC4 expression plasmid:

Human MC4 (hMC4) cDNA is cloned in a similar way as hMC3 cDNA by PCR using human fetal brain cDNA (Clontech Cat. # 7402-1) as a template. The hMC4 cDNA PCR product is digested with EcoRI/Xbal, and then subcloned into pCIneo (Promega Cat. # El 841) and sequenced. The resulting hMC4R plasmid has two mutations, which are then corrected to create the hMC4 cDNA encoding the correct hMC4 protein. The corrected hMC4 cDNA is then subcloned into pcDNA3.1 to generate expression plasmid pCDNA3-hMC4. Construction of human MC5 expression plasmid: Human MC5 cDNA is cloned by PCR using human genomic DNA (Clontech

Cat. # 6550-1) as a template. A forward hMC5 gene-specific primer containing initiation codon (ATG) and HindIII site and a reverse hMC5 gene specific primer containing a stop

codon and Xbal site are used in the PCR. The full-length hMC5 cDNA generated by PCR is cloned into pUC18/SmaI plasmid (Pharmacia Cat. # 27-5266-01), and the correct hMC5 cDNA is confirmed by DNA sequencing. The sequenced pUC18hMC5 is digested with EcoRI and Xbal, and the hMC5 cDNA fragment is then subcloned into pcDNA3.1 (Invitrogen Cat. # V790-20) to generate expression plasmid pCDNA3-hMC5. Stable HEK-293 cells expressing human MCRs:

Stable 293 cells expressing all hMCRs are generated by co-transfecting HEK-293 cells with pCDNA3-hMC4R and a CRE-luciferase reporter plasmid following the protocol of Lipofectamine Plus Reagent (Invitrogen, Cat. # 10964-013). For selection of stable transfectants, Genticin (G418) at a concentration of 300 μg/mL is added to the media 48 hours after the start of transfection. After 2-3 weeks, 40-50 of isolated clones are selected, propagated, and assayed for luciferase activity using a Luciferase Reporter Gene Assay Kit (Roche, Cat. # 1814036). Around five stable clones with highly stimulated luciferase activities of 10 nM NDP-αMSH are established.

Example 2: Melanocortin Receptor Whole Cell cAMP Accumulation Assay Hank's Balanced Salt Solution without phenol red (HBSS-092), 1 M HEPES, Dulbecco's Modified Eagle Media (DMEM), Fetal Bovine Serum (FBS), Antibiotic/Antimycotic Solution, and sodium acetate are obtained from GibcoBRL. Triton X-100, ascorbic acid, cAMP, and 3-isobutyl-l-methyl-xanthine (IBMX) are purchased from Sigma. Bovine Serum Albumin (BSA) is obtained from Roche. SPA PVT antibody-binding beads type II anti-sheep beads and 125 I cAMP are obtained from Amersham. Anti-goat cAMP antibody is obtained from ICN. Enzyme Free Cell Dissociation Solution Hank's based is obtained from Specialty Media. NDP-αMSH is obtained from Calbiochem. Dimethylsulfoxide (DMSO) is obtained from Aldrich. Compound Preparation:

In the agonist assay, compounds are prepared as 10 mM solutions, and NDP- αMSH (control) is prepared as 33.3 μM stock solutions in 100% DMSO. These solutions are serially diluted in 100% DMSO. The compound plate is further diluted in compound dilution buffer (HBSS-092, 1 mM ascorbic acid, 1 mM IBMX, 0.6% DMSO, 0.1% BSA) to yield a final concentration range in the assay between 600 nM-6 pM for compound and

100 nM-1 pM for NDP-αMSH control in 0.5% DMSO. Twenty μL of compound solution are transferred from this plate into four PET 96-well plates (all assays are performed in duplicate for each receptor). Cell Culture and Cell Stimulation: HEK 293 cells stably transfected with the human MC3R or MC4R are grown in

DMEM containing 10 % FBS and 1% Antibiotic/ Antimycotic Solution. On the day of the assay, the cells are dislodged with enzyme free cell dissociation solution and re-suspended in cell buffer (HBSS-092, 0.1% BSA, 10 mM HEPES) at 1 x 10 6 cells/mL. Forty μL of cell suspension are added per well to PET 96-well plates containing 20 μL of diluted compound or control. Plates are incubated at 37°C in a waterbath for 20 minutes. The assay is stopped by adding 50 μL Quench Buffer (50 mM sodium acetate, 0.25% Triton X-100). Determination of cAMP concentrations:

Radioligand binding assays are run in SPA buffer (50 mM sodium acetate, 0.1% BSA). The beads, antibody, and radioligand are diluted in SPA buffer to provide sufficient volume for each 96-well plate. To each quenched assay well is added 100 μL cocktail containing 33.33 μL of beads, 33.33 μL antibody, and 33.33 μL 125 I-CAMP. This is based on a final concentration of 6.3 mg/mL beads, 0.65% anti-goat antibody, and 61 pM of 125 I-CAMP (containing 25,000-30,000 CPM) in a final assay volume of 210 μL. The.plates are counted in a Wallac MicroBeta counter after a 12-hour incubation.

The data are converted to pmol of cAMP using a standard curve assayed under the same conditions. The data are analyzed using Activity Base software to generate agonist potencies (EC50), and percent relative efficacy data compared to NDP-αMSH.

Example 3: PEGylation of a Carboxylic Acid Moiety in MC4R or MC3R Agonist Peptides

PEGylation reactions are run under conditions that permit the formation of a stable covalent linkage between the PEG reagent and the MC4R or MC3R peptide molecule. The PEGylated MC4R or MC3R agonist peptide is then isolated using ion exchange HPLC combined with size exclusion chromatography (desalting) or reversed- phase HPLC directly. PEGylated MC4R or MC3R peptide analogs are characterized using analytical RP-HPLC, ion exchange HPLC, and MALDI mass spectrometry.

Peptide A (47.0 mg, 0.04 mmole) and 41.6 mg of PyBOP dissolved in 20 mL of dry DMF. Then, 400 mg (0.08 mmole) of PEG reagent (mPEG-NH 2 , MW 5,000 Da, Cat No. 2M2V0H01, Nektar Therapeutics, San Carlos, CA) and 100 μL of DIEA are added to above solution under stirring. Dry DCM (dichloromethane, 10-30% v/v) could be added to improve the solubility of PEG reagent in the reaction mixture. The reaction is allowed to proceed overnight at room temperature with a constant gentle stirring. Solvents in the reaction mixture are removed under vacuum and the residue is acidified to an acidic pH with a small amount of TFA. The acidified residue is retaken in water and loaded onto cation exchange HPLC (HiPrep SP, 1.6 x 10 cm, Pharmacia Biotech). The fractions containing the peptide-PEG conjugate are pooled and lyophilized. The lyophilized material is dissolved in water and desalted on a size exclusion column (Sephadex G-25 SF, 2.6 x 34 cm). The purified PEGylated peptide (106 mg) is further characterized by analytical cation exchange HPLC and MALDI mass spectroscopy.