WO2008048691A2

WO2008048691A2 - Use of leptin for treating post-lipectomy ectopic fat deposition and other post-lipectomy associated disorders

Info

Publication number: WO2008048691A2
Application number: PCT/US2007/022413
Authority: WO
Inventors: Howard E. Greene; Andrew A. Young
Original assignee: Amylin Pharmaceuticals, Inc.
Priority date: 2006-10-18
Filing date: 2007-10-18
Publication date: 2008-04-24
Also published as: WO2008048691A3

Abstract

Leptin, leptin analogs, leptin derivatives, and other leptin agonists are used to treat subjects with post-lipectomy ectopic fat deposition and associated metabolic abnormalities or disorders. Such leptin, leptin analogs, derivatives, and other leptin agonists are also used in the preparation of medicaments for treating or preventing ectopic fat deposition and associated metabolic abnormalities or disorders in such subjects. Such ectopic fat deposition and metabolic abnormalities conditions or disorders include, but are not limited to, hepatic steatosis, hypertriglyceridemia, dyslipidemia, cardiovascular disease, insulin-resistance, and diabetes mellitus.

Description

USE OF LEPTIN FOR TREATING POST-LIPECTOMY ECTOPIC FAT DEPOSITION AND OTHER POST-LIPECTOMY ASSOCIATED DISORDERS

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 60/852,811, filed October 18, 2006, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Obesity is a growing health problem in the world. Excess weight and obesity are associated with numerous complications that increase morbidity and mortality. For example, abdominal obesity, manifested by increased waist circumference, increased abdominal subcutaneous fat, and increased visceral fat, is associated with insulin resistance and other metabolic risk factors for coronary heart disease (Kissebah et al. (1982) J. Clin. Endocrinol. Metab. 54:254-260). [0003] Liposuction, also known as lipoplasty or suction-assisted lipectomy, is a common aesthetic surgical procedure performed in the United States. Nearly 400,000 procedures were performed in 2002, for example. Advances in liposuction techniques make it possible to remove large amounts of body fat such that lipectomy procedures are being considered as treatment options for obesity and possibly for metabolic complications of obesity.

[0004] The metabolic effects of liposuction, however, are unclear because the results of studies have varied and interpretation of the data is confounded by lifestyle and weight changes that occurred among the subjects post-surgery, by variations in the volume of adipose tissue removed and the site of its removal, by differences in the methods used to assess insulin sensitivity, and by differences in the subjects' baseline weight and insulin sensitivity. See, for example, Gonzalez-Ortiz et al. (2002) Horm. Metab. Res. 34:446-449; Klein et al. (2004) N. Engl. J. Med. 350:2549-2557; Esposito et al. (2004) N. Engl. J. Med. 351 :1354-1357; Giugliano et al. (2004) Br. J. Plast. Surg. 57:190-194; Arner (2004) TV. Engl. J. Med. 351 :1354-1357; Busetto et al. (2004) N. Engl. J. Med. 350:1354-1357; Ersek et al. (2004) Aesthetic Plast. Surg. 28:120-122.

[0005] Investigations in animal models have demonstrated that metabolic abnormalities may be associated with fat loss (Gavrilova et al. (2000) J. Clin. Invest.

105: 271-278). For example, subcutaneous lipectomy in conjunction with a high fat diet in hamsters resulted in fat deposition in visceral adipose tissue and the liver, significant hypertriglyceridemia, and an elevated basal insulinemic index, components of the metabolic, insulin resistance syndrome X of obesity (Weber et al. (2000) Am. J.

Physiol. Regulatory Integrative Comp. Physiol. 279:R936-R943).

[0006] Methods and compositions are needed to ensure healthy fat metabolism and deposition following fat removal, as well as treating or preventing associated metabolic and other physiologic abnormalities and disorders associated with fat removal.

[0007] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

SUMMARY

[0008] Methods provided include the use of leptin in treating or preventing ectopic fat deposition following lipectomy. Provided also are methods for treating or preventing lipectomy-associated metabolic abnormalities and other lipectomy-associated physiologic abnormalities.

[0009] In one embodiment, leptin is used in subjects having reduced serum concentration of leptin following lipectomy. In some embodiments, recombinant human leptin or leptin analog or leptin derivative is used. In some embodiments, a leptin agonist is used. Leptin proteins may be administered subcutaneously or systemically, or through any other route.

[0010] In some embodiments, leptin is administered in combination with at least one other active agent for the prevention or treatment of post-lipectomy ectopic fat deposition, a lipectomy associated metabolic abnormality or other physiologic abnormality, or any combination thereof.

DETAILED DESCRIPTION

[0011] Provided herein are methods and composition useful in treatment and prevention of post-lipectomy ectopic fat deposition, post-lipectomy metabolic abnormalities, and other post-lipectomy physiologic abnormalities. The methods provided herein use the administration of a leptin or leptin analog or leptin derivative or other leptin agonist for the control, prevention or treatment of such conditions and abnormalities.

[0012] The adipocyte hormone leptin plays a central role in energy homeostasis (Zhang et al (1994) Nature 372:425-432; Pelleymounter et al (1995) Science 269:540-543). Therapeutic attempts using leptin have been in the treatment of obesity and in the treatment of lipoatrophy (Heymsfield et al. (1999) JAMA 282:1568-1575; Farooqi et al. (1999) N. Engl. J. Med. 341 :879-884; Farooqi et al. (2002) J. Clin. Invest. 110:1093-1103; U.S. Patent Application Publication No. 2005/0020496). [0013] In one embodiment, methods provided herein are used to reduce, lessen or eliminate ectopic fat deposits in a subject following lipectomy. In one embodiment, post-lipectomy ectopic fat deposits are reduced, lessened or eliminated following administration of the leptin protein, or analog or derivative thereof, or other leptin agonist. An effective amount of leptin protein, or analog or derivative thereof, or other leptin agonist as used in this embodiment is an amount effective to reduce ectopic fat deposits in the subject. In one instance, post-lipectomy ectopic fat deposition is reduced by at least about 5%, 10%, 15%, 20% 25%, 30%, or 35% in a subject. Alternatively, the amount of ectopic fat is proportionally reduced 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in comparison to subcutaneous fat in a subject.

[0014] Ectopic fat deposition following lipectomy may occur anywhere in the body outside classical adipose tissue depots. Areas of ectopic fat deposition include, but are not limited to, liver, heart, pancreas, kidneys, blood vessels, and skeletal muscle. Ectopic fat can be measured in a subject using any method available for measuring ectopic fat.

[0015] In another embodiment, methods provided herein are used to prevent the accumulation or lessen the incidence of ectopic fat deposition in a subject following lipectomy, wherein the method comprises administering, to a post-lipectomy subject in need thereof, a leptin protein, or analog or derivative thereof, or other leptin agonist in an amount effective to prevent accumulation of ectopic fat or to lessen the incidence of ectopic fat deposition in the subject.

[0016] In another embodiment, methods are provided for producing a more favorable fat distribution in a subject following lipectomy, where the method comprises administering to a post-lipectomy subject a leptin protein, or analog or derivative thereof, or other leptin agonist in an amount effective to produce a favorable fat distribution. In one embodiment, administration of a leptin protein, or analog or derivative thereof, or other leptin agonist reduces the amount of visceral fat or ectopic fat, or both, in the subject. Such methods result in a higher ratio of subcutaneous fat to visceral fat or ectopic fat. Such prevention or reduction of ectopic fat deposition following lipectomy may result in a reduced risk of the development of cardiovascular diseases, hepatic steatosis, metabolic syndrome, or any combination thereof. [0017] In one embodiment, methods provided herein are used to treat metabolic abnormalities in a subject following lipectomy. In one embodiment, post-lipectomy metabolic abnormalities are ameliorated following administration of the leptin protein, or analog or derivative thereof, or other leptin agonist. An effective amount of leptin protein, or analog or derivative thereof, or other leptin agonist as used in this embodiment is an amount effective to treat post-lipectomy metabolic abnormalities in the subject. Post-lipectomy metabolic abnormalities include, but are not limited to, hyperglycemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis, and insulin resistance. Post-lipectomy metabolic abnormalities may also include diabetes mellitus and Syndrome X. The diagnosis of such metabolic abnormalities is generally based on clinical grounds well known to one of ordinary skill in the art. Diagnostic signals and biological markers to monitor the metabolic abnormalities are known in the art and include, but are not limited to serum glucose levels, serum insulin levels, glycosylated hemoglobin HbA₁C levels, and serum lipid levels including free fatty acid levels and fasting triglyceride levels.

[0018] In one embodiment, methods provided herein are used to treat other physiologic abnormalities in a subject following lipectomy. In one embodiment, post- lipectomy physiologic abnormalities are ameliorated following administration of the leptin protein, or analog or derivative thereof, or other leptin agonist. An effective amount of leptin protein, or analog or derivative thereof, or other leptin agonist as used in this embodiment is an amount effective to treat post-lipectomy physiologic abnormalities in the subject. Post-lipectomy physiologic abnormalities include, but are not limited to, reproduction dysfunction and neuroendocrine dysfunction, for example, amenorrhea (including hypothalamic amenorrhea), hypogonadism, hypothalamic dysfunction, and infertility (including females incapable of ovulation or conception and males with insufficient sperm count). Post-lipectomy physiologic abnormalities may also include decreased immune function, for example, suppressed lymphocyte subpopulations, suppressed lymphoproliferative responses, and suppressed ThI immunity. Post-lipectomy physiologic abnormalities may also include abnormalities in bone function and bone metabolism, for example, decreased bone mineral density. The diagnosis of such abnormalities is generally based on clinical grounds well known to one of ordinary skill in the art. Diagnostic signals and biological markers to monitor the abnormalities are known in the art. [0019] In another aspect, the methods provided herein may use a leptin protein, or analog or derivative thereof, or other leptin agonist as part of a combination therapy for the treatment or prevention of post-lipectomy ectopic fat deposition and post- lipectomy metabolic and physiologic abnormalities. Exemplary active agents used as part of such a combination therapy include, but are not limited to, an amylin or an amylin agonist, an anticonvulsant agent such as topiramate, an anorexigenic agent such as sibutramine and phentermine, a cannabinoid receptor antagonist such as rimonabant, an angiogenic factor such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and analogs thereof, and a satiety agent such as bombesin, gastrin-releasing peptide (GRP), and cholecystokinin (CCK) or a CCK agonist.

[0020] In some embodiments, the leptin protein, or analog or derivative thereof, or other leptin agonist is used in combination with at least one other active agent. In some embodiments, the leptin protein, or analog or derivative thereof, or other leptin agonist is used in combination with at least two other active agents. In some embodiments, the use of leptin protein, or analog or derivative thereof, or other leptin agonist in combination with at least one other such agent results in a synergistic effect for the reduction of post-lipectomy ectopic fat deposition, post-lipectomy metabolic abnormality, post-lipectomy physiologic abnormality or any combination thereof. In some embodiments, the use of leptin protein, or analog or derivative thereof, or other leptin agonist in combination with at least one other such agent results in a lower dosage requirement for at least one of the compounds, with the same effect in treating or preventing post-lipectomy ectopic fat deposition or post-lipectomy metabolic or physiologic abnormalities, or any combination thereof. [0021] As used herein, "lipectomy" refers to removal of fat tissue and cells from the body, generally through a surgical procedure. As used herein, "lipectomy" includes, but is not limited to, procedures referred to as lipoplasty, suction-assisted lipoectomy (SAL), liposuction, and large-volume liposuction. Lipectomy generally refers to removal of localized subcutaneous fat deposits but may also refer to the removal of intraperitoneal or visceral fatty tissue.

[0022] While "obesity" is generally defined as a body mass index over 30, for purposes of this disclosure, any subject, including those with a body mass index of less than 30, who needs or wishes to reduce body weight or body fat is included in the scope of "obese." Thus, subjects with a BMI of less than 30 but 25 and above (considered overweight) or below 25 are also included in the subjects of the invention. Morbid obesity refers to a BMI of 40 or greater. In one embodiment, a "subject in need thereof is obese. Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes) can benefit from this method.

[0023] A "subject" may include any mammal, including humans. Subjects who may benefit from the methods disclosed herein may be overweight or obese; however, they may also be lean. Subjects who may benefit from the methods disclosed herein may be desirous of losing weigh, losing body fat, altering body fat deposition, or any combination thereof. They may have a metabolic disorder or condition in addition to these conditions. Exemplary metabolic disorders include diabetes, metabolic syndrome X, insulin-resistance, and dyslipidemia.

[0024] By "fat distribution" is meant the location of fat deposits in the body. Such locations of fat deposition include subcutaneous, visceral and ectopic fat depots. [0025] By "subcutaneous fat" is meant the deposit of lipids just below the skin's surface. The amount of subcutaneous fat in a subject can be measured using any method available for the measurement of subcutaneous fat. Methods of measuring subcutaneous fat are known in the art, for example, those described in U.S. Pat. No. 6,530,886, the entirety of which is incorporated herein by reference. [0026] By "visceral fat" is meant the deposit of fat as intra-abdominal adipose tissue. Visceral fat surrounds vital organs and can be metabolized by the liver to produce blood cholesterol. Visceral fat has been associated with increased risks of conditions such as polycystic ovary syndrome, metabolic syndrome and cardiovascular diseases. [0027] By "ectopic fat storage" is meant lipid deposits within and around tissues and organs that constitute the lean body mass (e.g., skeletal muscle, heart, liver, pancreas, kidneys, blood vessels). Generally, ectopic fat storage is an accumulation of lipids outside classical adipose tissue depots in the body.

[0028] As used herein, "lean mass" or "lean body mass" refers to muscle and bone. Lean body mass does not necessarily indicate fat free mass. Lean body mass contains a small percentage of fat (roughly 3%) within the central nervous system (brain and spinal cord), marrow of bones, and internal organs. Lean body mass is measured in terms of density.

[0029] Methods of measuring fat mass and lean mass are known in the art and include, but are not limited to, underwater weighing, air displacement plethysmography, bioelectric impedance, x-ray, dual-energy x-ray absorptiometry (DEXA) scans, MRIs and CT scans. For example, body fat may be determined using DEXA (Hologic QDR 4500, Hologic, Inc., Bedford, MA) (Lambrinoudaki et al. (1998) Metabolism 47:1379-1382). Axial Tl weighted MR scans of the liver may be obtained on a 1.5 tesla scanner (General Electric Medical Systems, Milwaukee, WI) (Abate et al. (1994) J. Lipid Res. 35:1490-1496). Liver volumes may be calculated using the MEDx image analysis software package (Sensor Systems, Inc., Sterling, VA).

[0030] As used herein, and as well-understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. "Treating" or "palliating" a disease, disorder, or condition means that the extent, undesirable clinical manifestations of a condition, or both, of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. For purposes of the methods disclosed herein, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disorder, stabilized (i.e., not worsening) state of disorder, delay or slowing of disorder progression, amelioration or palliation of the disorder, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Further, treating does not necessarily occur by administration of one dose, but often occurs upon administration of a series of doses. Thus, a therapeutically effective amount, an amount sufficient to palliate, or an amount sufficient to treat a disease, disorder, or condition may be administered in one or more administrations.

[0031] As used herein, the singular form "a", "an", and "the" includes plural references unless otherwise indicated or clear from context. For example, as will be apparent from context, "a" leptin analog can include one or more leptin analogs. [0032] As described herein, leptin proteins, and analogs and derivatives thereof, and other leptin agonists are known in the art. Exemplary leptin proteins or analogs for use in the methods provided include, but are not limited to, the amino acid sequence for mature, recombinant methionyl human leptins presented herein as SEQ ED NO.:1 and SEQ ID NO.:2. In SEQ ID NO:1 (herein called rmetHu-Leptin 1-146), a methionyl residue is located at position 1 and the first amino acid of the mature leptin protein is valine (at position 2) and the amino acid sequence is: MVPIQKVQD DTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPI LTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLL HVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVA LSRLQGSLQDMLWQLDLSPGC. SEQ ID NO:2 (herein called rmetHu-Leptin 1-145) comprises a natural variant of human leptin, which has 145 amino acids, and, as compared to Hu-Leptin 1-146, has a glutamine absent at position 28. The amino acid sequence of SEQ ID NO.:2 is as follows: MVPIQKVQDD TKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILT LSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLH VLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVAL SRLQGSLQDMLWQLDLSPGC.

[0033] Exemplary leptin proteins, analogs, derivatives, preparations, formulations, pharmaceutical composition, doses, and administration routes have previously been described in the following U.S. and PCT Application Publications: U.S. Pat. Nos.5,935,810; 6,001,968; 6,429,290; 6,350,730; 6,936,439; 6,420,339; 6,541,033; U.S. Pat. Publication Nos.2005/0176107; 2005/0163799; and PCT Publication Nos. WO 96/05309; WO 96/40912; WO 97/06816; WO 97/18833; WO 97/38014; WO 98/08512; WO 98/28427; WO 98/46257; WO 00/20872; WO 00/09165; WO 00/47741; and WO 00/21574. The contents of each is hereby incorporated by reference.

[0034] Other exemplary leptin proteins, analogs and related molecules are reported in the following publications; however, no representation is made with regard to the activity of any composition reported: U.S. Pat. Nos. 5,521,283; 5,525,705; 5,532,336; 5,552,522; 5,552,523; 5,552,524; 5,554,727; 5,559,208; 5,563,243; 5,563,244; 5,563,245; 5,567,678; 5,567,803; 5,569,743; 5,569,744; 5,574,133; 5,580,954; 5,594,101; 5,594,104; 5,605,886; 5,614,379; 5,691,309; 5,719,266 ; PCT Publication Nos. WO96/23513; WO96/23514; WO96/23515; WO96/23516; WO96/23517; WO96/23518; WO96/23519; WO96/34111 ; WO96 37517; WO96/27385; WO97/00886; WO97/20933; WO97/16550; WO96/35787; WO96/34885; WO97/46585; WO96/22308; European Patent Publication Nos. EP 725078; EP 725079; EP 744408; EP 745610; EP 835879; EP 736599; EP 741187. To the extent these references provide for useful leptin proteins or analogs, or associated compositions or methods, such compositions and/or methods may be used in conjunction with the present methods. With this proviso, these publications are herein incorporated by reference.

[0035] By "analog" is meant a protein whose sequence is derived from that of leptin including insertions, substitutions, extensions, and/or deletions, having at least some amino acid identity to leptin or region of a leptin protein. Leptin analogs may have at least 50 or 55% amino acid sequence identity with a native protein, or at least 60, 65, 70, 75, 80, 85, 90, 95, or 99% amino acid sequence identity with a native leptin. In one embodiment, such analogs may comprise conservative or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms). Analogs, as herein defined, also include derivatives. Exemplary leptin analogs are known in the art and described, for example, in PCT Publication No. WO 96/05309, incorporated herein by reference.

[0036] A "derivative" is defined as a molecule having the amino acid sequence of a native leptin or analog, but additionally having a chemical modification of one or more of its amino acid side groups, α-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine ε-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, constrained alkyls {e.g. branched, cyclic, fused, adamantyl) and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, constrained alkyls {e.g. branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily- skilled synthetic chemist. The α-carbon of an amino acid may be mono- or dimethylated.

[0037] An "agonist" of leptin refers to a compound that mimics or elicits one or more effects or biological activities of leptin in vitro or in vivo. The effects of leptin include, but are not limited to, the ability to directly or indirectly interact or bind with a receptor that is activated or deactivated by leptin. Exemplary leptin agonists include small molecule compounds such as CBT-001452 (Cambridge Biotechnology Ltd.). [0038] As described herein, methods are provided which use the leptin agent in combination therapy with at least one other active agent to treat or prevention of post- lipectomy ectopic fat deposition, a post-lipectomy metabolic abnormality, a post- lipectomy physiologic disorder, or any combination thereof. An example of an agent for use in combination with the leptin agent is an amylin or an amylin agonist. Exemplary amylin and amylin agonists include, but are not limited to, those described in U.S. Pat. Nos. 5,686,411, 6,087,334, 6,114,304, and 6,410,511, and PCT Application Publication Nos. WO 2006/083254, WO 2006/052608, WO 2006/042242, WO 2004/048547, and WO 93/10146.

[0039] Another example of an active agent for use in combination with leptin, or an analog or derivative thereof, or other leptin agonist is an anticonvulsant agent such as topiramate (e.g., TOPIMAX® (Ortho McNeil Pharmaceuticals)). See, for example, U.S. Pat. No. 4,513,006.

[0040] Anorexigenic agents for use in combination therapy with leptin , or an analog or derivative thereof, or other leptin agonist include, but are not limited to, selective serotonin re-uptake inhibitors such as sibutramine (e.g., MERJDIA® (Abbott Laboratories)) and, for example, those described in U.S. Pat. Nos. 4,746,680, 5,436,272, 6,365,633 and PCT Patent Application Publication No. WO 01/062341. The anorexigenic agents also include catecholaminergic agonists such as phentermine. Phentermine is classically referred to as a catecholaminergic agonist as it actually interacts with noradrenalin and 5-hydroxytrytamine (serotonin) receptors. [0041] Another example of an active agent for use in combination with leptin, or an analog or derivative thereof, or other leptin agonist is an antagonists/inverse agonist of the central cannabinoid receptors (the CB-I receptors), including, but not limited to, rimonabant (Sanofi Synthelabo), and SR-147778 (Sanofi Synthelabo). See, for example, in U.S. Pat. Nos. 6,344,474 and 5,624,941; European Patent Application Nos. EP-656 354 and EP-658546; and PCT Application Publication Nos. WO 03/040105, WO 03/18060, WO 01/58869, WO 01/58450, and WO 98/43635. [0042] Angiogenic factors for use in combination therapy with leptin, or an analog or derivative thereof, or other leptin agonist include, but are not limited to, FGF-2 and VEGF, and analogs of each.

[0043] Another example of an active agent for use in combination with leptin, or an analog or derivative thereof, or other leptin agonist is TGF-β and analogs thereof. [0044] Satiety agents for use in combination therapy with leptin, or an analog or derivative thereof, or other leptin agonist include, but are not limited to, CCK, CCK agonists, bombesin, bombesin agonists, GRPs and GRP agonists. Exemplary cholecystokinin-A (CCK-A) agonists include, but are not limited to, those described U.S. Pat. No. 5,739,106. Specific CCK-A agonists include, but are not limited to, AR-R 15849, GI 181771, JMV-180, A-71378, A-71623 and SR146131. Bombesin, a tetradeca-neuropeptide analogous to mammalian GRP, stimulates release of a variety of gastrointestinal hormones and has a satiety effect in humans (see, for example, Debas et al. (1991) Am. Surg. 161 :243 249; Muurahainen et al. (1993) Am. J. Physiol. 264:R350-R354; Lee et al. (1994) Neurosci. Biobehav. Rev. 18:313 232; Anastasi et al. (1971) Experientia 27:166-167; McDonald et al. (1979) Biochem. Biophys. Res. Commun. 90:227-233).

[0045] In general, with respect to an amino acid sequence, the term "modification" includes substitutions, insertions, elongations, deletions, and derivatizations alone or in combination. In some embodiments, the leptin protein, analog or derivative may include one or more modifications of a "non-essential" amino acid residue. In this context, a "non-essential" amino acid residue is a residue that can be altered, e.g., deleted or substituted, in the novel amino acid sequence without abolishing or substantially reducing the activity (e.g., the agonist activity) of the protein (e.g., the analog protein). In some embodiments, the proteins may include one or more modifications of an "essential" amino acid residue. In this context, an "essential" amino acid residue is a residue that when altered, e.g., deleted or substituted, in the novel amino acid sequence the activity of the reference peptide is substantially reduced or abolished. In such embodiments where an essential amino acid residue is altered, the modified protein may possess an activity of leptin of use in the methods provided. The substitutions, insertions and deletions may be at the N-terminal or C- terminal end, or may be at internal portions of the protein.

[0046] Substitutions include conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain, or physicochemical characteristics (e.g., electrostatic, hydrogen bonding, isosteric, hydrophobic features). The amino acids may be naturally occurring or nonnatural (unnatural). Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Substitutions may also include non-conservative changes.

[0047] By "amino acid" or "amino acid residue" is meant natural amino acids, unnatural amino acids, and modified amino acid. Unless stated to the contrary, any reference to an amino acid, generally or specifically by name, includes reference to both the D and the L stereoisomers if their structure allow such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (GIn), glutamic acid (GIu), glycine (GIy), histidine (His), isoleucine (He), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (VaI). Unnatural amino acids include, but are not limited to, homolysine, homoarginine, homoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4- aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4- diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo- hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N- methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N- methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid, pyroglutamate, and thioproline. Additional unnatural amino acids include modified amino acid residues which are chemically blocked, reversibly or irreversibly, or chemically modified on their N-terminal amino group or their side chain groups, as for example, N-methylated D and L amino acids or residues wherein the side chain functional groups are chemically modified to another functional group. For example, modified amino acids include methionine sulfoxide; methionine sulfone; aspartic acid- (beta-methyl ester), a modified amino acid of aspartic acid; N- ethyl glycine, a modified amino acid of glycine; or alanine carboxamide, a modified amino acid of alanine. Additional residues that can be incorporated are described in Sandberg et al. (1998) J. Med. Chem. 41 :2481-2491.

[0048] "Sequence identity", as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" can also mean the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Parti, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG (Devereux et al. (1984) Nucleic Acids Research 12:387; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson (1994) Trends in Biotechnology 12:76-80; Birren et al. (1997) Genome Analysis 1 :543-559). The BLAST X program is publicly available from NCBI and other sources {BLAST Manual, Altschul, S., et al, NCBI NLM NIH, Bethesda, MD 20894; Altschul et al (1990) J. MoI. Biol. 215:403-410). The well known Smith Waterman algorithm can also be used to determine identity.

[0049] Parameters for polypeptide sequence comparison typically include the following: Algorithm: Needleman and Wunsch (1970) J. MoI. Biol. 48:443-453; Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff (1992) Proc. Natl. Acad. ScL USA 89:10915-10919; Gap Penalty: 12; Gap Length Penalty: 4. A program that can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group ("GCG"), Madison, WI. The above parameters along with no penalty for end gap are the default parameters for peptide comparisons, hi one embodiment the BLASTP program of NCBI is used with the default parameters of no compositional adjustment, expect value of 10, word size of 3, BLOSUM62 matrix, gap extension cost of 11 , end gap extension cost of 1 , dropoff (X) for blast extension (in bits) 7, X dropoff value for gapped alignment (in bits) 15, and final X dropoff value for gapped alignment (in bits) 25.

[0050] Parameters for nucleic acid molecule sequence comparison include the following: Algorithm: Needleman and Wunsch (1970) J. MoI. Bio. 48:443-453; Comparison matrix: matches - +10; mismatches = 0; Gap Penalty: 50; Gap Length Penalty: 3. As used herein, "% identity" is determined using the above parameters as the default parameters for nucleic acid molecule sequence comparisons and the "gap" program from GCG, version 10.2.

[0051] Derivatives of the leptin proteins or analogs are also known in the art. Such derivatives include leptin proteins and analogs thereof conjugated to one or more water soluble polymer molecules, such as polyethylene glycol (PEG) or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or by the addition of polyamino acids, such as poly-his, poly-arg, poly-lys, poly-ala, and combinations of polyamino acids, such as poly-his-ala, poly-arg-ala, and poly-lys-ala. Modifications to the proteins or analogs thereof can also include small molecule substituents, such as short alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic groups. As described herein, such polymer-conjugations and small molecule substituent modifications may occur singularly at the N- or C-terminus or at the side chains of amino acid residues within the protein. Alternatively, there may be multiple sites of derivatization along the protein. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine may provide additional sites for derivatization. In some embodiments, the proteins may be conjugated to one, two, or three polymer molecules.

[0052] Exemplary water soluble polymer molecules will have a molecular weight ranging from about 500 to about 20,000 Daltons. In some instances, the water soluble polymer molecules are linked to an amino, carboxyl, or thiol group, and may be linked by N or C terminus, or at the side chains of lysine, aspartic acid, glutamic acid, or cysteine. Alternatively, the water soluble polymer molecules may be linked with diamine and dicarboxylic groups. In some embodiments, the proteins are linked to one, two, or three PEG molecules through an epsilon amino group on a lysine amino acid.

[0053] The proteins disclosed herein may be produced by recombinant techniques well known in the art. See, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d ed. , Cold Spring Harbor, NY. The proteins produced by recombinant technologies may be expressed from a polynucleotide. One skilled in the art will appreciate that the polynucleotides, including DNA and RNA, that encode such the various fragments of the peptides may be obtained from the wild-type cDNA, taking into consideration the degeneracy of codon usage, or may be engineered as desired. These polynucleotide sequences may incorporate codons facilitating transcription and translation of mRNA in microbial hosts. Such manufacturing sequences may readily be constructed according to the methods well known in the art. The polynucleotides above may also optionally encode an N-terminal methionyl residue. The polynucleotides above may also optionally encode a C-terminal glycyl residue for proper amide formation. Non-peptide compounds useful in composition and methods provided herein may be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids may be prepared using methods known in the art. See, e.g., Bartlett et al. (1986) Bioorg. Chem. 14: 356-377. [0054] A variety of cell types may be used to contain and express a peptide coding sequence including, for example, bacteria, yeast, algae, insect cells, plant cells, and animal cells such as mammalian and avian cells. A variety of expression vector/host systems may be used, including, but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus (CaMV); tobacco mosaic virus (TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells and cell lines that are useful in recombinant protein productions include, but are not limited to, VERO (African green monkey kidney) cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI38 (human lung fibroblasts), baby hamster kidney (BHK) cells, HepG2, 3T3, RIN, Madin-Darby canine kidney epithelial (MDCK) cells, A549, PC 12, K562 and 293 cells. Exemplary protocols for the recombinant expression of polypeptides are well known in the art. [0055] The proteins described herein may be prepared using chemical peptide synthesis techniques known in the art, e.g. , using an automated or semi-automated peptide synthesizer, standard recombinant techniques, or both. Likewise, the derivatives of the proteins may be produced using standard chemical, biochemical, or in vivo methodologies. The proteins may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automated synthesizers are commercially available and may be used in accordance with known protocols. See, e.g., Stewart et al. (1984) Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.; Tarn et al. (1983) J. Am. Chem. Soc. 105: 6442; Merrifield (1986) Science 232: 341-347; and Barany et al. (1979) The Peptides, Gross et al, eds., Academic Press, NY, 1-284.

[0056] Proteins described herein may be produced using a combination of both automated protein synthesis and recombinant techniques. For example, a protein may contain a combination of modifications including deletion, substitution, and insertion by PEGylation. Such a protein may be produced in stages. For example, in the first stage, an intermediate protein containing the modifications of deletion, substitution, insertion, and any combination thereof, may be produced by recombinant techniques as described. Then, after an optional purification step, the intermediate protein is PEGylated through chemical modification with an appropriate PEGylating reagent (e.g., from Nektar Therapeutics, San Carlos, CA) to yield the desired protein. One skilled in the art will appreciate that the above-described procedure may be generalized to apply to a protein containing a combination of modifications selected from deletion, substitution, insertion, derivation, and other means of modification well known in the art and contemplated herein.

[0057]ιProtein described herein may also be produced using chemical ligation schemes known in the art, including those described, for example, in U.S. Application Publication Nos. 2003-0191291, 2003-0208046, and 2004-0115774. Chemical ligation refers to a chemoselective reaction involving the covalent joining of two chemical moieties, each of which moieties bears a mutually reactive functional group that is uniquely capable of forming a non-reversible covalent bond with the other. Unique, mutually reactive, functional groups present on the first and second components can be used to render the ligation reaction chemoselective. For example, the chemical ligation of peptides and polypeptides involves the chemoselective reaction of peptide or polypeptide segments bearing compatible unique, mutually reactive, C-terminal and N-terminal amino acid residues. Chemical ligation includes covalent ligation of (1) a first peptide or polypeptide bearing a uniquely reactive C- terminal group with (2) a second peptide or polypeptide bearing a uniquely reactive N-terminal group, where the C-terminal and N-terminal reactive groups form a nonreversible covalent bond therein between. It also includes N-terminal to N-terminal and C-terminal to C-terminal ligation. In particular, chemical ligation includes any chemoselective reaction chemistry that can be applied to ligation of unprotected peptide segments. Several different chemistries have been utilized for this purpose, examples of which include native chemical ligation, oxime forming chemical ligation, thioester forming ligation (Schnolzer et al. (1992) Science 256:221-225; Gieselman et al. (2001) Org. Lett. 3:1331-1334), thioether forming ligation (Englebretsen et al. (1995) Tot. Leffs. 36:8871-8874), hydrazone forming ligation (Gaertner, et al. (1994) Bioconj. Chem. 5:333-338), and thiazolidine forming ligation and oxazolidine forming ligation (Zhang et al. (1998) Proc. Natl. Acad. ScL USA 95:9184-9189; PCT Publication No. WO 95/00846; U.S. Pat. No. 5,589,356); and Staudinger amide forming chemical ligation (Saxon et al. (2000) Org. Leff. 2:2141-2143). [0058] As used herein, the term "purified protein" is intended to refer to a composition, isolated from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Generally, "purified" will refer to a protein composition that has been subjected to fractionation to remove various other components, and which composition substantially retains a biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition. [0059] It may be desirable to purify the peptides generated by the methods described herein. Peptide purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to protein and non- protein fractions. Having separated the protein from non-proteins, the protein of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Purification techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies, and the like; heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method of purifying peptides is reverse phase HPLC, followed by characterization of purified product by liquid chromatography/mass spectrometry (LC/MS) and Matrix- Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Additional confirmation of purity is obtained by determining amino acid analysis. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0060] There is no general requirement that the protein always be provided in its most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation- exchange column chromatography performed, utilizing an HPLC apparatus, will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of the peptide. In some embodiments, a combination of anion exchange and immunoaffinity chromatography may be used to produce purified protein compositions described herein.

[0061] One skilled in the art will be able to ascertain effective dosages by administering leptin, leptin analog, leptin derivative, or other leptin agonist and observing the desired therapeutic effect. In some embodiments, the goal of leptin therapy is to reduce ectopic fat deposition following lipectomy. In some instances, this goal can be reached by administration of leptin to achieve near physiological concentrations of leptin in the plasma. It is estimated that the physiological replacement dose of leptin is about 0.02 mg per kilogram of body weight per day for males of all ages, about 0.03 mg per kilogram per day for females under 18 years and about 0.04 mg per kilogram per day for adult females. When attempting to achieve near physiological concentrations of leptin, one may, for example, treat a patient with 50 percent of the estimated replacement dose for the first month of treatment, 100 percent of the replacement dose for the second month of treatment, 200 percent of the replacement dose for the third month of treatment, etc. During the course of leptin therapy, one can measure certain biochemical markers to monitor therapeutic effect of the leptin treatment including, for example, serum glucose levels, serum free fatty acid (FFA) levels, serum insulin levels, glycosylated hemoglobin (HbA₁C) levels and triglyceride (fasting) levels. Such biochemical markers can be monitored by any method known in the art. For example, serum glucose and triglyceride levels may be determined by standard methods using automated Hitachi equipment (Boehringer Mannheim, Indianapolis, IN.) and using Beckman Instrument (Beckman, CA). HbAiC levels may be determined by ion-exchange high-pressure liquid chromatography (Bio-Rad Laboratories Inc., Hercules, CA). Serum FFA levels may be determined with a commercial kit (Wako, Richmond, VA). Serum insulin levels may be determined by immunoassays using reagents provided by Abbott IMx® Instrument (Abbott Park, IL) and a commercial kit (Linco Research, Inc., St. Charles, MO).

[0062] Alternatively, leptin levels in a biological sample, e.g., plasma, serum, blood, may be determined by any method known in the art. For example, serum leptin levels may be determined by immunoassays using a commercial kit (Linco Research, Inc., St. Charles, MO). In general, a diagnostic assay for measuring the amount of leptin in the sample may first be used to determine endogenous levels of protein. Such diagnostic tools may be in the form of an antibody assay, such as an antibody sandwich assay or a standard enzyme-linked immunosorbent assay (ELISA). Any antibody that can specifically detect leptin may be used in the ELISA method. In certain embodiments, the ELISA method may use antibodies which specifically react with native human leptin, and are sensitive to detect leptin quantities of equal to or below 5 ng/ml of serum or other biological sample. The ELISA method may use a purified rat monoclonal anti-rmetHu-Leptin antibody for capturing leptin from serum. Affinity purified rabbit anti-rmetHu-leptin polyclonal antibody conjugated to horseradish peroxidase may also be used to detect captured leptin. The limit of detection of the assay using these antibodies may be in the range of 0.5-0.8 ng/ml. [0063] The amount of endogenous leptin is quantified initially following the lipectomy procedure, and a baseline is determined. The therapeutic dosages are determined as the quantification of endogenous and exogenous leptin protein (that is, leptin, leptin analog or leptin derivative found within the body, either self-produced or administered). Monitoring the leptin levels of a subject is continued over the course of therapy.

[0064] In some cases, the timing for determining the baseline leptin levels in a subject is after an 8-12 hour fast such as during morning hours. Baseline leptin levels may not be confounded by raising levels, such as after a meal, or due to sleep cycle rise in leptin seen in most individuals (e.g., 3:00 a.m. rise in leptin levels). Such baseline levels may be used, such as observation of nocturnal elevation of leptin levels, but those levels should be compared against similar levels in similarly situated subjects. [0065] The present disclosure also provides methods of using pharmaceutical compositions of leptin, leptin analog, leptin derivative, or other leptin agonist. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. Exemplary methods of administering the leptin proteins include subcutaneously, systemically and by gene therapy methods.

[0066] In general, pharmaceutical compositions provided comprise effective amounts of leptin, leptin analog, leptin derivative, or other leptin agonist together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

[0067] To aid dissolution of the therapeutic leptin, leptin analog or leptin derivative protein into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios. [0068] Additives that potentially enhance uptake of the leptin, leptin analog, leptin derivative protein, or other leptin agonist are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

[0069] In some embodiments, a controlled release formulation is used. For example, the leptin, leptin analog, leptin derivative protein, or other leptin agonist may be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the leptin, leptin analog, leptin derivative protein, or other leptin agonist is enclosed in a semi-permeable membrane, which allows water to enter and push the protein out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

[0070] In certain embodiments, methods are provided for reducing post-lipectomy ectopic fat deposition in a subject comprising administering to the subject an effective amount of a leptin protein, analog or derivative thereof, or other leptin agonist in a bolus dose one or more times a day. A bolus dose is an intermittent dosage of medicine (as opposed to a continuous infusion). A subject can be administered one or more bolus doses per day. The bolus dose can be the same no matter when it is administered to the subject, or can be adjusted such that the subject is administered a larger bolus dose at certain times of the day as compared to others. Administration of a leptin protein, analog or derivative thereof, or other leptin agonist in certain formulations, e.g., sustained release formulations, a bolus dose can be administered less frequently, for example, once every three days, once per week, twice a month, once every month. Furthermore, the time between bolus doses is preferably long enough to allow the drug administered in the previous bolus dose to clear the subject's blood stream. In some embodiments, a leptin analog or leptin derivative having a longer half-life than leptin protein are administered.

[0071] In other embodiments, methods are provided for reducing post-lipectomy ectopic fat deposition in a subject comprising administering to the subject an effective amount of a leptin protein, or analog or derivative thereof, or other leptin agonist in continuous doses. By continuous dose it is intended to mean the continuous infusion of the drug by, for example, intravenous injection or a transdermal patch. Alternatively, a continuous dose can be administered orally in the form of a controlled release capsule or tablet which releases the drug into the subjects system over a period of time. When administered by a continuous dose, the drug is released over a period of about 1 hour, more preferably the drug is released over a period of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours.

[0072] In embodiments where the methods comprise administration of the leptin protein, or analog or derivative thereof, or other leptin agonist as part of a combination therapy, administration of the agents "in combination" should be understood to mean providing each of the agents to a subject in need of treatment. Administration of the agents could occur as a single pharmaceutical dosage formulation containing all of the intended active agents or separately with each intended agent in its own dosage formulation.

[0073] For example, a leptin protein, leptin analog leptin derivative, or other leptin agonist may be combined with one or more other active agents, in a unitary dosage form, or in separate dosage forms intended for simultaneous or sequential administration to a subject in need. In one embodiment, the a leptin protein, leptin analog, leptin derivative, or other leptin agonist is co-administered with the second active agent, e.g., as a single administration with a second active agent, simultaneously as separate doses, or as sequentially administered where the administration of the compounds may be separated in time by seconds, minutes, or hours. Sequential administration may also include administration of a first course of an active agent, e.g., a leptin protein, leptin analog, leptin derivative, or other leptin agonist, followed by at least one course of another active agent. The treatment courses may or may not overlap.

[0074] When administered sequentially, the combination may be administered in two or more administrations. In an alternative embodiment, it is possible to administer one or more a leptin protein, leptin analog, leptin derivative, or other leptin agonist and one or more additional active ingredients by different routes. The skilled artisan will also recognize that a variety of active ingredients may be administered in combination with a leptin protein, leptin analog, leptin derivative, or other leptin agonist that may act to augment or synergistically enhance the prevention or treatment of post-lipectomy ectopic fat deposition and post-lipectomy associated metabolic and other physiologic abnormalities. [0075] Where separate dosage formulations are used, the individual active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially prior to or subsequent to the administration of the other active agent of the method. In some embodiments, administration in combination involves administration of separate dosage formulations during overlapping intervals. For example, active agent 1 is administered from day 1 through day 30 and active agent 2 is administered from day 20 through day 50. In other embodiments, administration in combination involves administration of separate dosage formulations in sequential, nonoverlapping intervals. For example, active agent 1 is administered from day 1 through day 30 and active agent 2 is administered from day 35 through day 50. The combination therapy methods provided are therefore to be understood to include all such regimes of simultaneous, alternating, or completely separate treatments over the total treatment course, and the terms "administration in combination" and "administering in combination" are to be interpreted accordingly.

[0076] In accordance with the treatment methods, treatment modalities, administration methods, administration modalities, pharmaceutical compositions, and formulations provided herein, a leptin, a leptin analog, a leptin derivative, or other leptin agonist may be used in the preparation of a medicament for treating or preventing post lipectomy fat deposition or post-lipectomy associated metabolic and other physiologic abnormalities in a subject. Exemplary such post-lipectomy associated metabolic and other physiologic abnormalities include hyperglycemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis, insulin resistance, diabetes, hepatic steatosis, and the like.

[0077] According to the methods and uses provided herein, when used in combination therapy with at least one other active agent, a leptin protein, leptin analog, leptin derivative, or other leptin agonist may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art. [0078] While the foregoing description discloses the present invention, it will be understood that the practice of the present invention encompasses all of the usual variations, adaptations, or modifications as being within the scope of the claimed invention. Therefore, descriptions should not be construed as limiting the scope of the invention.

Claims

CLAIMSWe claim:

1. A method of treating or preventing post-lipectomy ectopic fat deposition in a subject comprising administering to a subject in need thereof an effective amount of leptin, leptin analog, leptin derivative, or other leptin agonist.

2. The method of claim 1, wherein the subject has ectopic fat deposition following suction-assisted lipectomy.

3. The method of any of claims 1 and 2, wherein the subject has a post-lipectomy metabolic abnormality.

4. The method of claim 3, wherein the metabolic abnormality is selected from a group consisting of hyperglycemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis, and insulin resistance.

5. The method of any of claims 3 and 4, wherein the metabolic abnormality comprises diabetes.

6. The method of any of claims 1, 2, 3, 4, and 5, wherein the subject has a post- lipectomy hepatic steatosis.

7. The method of any of claims 1, 2, 3, 4, 5, and 6,wherein said leptin, leptin analog, leptin derivative, or other leptin agonist is administered subcutaneously.

8. The method of any of claims 1, 2, 3, 4, 5, 6, and 7, wherein said leptin, leptin analog, leptin derivative, or other leptin agonist is administered together with a pharmaceutically acceptable carrier.

9. The method of any of claims 1, 2, 3, 4, 5, 6, 7, and 8, wherein said leptin is recombinant human leptin.

10. The method any of claims 1, 2, 3, 4, 5, 6, 7, 8, and 9, wherein the leptin is selected from the group consisting of recombinant human leptin of SEQ ID NO: 1 and SEQ ID NO: 2.

11. The method of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, further comprising administering an effective amount of at least one other active agent in combination with the leptin, leptin analog, leptin derivative, or other leptin agonist.

12. Use of leptin, leptin analog, leptin derivative, or other leptin agonist in the preparation of a medicament for treating or preventing post-lipectomy ectopic fat deposition in a subject.

13. The use of claim 12, wherein the subject has ectopic fat deposition following suction-assisted lipectomy.

14. The use of any of claims 12 and 13, wherein the subject has a post-lipectomy metabolic abnormality.

15. The use of claim 14 wherein the metabolic abnormality is selected from a group consisting of hyperglycemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis, and insulin resistance.

16. The use of any of claims 14 and 15, wherein the metabolic abnormality comprises diabetes.

17. The use of any of claims 12, 13, 14, 15, and 16, wherein the subject has a post- lipectomy hepatic steatosis.

18. The use of any of claims 12, 13, 14, 15, 16, and 17, wherein said leptin, leptin analog, leptin derivative, or other leptin agonist is administered subcutaneously.

19. The use of any of claims 12, 13, 14, 15, 16, 17, and 18, wherein said leptin, leptin analog, leptin derivative, or other leptin agonist is administered together with a pharmaceutically acceptable carrier.

20. The use of any of claims 12, 13, 14, 15, 16, 17, 18, and 19, wherein said leptin is recombinant human leptin.

21. The use any of claims 12, 13, 14, 15, 16, 17, 18, 19, and 20, wherein the leptin is selected from the group consisting of recombinant human leptin of SEQ ID NO: 1 and SEQ BD NO: 2.

22. The use of any of claims 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, further comprising administering an effective amount of at least one other active agent in combination with the leptin, leptin analog, leptin derivative, or other leptin agonist.