WO2011050008A2

WO2011050008A2 - Combination therapy comprising administration of an amylinomimetic and a pyy peptidomimetic for effecting weight loss and for treating obesity and related metabolic conditions and disorders

Info

Publication number: WO2011050008A2
Application number: PCT/US2010/053278
Authority: WO
Inventors: Jonathan Todd Coffey; David G. Parkes; Jonathan David Roth
Original assignee: Amylin Pharmaceuticals, Inc.
Priority date: 2009-10-19
Filing date: 2010-10-19
Publication date: 2011-04-28
Also published as: WO2011050008A9

Abstract

Methods for treating obesity and obesity related disorders are disclosed. These methods include the use of at least: a first anti-obesity agent, or a derivative thereof; and a second anti-obesity agent or a derivative thereof.

Description

COMBINATION THERAPY COMPRISING ADMINISTRATION OF AN AMYLINOMIMETIC AND A PYY PEPTIDOMIMETIC FOR EFFECTING WEIGHT LOSS AND FOR TREATING OBESITY AND RELATED METABOLIC

CONDITIONS AND DISORDERS FIELD OF THE INVENTION

The present invention relates to the medical field and in particular to the field of health, diet and nutrition. The invention relates to the use of anti-obesity agents.

BACKGROUND

Obesity and its associated disorders are common and very serious public health problems in the United States and throughout the world. It is estimated that about 64% of Americans are overweight or obese (roughly about 97 million adults) and it is generally believed that these numbers are increasing. People who are overweight or obese are considered those with a Body Mass Index (BMI) equal to or greater than 25. BMI is a mathematical formula commonly used to express the relationship of weight-to-height; a person's body weight in kilograms is divided by the square of his or her height in meters (i.e., wt/(ht)²). In a human healthcare setting, individuals with a BMI of 25 to 29.9 are generally considered overweight, while individuals with a BMI of 30 or more are generally considered obese. Morbid obesity refers to a BMI of 40 or greater. According to the NIH Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, all adults (aged 18 years or older) who have a BMI of 25 or more are considered at risk for premature death and disability as a consequence of overweight and obesity.

Upper body obesity is the strongest risk factor known for type 2 diabetes mellitus and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, cancers of the breast, prostate, and colon, and increased incidence of complications of general anesthesia (see, e.g., Kopelman, Nature 404: 635-43 (2000)). Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or "Syndrome X" and metabolic syndrome. The worldwide medical cost of obesity and associated disorders is enormous. Being obese or overweight may substantially increase the risk of morbidity from hypertension; dyslipidemia; type 2 diabetes; coronary heart disease; stroke; gallbladder disease; osteoarthritis; sleep apnea and respiratory problems; and endometrial, breast, prostate, and colon cancers. Higher body weights are also associated with increases in all- cause mortality. Furthermore, being obese or overweight may cause a person to have a negative self-image about him or her self.

Obesity reduces life-span and carries a serious risk of the co-morbidities listed above, as well disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injury, and thromboembolic disease (Rissanen et ah, Br. Med. J. 301 : 835-7 (1990)).

The pathogenesis of obesity is believed to be multifactorial. A problem is that, in obese subjects, nutrient availability and energy expenditure do not come into balance until there is excess adipose tissue. The central nervous system (CNS) controls energy balance and coordinates a variety of behavioral, autonomic and endocrine activities appropriate to the metabolic status of the animal. The mechanisms or systems that control these activities are broadly distributed across the forebrain (e.g., hypothalamus), hindbrain (e.g., brainstem), and spinal cord. Ultimately, metabolic (i.e., fuel availability) and cognitive (i.e., learned preferences) information from these systems is integrated and the decision to engage in appetitive (food seeking) and consummatory (ingestion) behaviors is either turned on (meal procurement and initiation) or turned off (meal termination). The hypothalamus is thought to be principally responsible for integrating these signals and then issuing commands to the brainstem. Brainstem nuclei that control the elements of the

consummatory motor control system (e.g., muscles responsible for chewing and

swallowing). As such, these CNS nuclei have literally been referred to as constituting the "final common pathway" for ingestive behavior.

Neuroanatomical and pharmacological evidence support that signals of energy and nutritional homeostasis integrate in forebrain nuclei and that the consummatory motor control system resides in brainstem nuclei, probably in regions surrounding the trigeminal motor nucleus. There are extensive reciprocal connection between the hypothalamus and brainstem. A variety of CNS-directed anti-obesity therapeutics (e.g., small molecules and peptides) focus predominantly upon forebrain substrates residing in the hypothalamus and/or upon hindbrain substrates residing in the brainstem. Numerous such signals, of energy and nutritional status have been identified in vivo, such as peptide hormones. Particular areas of the forebrain (telencephalonic- and diencephalonic-derived constituents of the brain) and hindbrain or brainstem (including the midbrain, pons and medulla) have been identified as being involved in controlling energy balance. Forebrain structures or nuclei residing in the hypothalamus involved in food intake and/or body weight modulation include, for example, the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), the ventromedial nucleus (VMH), and the lateral hypothalamus nucleus (LHA). Hindbrain structures or nuclei residing in the brainstem involved in food intake and/or body weight modulation include, for example, the nucleus of the solitary tract (NST), the area postrema (AP), and the lateral parabrachial nucleus (1PBN). Brainstem nuclei that control the elements of the consummatory motor control system are likely controlled by primary or second order projections from brainstem regions like the NST, AP, and 1PBN. It is noteworthy that the AP, NST and 1PBN have all been shown to (collectively and independently) possess their own integrative abilities.

A variety of CNS-directed anti-obesity agents act upon these forebrain structures residing in the hypothalamus involved in food intake and/or body weight modulation. In addition, CNS-directed anti-obesity agents act upon hindbrain structures residing in the brainstem involved in food intake and/or body weight modulation. Examples of such anti- obesity agents are described herein. See Table 1 for examples. Such agents include, for example, neuropeptide Yl (NPY1) receptor antagonists, NPY5 receptor antagonists, leptin and leptin agonists, ciliary neurotrophic factor (CNTF) and CNTF agonists, melanin- concentrating hormone (MHC) and MCH antagonists, melacortins (MC) and MC agonists, cannabinoid receptor (CB-1) antagonists, serotonin (5-HT) and 5-HT agonists, peptide YY (PYY), and PYY agonists, exendin and exendin agonists, GLP-1 and GLP-1 agonist, DPP- IV inhibitors, ghrelin and ghrelin antagonists, cholecystokinin (CCK) and CCK agonists, and amylin and amylin agonists.

Table 1. Individual anti-obesity targets and location

concentrating (ARC/PV )

hormone (MCH)

Melanocortins (MC) Forebrain Agonists decrease MC4 agonists

(PV /ARC) intake

Cannabinoids (CB) Forebrain Increase intake Cannabinoid receptor

(widespread) antagonists

Serotonin (5-HT) Forebrain (VMH) Decrease intake 5-HT2C agonists

Peptide YY (PYY) Forebrain (ARC) Decrease intake PYY(3-36) agonists

Glucagon-like Forebrain (PVN) Decrease intake Exenatide and other peptide- 1 (GLP-1) GLP-1 ligands, DPP- IV inhibitors

Ghrelin Forebrain (ARC) Increase intake Ghrelin antagonists

Cholecystokinin Hindbrain (AP) Decrease intake CCK agonists (CCK)

Amylin Hindbrain (AP) Decrease intake Amylin agonists,

Pramlintide, amylin analogs

Nonetheless, efforts to exploit such hormones and agents for use as pharmacologic agents for treating obesity and related disorders have often been confounded by limited potency, efficacy, and/or limited stability and circulation half-life of the native hormones and agonists, such that the doses of such that are required to achieve acceptable biological and clinical efficacy often give rise to untoward and offsetting side effects, such as nausea and emesis often offset any therapeutic benefit that may otherwise be achieve such treatment.

Accordingly, there exists a need for the provision of more potent and efficacious anti-obesity agents, such as PYY peptidomimetics and amylinomimetics, or derivatives thereof, which may co-administered so as to optimize the beneficial properties of the respective reference compounds with regard to treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing gastric emptying, reducing adiposity, reducing caloric efficiency, inducing satiety, and the like. There also exists a need to provide methods of new therapies comprising the administration of agents useful in weight reduction and/or weight maintenance in a subject, at dosages which elicit fat-specific weight loss at dosages that provide mitigation of untoward side effects, such as nausea and emesis. Such therapies would lead to improved efficacy with regard to weight loss and decrease of weight gain in treated subjects, as well as result in improved increase subject compliance and, ultimately, improved health benefits and outcomes in the overweight and obese patient populations.

All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety and for all purposes.

SUMMARY

Provided are, for example, methods of treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing gastric emptying, reducing adiposity, reducing caloric efficiency, inducing satiety, and the like, as described herein throughout, in subjects, comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a second anti-obesity agent, wherein: said first anti-obesity agent comprises an amylinomimetic having an amino acid amino acid sequence set out in SEQ ID NO: l, or a derivative thereof; and said second anti-obesity agent comprises a PYY peptidomimetic having an amino acid sequence set out in SEQ ID NO:2, or a derivative thereof. The methods are also useful in treating, controlling, and ameliorating co-morbidities and conditions associated with obesity and overweight, diabetes mellitus, gestational diabetes, type I diabetes, type II diabetes, insulin-resistance syndrome, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, cardiovascular disease, polycystic ovary syndrome, and metabolic syndrome.

The methods further provide supra-additive potency and/or efficacy in reducing body weight, reducing body weight gain, reducing food intake, reducing nutrient availability, reducing adiposity, treating obesity, and treating overweight, relative to the potency and or efficacy achieved by the administration of either anti-obesity agent alone. Moreover, the supra-additive effects provided by the disclosed methods afford the use of dose-sparing amount of one or both of the anti-obesity agents, such that much lower dosages and amounts of either anti-obesity agent or both anti-obesity agents such are required in order to elicit the benefits of the combined administration, relative to the dose that is required to be provided for each or either agent when administered alone. Thus, not only do these supra-additive or synergistic (used interchangeably throughout) properties yield the potencies and efficacies with regard to the benefits mentioned above, such properties also mitigate the tendency and prevalence of untoward and often confounding side effects that have been reported with monotherapeutic use of either or both agents or to corresponding native or reference peptides (e.g., amylin, Pramlintide, PYY(l-36), and PYY(3-36) (see, e.g., Gantz et al, J. Clin. Endocrinol. Metab. , Vol. 92(5), pp. 1754-177 (2007).

In certain embodiments, the disclosed methods further comprise the administration to a subject at least one further anti-obesity agent selected from the group consisting of a NPY1 receptor antagonist, an NPY5 receptor antagonist, an NPY2 receptor agonist, an NPY4 receptor agonist, a leptin, a recombinant leptin, a leptin derivative, a leptin agonist, a CNTF, a CNTF agonist/modulator, a CNTF derivative, a MCH1R antagonist, a MCH2R antagonist, a melanocortin 4 agonist, a MC4 receptor agonist, a cannabinoid receptor (CB- 1) antagonist/inverse agonist, a ghrelin antagonist, a 5HT2c agonist, a serotonin reuptake inhibitor, a serotonin transport inhibitor, an exendin, an exendin derivative, an exendin agonist, a GLP-1, a GLP-1 analog, a GLP-1 agonist, a DPP-IV inhibitor, an opioid antagonist, an orexin antagonist, a metabotropic glutamate subtype 5 receptor antagonist, a histamine 3 antagonist/inverse agonist, topiramate, a CCK, a CCK analog, a CCK agonist, an amylin, an amylin analog, and an amylin agonist. In certain embodiments, a further anti- obesity agent administered is phentermine, rimonabant, sibutramine or Pramlintide (human ²⁵'²⁸'²⁹Pro-amylin).

In certain embodiments, provided are methods whereby the subject reduces body weight by least 10%, the subject reduces body fat mass, the subject reduces percent fat the subject loses ectopic fat, or any combination thereof.

In some embodiments, the methods are direct to a subject who suffers from obesity, an obesity-related disorder, an obesity related disease, an obesity-related condition, diabetes, insulin-resistance syndrome, nonalcoholic steatohepatitis, a cardiovascular disease, polycystic ovary syndrome, metabolic syndrome or a desire to lose body weight.

In one aspect, administration of the anti-obesity agents in combination may be simultaneous, concurrent, or sequential administration.

Pharmaceutical compositions comprising one or both of the anti-obesity agents of the invention are also provided, as well as uses and treatment methods employing such pharmaceutical compositions. In certain embodiments, such pharmaceutical compositions comprise sub-optimal doses or dose-sparing amounts of one or both of the first and second obesity agents are provided, as well as formulations comprising such. In yet another aspect, uses of the anti-obesity agents and compositions are provided for the manufacture of a medicament useful for treating the conditions and for providing the beneficial effects that are disclosed throughout.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts reduction in food intake upon IP administration of human PYY(3- 36), (the amino acid sequence of which is

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO:4)) (1 mg/kg) and rat amylin (the amino acid sequence of which is

KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY (SEQ ID NO: 15)) (0.01 mg/kg) to Harlan Sprague Dawley (HSD) rats (left panel), and reduction in body weight with subcutaneous (s.c.) infusion via minipump of amylin (100 μg/kg/d) +/- PYY(3-36) (200 μg/kg/d) in diet induced obesity (DlO)-prone rats (right panel).

Figure 2 depicts response surface analysis of the effects of amylin and PYY(3-36) on body weight (left panel) and food intake (right panel) in DIO-prone rats. The response surfaces reflect the predicted response surface for mean change in body weight (vehicle corrected) (right panel) and food intake (vehicle corrected) (left panel) for amylin administered at (0, 4, 20, and 100 μg kg·d) and PYY(3-36) administered (0, 200, and 400 μg/kg·d), alone and in combination (right panel). Left panel: amylin, p<0.001 ; PYY(3-36), p=0.0003; synergy, p=0.27. Right panel: amylin, pO.011 ; PYY(3-36), p=0.0004; synergy, p=0.022.

Figure 3 is a three dimensional plot depicting mean percentage weight loss (vehicle- corrected) observed in DIO rats upon s.c. continuous infusion of Davalintide (infused at a rate of 0, 1.25, 2.5 or5 μg/kg/d, as indicated) and SEQ ID NO:2 (infused at a rate of 0, 50 and 200 μg/kg/d, as indicated), alone and in combination.

Figure 4 is a three dimensional plot depicting mean percent inhibition of food intake observed in DIO rats upon s.c. continuous infusion of Davalintide (infused at a rate of 0, 1.25, 2.5 or5 μg/kg/d, as indicated) and SEQ ID NO:2 (infused at a rate of 0, 50 and 200 μg/kg/d, as indicated), alone and in combination.

Figure 5 depicts response surface analysis of the effects of Davalintide and SEQ ID NO:2 on weight loss (vehicle-corrected) in DIO-prone rats, alone and in combination. Both Davalintide and SEQ ID NO:2 were infused s.c. using separate minipumps at infusion rates of 0, 1.25, 2.5 or 5 μg/kg/d, and 0, 50 and 200 μg/kg/d, respectfully, as indicated.

Figure 6 depicts response surface analysis of the effects of Davalintide and SEQ ID NO:2 on percent inhibition of cumulative food intake (vehicle-corrected) in DIO-prone rats, alone and in combination. Both Davalintide and SEQ ID NO:2 were infused s.c. using separate minipumps at infusion rates of 0, 1.25, 2.5 or 5 μg/kg/d, and 0, 50 and 200 μg/kg/d, respectfully, as indicated.

Figure 7 depicts the cumulative food intake (left panel), change in body weight (vehicle corrected; center panel), and adiposity (percent fat; right panel) of s.c. infusion in DIO-prone rats of Davalintide and SEQ ID NO:2 at the highest doses used in Figures 5 and 6, alone and in combination.

Figure 8 provides a summary of the percent of baseline body weight observed upon s.c. infusion in DIO-prone rats of Davalintide and SEQ ID NO:2 at the highest doses used in Figures 5 and 6, alone and in combination.

Figure 9 provides a summary of statistical results as derived from the results disclosed in the Examples and depicted in Figures 1 through 8.

DETAILED DESCRIPTION

It has now been discovered that combined administration of at least: a first anti- obesity agent, which is an amylinomimetic peptide comprising the amino acid sequence set forth in SEQ ID NO: l : KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (also referred to as Davalintide) (see, e.g., WO2006/083254 and US 2008/0274952, or a derivative thereof; with a second anti-obesity agent, which is a Peptide YY (PYY) peptidomimetic comprising the amino acid sequence set forth in SEQ ID NO: 2:

PKPEHPGEDASPEELARYYASLRAYINLITRQRY (see, e.g., WO2006/066024 and

US2006/0135747, or a derivative thereof; is beneficial and advantageous in treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing gastric emptying, reducing adiposity, reducing caloric efficiency, inducing satiety, and the like, as described herein throughout, when administered to overweight or obese subjects.

Additionally, the combined administration of such anti-obesity agents has been surprisingly discovered to be more effective in treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing gastric emptying, reducing adiposity, reducing caloric efficiency, inducing satiety, and the like, in overweight or obese subjects than the additive effectiveness of each anti-obesity agent when each anti-obesity agent is administered alone. Hence, the combined administration of both agents results in supra-additive, also referred to as synergistic, effects with regard to for example, treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing gastric emptying, reducing adiposity, reducing caloric efficiency, inducing satiety, and the like in such overweight or obese subjects.

It has been further discovered that sub-optimal doses of at least one of the anti- obesity agents, as well as both anti-obesity agents, may be administered in combination in order to elicit efficacious and/or potent weight loss, reduction in weight gain, reduction in food intake, reduction in nutrient availability, reduction in gastric emptying, reduction in caloric efficiency, induction of satiety, and/or reduction in adiposity. Such effects are observed, for example, upon administration of Davalintide (SEQ ID NO: l) and SEQ ID NO:2 at as little as one twentieth (l/20^th) and one-half (1/2), respectively, of the corresponding effective doses of the native hormones (i.e., amylin and PYY(3-36)), that are required to be administered to a subject in order to achieve the same effects at s substantially the same potency and/or efficacy . Moreover, the weight loss observed by coadministration of Davalintide and SEQ ID NO: 2 is fat-specific (e.g., lean-sparing), insofar as the adiposity (i.e. percentage body fat) of treated subjects is significantly decreased relative to controls or to either compound when administered alone.

These surprising benefits achieved by the combined administration of the disclosed anti-obesity agents provide additional advantages, such as allowing for dose-sparing amounts of the first anti-obesity agent, the second anti-obesity agent, or both the first anti- obesity and the second anti-obesity agent to be administered in accordance with the disclosed and claimed invention in a dose-sparing amount in order to achieve similar or superior potency, efficacy, plasma concentration, bioavailability, plasma half-life, and/or pharmacokinetic (PK) characteristics, relative to the amount of either or both of the reference peptides (e.g., amylin (SEQ ID NO: 15) and PYY(3-36)(SEQ ID NO:4), or even relative to the amounts of SEQ ID NO: 1 or SEQ ID NO: 2 when administered as monotherapies. Further, benefits that flow from this dose-sparing advantage include, inter alia: lower manufacturing cost and costs of goods; lower/smaller administration doses and volumes, mitigation of untoward side-effects associated with administration of the reference peptides, such as nausea and emesis, which have been reported when each of Davalintide and SEQ ID NO:2, as well as the native reference compounds and/or certain agonists thereof have been administered as monotherapies, and potential for hypoglycemia as may occur when for example Pramlintide is administered, which is an amylin agonist when given to type II diabetics who may happen to be obese or overweight;); enhanced subject compliance; and, ultimately improved subject outcomes.

In certain embodiments, the methods comprise at least one further anti-obesity agent selected from the group consisting of: a PYl receptor antagonist, an NPY5 receptor antagonist, an NPY2 receptor agonist, an NPY4 receptor agonist, a CNTF (e.g.,

ΑΧΟΚΓΝΕ®), a CNTF agonist/modulator, a CNTF derivative, a MCH1R antagonist, a MCH2R antagonist (e.g., rimonabant), a melanocortin 4 agonist, a MC4 receptor agonist, a cannabinoid receptor (CB-1) antagonist/inverse agonist, a ghrelin antagonist, a 5HT2c agonist, a serotonin reuptake inhibitor, a serotonin transport inhibitor, an exendin, an exendin derivative, an exendin agonist, a GLP-1, a GLP-1 analog, a GLP-1 agonist, a DPP- IV inhibitor, an opioid antagonist, an orexin antagonist, a metabotropic glutamate subtype 5 receptor antagonist, a histamine 3 antagonist/inverse agonist, topiramate, a CCK, a CCK analog, a CCK agonist and a PYY(3-36), a PYY(3-36) analog, and a PYY(3-36) agonist; and derivatives thereof.

Definitions

An "anti-obesity agent" is a compound that is able, for example, to treat obesity in a subject, to treat overweight in a subject, to reduce nutrient availability to a subject, to reduce food intake by a subject, to reduce body weight of a subject, or to reduce body weight gain by a subject, to reduce adiposity in a subject, to reduce gastric emptying in a subject, to reduce caloric efficiency in a subject, and/or to induce satiety in a subject, upon

administration of such an anti-obesity agent to such a subject. In certain embodiments such subjects are overweight subjects; in other embodiments, such subjects are obese subjects. Exemplary anti-obesity agents are: an amylinomimetic having the amino acid set out in SEQ ID NO: 1, also referred to as Davalintide, or a derivative thereof,; and a PYY peptidomimetic having the amino acid sequence set out in SEQ ID NO:2, or a derivative thereof.

As used herein, an anti-obesity agent that "acts on a forebrain structure involved in food intake and/or body weight modulation" stimulates or suppresses activity of a particular region, e.g., particular nuclei and/or neuronal circuits, in the forebrain. This forebrain stimulation or suppression leads to a reduction in nutrient availability to the body. An anti- obesity agent that "acts on a hindbrain structure involved in food intake and/or body weight modulation" stimulates or suppresses activity of a particular region, e.g., particular nuclei and/or neuronal circuits, in the hindbrain. This hindbrain stimulation or suppression results in a reduction in nutrient availability to the body.

"Reduced nutrient availability" is meant to include any means by which the body reduces the nutrients available to the body to store as fat. In other words, reducing nutrient availability may be accomplished by means that include, but are not limited to, reducing food intake, reducing appetite, increasing satiety, affecting food choice/taste aversion, increasing metabolism, and/or decreasing or inhibiting food absorption, and reducing or delaying gastric emptying.

"Obesity", "overweight", "being obese", or "being overweight" generally refers to mammals, such as humans, having a weight that is greater than what they should have, which may be determined by, e.g., physical appearance, body mass index (BMI), waist-to- hip circumference ratios, skinfold thickness, and waist circumference. BMI is a calculation based on an individual's sex, weight, and height. The Centers for Disease Control and Prevention define overweight as an adult human having a BMI of 25 to 29.9; and define obese as an adult human having a BMI of 30 or higher. The Centers for Disease Control and Prevention state that a person with a waist-to-hip ratio greater than 1.0 is overweight. For purposes of this disclosure, any subject, including those with a BMI of less than 30, who needs or wishes to reduce body weight, reduce nutrient availability, reduce food intake, reduce adiposity, reduce caloric efficiency, or reduce or prevent body weight gain is included in the scope of "obese." Thus, subjects with a BMI between less than 30 and 25 or above (considered overweight) or below 25 are also included in the subjects who may benefit from and be treated in accordance with the disclosed and claimed the invention. Additionally, morbid obesity refers to a BMI of 40 or greater.

With regard to the methods disclosed throughout, as used herein, a "subject in need thereof includes subjects who are overweight, who are or obese, or who are morbidly obese, or who are otherwise desirous of reducing body weight, reducing weight gain, reducing food intake, weight, . In addition, subjects who are insulin resistant, glucose intolerant, or have any form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes), and/or have a co-morbidity or condition that is associated with obesity or overweight as disclosed throughout can benefit from such methods.

A "sub-optimal dose" is meant a dose or dosage amount of an a first or a second anti-obesity agent, which does not provide an acceptable or desirable efficacy, potency, or outcome with regard to reduction in body weight , reduction in body weight gain, reduction in food intake, reduction in nutrient availability, reduction in body fat, reduction in fat mass, reduction in percent body fat, reduction in adiposity, reduction in caloric efficiency, or reduction or delay of gastric emptying, when administered alone, but affords such acceptable or desirable efficacy, potency, or outcome when administered in combination. In certain embodiments, such a sub-optimal dose comprises a supra-additive or synergistic, efficacy, potency, or outcome when administered in combination. In certain embodiments, such a sub-optimal dose may alternatively or also constitute a dose-sparing amount.

Accordingly, smaller or lower doses or dosage amounts of one or both of the first and second anti-obesity agents may be advantageously administered to an overweight or obese subject in order to achieve one or more of the therapeutic benefits described herein, which one or more benefits would not be realized in such a subject if such a sub-optimal dose of either anti-obesity agent were administered as a monotherapy.

A "supra-additive amount" or "a synergistic amount" (used interchangeably herein throughout) of an anti-obesity agent means an amount of such an anti-obesity agent that, when administered in combination with an amount of another anti-obesity agent, provides at least one of the above-mentioned effects such that the effect achieved thereby is greater than the additive effect that is observed upon administration of each anti-obesity agent when administered alone (e.g., as a monotherapy).

A "dose-sparing amount" of an anti-obesity agent, is meant a therapeutically effective amount or dose of anti-obesity agent, such as Davalintide or SEQ ID NO:2, or derivatives thereof, that provides supra-additive or synergistic effect, as described above, upon combined administration the another anti-obesity agent, such that the dosage or amount required for such supra-additivity or synergism is substantially lower that the corresponding amount of the corresponding native hormone, either alone or in combination with another native hormone. For example, as provided herein, a dose-sparing amount or dose of Davalintide may be as little as about one-twentieth the corresponding

therapeutically effective amount of amylin; and a dose-sparing amount or dose of SEQ ID NO:2 may be as little as about one-half the corresponding therapeutically effective amount of PYY(3-36); that would be required of the two native hormones, administered in combination, to achieve a substantially the same effect.,

A "subject" is meant to include any animal, including humans, primates, and other mammals including rats, mice, pets such as cats, dogs, livestock such as horses, cattle, sheep and goats, as well as chicken, turkey and any other animal for which body weight or altering body composition may be an issue. In certain embodiments, the subject is a human.

In other embodiments, the subject is an overweight human or an obese human.

By "metabolic rate" is meant the amount of energy liberated/expended per unit of time. Metabolism per unit time can be estimated by food consumption, energy released as heat, or oxygen used in metabolic processes. It is generally desirable to have a higher metabolic rate when one wants to lose weight. For example, a person with a high metabolic rate may be able to expend more energy (e.g., the body burns more calories) to perform an activity than a person with a low metabolic rate for that activity.

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.

Methods of measuring fat mass and lean mass include, but are not limited to, underwater weighing, air displacement plethysmograph, x-ray, DEXA scans, MRIs and CT scans. In certain embodiments, fat mass and lean mass is measured using underwater weighing as known in the art.

By "fat distribution" is meant the location of fat deposits in the body. Such locations of fat deposition include, for example, subcutaneous, visceral and ectopic fat depots.

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. Patent No. 6,530,886, the entirety of which is incorporated herein by reference.

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.

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.

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 and/or undesirable clinical manifestations of a condition, 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 example, in treating obesity, a decrease in body weight, e.g., at least a 5% decrease in body weight, is an example of a desirable treatment result. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, 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 .

As used herein, the term "therapeutically effective amount" means the amount of the active compounds in the composition that will elicit the biological or medical response in a tissue, system, subject, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disorder being treated. The novel methods of treatment of this invention are for disorders known to those skilled in the art.

As used herein, the term "prophylactically effective amount" means the amount of the active compounds in the composition that will elicit the biological or medical response in a tissue, system, subject, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, to prevent the onset of obesity or an obesity-related disorder, condition or disease in subjects as risk for obesity or the obesity-related disorder, condition or disease.

As used herein, the singular form "a", "an", and "the" includes plural references unless otherwise indicated or clear from context.

As used herein, the term "peptidomimetic" refers to a character of an anti-obesity agent or a derivative thereof, as described throughout, that is peptidic in nature, or is a derivative which comprises a peptide to which a chemical modification has been made, as described below, and which displays physical, chemical, biochemical, and/or biological characteristics of a reference peptide, such as a native hormone, such as an amylin or a PYY. As used herein, an "amylinomimetic" refers to a peptidomimetic that is based on a reference amylin or an peptidic amylin agonist, such as Pramlintide, salmon calcitonin, human amylin, rat amylin, etc., and which displays physical, chemical, biochemical, and/or biological characteristics that have been observed for such an amylin or peptidic amylin agonist. As used herein, a "PYY peptidomimetic" refers to a peptidomimetic that is based on a reference PYY or a peptidic PYY agonist and which displays physical, chemical, biochemical, and/or biological characteristics that have been observed for such a PYY or a peptidic PYY agonist.

A "derivative" is defined as a peptide, such as a peptide set forth in SEQ ID NO: 1 or SEQ ID NO:2, as described throughout, to which a chemical modification has been made of one or more of its amino acid side groups, a-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, alkylation, acylation, ester formation, amide formation, maleimide coupling, 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, and N- acyl modifications. Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications, such as alkyl acyls, branched alkylacyls, alkylaryl-acyls. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, arylamide, alkylarylamide 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 a-carbon of an amino acid may be mono- or dimethylated.

Such derivatives include anti-obesity agents of the invention 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), by the addition of polyamino acids, such as poly -his, poly-arg, poly-lys, and poly-ala, or by addition of small molecule substituents include short alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic groups. In some embodiments, the water soluble polymer molecules will have a molecular weight ranging from about 500 to about 20,000 Daltons. Such polymer-conjugations may occur singularly at the N- or C-terminus or at the side chains of amino acid residues within the sequence of the anti-obesity agents.

Alternatively, there may be multiple sites of derivatization along the anti-obesity agent polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine may provide additional sites for derivatization. See, e.g., U.S. Patent Nos. 5,824,784 and 5,824,778. In some embodiments, the PYY analog polypeptides may be conjugated to one, two, or three polymer molecules.

In some embodiments, the water soluble polymer molecules are linked to an amino, carboxyl, or thiol group, and may be linked by N or C termini, 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 anti-obesity agents are conjugated to one, two, or three PEG molecules through an epsilon amino group on a lysine amino acid.

Anti-obesity agent derivatives also include anti-obesity agents with chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. The chemical alterations may occur singularly at the N- or C-terminus or at the side chains of amino acid residues within the sequence of the anti-obesity agent polypeptides. In one embodiment, the C-terminus of these peptides may have a free -OH or -NH2 group. In another embodiment, the N-terminal end may be capped with an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group ("isocap"), an octanyl group, an octyl glycine group (denoted as "G(Oct)" or "octylGly"), an 8-aminooctanic acid group, a dansyl, and/or a Fmoc group. In some embodiments, cyclization can be through the formation of disulfide bridges.

Alternatively, there may be multiple sites of chemical alteration along the PYY analog polypeptide.

In certain embodiments, anti-obesity agent polypeptides are chemically altered to include a Bolton-Hunter group. Bolton-Hunter reagents are known in the art

("Radioimmunoassay and related methods," A.E. Bolton and W.M. Hunter, Chapter 26 of Handbook of Experimental Immunology, Volume I, Immunochemistry, edited by D.M.

Weir, Blackwell Scientific Publications, 1986), and may be used to introduce tyrosine-like moieties with a neutral linkage, through amino-terminal a-amino groups or ε-amino groups of lysine. In some embodiments, the N-terminal end of an anti-obesity agent polypeptide is modified with a Bolton-Hunter group. In some embodiments, an internal lysine residue is modified with a Bolton-Hunter group. In some embodiments, there may be multiple sites of Bolton-Hunter modification along the anti-obesity agent polypeptide. Bolton-Hunter reagents used for polypeptide modification are commercially available, and may include, but are not limited to, water-soluble Bolton-Hunter reagent, Sulfosuccinimidyl-3-[4- hydrophenyljpropionate (Pierce Biotechnology, Inc., Rockford, IL) and Bolton-Hunter reagent-2, N-Succinimidyl 3-(4-hydroxy-3-iodophenyl) Priopionate (Wako Pure Chemical Industries, Ltd., Japan, catalog # 199-09341). An exemplary Bolton-Hunter group conjugated through an amide linkage to an anti-obesity agent polypeptide is illustrated below, wherein the dashed line passes through the amide bond:

Anti-obesity agent polypeptides may be iodinated (such as radiolabeled with I) before or after Bolton-Hunter modification.

In order to prepare derivatives of the anti-obesity agents in accordance with the invention, the polypeptides of the invention may include one or more modifications of a

"non-essential" amino acid residue. In the context of the invention, a "non-essential" amino acid residue is a residue that can be altered, e.g., derivatized, without abolishing or substantially reducing the activity (e.g., the agonist activity) of the polypeptide (e.g., the analog polypeptide). The polypeptides of the invention may include derivatizations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues; of these, one or more amino acid residues may be non-essential amino acid residues. Additionally, the polypeptides of the invention may be derivatized such that they include additions of at least of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids without abolishing or substantially reducing the activity of the polypeptide. Additionally, such non-essential amino acid residues may be substitutes with an amino acid residue that is amenable to derivatization as described throughout.

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 (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). 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 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-ethylglycine, 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, J. Med. Chem. 41: 2481-91, 1998.

Methods of the Invention

In a general aspect, the invention provides methods for treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing or delaying gastric emptying, reducing adiposity, reducing caloric efficiency, or inducing satiety through administration of a combination of a first anti-obesity agent, which comprises the amino acid sequence set for the in SEQ ID NO: l, or a derivative thereof, and a second anti-obesity agent, which comprises the amino acid sequence set forth in SEQ ID NO:2, or a derivative thereof. Thus, the invention provides methods for treating obesity and obesity-related diseases, disorders, and/or conditions that would benefit from a reduction in nutrient availability, reduction in food intake, reduction in body weight, reduction in weight gain, reduction of adiposity, reduction in body fat, reduction in percent body fat, reduction or delay in gastric emptying, and the like, as disclosed inter alia. Given the increase in effectiveness when used in combinations, methods of the invention may allow for administration of lower dosages of one or more of the anti-obesity agents used in combination as compared to the use of the agent alone, such as in a monotherapy. Such dosages may comprise sub-optimal dosages as described throughout. Such dosages may also comprise dose-sparing dosages as described throughout.

The methods of the invention provide administration of a combination of anti- obesity agents. 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 anti-obesity agents or in separately with each intended agent in its own dosage formulation.

Where separate dosage formulations are used, the individual anti-obesity 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 anti-obesity agent of the method. In some embodiments, administration in combination involves administration of separate dosage formulations during overlapping intervals. For example, anti-obesity agent 1 is administered from day 1 through day 30 and anti-obesity 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, anti-obesity agent 1 is administered from day 1 through day 30 and anti-obesity agent 2 is administered from day 35 through day 50. The instant invention is 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," "administering," "administration in combination" and "administering in combination" are to be interpreted accordingly.

In certain embodiments, the invention provides methods for reducing nutrient availability through administration of at least one anti-obesity agent that acts upon forebrain structures involved in food intake and/or body weight modulation in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake and/or body weight modulation. In some cases, the methods of the invention increase or enhance the effectiveness an anti-obesity agent that has limited effectiveness, if any, when used alone (monotherapy). In such cases, the methods of the invention increase or enhance the effectiveness an anti-obesity agent by, for example, preventing or delaying loss of effectiveness by continued use or increasing potency. Methods of the invention may allow for administration of lower dosages of one or more of the anti-obesity agents used in combination as compared to the use of either agent alone.

In one aspect, methods of the invention provide a synergistic anti-obesity effect among the administered agents. Accordingly, in certain embodiments, administration of a combination of anti-obesity agents results in an effect, e.g., a reduction in nutrient availability, reduction in body weight, reduction in food intake, increase in metabolism, which is greater than the combination of the results of administration of the anti-obesity agent alone (monotherapy).

In another aspect of the invention, methods are provided which reduce or eliminate a subject's resistance to an anti-obesity agent so that when the agent is administered, it will be able to elicit an anti-obesity response (e.g., reduce nutrient availability, reduce weight, reduce fat mass, reduce percent body fat, effect body weight loss, and the like).

In certain embodiments, the invention is directed to the delivery of a first anti- obesity agent that acts upon hindbrain structures involved in food intake and/or body weight modulation to prime the body before administration of a second anti-obesity agent that acts upon forebrain structures involved in food intake and/or body weight modulation. In certain embodiments, the administration of the first agent is for a number of days, weeks or even months before the administration of the second agent. At this point, the second agent may be administered alone or in combination with the first agent.

In another aspect of the present invention, methods for reducing the risk of developing metabolic disorders are provided, where the method comprises administering to the subject a combination of anti-obesity agents in effective amounts to reduce the weight of a subject.

In some embodiments of the invention, methods of the invention are used to increase the metabolic rate in a subject, decrease a reduction in the metabolic rate in a subject, or preserve the metabolic rate in a subject. In certain embodiments, the metabolic rate may involve the preferential use of the body's fat as an energy source over lean body tissue. In one aspect, lean body mass is not decreased following administration of the combination of anti-obesity agents. In another aspect, a reduction in the lean body mass is lessened or prevented following administration of the combination of anti-obesity agents. In still another aspect, lean body mass is increased following administration of the combination of anti-obesity agents. Such preference for fat as the energy source may be determined by comparing the amount of fatty tissue to lean body tissue, ascertained by measuring total body weight and fat content at the beginning and end of the treatment period. An increase in metabolic rate is a higher level of the use of calories or another energy source by a subject over a period of time compared with the level of use of calories or other energy source by the subject over another period of time under substantially similar or identical conditions without administration of the combination of anti-obesity agents. In certain embodiments, the metabolic rate is increased at least about 5% in a subject, in other embodiments, the metabolic rate is increased at least about 10%, 15%, 20% 25%, 30%, or 35% in a subject compared with the level of use of calories or other energy source by the subject over another period of time under substantially similar or identical conditions without administration of the combination of anti-obesity agents. The increase in metabolic rate can be measured using a respiratory calorimeter, for example. An effective amount of the anti-obesity agents as used in these embodiments is an amount of each agent effective to increase the metabolic rate in a subject when administered in combination compared to a subject not receiving the agents or only one of the agents.

In another embodiment, a method is provided to reduce a decrease in metabolic rate in a subject. Such a decrease in metabolic rate can be the result of any condition or nutritional or physical regimen that leads to a reduction in metabolic rate, for example, due to a reduced calorie diet, a restricted diet, or weight loss. A restricted diet includes allowances or prohibitions, or both on the types of food or the amounts of food or both permitted in a diet, not necessarily based on calories. For example, as in individual diets, the body compensates with a reduced metabolic rate based on the lower caloric intake. In essence, the body down-regulates the requirement for food, thereby subsisting on less food. As dieting continues, the threshold for caloric intake is reduced. When dieting has ended, the individual typically gains weight while eating a normal diet because of the lowered caloric intake threshold and lower-basal metabolic rate (NIH Technology Assessment

Conference Panel (1992) Ann. Intern. Med. 1 16:942-949; Wadden (1993) Ann. Intern. Med. 1 19:688-693). In one aspect, a method is provided to reduce the loss of metabolic rate in a subject, where the loss of metabolic rate is the result of a reduced calorie diet or weight loss. By using such a method, the subject's reduction in metabolic rate is decreased by at least about 10%, 15%, 20% 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in a subject. For such methods, it may be desirable to administer the combination of anti-obesity agents at the time the condition or nutritional or physical regimen is initiated which leads to a loss or reduction in metabolic rate. However, it is also contemplated that administration of the agents is commenced before the condition or nutritional or physical regimen is initiated. In one instance, metabolic rate is measured using a respiratory calorimeter. An effective amount of the anti-obesity agents of as used in this embodiment is an amount of each agent effective to decrease the reduction of the metabolic rate in a subject when administered in combination.

In another aspect, methods for reducing metabolic plateaus are provided, where a method comprises administering effective amounts of anti-obesity agents in combination to a subject. In certain embodiments, the subject is losing weight, or has lost weight, for example, due to a reduced calorie diet, increased exercise or a combination thereof. By "metabolic plateau" is meant time intervals of steady metabolic rate while the body adjusts to changes in caloric or energy input. Changes in caloric input or expenditure can be the result of, for example, reduced calorie diets or increased physical activity. Such plateaus can be observed, for example, during a weight loss regimen when weight loss slows or stops. In certain embodiments, a method of the present invention reduces the duration of a metabolic plateau in a subject compared with the duration of metabolic plateaus in an otherwise identical subject over the same period of time under substantially similar or identical conditions without administration of the combination of anti-obesity agents. In other embodiments, a method of the present invention reduces the frequency of metabolic plateaus compared with the frequency of metabolic plateaus in an otherwise identical subject over the same period of time under substantially similar or identical conditions without administration of the combination of anti-obesity agents. In still other

embodiments, a method of the present invention delays the onset of a metabolic plateau compared with the onset of a metabolic plateau in an otherwise identical subject over the same period of time under substantially similar or identical conditions without

administration of the combination of anti-obesity agents. In certain embodiments, metabolic plateaus are identified by charting periods of reduced or no weight loss. In certain embodiments, at least one metabolic plateau is reduced. In other embodiments, at least two, three, four, five, six, seven, eight, nine, or ten metabolic plateaus are reduced. In another aspect, metabolic plateaus are delayed one day as compared to a subject not administered the combination of anti-obesity agents under identical or similar conditions. In other aspects, metabolic plateaus are delayed 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks or 3 weeks in a subject.

In yet other embodiments, a method is provided to preserve the metabolic rate in a subject. In certain embodiments, the subject may be at risk of losing metabolic rate, for example, due to the initiation of a reduced calorie diet, restricted diet, or anticipated weight loss. A preservation of metabolic rate is a maintenance of the level of the use of calories or another energy source by a subject over a period of time compared with the level of use of calories or other energy source by an otherwise identical subject over the same period of time under substantially similar or identical conditions without administration of the combination of anti-obesity agents. In one aspect, the metabolic rate is maintained within 15% of the subject's metabolic rate prior to the initiation of the event that results in the decrease in metabolic rate. In other aspects, the metabolic rate is maintained within 10%, within 7%, within 5%, within 3% or less of the subject's metabolic rate. In one aspect, the combination of anti-obesity agents is administered at the initiation of a reduced calorie diet, restricted diet, or exercise regimen.

Metabolic rates can be assessed using any method available for determining such rates, for example by using a respiratory calorimeter. Such methods and devices for assaying metabolic rates are known in the art and are described, for example, in U.S. Patent Nos. 4,572,208, 4,856,531, 6,468,222, 6,616,615, 6,013,009, and 6,475,158. Alternatively, the metabolic rate of an animal can be assessed by measuring the amount of lean tissue versus fatty tissue catabolized by the animal following the diet period. Thus, total body weight and fat content can be measured at the end of the dietary period. In rats, a frequently used method to determine total body fat is to surgically remove and weigh the

retroperitoneal fat pad, a body of fat located in the retroperitoneum, the area between the posterior abdominal wall and the posterior parietal peritoneum. The pad weight is considered to be directly related to percent body fat of the animal. Since the relationship between body weight and body fat in rats is linear, obese animals have a correspondingly higher percent of body fat and retroperitoneal fat pad weight.

In another aspect of the present invention, methods for reducing fat mass, adiposity, or percent fat, as described, for instance, in the Examples, by, for example, increasing the metabolic rate in a subject are provided, where the methods comprise administering a combination of anti-obesity agents in amounts effective to reduce fat mass by increasing the subject's metabolic rate. Fat mass can be expressed as a percentage of the total body mass. In some aspects, the fat mass is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% over the course of treatment. In one aspect, the subject's lean mass is not decreased over the course of the treatment. In another aspect, the subject's lean mass is maintained or increased over the course of the treatment. In another aspect, the subject is on a reduced calorie diet or restricted diet. By "reduced calorie diet" is meant that the subject is ingesting fewer calories per day than compared to the same subject's normal diet. In one instance, the subject is consuming at least 50 fewer calories per day. In other instances, the subject is consuming at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 fewer calories per day.

In certain embodiments of the present invention, a method for altering the fat distribution in a subject is provided where the method comprises administering a combination of anti-obesity agents in amounts effective to alter fat distribution in the subject. In one aspect, the alteration results from an increased metabolism of visceral or ectopic fat, or both in the subject. In some embodiments, the method involves the metabolism of visceral or ectopic fat or both at a rate of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% greater than for subcutaneous fat. In one aspect, the methods result in a favorable fat distribution. In certain embodiments, favorable fat distribution is an increased ratio of subcutaneous fat to visceral fat, ectopic fat, or both. In one aspect, the method involves an increase in lean body mass, for example, as a result of an increase in muscle cell mass.

In other embodiments, methods for reducing the amount of subcutaneous fat in a subject are provided, wherein the method comprises administering, to a subject in need thereof, a combination of anti-obesity agents in amounts effective to reduce the amount of subcutaneous fat in the subject. In one instance, the amount of subcutaneous fat is reduced in a subject by at least about 5%. In other instances, the amount of subcutaneous fat is reduced by at least about 10%, 15%, 20%, 25%, 30% 40%, or 50% compared to the subject prior to administration of the anti-obesity agents.

The methods described herein can be used to reduce the amount of visceral fat in a subject. In one instance, the visceral fat is reduced in a subject by at least about 5%. In other instances, the visceral fat is reduced in the subject by at least about 10%, 15%, 20%, 25%, 30% 40%, or 50% compared to the subject prior to administration of the combination of anti-obesity agents. Visceral fat can be measured through any means available to determine the amount of visceral fat in a subject. Such methods include, for example, abdominal tomography by means of CT scanning and MRI. Other methods for determining visceral fat are described, for example, in U.S. Patent Nos. 6,864,415, 6,850,797, and 6,487,445.

In certain embodiments, a method for preventing the accumulation of ectopic fat or reducing the amount of ectopic fat in a subject is provided, wherein the method comprises administering, to a subject in need thereof, a combination of anti-obesity agents in amounts effective to prevent accumulation of ectopic fat or to reduce the amount of ectopic fat in the subject. In one instance, the amount of ectopic fat is reduced in a subject by at least about 5% compared to the subject prior to administration of the combination of anti-obesity agents. In other instances, the amount of ectopic fat is reduced in a subject by at least about 10%, or by at least about 15%, 20%, 25%, 30% 40%, or 50%. 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. Ectopic fat can be measured in a subject using any method available for measuring ectopic fat.

In other embodiments, methods are provided for producing a more favorable fat distribution in a subject, where the method comprises administering to a subject a combination of anti-obesity agents in amounts effective to produce a favorable fat distribution. In certain embodiments, administration of a combination of anti-obesity agents reduces the amount of visceral fat or ectopic fat, or both, in a subject. For example, administration of a combination of anti-obesity agents, where at least one anti-obesity agent that acts upon forebrain structures involved in food intake or body weight modulation or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake or body weight modulation or both. In certain embodiments, the methods preferentially reduce the amount of visceral or ectopic fat, or a combination of both, over the reduction in subcutaneous fat. Such methods result in a higher ratio of subcutaneous fat to visceral fat or ectopic fat. Such improved ratios may result in a reduced risk of the development of cardiovascular diseases, polycystic ovary syndrome, metabolic syndrome, or any combinations thereof. In certain embodiments, ectopic or visceral fat is metabolized at a rate 5% greater than subcutaneous fat. In other embodiments, ectopic or visceral fat is metabolized at a rate at least 10% 15%, 20%, 25%, 30% 50%, 60%, 70%, 80%, 90%, or 100% greater than subcutaneous fat.

In still another aspect, methods of the invention include the use of a therapeutically effective amount of a combination of anti-obesity agents administered in combination with glucocortico steroids. Glucocortico steroids have the adverse effect of increasing fat mass and decreasing lean mass. Accordingly, it is contemplated that the anti-obesity agent combination can be used in conjunction with glucocortico steroids under conditions where glucocortico steroid use is beneficial.

Also provided are methods to reduce weight in a morbidly obese subject by first reducing the subject's weight to a level below that of being morbidly obese, then administering to the subject a combination of anti-obesity agents in effective amounts to further reduce the subject's weight. Methods for reducing a subject's weight to below that of morbid obesity include reducing caloric intake, increasing physical activity, drug therapy, bariatric surgery, such as gastric bypass surgery, or any combinations of the preceding methods. In one aspect, administering the combination of anti-obesity agents further reduces the weight of the subject. In other embodiments, methods are provided for reducing the body mass index in a subject having a body mass index of 40 or less by administering a combination of anti-obesity agents in effective amounts to further reduce the subject's weight.

By reducing weight it is meant that the subject loses a portion of his/her total body weight over the course of treatment, whether the course of treatment be days, weeks, months or years. Alternatively, reducing weight can be defined as a decrease in proportion of fat mass to lean mass (in other words, the subject has lost fat mass, but maintained or gained lean mass, without necessarily a corresponding loss in total body weight). An effective amount of the anti-obesity agents administered in combination in these

embodiments is an amount effective to reduce a subject's body weight over the course of the treatment, or alternatively an amount effective to reduce the subject's percentage of fat mass over the course of the treatment. In certain embodiments, the subject's body weight is reduced, over the course of treatment, by at least about 1%, by at least about 5%, by at least about 10%, by at least about 15%, or by at least about 20%. Alternatively, the subject's percentage of fat mass is reduced, over the course of treatment, by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%.

In certain embodiments, methods of reducing nutrient availability, e.g., reducing weight, in a subject comprise administering to the subject an effective amount of the anti- obesity agents 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 an agent 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 other embodiments, methods of reducing nutrient availability, e.g., reducing weight, in a subject comprise administering to the subject an effective amount of the anti- obesity agents 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 subject's system over a period of time. When administered by a continuous dose, the drug is released over a period of about 1 hour, in some cases the drug is released over a period of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours.

By "administered in combination" is meant that the anti-obesity agents are administered as a single administration, simultaneously as separate doses, or as sequentially administered. Sequential administration refers to administering one of the anti-obesity agents either before or after an anti-obesity agent. In certain embodiments, the first anti- obesity agent is administered about 30 minutes before or after the at least one other anti- obesity agent, in other embodiments about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before or after the at least one other anti-obesity agents. Any of the administered anti-obesity agents can be administered as a bolus dose or as a continuous dose.

The present invention is further directed to methods of increasing thermogenesis in a subject, the method comprising administering to a subject in need thereof an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both. Thermogenesis is the process of liberating calories as heat by increasing the body's metabolic rate. Thermogenesis is activated by mechanisms, including supplements, nutrition, exercise, and exposure to cold.

The present invention is yet further directed to methods of increasing oxidative metabolism in a subject, the method comprising administering to a subject in need thereof an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with

administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both. Oxidative metabolism is the process by which oxygen is used to make energy from carbohydrates (sugars).

In another aspect, a method of inducing a feeling of fullness in a subject is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In yet another aspect, a method of controlling hunger in a subject is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In yet a further aspect, a method of prolonging a feeling of satiation in a subject is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In yet a further aspect, a method of reducing caloric intake by reducing the size of a meal is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In another aspect, a method of controlling food intake is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In yet another aspect, a method for ensuring or assisting in compliance with a reduced calorie or restrictive diet is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with

administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In a further aspect, a method of adjusting a subject's set point so that the body's propensity for homeostasis is adjusted to a healthier set point is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject.

In yet a further aspect, a method of maintaining weight loss or maintaining the weight lost is provided, wherein the method comprises administering an effective amount of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti- obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both to said subject. In other embodiments of this aspect of the invention, the weight loss is maintained by re-setting the subject's set point.

Furthermore, in certain embodiments, administration of the anti-obesity agents in combination results in a synergistic effect in any of the methods described herein. In addition, in certain embodiments, administration of the anti-obesity agents in combination results in a lower dosage requirement for at least one of the agents, with the same effect.

In certain embodiments, methods of the invention are of use in treating and/or preventing metabolic conditions or disorders that benefit from a reduction in nutrient availability. Accordingly, these methods may be useful in treating and/or preventing of obesity, diabetes (e.g., type 2 or non-insulin dependent diabetes, type 1 diabetes, and gestational diabetes), eating disorders, insulin-resistance syndrome, and cardiovascular disease.

In certain embodiments, methods of use in altering fat distribution, reducing fat mass, or both in a subject are provided. Accordingly, subjects for whom altering body composition is of benefit can also benefit from the present methods. Altered body composition, as intended herein, includes loss or maintenance of body fat, with

minimization of loss, maintenance, or gain of lean body mass. In such situations, weight may increase as well as decrease. Accordingly, subjects may be lean, overweight, or obese as these terms are generally used in the art. Methods of the invention may also include reducing fat in non-adipose tissue while sparing lean mass. Uses for this method include treating diseases such as nonalcoholic steatohepatitis (NASH) or lipodystrophy.

Methods described herein use the administration of at least one anti-obesity agent that acts upon forebrain structures involved in food intake, body weight modulation, or both in combination with administration of at least one anti-obesity agent that acts upon hindbrain structures involved in food intake, body weight modulation, or both for the control, prevention and/or treatment of such conditions or disorders. With regard to the administration of weight-inducing agents, the weight-inducing agents are administered as a single administration, simultaneously as separate doses, or as sequentially administered. Where separate dosage formulations are used, the individual weight-inducing agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, e.g., sequentially prior to or subsequent to the

administration of the other weight-inducing agent of the method. In certain embodiments, the first weight-inducing agent is administered about 30 minutes before or after the at least one other weight-inducing agent, in other embodiments about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before or after the at least one other weight-inducing agents. In some embodiments, administration in combination involves administration of separate dosage formulations during overlapping intervals. For example, weight-inducing agent 1 is administered from day 1 through day 30 and weight-inducing 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, weight-inducing agent 1 is administered from day 1 through day 30 and weight- inducing agent 2 is administered from day 35 through day 50. The instant invention is therefore to be understood to include all such regimes of simultaneous, alternating, or completely separate treatments over the total treatment course. Any of the administered weight-inducing agents can be administered as a bolus dose or as a continuous dose.

Furthermore, in certain embodiments, administration of the weight-inducing agents in combination results in a synergistic effect in any of the aspects of the invention. In addition, in certain embodiments, administration of the weight-inducing agents in combination results in a lower dosage requirement for at least one of the agents, with the same effect.

Further anti-obesity agents that may be employed in the disclosed and claimed methods in combination with the first and second anti-obesity agents, such as a third anti- obesity agent, a fourth anti-obesity agent, and so on, include leptin, leptin derivatives, recombinant leptin, metreleptin, and leptin agonists. Leptin (derived from Greek leptos, meaning thin) is a hormone produced predominantly by fat cells. In obese humans, leptin blood levels generally correlate with the amount of fat stored in the body. Generally, the greater the amount of fat, the greater the amount of leptin. Serum leptin levels

concentrations in the majority of humans with obesity are high, and without wishing to be bound by theory, a state of leptin resistance is thought to exist (Mantzoros et al. (2000) J. Clin. Endocrinol. Metab. 85:4000-4002). Despite therapeutic attempts at using leptin to treat obesity, the effect of recombinant human leptin has been limited, if any, in causing weight loss in obese individuals. Exceptions to this have included the treatment of individuals with congenital leptin deficiency and the treatment of individuals with lipoatrophy. See, for example, Heymsfield et al. (1999) JAMA 282:1568-1575, Farooqi et al. (1999) N. Engl. J. Med.341:879-884, and U.S. Pat. Publication No.2005/0020496.

Additionally, as disclosed in, e.g., WO2009/064298 and US2008/0207512, amylin agonism has been shown to, for example, rescue leptin responsiveness in leptin resistant subjects, such as obese or overweight subjects, thereby eliciting substantial weight loss, reductions in food intake, reduction in nutrient availability, etc. when amylin agonists, are administered in combination with leptin agonists or leptin derivatives to overweight or obese subjects.

Accordingly, it is believed that the administration of a leptin agonist or leptin derivative as a further anti-obesity agent in accordance with the disclosed and claimed methods will elicit further advantages with regard to efficacy and potency of the effects described herein when such methods are practiced. In certain embodiments, it is believed that because of the synergistic and dose-sparing benefits afforded by the administration of the first and second anti-obesity agents as disclosed and claimed herein, the further administration of a sub- optimal or dose-sparing amount of a leptin agonist or a leptin derivative will yield substantially greater efficacy and/or potency with regard to the effects described throughout.

Accordingly, exemplary leptins, leptin derivatives, recombinant leptins, and leptin agonists for use in the methods and compositions described herein include, but are not limited to, the amino acid sequence for mature, recombinant methionyl human leptin (herein called rmetHu-Leptin 1-146 or Metreleptin) having the amino acid sequence: M V P I Q K VQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPI LTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHV LAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQ GSLQDMLWQLDLSPGC (SEQ ID NO:5).

Leptin proteins and leptin protein containing compositions appropriate for use in accordance with the disclose and claimed methods and pharmaceutical compositions are known in the art and include, but are not limited to pegylated recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen) (herein called rmetHu-Leptin 1-146 or metreleptin). Leptin proteins, analogs, derivatives, preparations, formulations, pharmaceutical compositions, doses, and administration routes have previously been described in the following patent publications and are hereby incorporated by reference in their entirety and for all purposes: U.S. Pat. Nos.5,552,524; 5,552,523; 5,552,522; 5,521,283, 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 Application Publication Nos. WO 96/05309, WO 96/40912; WO 97/06816, WO 00/20872, WO 97/18833, WO 97/38014, WO 98/08512, WO 98/28427, WO 98/46257, WO 00/09165, WO 00/47741, and WO 00/21574.

Leptin agonists that may be employed as further anti-obesity agent as disclosed and claimed are available in the art. For example, leptin agonists are described in U.S. Pat. Publication Nos. 2004/0072219, 2003/049693, 2003/0166847, 2003/0092126, and U.S. Pat. Nos. 6,777,388 and 6,936,439. Leptin antagonists are described e.g. in U.S. Pat.

Publication Nos. 2004/0048773, 2002/0160935 and U.S. Pat. No. 6,399,745. Means for testing for leptin agonism or antagonism are described e.g. in U.S. Pat. Nos. 6,007,998 and 5,856,098. These patents are exemplary and are incorporated herein by reference in their entirety and for all purposes.

Additional further anti-obesity agents for use in the present invention also include exendin peptide hormones and exendin agonists. Native exendin peptide hormones are known in art, as are functional peptide analogs and derivatives. Certain native peptides, peptide analogs and derivatives are described herein, however it should be recognized that any known exendin peptides that exhibit hormonal activity known in the art may be used in conjunction with the present invention. Exemplary exendin peptides include exendin-3 (His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe He Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO:6)) and exendin-4 (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe He Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO:7)).

Any exendin peptide analog or derivative known in the art may be used in conjunction with the present invention. In certain embodiments, the exendin peptide analogs and derivatives have at least one hormonal activity of a native exendin peptide. In certain embodiments, the exendin peptide analogs are agonists of a receptor which a native exendin peptide is capable of specifically binding. Exendin compounds include exendin peptide analogs in which one or more naturally occurring amino acids are eliminated or replaced with another amino acid(s). As known in the art, such exendin analogs are may be amidated or may be in the acid form.

In certain embodiments, an exendin analog can have one or more amino acid substitutions, deletions, inversion, or additions compared to a native or naturally occurring exendin. Thus, exendin analogs can have an amino acid sequence that has one or more amino acid substitutions, additions or deletions as compared with a naturally occurring exendin, for example, exendin-4. In certain embodiments, an exendin analog has an amino acid sequence that has about 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less substitutions, additions, or deletions as compared to a naturally occurring exendin, such as exendin-4

Exemplary exendin compounds include agonist analogs of exendin-4, including, but are not limited to, ¹⁴Leu,²⁵Phe-exendin-4 (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe He Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO: 8), ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4 (His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe He Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO: 9)), and

1⁴Leu,²²Ala,²⁵Phe-exendin-4 (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Ala He Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO: 10)). Other exemplary exendin analogs include, but are not limited to, exendin-4 (1-30) (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe He Glu Trp Leu Lys Asn Gly Gly (SEQ ID NO: 11)), exendin-4 (1-28) amide (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu Glu Ala Val Arg Leu Phe He Glu Trp Leu Lys Asn-NH2 (SEQ ID NO: 12)),

14Leu,25Phe exendin-4 (1-28) amide (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe He Glu Phe Leu Lys Asn-NH2 (SEQ ID

NO: 13)), and 14Leu,22Ala,25Phe exendin-4 (1-28) amide (His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Ala He Glu Phe Leu Lys Asn- NH2 (SEQ ID NO: 14)).

Additional exemplary exendin agonists are described in U.S. patent application Ser.

No. 10/181,102 and PCT Application No. PCT/US98/16387, both which claim the benefit of U.S. patent application Ser. No. 60/055,404, filed Aug. 8, 1997, all of which are herein incorporated by reference. Exemplary exendin agonists include compounds of the formula (I), formula (II) and formula (III) of U.S. patent application Ser. No. 10/181,102 and PCT Application No. PCT/US98/16387.

Other exendin agonists are described in U.S. patent application Ser. No. 09/554,533 and PCT Application Serial No. PCT/US98/24210, both of which claim the benefit of U.S. Provisional Application No. 60/065,442 filed Nov. 14, 1997, all of which are herein incorporated by reference. Still other exendin agonists are described in U.S. patent application Ser. No. 09/554,531 and PCT Application Serial No. PCT/US98/24273, both of which claim the benefit of U.S. Provisional Application No. 60/066,029 filed Nov. 14, 1997, all of which are herein incorporated by reference. Still other exendin agonists are described in PCT Application Serial No. PCT/US97/14199, filed Aug. 8, 1997, which is a continuation-in-part of U.S. patent application Ser. No. 08/694,954 filed Aug. 8, 1996, both of which are hereby incorporated by reference. Still other exendin agonists are described in U.S. Patent No. 6,956,026, which claims priority to U.S. Provisional Application No.

60/034,905 filed Jan. 7, 1997, both of which are hereby incorporated by reference. Yet other exendin analogs and derivatives are described in US 2004/0209803 Al, filed

December 19, 2003, which is hereby incorporated by reference.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions include ciliary neurotrophic factor (CNTF), CNTF-related polypeptides, modified CNTF polypeptides, CNTF agonists, and CNTF analogs, including, but not limited to AXOKINE® (Regeneron). CNTF, CNTF-related polypeptides, and CNTF and/or CNTF-related polypeptide containing compositions appropriate for use in the methods of the invention are known in the art. CNTF polypeptides, CNTF-related polypeptides, modified CNTF polypeptides, CNTF agonists, analogs, derivatives, preparations, formulations, pharmaceutical compositions, doses, and administration routes have previously been described, for example, in U.S. Pat. Nos. 6,680,291 and 6,767,894, and in PCT Application Publication Nos. WO 94/09134, WO 98/22128, and WO 99/43813, which are hereby incorporated by reference in their entirety.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include serotonin (5HT) transport inhibitors, including, but not limited to, paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine. Anti-obesity agents in the present invention also include selective serotonin reuptake inhibitors, including, but not limited to dexfenfluramine, fluoxetine, sibutramine (e.g., MERIDIA®) and those described in U.S. Pat. No. 6,365,633 and PCT Patent Application Publication Nos. WO 01/27060 and WO 01/162341, which are hereby incorporated by reference in their entirety. Such 5HT transport inhibitors and serotonin reuptake inhibitors, analogs, derivatives, preparations, formulations, pharmaceutical compositions, doses, and administration routes have previously been described.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include selective serotonin agonists and selective 5-HT2C receptor agonists, including, but not limited to, U.S. Pat. No. 3,914,250; and PCT Application Publication Nos. WO 02/36596, WO 02/48124, WO 02/10169, WO 01/66548, WO

02/44152; WO 02/51844, WO 02/40456, and WO 02/40457, which are hereby incorporated by reference in their entirety. Such selective serotonin agonists and 5-HT2C receptor agonists, compositions containing such agonists, and administration routes appropriate for use in the methods of the invention are known in the art. See, for example, Halford et al. (2005) Curr. Drug Targets 6:201-213 and Weintraub et al. (1984) Arch. Intern. Med.

144: 1143-1148.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include antagonists/inverse agonists of the central cannabinoid receptors (the CB-1 receptors), including, but not limited to, rimonabant (Sanofi

Synthelabo), and SR-147778 (Sanofi Synthelabo). CB-1 antagonists/inverse agonists, derivatives, preparations, formulations, pharmaceutical compositions, doses, and administration routes have previously been described, for example, in U.S. Pat. Nos.

6,344,474, 6,028,084, 5,747,524, 5,596,106, 5,532,237, 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,624,941; European Patent Application Nos. EP-656 354 and EP- 658546; and PCT Application Publication Nos. WO 96/33159, WO 98/33765,

W098/43636, W098/43635, WO 01/09120, W098/31227, W098/41519, WO98/37061, WO00/10967, WO00/10968, WO97/29079, WO99/02499, WO 01/58869, and WO

02/076949, which are hereby incorporated by reference in their entirety.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include melanocortins and melanocortin agonists. Melanacortins are peptides from the pro-opiomelanocortin gene, including a-melanocyte-stimulating hormone (a-MSH) and adrenocorticotrophic hormone (ACTH), and five melanocortin receptors are known, MC1-5R. MC4R appears to play a role in energy balance and obesity. See, for example, Anderson et al. (2001) Expert Opin. Ther. Patents 11: 1583-1592, Speake et al.

(2002) Expert Opin. Ther. Patents 12: 1631-1638, Bednarek et al. (2004) Expert Opin. Ther. Patents 14:327-336. Melanocortin agonists, including, but not limited to, MC4R agonists, and composition containing such agonist appropriate for use in the methods of the invention are known in the art. MCR agonists, MC4R agonists, derivatives, preparations, formulation, pharmaceutical compositions, doses, and administration routes have previously been described, for example, in the following PCT patent applications, which are hereby incorporated by reference in their entirety: WO 03/007949, WO 02/068388, WO

02/068387, WO 02/067869, WO 03/040117, WO 03/066587, WO 03/068738, WO

03/094918, and WO 03/031410. Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include metabotropic glutamate subtype 5 receptor (mGluR5) antagonists, including, but are not limited to, compounds such as 2-methyl-6- (phenylethynyl)-pyridine (MPEP) and (3-[(2-methyl-l,3-thiazol-4-yl)ethynyl]pyridine) (MTEP) and those compounds described in Anderson et al. (2003) J. Eur. J. Pharmacol. 473:35-40; Cosford et al. (2003) Bioorg. Med. Chem. Lett. 13(3):351-4; and Anderson et al. (2002) J. Pharmacol. Exp. Ther. 303: 1044-1051.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include topiramate (TOPIMAX® (Ortho McNeil Pharmaceuticals), indicated as an anti-convulsant and an anti-convulsant, but also shown to increase weight loss.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include neuropeptide Yl (NPYl) antagonists and NPY5 antagonists. NPYl and NPY5 antagonists are known in the art. See, for example Duhault et al. (2000) Can. J Physiol. Pharm. 78: 173-185, and U.S. Pat. Nos. 6,124,331, 6,214,853, and

6,340,683. NPYl and NPY5 antagonists, derivatives, preparations, formulation, pharmaceutical compositions, doses, and administration routes have previously been described. NPYl antagonists useful in the present invention, include: U.S. Pat. No.

6,001,836; and PCT Application Publication Nos. WO 96/14307, WO 01/23387, WO 99/51600, WO 01/85690, WO 01/85098, WO 01/85173, and WO 01/89528, which are hereby incorporated by reference in their entirety. NPY5 antagonists useful in the present invention, include, but are not limited to, the compounds described in: U.S. Pat. Nos.

6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,337,332, 6,329,395, 6,340,683, 6,326,375, and 6,335,345; European Patent Nos. EP -01010691, and EP-01044970; and PCT Patent Publication Nos. WO 97/19682, WO 97/20820, WO 97/20821, WO 97/20822, WO

97/20823, WO 98/27063, WO 00/64880, WO 00/68197, WO 00/69849, WO 01/09120, WO 01/85714, WO 01/85730, WO 01/07409, WO 01/02379, WO 01/02379, WO 01/23388, WO,01/23389, WO 01/44201, WO 01/62737, WO 01/62738, WO 01/09120, WO 02/22592, WO 0248152, WO 02/49648, and WO 01/14376.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include melanin-concentrating hormone (MCH) antagonists including melanin-concentrating hormone 1 receptor (MCH1R) antagonists, such as T- 226296 (Takeda) and melanin-concentrating hormone 2 receptor (MCH2R) antagonists. MCH receptor antagonists, derivatives, preparations, formulation, pharmaceutical compositions, doses, and administration routes have previously been described, for example, in U.S. Patent Application Publication Nos. 2005/0009815, 2005/0026915, 2004/0152742, 2004/0209865; PCT Patent Application Publication Nos. WO 01/82925, WO 01/87834, WO 02/06245, WO 02/04433, and WO 02/51809; and Japanese Patent Application No. JP 13226269, which are hereby incorporated by reference in their entirety.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include opioid antagonists, including, but not limited to those described in PCT Application No. WO 00/21509. Specific opioid antagonists useful in the present invention include, but are not limited to, nalmefene (REVEX®), 3- methoxynaltrexone naloxone, naltrexone, naloxonazine, beta-funaltrexamine, deltal ([D- Ala2,Leu5,Cys6]-enkephalin (DALCE), naltrindole isothiocyanate, and nor- binaltorphamine.

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include orexin antagonists, including, but not limited to, those described in PCT Patent Application Nos. WO 01/96302, WO 01/68609, WO 02/51232, and WO 02/51838. Specific orexin antagonists useful in the present invention include, but are not limited to, SB-334867-A.

Additional further anti-obesity agents for use in the present invention also include histamine 3 (H3) antagonist/inverse agonists including but not limited to, those described in PCT Application No. WO 02/15905, 0-[3-(lH-imidazol-4-yl)propanol]carbamates (Kiec- Kononowicz et al. (2000) Pharmazie 55:349-355), piperidine-containing histamine H3- receptor antagonists (Lazewska et al. (2001) Pharmazie 56:927-932), benzophenone derivatives and related compounds (Sasse et al. (2001) Arch. Pharm. (Weinheim) 334:45- 52), substituted N-phenylcarbamates (Reidemeister et al. (2000) Pharmazie 55:83-86), and proxifan derivatives (Sasse et al. (2000) J. Med. Chem. 43:3335-3343). Specific H3 antagonists/inverse agonists useful in the present invention include, but are not limited to, thioperamide, 3-(lH-imidazol-4-yl)propyl N-(4-pentenyl)carbamate, clobenpropit, iodophenpropit, imoproxifan, and GT2394 (Gliatech).

Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include cholecystokinin (CCK) and CCK agonists. Cholecystokinin- A (CCK-A) agonists useful in the present invention 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. Additional further anti-obesity agents for use in the disclosed and claimed methods and compositions also include ghrelin antagonists such as those described in PCT

Application Publication Nos. WO 01/87335 and WO 02/08250. Ghrelin antagonists are also known as GHS (growth hormone secretagogue receptor) antagonists. The

compositions and methods of the present invention therefore comprehend the use GHS antagonists in place of ghrelin antagonists.

Activity of the first anti-obesity agent, second anti-obesity agent, a further anti- obesity agent, and/or derivatives of such, as mentioned throughout, can be confirmed and quantified by performing various screening assays, including: a nucleus accumbens receptor binding assay, followed by a soleus muscle assay; a gastric emptying assay; an amylin receptor binding assay; an amylin receptor activity assay; a calcitonin receptor assay; a calcitonin receptor activity assay; a calcitonin gene related protein receptor binding assay; a calcitonin gene related protein receptor activity assay; a Y receptor (such as a Y2, Y5, or Y7) binding assay; Y receptor (such as a Y2, Y5, or Y7) activity assay; a leptin receptor binding assay; a leptin receptor activity assay; a GLP-1 receptor binding assay;a GLP-1 receptor activity assay; a food intake assay; a caloric efficiency assay; a metabolic rate assay; an energy expenditure assay; a body weight assay; a weight loss assay; or by the ability to induce hypocalcemia or reduce postprandial hyperglycemia in mammals; as is understood and available in the art.

Preparation of compounds

In one exemplary method of making the compounds, compounds of the invention may be prepared using standard solid-phase peptide synthesis techniques and preferably an automated or semiautomated peptide synthesizer. Typically, using such techniques, an a - N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1 -hydroxybenzotriazole in the presence of a base such as diisopropylethylamine. The a-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, with t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) being preferred herein. Other methods of synthesizing or expressing amylin and amylin agonists and purifying them are known to the skilled artisan.

Dosage/Formulation

Anti-obesity agents and further anti-obesity agents (herein referred to in this section as the "compounds") may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. These pharmaceutical compounds may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang et al. (1988) J. of Parenteral Set and Tech., Technical Report No. 10, Supp. 42:2S.

In general, the compounds may be formulated into a stable, safe pharmaceutical composition for administration to a patient. Pharmaceutical formulations contemplated for use in the methods of the invention may comprise approximately 0.01 to 1.0% (w/v), in certain cases 0.05 to 1.0%, of the compound, approximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate or glutamate buffer allowing a pH of the final composition of from about 3.0 to about 7.0; approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol tonicifier and, optionally, approximately 0.005 to 1.0% (w/v) of a preservative selected from the group consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol. Such a preservative is generally included if the formulated peptide is to be included in a multiple use product.

In particular embodiments of the present invention, a pharmaceutical formulation of the present invention may contain a range of concentrations of the compound, e.g., between about 0.01% to about 98% w/w, or between about 1 to about 98% w/w, or preferably between 80% and 90% w/w, or preferably between about 0.01% to about 50% w/w, or more preferably between about 10% to about 25% w/w in these embodiments. A sufficient amount of water for injection may be used to obtain the desired concentration of solution.

Additional tonicifying agents such as sodium chloride, as well as other known excipients, may also be present, if desired. In some cases, such excipients are useful in maintenance of the overall tonicity of the compound. An excipient may be included in the presently described formulations at various concentrations. For example, an excipient may be included in the concentration range from about 0.02% to about 20% w/w, preferably between about 0.02% and 0.5% w/w, about 0.02% to about 10% w/w, or about 1 % to about 20% w/w. In addition, similar to the present formulations themselves, an excipient may be included in solid (including powdered), liquid, semi-solid or gel form.

The pharmaceutical formulations may be composed in various forms, e.g., solid, liquid, semisolid or liquid. The term "solid", as used herein, is meant to encompass all normal uses of this term including, for example, powders and lyophilized formulations. The presently described formulations may be lyophilized.

The terms buffer, buffer solution and buffered solution, when used with reference to hydrogen-ion concentration or pH, refer to the ability of a system, particularly an aqueous solution, to resist a change of pH on adding acid or alkali, or on dilution with a solvent. Characteristic of buffered solutions, which undergo small changes of pH on addition of acid or base, is the presence either of a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base. An example of the former system is acetic acid and sodium acetate. The change of pH is slight as long as the amount of hydronium or hydroxyl ion added does not exceed the capacity of the buffer system to neutralize it.

As described herein, a variety of liquid vehicles are suitable for use in the formulations of peptidic anti-obesity agents, for example, water or an aqueous/organic solvent mixture or suspension.

The stability of a peptide formulation for use in the present invention is enhanced by maintaining the pH of the formulation in the range of about 3.0 to about 7.0 when in liquid form. In certain embodiments, the pH of the formulation is maintained in the range of about 3.5 to 5.0, or about 3.5 to 6.5, in some embodiments from about 3.7 to 4.3, or about 3.8 to 4.2. In some embodiments, pH may be about 4.0. While not seeking to be bound by this theory, it is presently understood that where the pH of the pharmaceutical formulation exceeds 5.5, chemical degradation of the peptide may be accelerated such that the shelf life is less than about two years.

In certain embodiments, the buffer with the anti-obesity agents is an acetate buffer (preferably at a final formulation concentration of from about 1 -5 to about 60 mM), phosphate buffer (preferably at a final formulation concentration of from about 1 -5 to about to about 30 mM) or glutamate buffer (preferably at a final formulation concentration of from about 1-5 to about to about 60 mM). In some embodiments, the buffer is acetate (preferably at a final formulation concentration of from about 5 to about 30 mM).

A stabilizer may be included in the formulations of anti-obesity agents but, and importantly, is not necessarily needed. If included, however, a stabilizer useful in the practice of the present invention is a carbohydrate or a polyhydric alcohol. A suitable stabilizer useful in the practice of the present invention is approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol. The polyhydric alcohols and carbohydrates share the same feature in their backbones, i.e., -CHOH-CHOH-, which is responsible for stabilizing the proteins. The polyhydric alcohols include such compounds as sorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). These compounds are straight-chain molecules. The carbohydrates, such as mannose, ribose, sucrose, fructose, trehalose, maltose, inositol, and lactose, on the other hand, are cyclic molecules that may contain a keto or aldehyde group. These two classes of compounds have been demonstrated to be effective in stabilizing protein against denaturation caused by elevated temperature and by freeze-thaw or freeze-drying processes. Suitable carbohydrates include: galactose, arabinose, lactose or any other carbohydrate which does not have an adverse affect on a diabetic patient, i.e., the carbohydrate is not metabolized to form unacceptably large concentrations of glucose in the blood. Such carbohydrates are well known in the art as suitable for diabetics. Sucrose and fructose are suitable for use with the compound in non- diabetic applications (e.g. treating obesity).

In certain embodiments, if a stabilizer is included, the compound is stabilized with a polyhydric alcohol such as sorbitol, mannitol, inositol, glycerol, xylitol, and

polypropylene/ethylene glycol copolymer, as well as various polyethylene glycols (PEG) of molecular weight 200, 400, 1450, 3350, 4000, 6000, and 8000). Mannitol is the preferred polyhydric alcohol in some embodiments. Another useful feature of the lyophilized formulations of the present invention is the maintenance of the tonicity of the lyophilized formulations described herein with the same formulation component that serves to maintain their stability. In some embodiments, mannitol is the preferred polyhydric alcohol used for this purpose.

The United States Pharmacopeia (USP) states that anti-microbial agents in bacteriostatic or fungistatic concentrations must be added to preparations contained in multiple dose containers. They must be present in adequate concentration at the time of use to prevent the multiplication of microorganisms inadvertently introduced into the preparation while withdrawing a portion of the contents with a hypodermic needle and syringe, or using other invasive means for delivery, such as pen injectors. Antimicrobial agents should be evaluated to ensure compatibility with all other components of the formula, and their activity should be evaluated in the total formula to ensure that a particular agent that is effective in one formulation is not ineffective in another. It is not uncommon to find that a particular antimicrobial agent will be effective in one formulation but not effective in another formulation.

A preservative is, in the common pharmaceutical sense, a substance that prevents or inhibits microbial growth and may be added to pharmaceutical formulations for this purpose to avoid consequent spoilage of the formulation by microorganisms. While the amount of the preservative is not great, it may nevertheless affect the overall stability of the peptide.

While the preservative for use in the pharmaceutical compositions can range from 0.005 to 1.0% (w/v), in some embodiments range for each preservative, alone or in combination with others, is: benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%- 0.03%) parabens. The parabens are lower alkyl esters of para-hydroxybenzoic acid. A detailed description of each preservative is set forth in Remington's Pharmaceutical Sciences by Martin.

With regard to compounds which do have such a tendency when in liquid form, a surfactant should be used in their formulation. These formulations may then be lyophilized. Surfactants frequently cause denaturation of protein, both of hydrophobic disruption and by salt bridge separation. Relatively low concentrations of surfactant may exert a potent denaturing activity, because of the strong interactions between surfactant moieties and the reactive sites on proteins. However, judicious use of this interaction can stabilize proteins against interfacial or surface denaturation. Surfactants which could further stabilize the peptide may optionally be present in the range of about 0.001 to 0.3% (w/v) of the total formulation and include polysorbate 80 (i.e., polyoxyethylene(20) sorbitan monooleate), CHAPS® (i.e., 3-[(3-cholamidopropyl) dimethylammonio] 1-propanesulfonate), Brij® (e.g., Brij 35, which is (polyoxyethylene (23) lauryl ether), poloxamer, or another non-ionic surfactant.

It may also be desirable to add sodium chloride or other salt to adjust the tonicity of the pharmaceutical formulation, depending on the tonicifier selected. However, this is optional and depends on the particular formulation selected. Parenteral formulations preferably may be isotonic or substantially isotonic.

A preferred vehicle for parenteral products is water. Water of suitable quality for parenteral administration can be prepared either by distillation or by reverse osmosis.

Water for injection is the preferred aqueous vehicle for use in the pharmaceutical formulations. It is possible that other ingredients may be present in the pharmaceutical formulations. Such additional ingredients may include, e.g., wetting agents, emulsifiers, oils, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Additionally, polymer solutions, or mixtures with polymers provide the opportunity for controlled release of the peptide. Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

Containers are also an integral part of the formulation of an injection and may be considered a component, for there is no container that is totally inert, or does not in some way affect the liquid it contains, particularly if the liquid is aqueous. Therefore, the selection of a container for a particular injection must be based on a consideration of the composition of the container, as well as of the solution, and the treatment to which it will be subjected. Adsorption of the peptide to the glass surface of the vial can also be minimized, if necessary, by use of borosilicate glass, for example, Wheaton Type I borosilicate glass #33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.). Other vendors of similar borosilicate glass vials and cartridges acceptable for manufacture include Kimbel Glass Co., West Co., Bunder Glas GMBH and Forma Vitrum. The biological and chemical properties of the compound may be stabilized by formulation and lyophilization in a Wheaton Type I- 33 borosilicate serum vial to a final concentration of 0.1 mg/ml and 10 mg/ml of the compound in the presence of 5% mannitol, and 0.02% Tween 80.

For formulations to be delivered by injection, in order to permit introduction of a needle from a hypodermic syringe into a multiple-dose vial and provide for resealing as soon as the needle is withdrawn, the open end of each vial is preferably sealed with a rubber stopper closure held in place by an aluminum band.

Stoppers for glass vials, such as, West 4416/50, 4416/50 (Teflon faced) and

4406/40, Abbott 5139 or any equivalent stopper can be used as the closure for

pharmaceutical for injection. For formulations comprising peptidic anti-obesity agents, these stoppers are compatible with the peptide as well as the other components of the formulation. The inventors have also discovered that these stoppers pass the stopper integrity test when tested using patient use patterns, e.g., the stopper can withstand at least about 100 injections. Alternatively, the peptide can be lyophilized in to vials, syringes or cartridges for subsequent reconstitution. Liquid formulations of the present invention can be filled into one or two chambered cartridges, or one or two chamber syringes.

Each of the components of the pharmaceutical formulation described above is known in the art and is described in Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2nd ed., Avis et al. Ed., Mercel Dekker, New York, N.Y. 1992, which is

incorporated by reference in its entirety herein.

The manufacturing process for the above liquid formulations generally involves compounding, sterile filtration and filling steps. The compounding procedure involves dissolution of ingredients in a specific order (preservative followed by stabilizer/tonicity agents, buffers and peptide) or dissolving at the same time.

Alternative formulations, e.g., non-parenteral, may not require sterilization.

However, if sterilization is desired or necessary, any suitable sterilization process can be used in developing the peptide pharmaceutical formulation of the present invention.

Typical sterilization processes include filtration, steam (moist heat), dry heat, gases (e.g., ethylene oxide, formaldehyde, chlorine dioxide, propylene oxide, beta-propiolacctone, ozone, chloropicrin, peracetic acid methyl bromide and the like), exposure to a radiation source, and aseptic handling. Filtration is the preferred method of sterilization for liquid formulations of the present invention. The sterile filtration involves filtration through 0.45 μηι and 0.22 μηι (1 or 2) which may be connected in series. After filtration, the solution is filled into appropriate vials or containers.

In certain embodiments, the anti-obesity agents are administered peripherally to the subjects. In some embodiments, the liquid pharmaceutical formulations of the present invention are intended for parenteral administration. Suitable routes of administration include intramuscular, intravenous, subcutaneous, intradermal, intraarticular, intrathecal and the like. In some embodiments, the subcutaneous route of administration is preferred. In certain embodiments, mucosal delivery is also preferred. These routes include, but are not limited to, oral, nasal, sublingual, pulmonary and buccal routes which may include administration of the peptide in liquid, semi-solid or solid form. For formulations comprising peptidic anti-obesity agents, administration via these routes requires

substantially more peptide to obtain the desired biological effects due to decreased bioavailability compared to parenteral delivery. In addition, parenteral controlled release delivery can be achieved by forming polymeric microcapsules, matrices, solutions, implants and devices and administering them parenterally or by surgical means. Examples of controlled release formulations are described in U.S. Patent Nos. 6,368,630, 6,379,704, and 5,766,627, which are incorporated herein by reference. These dosage forms may have a lower bioavailability due to entrapment of some of the peptide in the polymer matrix or device. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,842.

The compounds may be provided in dosage unit form containing an amount of the one or more of the anti-obesity agents that will be therapeutically effective in one or multiple doses.

As will be recognized by those in the field, an effective amount of the anti-obesity agents will vary with many factors including the age and weight of the patient, the patient's physical condition, the condition to be treated, and other factors. An effective amount of the anti-obesity agents will also vary with the particular combination administered. As described herein, administration of the agents in combination may allow for a reduced amount of any of the administered agents to be an effective amount.

However, typical doses may contain from a lower limit of about 1 μg, 5 μg, 10 μg, 50 μg to 100 μg to an upper limit of about 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg or 100 mg of the anti-obesity agent per day or per kilogram per day. Also contemplated are other dose ranges such as 0.1 μg to 1 mg of the anti-obesity agent per dose. The doses per day may be delivered in discrete unit doses, provided continuously in a 24 hour period or any portion of that the 24 hours. The number of doses per day may be from 1 to about 4 per day, although it could be more. Continuous delivery can be in the form of continuous infusions. The terms "QID," "TID," "BID" and "QD" refer to administration 4, 3, 2 and 1 times per day, respectively. Exemplary doses and infusion rates include from 0.005 nmol/kg to about 20 nmol/kg per discrete dose or from about 0.01/pmol/kg/min to about 10 pmol/kg/min in a continuous infusion. These doses and infusions can be delivered by intravenous administration (i.v.) or subcutaneous administration (s.c). Exemplary total dose/delivery of the pharmaceutical composition given i.v. may be about 2 μg to about 8 mg per day, whereas total dose/delivery of the pharmaceutical composition given s.c may be about 6 μg to about 6 mg per day.

Anti-obesity agents may be administered, for example, at a daily dosage of from about 0.01 mg/kg to about 20 mg/kg, in some cases, from about 0.01 mg/kg to about 0.3 mg/kg. Administration may be by injection of a single dose or in divided doses.

Sibutramine may be administered, for example, at a daily dosage of from about 0.01 mg/kg to about 10 mg/kg, in some cases from about 0.01 mg/kg to about 1 mg/kg in a single dose or in divided doses 2 to 3 times per day, or in sustained release form. In some instances, sibutramine may be administered the single daily dose of 5 mg, 10 mg, 15 mg, 20 mg or 30 mg orally.

Rimonabant may be administered, for example, at a daily dosage of from about 0.01 mg/kg to about 8 mg/kg, in some instances from about 0.3 mg/kg to about 3 mg/kg of body weight in a single dose or in divided doses 2 to 3 times per day, or in sustained release form.

Administration of anti-obesity agents and derivatives thereof in accordance with the disclosed and claimed invention can be by oral route. Without wishing to be bound by any theory, it is believed that the anti-obesity agents and derivatives thereof described herein may be orally available. It has been reported that tight junctions between intestinal cells opened by absorption enhancers are less than 20 nm wide. See e.g., Chao et al., 1998, J.

Drug Targeting, 6:37-43. Accordingly, it is believed that a sufficiently small (for example, less than 10 kD, 15 kD, 20 kD, 30 kD, 40 kD) anti-obesity agent or derivative thereof may transit the gut in the portal system, thereby gaining access to the circulation. Oral delivery of the anti-obesity agents and derivatives thereof of the present invention may afford administration frequencies that are once daily, once other day, once every three days, once weekly, once in two weeks, one in three weeks, or even one a month. Oral delivery systems suitable for peptides, such as for calcitonin, insulin and growth hormone, can be used. In one embodiment the oral delivery system may have a relatively rapid uptake profile, e.g. from 1 to 4 hours. In another embodiment, the oral delivery system provides a longer uptake profile, e.g. from 4 to 8 hours or more.

The dosage and frequency (single or multiple doses) of compound administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., the disease responsive to compounds described herein); presence of other diseases or other health-related problems; kind of concurrent treatment; and complications from any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.

Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring one or more physiological parameters, including but not limited to blood sugar and body mass, and adjusting the dosage upwards or downwards, as described above and known in the art..

Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. In one embodiment of the invention, the dosage range is 0.001% to 10% w/v. In another embodiment, the dosage range is 0.1% to 5% w/v.

Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.

Toxicity

The ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between LD5₀ (the amount of compound lethal in 50% of the population) and ED5₀ (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al, In: THE PHARMACOLOGICAL BASIS OF

THERAPEUTICS, Ch. 1, p.l, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition and the particular method in which the compound is used.

Further embodiments include the following:

Embodiment 1. A method of treating obesity in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

said first anti-obesity agent comprises an amino acid amino acid sequence set out in SEQ ID NO: 1, or a derivative thereof; and

said second anti-obesity agent comprises an amino acid sequence set out in SEQ ID NO:2, or a derivative thereof.

Embodiment 2. A method of reducing body weight in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

said first anti-obesity agent comprises a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 1, or a derivative thereof; and

said second anti-obesity agent comprises a polypeptide comprising the amino acid sequence set out in SEQ ID NO:2, or a derivative thereof.

Embodiment 3. A method of reducing food intake in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

Embodiment 4. A method of reducing nutrient availability in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein: said first anti-obesity agent comprises an amino acid amino acid sequence set out in SEQ ID NO: 1, or a derivative thereof; and

Embodiment 5. The method according to any one of embodiments 1 through 4, wherein the subject experiences a reduction in body weight by at least 10%.

Embodiment 6. The method according to any one of embodiments 1 through 5, wherein the subject reduces food intake by least 10%.

Embodiment 7. The method according to any one of embodiments 1 through 6, wherein the subject reduces food intake by least 20%. Embodiment 8. The method of any one of embodiments 1 through 7, wherein said therapeutically effective amount comprises a sub-optimal dose of said first anti-obesity agent or a sub-optimal dose of said second anti-obesity agent.

Embodiment 9. The method of any one of embodiments 1 through 8, wherein said therapeutically effective amount comprises a sub-optimal dose of said first anti-obesity agent and a sub-optimal dose of said second anti-obesity agent.

Embodiment 10. The method according to any one of embodiments 1 through 9, wherein the effective amount of said first anti-obesity agent and the effective amount of said second anti-obesity agent each comprise an amount such that a greater amount of weight loss is achieved when both said agents are administered in combination than the amount of weight loss that is achieved when either said agent is administered alone.

Embodiment 11. The method according to any one of embodiments 1 through 10, wherein the effective amount of said first anti-obesity agent and the effective amount of said second anti-obesity agent each comprise an amount such that a greater reduction in food intake is achieved when both said agents are administered in combination than the reduction in food intake that is achieved when either said agent is administered alone. Embodiment 12. The method according to any one of embodiments 1 through 1 1, wherein said first anti-obesity agent and said second anti-obesity agent are administered at the same time. Embodiment 13. The method according to any one of embodiments 1 through 1 1, wherein said first anti-obesity agent and said second anti-obesity agent are administered at different times.

Embodiment 14. The method according to any one of embodiments 1 through 12, wherein said first anti-obesity agent and said second anti-obesity agent are mixed together prior to administration.

Embodiment 15. The method according to any one of embodiments 1 through 14 wherein said first anti-obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about one milligram per kilogram per day (1 mg/kg/day).

Embodiment 16. The method according to any one of embodiments 1 through 15 wherein said first anti-obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about five hundred micrograms per kilogram per day (500 μg/kg/day).

Embodiment 17. The method according to any one of embodiments 1 through 16 wherein said first anti-obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about two hundred fifty micrograms per kilogram per day (250 μg/kg/day).

Embodiment 18. The method according to any one of embodiments 1 through 17 wherein said first anti-obesity agent is administered at from about 0.1 microgram per kilogram per day (1 μg/kg/day) to about twenty mircograms per kilogram per day (10 μg/kg/day).

Embodiment 19. The method according to any one of embodiments 1 through 18 wherein said first anti-obesity agent is administered at about 1.25 micrograms per kilogram per day (1.25 μ£ξ/1^^^), about 2.5 micrograms per kilogram per day (2.5 μg/kg/day), or about 5 micrograms per kilogram per day (5 μg/kg/day).

Embodiment 20. The method according to any one of embodiments 1 through 19 wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about five milligrams per kilogram per day (5 mg/kg/day).

Embodiment 21. The method according to any one of embodiments 1 through 20, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about one milligram per kilogram per day (1 mg/kg/day).

Embodiment 22. The method according to any one of embodiments 1 through 21, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10μg/kg/day), to about 500 micrograms per kilogram per day (500 μg/kg/day).

Embodiment 23. The method according to any one of embodiments 1 through 22, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about 250 micrograms per kilogram per day (250 μg/kg/day).

Embodiment 24. The method according to any one of embodiments 1 through 23, wherein said second anti-obesity agent is administered at from about 45 micrograms per kilogram per day (45 μg/kg/day), to about 200 micrograms per kilogram per day (200 μg/kg/day).

Embodiment 25. The method according to any one of embodiments 1 through 24, wherein said second anti-obesity agent is administered at about 50 micrograms per kilogram per day (50 μg/kg/day) or about 200 micrograms per kilogram per day (200 μg/kg/day).

Embodiment 26. The method according to any one of embodiments 1 through 25, wherein: said first anti-obesity agent is administered at about 1.25 micrograms per kilogram per day (1.25 μg/kg/day), about 2.5 micrograms per kilogram per day (2.5 μg/kg/day), or about 5 micrograms per kilogram per day (5 μg/kg/day); and

said second anti-obesity agent is administered at about 50 micrograms per kilogram per day (50 μg/kg/day) or about 200 micrograms per kilogram per day (200 μg/kg/day).

Embodiment 27. The method according to any one of embodiments 1 through 26 wherein said first anti-obesity agent comprises a derivative of said amino acid sequence set out in SEQ ID NO: 1, wherein said derivative comprises a polymer moiety attached to said amino acid sequence.

Embodiment 28. The method according to embodiment 27 wherein said polymer moiety comprises a fatty acyl moiety or a polyethylene glycol moiety.

Embodiment 29. The method according to any one of embodiments 1 through 26 wherein said second anti-obesity agent comprises a derivative of said amino acid sequence set out in SEQ ID NO: 2, wherein said derivative comprises a polymer moiety attached to said amino acid sequence.

Embodiment 30. The method according to embodiment 29 wherein said polymer moiety comprises a fatty acyl moiety or a polyethylene glycol moiety.

Embodiment 31. The method according to any one of embodiments 1 through 28 wherein said administration is performed by providing three daily doses of each of said first anti-obesity agent and said second anti-obesity agent.

Embodiment 32. The method according to any one of embodiments 1 through 28 wherein said administration is performed by providing one daily dose of each of said first anti-obesity agent and said second anti-obesity agent.

Embodiment 33. The method according to any one of embodiments 1 through 28 wherein said administration is performed by providing one weekly dose of each of said first anti-obesity agent and said second anti-obesity agent. Embodiment 34. The method according to any one of embodiments 1 through 28 wherein said administration is performed by providing one bi-monthly dose of each of said first anti-obesity agent and said second anti-obesity agent. Embodiment 35. The method of any one of embodiments 1 through 34 wherein at least one of said anti-obesity agents comprises a liquid formulation.

Embodiment 36. The method of any one of embodiments 1 through 34 wherein at least one of said anti-obesity agents comprises a dry formulation.

Embodiment 37. The method of any one of embodiments 1 through 36 wherein said anti-obesity agents comprise separate formulations but are packaged together.

Embodiment 38. The method according to any one of embodiments 1 to 37 further comprising at least one further anti-obesity agent selected from the group consisting of: a NPY1 receptor antagonist, an NPY5 receptor antagonist, an NPY2 receptor agonist, an NPY4 receptor agonist, a CNTF, a CNTF agonist/modulator, a CNTF derivative, a MCHIR antagonist, a MCH2R antagonist, a melanocortin 4 agonist, a MC4 receptor agonist, a cannabinoid receptor (CB-1) antagonist/inverse agonist, a ghrelin antagonist, a 5HT2c agonist, a serotonin reuptake inhibitor, a serotonin transport inhibitor, an exendin, an exendin derivative, an exendin agonist, a GLP-1, a GLP-1 analog, a GLP-1 agonist, a DPP- IV inhibitor, an opioid antagonist, an orexin antagonist, a metabotropic glutamate subtype 5 receptor antagonist, a histamine 3 antagonist/inverse agonist, topiramate, a CCK, a CCK analog, a CCK agonist and a PYY(3-36), a PYY(3-36) analog, and a PYY(3-36) agonist; and derivatives thereof.

Embodiment 39. The method according to embodiment 38, wherein the further at least one anti-obesity agent is phentermine, rimonabant, sibutramine or topiramate. Embodiment 40. The method according to any one of embodiments 1 to 39 wherein body fat mass, percent body fat, or adiposity of the subject is reduced.

Embodiment 41. The method according to any one of embodiments 1 to 40 wherein lean mass or percent lean mass of the subject is preserved or increased. Embodiment 42. The method according to any one of embodiments 1 to 41, wherein the subject has at least one condition selected from the group consisting of obesity, overweight a co-morbidity of obesity, a co-morbidity of overweight, an obesity -related condition, diabetes mellitus, gestational diabetes, type I diabetes, type II diabetes, insulin- resistance syndrome, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, cardiovascular disease, polycystic ovary syndrome, and metabolic syndrome.

Embodiment 43. The method according to any one of embodiments 1 to 42 wherein the BMI is greater than 25.

Embodiment 44. The method according to any one of embodiments 1 to 42 wherein the BMI is 25 to 45. Embodiment 45. The method according to any one of embodiments 1 to 42, wherein the BMI is 25 to 40.

Embodiment 46. The method according to any one of embodiments 1 to 42, wherein the BMI is 25 to 35.

Embodiment 47. The method according to any one of embodiments 1 to 42, wherein the BMI is 25 to

30. Embodiment 48. The method according to any one of embodiments 1 to 42, wherein the BMI is 30 to

45.

Embodiment 49. The method according to any one of embodiments 1 to 42, wherein the BMI is 30 to

40.

Embodiment 50. The method according to any one of embodiments 1 to 42, wherein the BMI is reduced to less than 30. Embodiment 51. The method according to any one of embodiments 1 to 42, wherein the BMI is reduced to less than 25. Embodiment 52. The method according to any one of embodiments 1 to 42, wherein the BMI is reduced to normal.

Embodiment 53. The method according to any one of embodiments 1 to 52, wherein weight loss is achieved within 4 weeks of treatment.

Embodiment 54. The method according to any one of embodiments 1 to 52 wherein weight loss is achieved within 8 weeks of treatment.

Embodiment 55. The method according to any one of embodiments 1 to 52, wherein weight loss is achieved within 12 weeks of treatment.

Embodiment 56. The method according to any one of embodiments 1 to 52, wherein weight loss is achieved within 20 weeks of treatment. Embodiment 57. The method according to any one of embodiments 1 to 52, wherein weight loss is achieved within 24 weeks of treatment.

Embodiment 58. The method according to any one of embodiments 1 to 57, wherein the subject is human.

Embodiment 59. The method according to any one of embodiments 1 to 58, wherein the subject is an obese human.

Embodiment 60. The method according to any one of embodiments 1 to 58, wherein the subject is a human adult female.

Embodiment 61. The method according to any one of embodiments 1 to 60, wherein the subject is an obese type II diabetic. Embodiment 62. The method according to any one of embodiments 1 to 59, wherein the weight loss is reduced by at least 12%.

Embodiment 63. The method according to any one of embodiments 1 to 62, wherein the weight loss is reduced by at least 15%.

Embodiment 64. The method according to any one of embodiments 1 to 62, wherein the weight loss is reduced by at least 10% within 8 weeks of treatment. Embodiment 65. The method according to any one of embodiments 1 to 62, wherein the weight loss is reduced by at least 10% within 12 weeks of treatment.

Embodiment 66. The method according to any one of embodiments 1 to 62, wherein the weight loss is reduced by at least 10% within 20 weeks of treatment.

Embodiment 67. The method according to any one of embodiments 1 to 62, wherein the weight loss is reduced by at least 15% within 40 weeks of treatment.

Embodiment 68. The method according to any one of embodiments 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered within 15 minutes prior to a meal.

Embodiment 69. The method according to any one of embodiments 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered within one hour prior to a meal.

Embodiment 70. The method according to any one of embodiments 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered within two hours prior to a meal.

Embodiment 71. The method according to any one of embodiments 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered prior to breakfast. Embodiment 72. The method according to any one of embodiments 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered prior to dinner.

Embodiment 73. The method according to any one of embodiments 1 to 72, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of 500 to 2000 pg/ml of said at least one anti-obesity agent.

Embodiment 74. The method according to any one of embodiments 1 to 73, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of 750 to 1500 pg/ml of said at least one anti- obesity agent.

Embodiment 75. The method according to any one of embodiments 1 to 74, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of about 1500 pg/ml of said at least one anti-obesity agent.

Embodiment 76. The method according to any one of embodiments 1 to 75, wherein said therapeutically effective amount of at least one of said first anti-obesity agent and said second anti-obesity comprises a dose-sparing amount.

Embodiment 77. The method according to any one of embodiments 1 to 76, wherein said therapeutically effective amount of said first anti-obesity agent and said second anti-obesity each comprises a dose-sparing amount.

Embodiment 78. The method according to any one of embodiments 1 to 77, wherein said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake. Embodiment 79. The method according to any one of embodiments 1 to 78, wherein said therapeutically effective amount of said second anti-obesity agent comprises an amount that is approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

Embodiment 80. The method according to any one of embodiments 1 to 79, wherein:

said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake; and

said therapeutically effective amount of said second anti-obesity agent comprises an amount that is approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

Embodiment 81. A pharmaceutical composition for use in the method of any of the embodiments 1 to 80, wherein the composition comprises an effective amount of a first anti- obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: 1; and a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID

NO:2.

Embodiment 82. A pharmaceutical composition for treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying, in a subject in need thereof, wherein said composition comprises an effective amount of a first anti-obesity agent and a second anti-obesity agent according to any one of

embodiments 1 through 81.

Embodiment 83. A pharmaceutical composition for the treatment of obesity, the treatment of overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying, in a subject in need thereof, wherein the composition comprises an effective amount of a first anti-obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: 1; and

a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID

NO:2 ;

and wherein the effective amount comprises an amount such that a greater amount of weight loss is achieved when the agents are administered in combination to said subject than the amount of weight loss achieved when either agent is administered alone.

Embodiment 84. The pharmaceutical composition of embodiment 83, wherein said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

Embodiment 85. The pharmaceutical composition of any one of embodiments 82 through 84, wherein said therapeutically effective amount of said second anti-obesity agent comprises an amount that is approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake. Embodiment 86. The pharmaceutical composition of any one of embodiments 82 through 85, wherein:

Embodiment 87. The pharmaceutical composition of any one of embodiments 82 through 86, wherein said pharmaceutical composition is contained in a device comprised of separate chambered cartridges. Embodiment 88. The pharmaceutical composition of any one of embodiments 82 through 86 wherein said pharmaceutical composition is contained in a device comprised of one chambered cartridge. Embodiment 89. The pharmaceutical composition of any one of embodiments 81 through 88, wherein said device further comprises a syringe.

Embodiment 90. The use of a composition comprising a first anti-obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: l; and

a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID

NO:2;

in the manufacture of a medicament for the treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying according to any one of embodiments 1 to 89.

The following Examples are provided to illustrate, but not limit, the invention.

EXAMPLES

EXAMPLE 1

Studies employing amylin and PYY(3-36):

Rat amylin (SEQ ID NO: 15) and human PYY(3-36) (SEQ ID NO:4), alone and in combination, were provided to HSD or DIO prone rats as described in Roth et al.

Endocrinology, Vol. 148(12), pp. 6054-6061 (2007).

Briefly, animals were housed individually in standard caging at 22 C in a 12-h light (lights on at 0500 h), 12-h dark (lights off at 1700 h) cycle. HSD rats were obtained from Harlan (Indianapolis, IN) and maintained on standard chow (7012; Harlan Teklad, Madison, WI). For 24-h feeding studies, HSD rats were fed a moderately high-fat diet (32% kcal from fat, D12266B; Research Diets, New Brunswick, NJ) for 10 d before study initiation. DIO-prone rats were adult male Levin rats obtained from Charles River Laboratories (Wilmington, MA). These rats were originally developed from a line of Crl:CD(SD)BR rats that are prone to becoming obese on a diet relatively high in fat and energy. DIO-prone rats were fattened ad libitum on a moderately high-fat diet (32% kcal from fat, D12266B; Research Diets) for 6 wk before and throughout drug treatment. DIO mice were adult male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) fattened on a high-fat diet (58% kcal from fat, D 12331; Research Diets) for 4 wk before and during drug treatment. All studies used rat amylin and humanPYY[3-36] obtained from Peptisynthia (Torrance, CA).

Twenty-four-hour food intake monitoring

HSD rats were habituated to the testing chamber, food hopper, and diet for 10 d before the study. Testing chambers (10.5 x 19 x 8 in., BioDAQ Food Intake Monitor; Research Diets) were equipped with a tunnel containing a food hopper at the end. Rats received daily ip injections of vehicle (10% dimethylsulfoxide in sterile water) before lights off on d 8, 9, and 10 of habituation. After habituation, rats (initial body weight was -441 g at time 0) were divided into treatment groups (seven to eight rats per group) of equal food consumption. On test day, rats received a single ip injection of vehicle or peptide (10 μg/kg amylin and/or 1000 μg/kg PYY(3-36)) 15 min before lights off and were placed immediately back into the BioDAQ Food Intake Monitor cages. These doses were chosen because previous data showed near-maximal anorexigenic effects in this model (data not shown). Food hoppers were weighed automatically every 5 sec throughout the experiment. Cumulative food intake (in grams) was monitored for 24 h.

Figure 1 shows the effects of amylin and PYY(3-36) on acute food intake and body weight in HSD and DIO-prone rats (left and right panels, respectively), alone and in combination. The results indicate that the combined administration of amylin and PYY(3- 36) (1 mg/kg) and amylin (0.01 mg/kg), respectively at results in non-synergistic, and at best, additive, reduction in food intake; similarly, reduction in body weight with subcutaneous (s.c.) infusion of amylin (100 μg/kg/d) +/- PYY(3-36) (200 μg/kg/d) results in non-synergistic, and at best, additive, reduction in body weight.

Figure 2

EXAMPLE 2

Studies employing Davalintide (SEQ ID NO:l) and SEQ ID NO:2:

Diet-induced obesity (DIO) in the in the Sprague-Dawley rat is a valuable model for the study of obesity and regulation of energy homeostasis. These rats were developed from a line of (Crl:CD®(SD)BR) rats that are prone to become obese on a diet relatively high in fat and energy. See, for example, Levin (1994) Am. J. Physiol. 267:R527-R535, Levin et al. (1997) Am. J. Physiol. 273:R725-R730. DIO male rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA). In-bred DIO (Levin) rats from Charles River Labs were housed individually in shoebox cages at 22°C on a 12/12 hour light dark cycle and maintained ad-libitum on a moderately high fat diet (32% kcal from fat; Research Diets D12266B) for approximately 6 weeks prior to and during drug treatment. After fattening period they had a mean body weight of 475.2g and a mean percent fat mass of 13.7%. Davalintide and SEQ ID NO:2 were each s.c. infused by osmotic mini-pump (Durect Corp, CA). Rats were implanted with one pump containing either Davalintide or vehicle and a second pump containing either SEQ ID NO:2 or vehicle. Davalintide and SEQ ID NO:2 were dissolved in 50% DMSO in sterile water.

Rats were implanted with one pump containing either Davalintide or vehicle and a second pump containing either SEQ ID NO:2 or vehicle. Rats were randomized to one of the 12 dose groups (n=5/group) resulting from full factorial design using 4 doses of

Davalintide (infused at a rate of 0, 1.25, 2.5 and 5 μg/kg/d) and 3 doses of SEQ ID NO:2 (infused at a rate of 0, 50 and 200 μg/kg/d). Body weight and food intake were recorded weekly for 4 weeks. Body composition (percent adiposity) was measured prior to and after drug treatment using NMR (Echo Medical Systems, Houston, TX).

Statistical Methods (Body Weight): Response surface methodology (RSM) was used for experimental design and analysis. A partial second-order linear model was used to model the observed % vehicle-corrected body weight (at day 28). This model contains effects for the intercept, linear effect of Davalintide, linear and quadratic effects of SEQ ID NO:2, and linear interaction effect for the Davalintide -SEQ ID NO:2 combination. A logarithmic transformation was taken on the dose of Davalintide to improve the model fit. The quadratic effect for Davalintide was not necessary and was dropped from this model. Using this model, a response surface that predicted vehicle-corrected weight loss over the entire dose region was created.

Statistical Methods (Food Intake): A full second-order linear model was used to model the observed cumulative (at day 28) food intake calculated as a percent of the vehicle group. This model contains effects for the intercept, linear and quadratic effects of Davalintide and SEQ ID NO:2, and a linear interaction effect for the Davalintide -SEQ ID NO:2 combination. A logarithmic transformation was applied to the dose of Davalintide to improve the model fit. Using this model, a response surface that predicted the change in percent vehicle cumulative food intake over the entire dose region was created.

The results reflected in Figures 3 and 4 reflect that for percent vehicle-corrected body weight at four weeks, the statistical model indicated a significant dose-response body weight reduction effect for Davalintide (p<0.0001), a marginally significant dose-response body weight effect for SEQ ID NO:2 (p=0.056 for linear term and p=0.09 for quadratic term), and a statistically significant synergy )p=0.03). The synergy between the two peptides indicated that the slope of the response surface changed as doses were increased; in this case the slope became steeper (i.e., weight loss increase) as the doses of Davalintide and SEQ ID NO:2 increased, suggesting that Davalintide in combination with SEQ ID NO:2 produced significantly greater weight loss than that expected by the sum of the two peptides. The predicted response surface and the interaction are demonstrated by combination doses that have greater weight loss than that predicted by the sum of the respective monotherapy arms (see, e.g., Figure 4). The partial second-order model fit to these data had a lack-of-fit p-value of 0.56, indicating that there was no evidence that the model needed additional terms to describe the response surface. The R² value for the model had a value of 0.78, indicating that most of the variability was explained by the model.

The results reflected in Figures 5 and 6 reflect that for percent vehicle cumulative food intake at four weeks, the statistical model indicated a significant dose-response effect for Davalintide (both linear and quadratic effects had p<0.001), a marginally significant dose-response effect for SEQ ID NO:2 (p=0.028 for linear term and p=0.09 for quadratic term), and a non-significant interaction (p=0.16). The slope of the response surface did not significantly change as the doses of the peptides were increased, suggesting that Davalintide in combination with SEQ ID NO:2 produced and additive or better (e.g., modestly supra- additive) reduction in food intake relative to that calculated to be the sum of the two peptides. The predicted response surface (see, e.g., Figure 6) shows that the cumulative food intake of the combinations was approximately equal to the sum of each monotherapy. The full second-order model fit to these data had a lack-of-fit p-value of 0.71, indicating that there was no evidence that the model needed additional terms to describe the response surface. The R² value for the model had a value of 0.73, indicating that most of the variability was explained by the model.

Figure 7 depicts the commutative food intake (left panel), cumulative body weight (center panel), and cumulative adiposity (i.e., percent fat; right panel) observed at the highest dose combination (i.e., 200 μg kg/day for SEQ ID NO:2 and 5 μg/kg/day for Davalintide). The results indicate that this combination dosage regiment elicits supra- additive decease in cumulative food intake, cumulative body weight and commutative adiposity (percent fat), as determined by comparison of measurements taken at the conclusion of the four-week study and measurements taken at baseline (i.e.), just prior to administration of compounds). Figure 8 depicts the change in body weight, reflected as percent of baseline body weight observed at the end of each week of the four- week study. The results indicate supra- additive body weight loss, for example, after the first week of treatment with the combination treatment of Davalintide and SEQ ID NO:2.

Figure 9 depicts a summary of statistical results derived from the results of the studies described in Examples 1 and 2 and depicted in Figure 1 through 8.

While the foregoing description discloses the present invention, with examples provided for the purpose of illustration, 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 and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of treating obesity in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

2. A method of reducing body weight in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

3. A method of reducing food intake in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

4. A method of reducing nutrient availability in a subject comprising peripherally administering a therapeutically effective amount of a first anti-obesity agent and a therapeutically effective amount of a second anti-obesity agent, wherein:

5. The method according to any one of claims 1 through 4, wherein the subject experiences a reduction in body weight by at least 10%.

6. The method according to any one of claims 1 through 5, wherein the subject reduces food intake by least 10%.

7. The method according to any one of claims 1 through 6, wherein the subject reduces food intake by least 20%.

8. The method of any one of claims 1 through 7, wherein said therapeutically effective amount comprises a sub-optimal dose of said first anti-obesity agent or a sub- optimal dose of said second anti-obesity agent.

9. The method of any one of claims 1 through 8, wherein said therapeutically effective amount comprises a sub-optimal dose of said first anti-obesity agent and a sub- optimal dose of said second anti-obesity agent.

10. The method according to any one of claims 1 through 9, wherein the effective amount of said first anti-obesity agent and the effective amount of said second anti-obesity agent each comprise an amount such that a greater amount of weight loss is achieved when both said agents are administered in combination than the amount of weight loss that is achieved when either said agent is administered alone.

1 1. The method according to any one of claims 1 through 10, wherein the effective amount of said first anti-obesity agent and the effective amount of said second anti-obesity agent each comprise an amount such that a greater reduction in food intake is achieved when both said agents are administered in combination than the reduction in food intake that is achieved when either said agent is administered alone.

12. The method according to any one of claims 1 through 1 1, wherein said first anti- obesity agent and said second anti-obesity agent are administered at the same time.

13. The method according to any one of claims 1 through 11, wherein said first anti- obesity agent and said second anti-obesity agent are administered at different times.

14. The method according to any one of claims 1 through 12, wherein said first anti- obesity agent and said second anti-obesity agent are mixed together prior to administration.

15. The method according to any one of claims 1 through 14 wherein said first anti- obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about one milligram per kilogram per day (1 mg/kg/day).

16. The method according to any one of claims 1 through 15 wherein said first anti- obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about five hundred micrograms per kilogram per day (500 μg/kg/day).

17. The method according to any one of claims 1 through 16 wherein said first anti- obesity agent is administered at from about 0.1 microgram per kilogram per day (0.1 μg/kg/day) to about two hundred fifty micrograms per kilogram per day (250 μg/kg/day).

18. The method according to any one of claims 1 through 17 wherein said first anti- obesity agent is administered at from about 0.1 microgram per kilogram per day (1 μg/kg/day) to about twenty mircograms per kilogram per day (10 μg/kg/day).

19. The method according to any one of claims 1 through 18 wherein said first anti- obesity agent is administered at about 1.25 micrograms per kilogram per day (1.25 μg/kg/day), about 2.5 micrograms per kilogram per day (2.5 μg/kg/day), or about 5 micrograms per kilogram per day (5 μg/kg/day).

20. The method according to any one of claims 1 through 19 wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about five milligrams per kilogram per day (5 mg/kg/day).

21. The method according to any one of claims 1 through 20, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about one milligram per kilogram per day (1 mg/kg/day).

22. The method according to any one of claims 1 through 21, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10μg/kg/day), to about 500 micrograms per kilogram per day (500 μg/kg/day).

23. The method according to any one of claims 1 through 22, wherein said second anti-obesity agent is administered at from about 10 micrograms per kilogram per day (10 μg/kg/day), to about 250 micrograms per kilogram per day (250 μg/kg/day).

24. The method according to any one of claims 1 through 23, wherein said second anti-obesity agent is administered at from about 45 micrograms per kilogram per day (45 μg/kg/day), to about 200 micrograms per kilogram per day (200 μg/kg/day).

25. The method according to any one of claims 1 through 24, wherein said second anti-obesity agent is administered at about 50 micrograms per kilogram per day (50 μg/kg/day) or about 200 micrograms per kilogram per day (200 μg/kg/day).

26. The method according to any one of claims 1 through 25, wherein:

said first anti-obesity agent is administered at about 1.25 micrograms per kilogram per day (1.25 μg/kg/day), about 2.5 micrograms per kilogram per day (2.5 μg/kg/day), or about 5 micrograms per kilogram per day (5 μg/kg/day); and

27. The method according to any one of claims 1 through 26 wherein said first anti- obesity agent comprises a derivative of said amino acid sequence set out in SEQ ID NO: 1, wherein said derivative comprises a polymer moiety attached to said amino acid sequence.

28. The method according to claim 27 wherein said polymer moiety comprises a fatty acyl moiety or a polyethylene glycol moiety.

29. The method according to any one of claims 1 through 26 wherein said second anti-obesity agent comprises a derivative of said amino acid sequence set out in SEQ ID NO: 2, wherein said derivative comprises a polymer moiety attached to said amino acid sequence.

30. The method according to claim 29 wherein said polymer moiety comprises a fatty acyl moiety or a polyethylene glycol moiety.

31. The method according to any one of claims 1 through 28 wherein said administration is performed by providing three daily doses of each of said first anti-obesity agent and said second anti-obesity agent.

32. The method according to any one of claims 1 through 28 wherein said administration is performed by providing one daily dose of each of said first anti-obesity agent and said second anti-obesity agent.

33. The method according to any one of claims 1 through 28 wherein said administration is performed by providing one weekly dose of each of said first anti-obesity agent and said second anti-obesity agent.

34. The method according to any one of claims 1 through 28 wherein said administration is performed by providing one bi-monthly dose of each of said first anti- obesity agent and said second anti-obesity agent.

35. The method of any one of claims 1 through 34 wherein at least one of said anti- obesity agents comprises a liquid formulation.

36. The method of any one of claims 1 through 34 wherein at least one of said anti- obesity agents comprises a dry formulation.

37. The method of any one of claims 1 through 36 wherein said anti-obesity agents comprise separate formulations but are packaged together.

38. The method according to any one of claims 1 to 37 further comprising at least one further anti-obesity agent selected from the group consisting of: a PYl receptor antagonist, an NPY5 receptor antagonist, an NPY2 receptor agonist, an NPY4 receptor agonist, a CNTF, a CNTF agonist/modulator, a CNTF derivative, a MCHIR antagonist, a MCH2R antagonist, a melanocortin 4 agonist, a MC4 receptor agonist, a cannabinoid receptor (CB-1) antagonist/inverse agonist, a ghrelin antagonist, a 5HT2c agonist, a serotonin reuptake inhibitor, a serotonin transport inhibitor, an exendin, an exendin derivative, an exendin agonist, a GLP-1, a GLP-1 analog, a GLP-1 agonist, a DPP-IV inhibitor, an opioid antagonist, an orexin antagonist, a metabotropic glutamate subtype 5 receptor antagonist, a histamine 3 antagonist/inverse agonist, topiramate, a CCK, a CCK analog, a CCK agonist and a PYY(3-36), a PYY(3-36) analog, and a PYY(3-36) agonist; and derivatives thereof.

39. The method according to claim 38, wherein the further at least one anti-obesity agent is phentermine, rimonabant, sibutramine or topiramate.

40. The method according to any one of claims 1 to 39 wherein body fat mass, percent body fat, or adiposity of the subject is reduced.

41. The method according to any one of claims 1 to 40 wherein lean mass or percent lean mass of the subject is preserved or increased.

42. The method according to any one of claims 1 to 41, wherein the subject has at least one condition selected from the group consisting of obesity, overweight a co-morbidity of obesity, a co-morbidity of overweight, an obesity-related condition, diabetes mellitus, gestational diabetes, type I diabetes, type II diabetes, insulin-resistance syndrome, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, cardiovascular disease, polycystic ovary syndrome, and metabolic syndrome.

43. The method according to any one of claims 1 to 42 wherein the BMI is greater than 25.

44. The method according to any one of claims 1 to 42 wherein the BMI is 25 to

45.

The method according to any one of claims 1 to 42, wherein the BMI

46. The method according to any one of claims 1 to 42, wherein the BMI is 25 to

35.

47. The method according to any one of claims 1 to 42, wherein the BMI is 25 to 30.

48. The method according to any one of claims 1 to 42, wherein the BMI is 30 to 45.

49. The method according to any one of claims 1 to 42, wherein the BMI is 30 to

40.

50. The method according to any one of claims 1 to 42, wherein the BMI is reduced to less than 30.

51. The method according to any one of claims 1 to 42, wherein the BMI is reduced to less than 25.

52. The method according to any one of claims 1 to 42, wherein the BMI is reduced to normal.

53. The method according to any one of claims 1 to 52, wherein weight loss is achieved within 4 weeks of treatment.

54. The method according to any one of claims 1 to 52 wherein weight loss is achieved within 8 weeks of treatment.

55. The method according to any one of claims 1 to 52, wherein weight loss is achieved within 12 weeks of treatment.

56. The method according to any one of claims 1 to 52, wherein weight loss is achieved within 20 weeks of treatment.

57. The method according to any one of claims 1 to 52, wherein weight loss is achieved within 24 weeks of treatment.

58. The method according to any one of claims 1 to 57, wherein the subject is human.

59. The method according to any one of claims 1 to 58, wherein the subject is an obese human.

60. The method according to any one of claims 1 to 58, wherein the subject is a human adult female.

61. The method according to any one of claims 1 to 60, wherein the subject is an obese type II diabetic.

62. The method according to any one of claims 1 to 59, wherein the weight loss is reduced by at least 12%.

63. The method according to any one of claims 1 to 62, wherein the weight loss is reduced by at least 15%.

64. The method according to any one of claims 1 to 62, wherein the weight loss is reduced by at least 10% within 8 weeks of treatment.

65. The method according to any one of claims 1 to 62, wherein the weight loss is reduced by at least 10% within 12 weeks of treatment.

66. The method according to any one of claims 1 to 62, wherein the weight loss is reduced by at least 10% within 20 weeks of treatment.

67. The method according to any one of claims 1 to 62, wherein the weight loss is reduced by at least 15% within 40 weeks of treatment.

68. The method according to any one of claims 1 to 67, wherein the first anti- obesity agent and the second anti-obesity agent are administered within 15 minutes prior to a meal.

69. The method according to any one of claims 1 to 67, wherein the first anti- obesity agent and the second anti-obesity agent are administered within one hour prior to a meal.

70. The method according to any one of claims 1 to 67, wherein the first anti- obesity agent and the second anti-obesity agent are administered within two hours prior to a meal.

71. The method according to any one of claims 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered prior to breakfast.

72. The method according to any one of claims 1 to 67, wherein the first anti-obesity agent and the second anti-obesity agent are administered prior to dinner.

73. The method according to any one of claims 1 to 72, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of 500 to 2000 pg/ml of said at least one anti-obesity agent.

74. The method according to any one of claims 1 to 73, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of 750 to 1500 pg/ml of said at least one anti-obesity agent.

75. The method according to any one of claims 1 to 74, wherein the effective amount of at least one of the first anti-obesity agent and the second anti-obesity agent achieves a blood plasma concentration of about 1500 pg/ml of said at least one anti-obesity agent.

76. The method according to any one of claims 1 to 75, wherein said therapeutically effective amount of at least one of said first anti-obesity agent and said second anti-obesity comprises a dose-sparing amount.

77. The method according to any one of claims 1 to 76, wherein said therapeutically effective amount of said first anti-obesity agent and said second anti-obesity each comprises a dose-sparing amount.

78. The method according to any one of claims 1 to 77, wherein said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

79. The method according to any one of claims 1 to 78, wherein said therapeutically effective amount of said second anti-obesity agent comprises an amount that is

approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

80. The method according to any one of claims 1 to 79, wherein:

81. A pharmaceutical composition for use in the method of any of the claims 1 to 80, wherein the composition comprises an effective amount of a first anti-obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: l; and

a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID

NO:2.

82. A pharmaceutical composition for treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying, in a subject in need thereof, wherein said composition comprises an effective amount of a first anti-obesity agent and a second anti-obesity agent according to any one of claims 1 through 81.

83. A pharmaceutical composition for the treatment of obesity, the treatment of overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying, in a subject in need thereof, wherein the composition comprises an effective amount of a first anti-obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: l; and

a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID

NO:2 ;

84. The pharmaceutical composition of claim 83, wherein said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

85. The pharmaceutical composition of any one of claims 82 through 84, wherein said therapeutically effective amount of said second anti-obesity agent comprises an amount that is approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

86. The pharmaceutical composition of any one of claims 82 through 85, wherein: said therapeutically effective amount of said first anti-obesity agent comprises an amount that is approximately one-twentieth the amount of amylin that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake; and said therapeutically effective amount of said second anti-obesity agent comprises an amount that is approximately one-half the amount of PYY(3-36) that is required to be administered to a subject in order to achieve substantially the same weight loss or reduction in food intake.

87. The pharmaceutical composition of any one of claims 82 through 86, wherein said pharmaceutical composition is contained in a device comprised of separate chambered cartridges.

88. The pharmaceutical composition of any one of claims 82 through 86 wherein said pharmaceutical composition is contained in a device comprised of one chambered cartridge.

89. The pharmaceutical composition of any one of claims 81 through 88, wherein said device further comprises a syringe.

90. The use of a composition comprising a first anti-obesity agent comprising an amino acid amino acid sequence set out in SEQ ID NO: 1; and

a second anti-obesity agent comprising an amino acid sequence set out in SEQ ID NO:2;

in the manufacture of a medicament for the treating obesity, treating overweight, reducing food intake, reducing body weight, reducing body weight gain, reducing nutrient availability, reducing adiposity, or reducing or delaying gastric emptying according to any one of claims 1 to 89.