Modification of feeding behaviour   

This disclosure was made in part with United States government support pursuant to grants RR00163, DK51730 and DK55819, from the National Institutes of Health. The United States government has certain rights in the disclosure.

This application claims the benefit of UK Application No. GB0200507.2 filed Jan. 10, 2002, and International Application PCT/US02/31944 which are both incorporated by reference in their entirety herein.

This application relates to the use of agents to control appetite, feeding, food intake, energy expenditure and calorie intake, particularly in the field of obesity.

According to the National Health and Nutrition Examination Survey (NHANES III, 1988 to 1994), between one third and one half of men and women in the United States are overweight. In the United States, sixty percent of men and fifty-one percent of women, of the age of 20 or older, are either overweight or obese. In addition, a large percentage of children in the United States are overweight or obese.

The cause of obesity is complex and multi-factorial. Increasing evidence suggests that obesity is not a simple problem of self-control but is a complex disorder involving appetite regulation and energy metabolism. In addition, obesity is associated with a variety of conditions associated with increased morbidity and mortality in a population. Although the etiology of obesity is not definitively established, genetic, metabolic, biochemical, cultural and psychosocial factors are believed to contribute. In general, obesity has been described as a condition in which excess body fat puts an individual at a health risk.

There is strong evidence that obesity is associated with increased morbidity and mortality. Disease risk, such as cardiovascular disease risk and type 2 diabetes disease risk, increases independently with increased body mass index (BMI). Indeed, this risk has been quantified as a five percent increase in the risk of cardiac disease for females, and a seven percent increase in the risk of cardiac disease for males, for each point of a BMI greater than 24.9 (see Kenchaiah et al., N. Engl. J. Med. 347:305, 2002; Massie, N. Engl. J. Med. 347:358, 2002). In addition, there is substantial evidence that weight loss in obese persons reduces important disease risk factors. Even a small weight loss, such as 10% of the initial body weight in both overweight and obese adults has been associated with a decrease in risk factors such as hypertension, hyperlipidemia, and hyperglycemia.

Although diet and exercise provide a simple process to decrease weight gain, overweight and obese individuals often cannot sufficiently control these factors to effectively lose weight. Pharmacotherapy is available; several weight loss drugs have been approved by the Food and Drug Administration that can be used as part of a comprehensive weight loss program. However, many of these drugs have serious adverse side effects. When less invasive methods have failed, and the patient is at high risk for obesity related morbidity or mortality, weight loss surgery is an option in carefully selected patients with clinically severe obesity. However, these treatments are high-risk, and suitable for use in only a limited number of patients. It is not only obese subjects who wish to lose weight. People with weight within the recommended range, for example, in the upper part of the recommended range, may wish to reduce their weight, to bring it closer to the ideal weight. Thus, a need remains for agents that can be used to effect weight loss in overweight and obese subjects.

SUMMARY

Disclosed herein are findings that peripheral administration of PYY, or an agonist thereof, to a subject results in decreased food intake, caloric intake, and appetite, and an alteration in energy metabolism. The subject can be any subject, including, but not limited to, a human subject. In several embodiments, the subject desires to lose weight, is obese, overweight, or suffers from a weight-related disorder. PYY3-36 can preferably be administered to the subject.

Also disclosed are findings that co-administration of PYY, or an agonist thereof, for example, PYY3-36, with GLP-1 or an agonist thereof to a subject results in a greater decrease in food intake, caloric intake, and appetite, than when PYY or an agonist thereof is administered alone. The effect is synergistic.

In one embodiment, a method is disclosed for decreasing calorie intake in a subject. The method includes peripherally administering a therapeutically effective amount of PYY or an agonist thereof and GLP-1 or an agonist thereof to the subject, thereby decreasing the calorie intake of the subject.

In another embodiment, a method is disclosed for decreasing appetite in a subject. The method includes peripherally administering a therapeutically effective amount of PYY or an agonist thereof and GLP-1 or an agonist thereof to the subject, thereby decreasing the appetite of the subject.

In a further embodiment, a method is disclosed for decreasing food intake in a subject. The method includes peripherally administering a therapeutically effective amount of PYY or an agonist thereof, and GLP-1 or an agonist thereof to the subject, thereby decreasing the food intake of the subject.

In yet another embodiment, a method is disclosed herein for increasing energy expenditure in a subject. The method includes peripherally administering a therapeutically effective amount of PYY or an agonist thereof, and GLP-1 or an agonist thereof to the subject, thereby increasing energy expenditure in the subject.

A method is also disclosed for decreasing calorie intake, food intake, or appetite in a human subject. The method includes peripherally injecting a therapeutically effective amount of PYY or an agonist thereof in a pharmaceutically acceptable carrier to the subject in a pulse dose, thereby decreasing the calorie intake, food intake, or appetite of the subject, and also administering GLP-1 or an agonist thereof.

In each method above, the GLP-1 or an agonist thereof may be administered simultaneously or substantially simultaneously as the PYY or agonist thereof, or sequentially, in any order. The PYY or agonist thereof and the GLP-1 or agonist thereof may be administered in a single pharmaceutical composition or in separate compositions, and they may be administered by the same route or my different routes.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

FIG. 1 is a set of diagrams and digital images showing the generation of transgenic mice expressing EGFP in ARC POMC neurons. FIG. 1a is a schematic diagram of the structure of the POMC-EGFP transgene. FIG. 1b is a digital image showing the identification of a single POMC neuron (arrowhead on recording electrode tip) by EGFP fluorescence (upper) and IR-DIC microscopy (lower) in a living ARC slice prior to electrophysiological recordings. FIG. 1c is a set of digital images showing the co-localization (bright, on right) of EGFP (left) and β-endorphin immunoreactivity (middle) in ARC POMC neurons. Scale bars: b & c, 50 μm. FIG. 1d is a set of diagrams showing the distribution of EGFP-positive neuronal soma throughout the ARC nucleus. ∘=5 cells, •=10 cells.

FIG. 2 is a tracing and graphs showing activation of MOP-Rs hyperpolarizes the EGFP-labeled POMC neurons by opening G protein-coupled inwardly-rectifying potassium channels. FIG. 2a is a tracing showing met-enkephalin hyperpolarizes POMC neurons and inhibits all action potentials. The horizontal bar indicates the time when 30 μM Met-Enk was bath-applied to the slice. FIG. 2b is a graph showing met-enkephalin current and reversal potential is shifted by extracellular K+ concentration. FIG. 2c is a graph showing met-enkephalin activates MOP-R5 on POMC neurons. A Met-Enk (30 μM) current was observed and the MOP-R specific antagonist CTAP (1 μM) was applied for 1 minute. Following CTAP Met-Enk elicited no current. The figure is representative of three experiments.

FIG. 3 are tracings and graphs demonstrating that leptin depolarizes POMC neurons via a non-specific cation channel, and decreases GABAergic tone onto POMC cells. FIG. 3a is a tracing demonstrating that leptin depolarizes POMC neurons and increases the frequency of action potentials within 1 to 10 minutes of addition. The figure is a representative example of recordings made from 77 POMC neurons. FIG. 3b is a graph showing that leptin causes a concentration dependent depolarization of POMC cells. The depolarization caused by leptin was determined at 0.1, 1, 10, 50, and 100 nM (EC50=5.9 nM) in (8, 7, 9, 3, 45) cells respectively. FIG. 3c is a graph showing that leptin depolarizes POMC cells by activating a nonspecific cation current. The figure is representative of the response in 10 cells. FIG. 3d is a graph showing that leptin decreases the frequency of IPSCs in POMC cells. The figure is an example of 5 cells in which leptin (100 nM) decreased the frequency of IPSCs. FIG. 3e is a tracing demonstrating that leptin had no effect on 5 adjacent non-fluorescent ARC neurons. FIG. 3f is a tracing showing that leptin hyperpolarized 5 non-fluorescent ARC neurons.

FIG. 4 is a set of images showing that the GABAergic inputs to POMC cells are from NPY neurons that co-express GABA. FIG. 4a is a graph showing that NPY decreases the frequency of mini IPSCs in POMC neurons. FIG. 4b is a graph demonstrating that D-Trp8-γMSH (7 nM), a dose that selectively activates MC3-R, increases the frequency of GABAergic IPSCs in POMC neurons. FIG. 4c is a tracing showing that D-Trp8-γMSH hyperpolarizes POMC neurons. FIGS. 4a, 4b and 4c are representative. FIG. 4d is a set of digital images demonstrating that expression of NPY in nerve terminals adjacent to POMC neurons in the ARC. NPY nerve terminals (black, arrowheads); POMC neuronal soma (grey). Scale bar, 10 μm. FIG. 4e is a digital image showing expression of GABA and NPY in nerve terminals synapsing onto POMC neurons in the ARC. GABA immunoreactivity (10 nm gold particles, arrowheads without tail) and NPY immunoreactivity (25 nm gold particles, arrows with tail) are in separate vesicle populations co-localized within synaptic boutons that make direct contact with the soma of POMC neurons (DAB contrasted with uranyl acetate and lead citrate, diffuse black in cytoplasm). Scale bar, 1 μm. FIG. 4f is a diagram of the model of NPY/GABA and POMC neurons in the ARC.

FIG. 5 is a set of graphs relating to the feeding response to PYY3-36 in rats. FIG. 5a is a bar graph of dark-phase feeding tabulating food intake after intraperitoneal injection of PYY3-36. Freely feeding rats were injected with PYY3-36 at the doses indicated (μg/100 g), or saline, just prior to ‘lights off’ and 4-hour cumulative food intake was measured. Results are the mean±s.e.m. (n=8 per group), *=p<0.05, **=p<0.01, ***=<0.001 compared to saline. FIG. 5b is a bar graph of food intake after intraperitoneal injection of PYY3-36. Fasted rats were injected with PYY3-36 at the doses indicated (μg/100 g), or saline, and 4-hour cumulative food intake was measured. Results are shown as the mean±s.e.m. (n=8 per group), *=p<0.05, **=p<0.01, ***=<0.001 compared to saline. FIG. 5c is a bar graph of cumulative food intake after intraperitoneal injection of saline or PYY3-36. Fasted rats were injected with either saline (closed bars) or PYY3-36 5 μg/100 g (open bars) and cumulative food intake measured at the time points indicated. Results are expressed as mean±s.e.m. (n=12 per group), **=p<0.01 compared to saline. FIG. 5d is a line graph of body weight gain during chronic treatment with PYY3-36. Rats were injected intraperitoneally with PYY3-36 5 μg/100 g (open squares) or saline (filled inverted triangles) twice daily for 7 days. Body weight gain was calculated each day. Results are expressed as mean±s.e.m. (n=12 per group)**=p<0.01 compared to saline.

FIG. 6 is a set of digital images of c-fos expression in Pomc-EGFP mice. FIGS. 6a and 6b are digital images of representative sections (bregma −1.4 mm22) of c-fos expression in the arcuate nucleus of Pomc-EGFP mice response to intraperitoneal saline (FIG. 6a) or PYY3-36 (5 μg/100 g) (FIG. 6b). Scale bar 100 μm. 3V, third ventricle; Arc, arcuate nucleus. FIGS. 6c and 6d are digital images of representative sections showing POMC-EGFP neurons (FIG. 6c) and c-fos immunoreactivity (FIG. 6d) either co-localizing (bright arrows) or alone (single darker arrow). Scale bar 25 μm.

FIG. 7 is a set of bar graphs relating to intra-arcuate PYY3-36 in rats and feeding effects of IP PYY3-36 in Y2r-null mice. FIG. 7a is a bar graph of food intake following intra-arcuate PYY3-36 injection. Fasted rats were injected with saline or PYY3-36 into the arcuate nucleus at the doses indicated. Post-injection 2-hour food intake was measured, **=p<0.01 compared to saline. FIGS. 7b and 7c are bar graphs of feeding response to PYY3-36 in Y2r-null mice following IP administration: wild type littermates mice (FIG. 7b) and Y2r-null mice (FIG. 7c), fasted for 24 hours, were injected with PYY3-36 at the doses indicated (μg/100 g), or saline, and 4-hour cumulative food intake was measured. Results are the mean±s.e.m. (n=5 per group), *=p<0.05, **=p<0.01 compared to saline.

FIG. 8 is a set of images relating to the electrophysiological and neuropeptide responses to PYY3-36 and Y2A. FIG. 8a is a tracing showing the effect of PYY3-36 (10 nM) on the frequency of action potentials in POMC neurons (whole-cell configuration recordings; n=22)*p<0.05. PYY3-38 was administered at time D for 3 minutes; baseline, −3 to 0 minute; PYY3-36, 2-5 minutes; and wash-out, 8-11 minutes. Inset shows a representative recording of membrane potential and action potential frequency. FIG. 8b is a graph of the effect if PYY3-38 (10 nM) on the frequency of action potentials in loose cell-attached patch recordings (n=8). Data from individual cells were normalized to the firing rate for the 200 s before PYY3-38 addition. FIG. 8c is a tracing and a graph of the effect of PYY3-36 (50 nM) on spontaneous IPSCs onto POMC neurons (n=13). Inset shows a representative recording of IPSCs before and after PYY3-36 (50 nM), respectively. Results in FIG. 8a-8c are expressed as mean∀s.e.m. FIGS. 8d and 8e are bar graphs showing NPY (FIG. 8d) and %-MSH (FIG. 8e) released from hypothalamic explants in response to Y2A. Hypothalamic slices were incubated with artificial CSF (aCSF), with or without 50 nM Y2A, for 45 minutes. Results are expressed as mean∀s.e.m. (n=40); **=p<0.01; ***=p<0.001 compared to saline.

FIG. 9 is a set of graphs showing the effect of PYY3-36 infusion on appetite and food intake in human subjects. FIG. 9a is a graph of the calorie intake from a “free-choice” buffet meal 2 hours after infusion with saline or PYY3-36. The thin lines indicate individual changes in calorie intake for each subject between saline and PYY3-36 administration. The thick line represents mean change between the two infusions (n=12). FIG. 9b is a graph of the 24-hour calorie intake following infusion with saline or PYY3-36. Total calorie intake, as assessed by food diaries, is shown for the 24-hour period following either saline or PYY3-36 infusion. Data is given as mean±s.e.m. (n=12), ***=p<0.0001 compared to saline. FIG. 9c is a graph of the appetite score (relative scale). Visual analogue scores (Raben et al., Br. J. Nutr. 73, 517-30, 1995) show perceived hunger during and after infusions. The results are presented as change from baseline scores and are the mean±s.e.m. for all 12 subjects.

FIG. 10 shows plasma PYY3-36 levels following subcutaneous administration of 10 nmols PYY3-36 (broken line) and 20 nmols PYY3-36 (solid line).

FIG. 11 shows the effect on 1-hour food intake in non-fasted rats of IP administration of GLP-1 in the presence or absence of concomitant exendin 9-39 in the arcuate nucleus of the rat. S=Saline, G=GLP-1 (25 nmol/kg), and Ex=exendin 9-39 (20 nmoles/kg).

FIG. 12 shows the effects of co-administration of PYY3-36 and GLP-1 on 1-hour food intake in non-fasted rats injected (intraperitoneally) prior to the onset of the dark phase. IP GLP-1 (30 μg/kg=1×G or 60 μg/kg=2×G in 500 μl saline) or IP PYY3-36 (3 μg/kg=1×P or 6 μg/kg=2×P in 500 μl saline). Co-administration (P+G group) is GLP-1 30 μg/kg and PYY3-363 μg/kg combined.

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

DETAILED DESCRIPTION

α-MSH: alpha melanocortin stimulating hormone Arc: arcuate nucleus EPSP: excitatory postsynaptic potential GABA: γaminobutyric acid GFP, EGFP: green fluorescent protein GLP-1: glucagons-like peptide-1 ICV: intracerebroventricular IP: intraperitoneal IPSC: inhibitory postsynaptic current kb: kilobase kg: kilogram MOP-R: μ-opiod receptor MV: millivolts NPY: neuropeptide Y Oxm: oxyntomodulin pmol: picomole POMC: proopiomelanocortin RIA: radioimmunoassay RPA: RNase protection assay s.e.m: standard error of the mean TH: tyrosine hydroxylase μM: micromolar V: volts Y2A: N-acetyl (Leu28, Leu31) NPY (24-36)

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Action potential: A rapidly propagated electrical message that speeds along an axon of a neuron and over the surface membrane of many muscle and glandular cells. In axons they are brief, travel at constant velocity, and maintain a constant amplitude. Like all electrical messages of the central nervous system, the action potential is a membrane potential change caused by the flow of ions through ion channels in the membrane. In one embodiment, an action potential is a regenerative wave of sodium permeability.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.

Anorexia: A lack or loss of the appetite for food. In one embodiment, anorexia is a result of “anorexia nervosa.” This is an eating disorder primarily affecting females, usually with onset in adolescence, characterized by refusal to maintain a normal minimal body weight, intense fear of gaining weight or becoming obese, and a disturbance of body image resulting in a feeling of being fat or having fat in certain areas even when extremely emaciated, undue reliance on body weight or shape for self-evaluation, and amenorrhea. Associated features often include denial of the illness and resistance to psychotherapy, depressive symptoms, markedly decreased libido, and obsessions or peculiar behavior regarding food, such as hoarding. The disorder is divided into two subtypes, a restricting type, in which weight loss is achieved primarily through diet or exercise, and a binge-eating/purging type, in which binge eating or purging behavior also occur regularly.

Antagonist: A substance that tends to nullify the action of another, as an agent that binds to a cell receptor without eliciting a biological response, blocking binding of substances that could elicit such responses.

Appetite: A natural desire, or longing for food. In one embodiment, appetite is measured by a survey to assess the desire for food. Increased appetite generally leads to increased feeding behavior.

Appetite Suppressants: Compounds that decrease the desire for food. Commercially available appetite suppressants include, but are not limited to, amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine fenfluramine, dexfenfluramine, and fluoxetine.

Binding: A specific interaction between two molecules, such that the two molecules interact. Binding can be specific and selective, so that one molecule is bound preferentially when compared to another molecule. In one embodiment, specific binding is identified by a disassociation constant (Kd).

Body Mass Index (BMI): A mathematical formula for measuring body mass, also sometimes called Quetelet\'s Index. BMI is calculated by dividing weight (in kg) by height2 (in meters). The current standards for both men and women accepted as “normal” are a BMI of 20-24.9 kg/m2. In one embodiment, a BMI of greater than 25 kg/m2 can be used to identify an obese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m2. Grade II obesity corresponds to a BMI of 30-40 kg/m2; and Grade III obesity corresponds to a BMI greater than 40 kg/m2 (Jequier, Am. J Clin. Nutr. 45:1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.

c-fos: The cellular homologue of the viral v-fos oncogene found in FBJ (Finkel-Biskis-Jinkins) and FBR murine osteosarcoma viruses (MSV). The human fos gene maps to chromosome 14q21-q31. Human fos has been identified as TIS-28.

C-fos is thought to have an important role in signal transduction, cell proliferation, and differentiation. It is a nuclear protein which, in combination with other transcription factors (for example, jun) acts as a trans-activating regulator of gene expression. C-fos is an immediate early response gene, which are believed to play a key role in the early response of cells to growth factors. C-fos is involved also in the control of cell growth and differentiation of embryonic hematopoietic cells and neuronal cells. The human c-fos coding amino acid and nucleic sequences are known (e.g., see Verma et al., Cold Spring Harb. Symp. Quant. Biol. 51, 949, 1986; GenBank Accession Nos. K00650 and M16287, and is available on the internet).

Caloric intake or calorie intake: The number of calories (energy) consumed by an individual.

Calorie: A unit of measurement in food. A standard calorie is defined as 4.184 absolute joules, or the amount of energy it takes to raise the temperature of one gram of water from 15 to 16° C. (or 1/100th the amount of energy needed to raise the temperature of one gram of water at one atmosphere pressure from 0° C. to 100° C.), food calories are actually equal to 1,000 standard calories (1 food calorie=1 kilocalorie).

Conservative variation: The replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

Non-limiting examples of conservative amino acid substitutions include those listed below:

Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Depolarization: An increase in the membrane potential of a cell. Certain stimuli reduce the charge across the plasma membrane. These can be electrical stimuli (which open voltage-gated channels), mechanical stimuli (which activate mechanically-gated channels) or certain neurotransmitters (which open ligand-gated channels). In each case, the facilitated diffusion of sodium into the cell increases the resting potential at that spot on the cell creating an excitatory postsynaptic potential (EPSP). Depolarizations can also be generated by decreasing the frequency of inhibitory postsynaptic currents (IPSCs), these are due to inhibitory neurotransmitters facilitating the influx of chloride ions into the cell, creating an IPSC. If the potential is increased to the threshold voltage (about −50 mV in mammalian neurons), an action potential is generated in the cell.

Diabetes: A failure of cells to transport endogenous glucose across their membranes either because of an endogenous deficiency of insulin and/or a defect in insulin sensitivity. Diabetes is a chronic syndrome of impaired carbohydrate, protein, and fat metabolism owing to insufficient secretion of insulin or to target tissue insulin resistance. It occurs in two major forms: insulin-dependent diabetes mellitus (IDDM, type I) and non-insulin dependent diabetes mellitus (NIDDM, type II) which differ in etiology, pathology, genetics, age of onset, and treatment.

The two major forms of diabetes are both characterized by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis. Diabetes type I, or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of β cells, which results in insufficient levels of endogenous insulin. Diabetes type II, or non-insulin dependent diabetes, results from a defect in both the body\'s sensitivity to insulin, and a relative deficiency in insulin production.

Exendin: A 39-amino acid peptide isolated from the salivary glands of the Gila monster (Heloderma suspectum) (Eng J et al J Biol Chem 267:7402-7405, 1992), see SEQ ID NO:341. Exendin is an example of an agonist at the GLP-1 receptor. Molecules derived from exendin-4 and that also have GLP-1 agonist activity are further examples of GLP-1 agonists.

Food intake: The amount of food consumed by an individual. Food intake can be measured by volume or by weight. In one embodiment, food intake is the total amount of food consumed by an individual. In another embodiment, food intake is the amount of proteins, fat, carbohydrates, cholesterol, vitamins, minerals, or any other food component, of the individual. “Protein intake” refers to the amount of protein consumed by an individual. Similarly, “fat intake,” “carbohydrate intake,” “cholesterol intake,” “vitamin intake,” and “mineral intake” refer to the amount of proteins, fat, carbohydrates, cholesterol, vitamins, or minerals consumed by an individual.

Glucagon-like peptide-1: Peptides produced by processing of preproglucogon, which is a 160 amino acid polypeptide, in the central nervous system (CNS) and the intestine. GLP-1 (1-37) is the initial product. GLP-1 (1-37) is amidated by post-translational processing to yield GLP-1 (1-36) NH, or is enzymatically processed to give GLP-1 (7-37) (SEQ ID NO: 338). GLP-1 (7-37) can be amidated to give GLP-1 (7-36) amide (SEQ ID NO: 339), which is the most biologically active form. GLP-1 is released into the circulation in response to nutrient intake. Intestinal cells secrete GLP-1 (7-37) and GLP-1 (7-36) amide in a ratio of 1 to 5.

GLP-1 receptors are found in the brainstem, arcuate nucleus and paraventricular nucleus. Physiological actions of GLP-1 in man include stimulation of insulin release, suppression of gastric acid secretion and slowing of gastric emptying.

A GLP-1 agonist is a peptide, small molecule, or chemical compound that preferentially binds to the GLP-1 receptor and stimulate the same biological activity as GLP-1. In one embodiment, an agonist for the GLP-1 receptor binds to the receptor with an equal or greater affinity than a GLP-1 peptide. In another embodiment, an agonist selectively binds the GLP-1 receptor, as compared to binding to another receptor.

One of skill in the art can readily determine the dissociation constant (Kd) value of a given compound. This value is dependent on the selectivity of the compound tested. For example, a compound with a Kd which is less than 10 nM is generally considered an excellent drug candidate. However, a compound that has a lower affinity, but is selective for the particular receptor, can also be a good drug candidate. In one specific, non-limiting example, an assay, such as a competition assay, is used to determine if a compound of interest is a GLP-1 receptor agonist.

GLP-1 agonists include GLP-1 related peptides and peptides that result from natural or synthetic enzymatic or chemical processing of a GLP-1 peptide or a related peptide.

Hyperpolarization: A decrease in the membrane potential of a cell. Inhibitory neurotransmitters inhibit the transmission of nerve impulses via hyperpolarization. This hyperpolarization is called an inhibitory postsynaptic potential (IPSP). Although the threshold voltage of the cell is unchanged, a hyperpolarized cell requires a stronger excitatory stimulus to reach threshold.

Inhibitory Postsynaptic Current: A current that inhibits an electrophysiological parameter of a postsynaptic cell. The potential of a postsynaptic cell can be analyzed to determine an effect on a presynaptic cell. In one embodiment, the postsynaptic cell is held in voltage clamp mode, and postsynaptic currents are recorded. If necessary, antagonists of other classes of current can be added. In one specific, non-limiting example, to record GABAergic IPSCs, blockers of excitatory channels or receptors can be added. The instantaneous frequency over time is then determined.

In one embodiment, IPSCs give a measure of the frequency of GABA release from an NPY neuron. Thus, as NPY neurons release GABA onto POMC neurons, measurement of IPSC frequency is a gauge of the inhibitory tone that POMC neurons are receiving, and can be used to assess the effect of an agonist of PYY.

Membrane potential: The electrical potential of the interior of the cell with respect to the environment, such as an external bath solution. One of skill in the art can readily assess the membrane potential of a cell, such as by using conventional whole cell techniques. Activation of a cell is associated with less negative membrane potentials (for example shifts from about −50 mV to about −40 mV). These changes in potential increase the likelihood of action potentials, and thus lead to an increase in the rate of action potentials.

The rate of action potentials can be assessed using many approaches, such as using conventional whole cell access, or using, for example, perforated-patch whole-cell and cell-attached configurations. In each event the absolute voltage or current is not assessed, rather the frequency of rapid deflections characteristic of action potentials is assessed, as a function of time (therefore this frequency is an instantaneous frequency, reported in “bins”). This time component can be related to the time at which a compound, such as a PYY agonist, is applied to the bath to analyze the effect of the compound, such as the PYY agonist, on action potential firing rate.

Neuropeptide Y (NPY): A 36-amino acid peptide that is a neuropeptide identified in the mammalian brain. NPY is believed to be an important regulator in both the central and peripheral nervous systems and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen, intestinal membranes, brain, aortic smooth muscle, kidney, testis, and placenta. In addition, binding sites have been reported in a number of rat and human cell lines.

Neuropeptide Y (NPY) receptor has structure/activity relationships within the pancreatic polypeptide family. This family includes NPY, which is synthesized primarily in neurons; peptide YY (PYY), which is synthesized primarily by endocrine cells in the gut; and pancreatic polypeptide (PP), which is synthesized primarily by endocrine cells in the pancreas. These 36 amino acid peptides have a compact helical structure involving an amino acid structure, termed a “PP-fold” in the middle of the peptide.

NPY binds to several receptors, including the Y1, Y2, Y3, Y4 (PP), Y5, Y6, and Y7 receptors. These receptors are recognized based on binding affinities, pharmacology, and sequence (if known). Most, if not all of these receptors are G protein coupled receptors. The Y1 receptor is generally considered to be postsynaptic and mediates many of the known actions of neuropeptide Y in the periphery. Originally, this receptor was described as having poor affinity for C-terminal fragments of neuropeptide Y, such as the 13-36 fragment, but interacts with the full length neuropeptide Y and peptide YY with equal affinity (e.g., see PCT publication WO 93/09227).

Pharmacologically, the Y2 receptor is distinguished from Y1 by exhibiting affinity for C-terminal fragments of neuropeptide Y. The Y2 receptor is most often differentiated by the affinity of neuropeptide Y(13-36), although the 3-36 fragment of neuropeptide Y and peptide YY provides improved affinity and selectivity (see Dumont et al., Society for Neuroscience Abstracts 19:726, 1993). Signal transmission through both the Y1 and the Y2 receptors are coupled to the inhibition of adenylate cyclase. Binding to the Y-2 receptor was also found to reduce the intracellular levels of calcium in the synapse by selective inhibition of N-type calcium channels. In addition, the Y-2 receptor, like the Y1 receptors, exhibits differential coupling to second messengers (see U.S. Pat. No. 6,355,478). Y2 receptors are found in a variety of brain regions, including the hippocampus, substantia nigra-lateralis, thalamus, hypothalamus, and brainstem. The human, murine, monkey and rat Y2 receptors have been cloned (e.g., see U.S. Pat. No. 6,420,352 and U.S. Pat. No. 6,355,478).

A Y2 receptor agonist is a peptide, small molecule, or chemical compound that preferentially binds to the Y2 receptor and stimulates intracellular signaling. In one embodiment, an agonist for the Y2 receptor binds to the receptor with an equal or greater affinity than NPY. In another embodiment, an agonist selectively binds the Y2 receptor, as compared to binding to another receptor.

One of skill in the art can readily determine the dissociation constant (Kd) value of a given compound. This value is dependent on the selectivity of the compound tested. For example, a compound with a Kd which is less than 10 nM is generally considered an excellent drug candidate. However, a compound that has a lower affinity, but is selective for the particular receptor, can also be a good drug candidate. In one specific, non-limiting example, an assay, such as a competition assay, is used to determine if a compound of interest is a Y2 receptor agonist. Assays useful for evaluating neuropeptide Y receptor antagonists are also well known in the art (see U.S. Pat. No. 5,284,839, which is herein incorporated by reference, and Walker et al., Journal of Neurosciences 8:2438-2446, 1988).

Normal Daily Diet: The average food intake for an individual of a given species. A normal daily diet can be expressed in terms of caloric intake, protein intake, carbohydrate intake, and/or fat intake. A normal daily diet in humans generally comprises the following: about 2,000, about 2,400, or about 2,800 to significantly more calories. In addition, a normal daily diet in humans generally includes about 12 g to about 45 g of protein, about 120 g to about 610 g of carbohydrate, and about 11 g to about 90 g of fat. A low calorie diet would be no more than about 85%, and preferably no more than about 70%, of the normal caloric intake of a human individual.

In animals, the caloric and nutrient requirements vary depending on the species and size of the animal. For example, in cats, the total caloric intake per pound, as well as the percent distribution of protein, carbohydrate and fat varies with the age of the cat and the reproductive state. A general guideline for cats, however, is 40 cal/lb/day (18.2 cal/kg/day). About 30% to about 40% should be protein, about 7% to about 10% should be from carbohydrate, and about 50% to about 62.5% should be derived from fat intake. One of skill in the art can readily identify the normal daily diet of an individual of any species.

Obesity: A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102:E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure. In one embodiment, the Body Mass Index (BMI) is used to assess obesity. In one embodiment, a BMI of 25.0 kg/m2 to 29.9 kg/m2 is overweight, while a BMI of 30 kg/m2 is obese.

In another embodiment, waist circumference is used to assess obesity. In this embodiment, in men a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese. Strong evidence shows that obesity affects both the morbidity and mortality of individuals. For example, an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001).

Overweight: An individual who weighs more than their ideal body weight. An overweight individual can be obese, but is not necessarily obese. In one embodiment, an overweight individual is any individual who desires to decrease their weight. In another embodiment, an overweight individual is an individual with a BMI of 25.0 kg/m2 to 29.9 kg/m2

Oxyntomodulin: A further peptide produced by post-translational processing of preproglucagon in the CNS and intestine, see SEQ ID NO: 340. Agonists of oxyntomodulin may be identified as described above for agonists of GLP-1 and of NPY. include oxyntomodulins of other species and also modified sequences, for example, sequences in which The term OXM used in this text also covers any analogue of the above OXM sequence, wherein the histidine residue at position 1 is maintained or replaced by an aromatic moiety carrying a positive charge or a derivative thereof, preferably wherein the moiety is an amino acid, more preferably wherein it is a histidine derivative, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the other amino acids in the above OXM sequence can be independently replaced by any other independently chosen amino acid, with the exception of histidine in position 1.

Any one or more (to 22) other alpha-amino acid residue in the sequence can be independently replaced by any other one alpha-amino acid residue. Preferably, any amino acid residue other than histidine is replaced with a conservative replacement as well known in the art i.e. replacing an amino acid with one of a similar chemical type such as replacing one hydrophobic amino acid with another.

As discussed above, 1 to 22 of the amino acids can be replaced. In addition to the replacement option above, this may be by a non-essential or modified or isomeric form of an amino acid. For example, 1 to 22 amino acids can be replaced by an isomeric form (for example a D-amino acid), or a modified amino acid, for example a nor-amino acid (such as norleucine or norvaline) or a non-essential amino acid (such as taurine). Furthermore, 1 to 22 amino acids may be replaced by a corresponding or different amino acid linked via its side chain (for example gamma-linked glutamic acid). For each of the replacements discussed above, the histidine residue at position 1 is unaltered or defined above.

In addition, 1, 2, 3, 4 or 5 of the amino acid residues can be removed from the OXM sequence with the exception of histidine at the 1 position (or as defined above). The deleted residues may be any 2, 3, 4 or 5 contiguous residues or entirely separate residues.

The C-terminus of the OXM sequence may be modified to add further amino acid residues or other moieties.

Pancreatic Polypeptide: A 36 amino acid peptide produced by the pancreas that is has homology to PYY and NPY.

Peripheral Administration: Administration outside of the central nervous system. Peripheral administration does not include direct administration to the brain. Peripheral administration includes, but is not limited to intravascular, intramuscular, subcutaneous, inhalation, oral, rectal, transdermal or intra-nasal administration

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “polypeptide fragment” refers to a portion of a polypeptide, for example such a fragment which exhibits at least one useful sequence in binding a receptor. The term “functional fragments of a polypeptide” refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional peptides can also include fusion proteins, in which the peptide of interest has been fused to another peptide that does not decrease its desired activity.

PYY: A peptide YY polypeptide obtained or derived from any species. Thus, PYY includes the human full length polypeptide (as set forth in SEQ ID NO: 1) and species variations of PYY, including e.g. murine, hamster, chicken, bovine, rat, and dog PYY (SEQ ID NOS: 5-12). In one embodiment, PYY agonists do not include NPY. PYY also includes PYY3-36. A “PYY agonist” is any compound which binds to a receptor that specifically binds PYY, and elicits an effect of PYY. In one embodiment, a PYY agonist is a compound that affects food intake, caloric intake, or appetite, and/or which binds specifically in a Y receptor assay or competes for binding with PYY, such as in a competitive binding assay with labeled PYY. PYY agonists include, but are not limited to, compounds that bind to the Y2 receptor.

Substantially purified: A polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. For example, the polypeptide may be at least 50%, 80% or 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.

Therapeutically effective amount: A dose sufficient to prevent advancement, or to cause regression of a disorder, or which is capable of relieving a sign or symptom of a disorder, or which is capable of achieving a desired result. In several embodiments, a therapeutically effect of PYY or an agonist thereof is an amount sufficient to inhibit or halt weight gain, or an amount sufficient to decrease appetite, or an amount sufficient to reduce caloric intake or food intake or increase energy expenditure.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

A method is disclosed herein for reducing food intake by peripherally administering to a subject a therapeutically effective amount of PYY or an agonist of PYY and GLP-1 or an agonist thereof. In one embodiment, administration of PYY, or an agonist of PYY, and GLP-1 or an agonist thereof, results in a decrease in the amount, either the total weight or the total volume of food. In other embodiment, administration of PYY, or an agonist thereof, and GLP-1 or an agonist thereof, results in a decrease of the intake of a food component, such as a decrease in the ingestion of lipids, carbohydrates, cholesterol, or proteins. In the any of the methods disclosed herein, a preferred compound, PYY3-36 can be administered. This disclosure includes the corresponding uses of PYY or an agonist thereof and GLP-1 or an agonist thereof, for the manufacture of a medicament or medicaments for the purposes set herein, and includes the use of PYY3-36.

A method is also disclosed herein for reducing caloric intake by peripherally administering to a subject a therapeutically effective amount of PYY or an agonist of PYY and GLP-1 or an agonist thereof. In one embodiment, total caloric intake is reduced by peripheral administration of a therapeutically effective amount of PYY and GLP-1 or an agonist thereof. In other embodiments, the caloric intake from the ingestion of a specific food component, such as, but not limited to, the ingestion of lipids, carbohydrates, cholesterol, or proteins, is reduced.

In an additional embodiment, a method is disclosed herein for reducing appetite by administering a therapeutically effective amount of PYY or an agonist thereof and GLP-1 or an agonist thereof. Appetite can be measured by any means known to one of skill in the art. For example, decreased appetite can be assessed by a psychological assessment. In this embodiment, administration of PYY and the other agent(s) results in a change in perceived hunger, satiety, and/or fullness. Hunger can be assessed by any means known to one of skill in the art. In one embodiment, hunger is assessed using psychological assays, such as by an assessment of hunger feelings and sensory perception using a questionnaire, such as, but not limited to, a Visual Analog Score (VAS) questionnaire (see the Examples section). In one specific, non-limiting example, hunger is assessed by answering questions relating to desire for food, drink, prospective food consumption, nausea, and perceptions relating to smell or taste.

In a further embodiment, a method is disclosed herein for altering energy metabolism in a subject. The method includes peripherally administering a therapeutically effective amount of PYY or an agonist thereof and GLP-1 or an agonist thereof, to the subject, thereby altering energy expenditure. Energy is burned in all physiological processes. The body can alter the rate of energy expenditure directly, by modulating the efficiency of those processes, or changing the number and nature of processes that are occurring. For example, during digestion the body expends energy moving food through the bowel, and digesting food, and within cells, the efficiency of cellular metabolism can be altered to produce more or less heat. In a further embodiment a method is disclosed herein for any and all manipulations of the arcuate circuitry described in this application, that alter food intake coordinately and reciprocally alter energy expenditure. Energy expenditure is a result of cellular metabolism, protein synthesis, metabolic rate, and calorie utilization. Thus, in this embodiment, peripheral administration of PYY and administration of GLP-1 or an agonist thereof, results in increased energy expenditure, and decreased efficiency of calorie utilization. In one embodiment, a therapeutically effective amount of PYY or an agonist and GLP-1 or an agonist thereof, thereof is administered to a subject, thereby increasing energy expenditure.

In several embodiments, PYY (e.g., PYY3-36) or an agonist thereof and GLP-1 or an agonist thereof, are used for weight control and treatment, reduction or prevention of obesity, in particular any one or more of the following: preventing and reducing weight gain; inducing and promoting weight loss; and reducing obesity as measured by the Body Mass Index. The disclosure further relates to the use of PYY or an agonist and GLP-1 or an agonist thereof, in control of any one or more of appetite, satiety and hunger, in particular any one or more of the following: reducing, suppressing and inhibiting appetite; inducing, increasing, enhancing and promoting satiety and sensations of satiety; and reducing, inhibiting and suppressing hunger and sensations of hunger. The disclosure further relates to the use of PYY an agonist thereof and GLP-1 or an agonist thereof, in maintaining any one or more of a desired body weight, a desired Body Mass Index, a desired appearance and good health.

The subject can be any subject, including both human and veterinary mammalian subjects. Thus, the subject can be a human, or can be a non-human primate, a farm animal such as swine, cattle, and poultry, a sport animal or pet such as dogs, cats, horses, hamsters, rodents, or a zoo animal such as lions, tigers, or bears.

Obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. A therapeutic drug useful in weight reduction of obese persons could have a profound beneficial effect on their health. Thus, the subject can be, but is not limited to, a subject who is overweight or obese. In one embodiment, the subject has, or is at risk of having, a disorder wherein obesity or being overweight is a risk factor for the disorder. Disorders of interest include, but are not limited to, cardiovascular disease, (including, but not limited to, hypertension, atherosclerosis, congestive heart failure, and dyslipidemia), stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as, but not limited to, polycystic ovarian syndrome, cancers (e.g., breast, prostate, colon, endometrial, kidney, and esophagus cancer), varicose veins, acnthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholithiasis, osteoarthritis, orthopedic injury, insulin resistance (such as, but not limited to, type 2 diabetes and syndrome X) and tromboembolic disease (see Kopelman, Nature 404:635-43; Rissanen et al., British Med. J. 301, 835, 1990).

Other associated disorders also include depression, anxiety, panic attacks, migraine headaches, PMS, chronic pain states, fibromyalgia, insomnia, impulsivity, obsessive compulsive disorder, and myoclonus. Obesity is a recognized risk factor for increased incidence of complications of general anesthesia. (See e.g., Kopelman, Nature 404:635-43, 2000). It reduces life span and carries a serious risk of co-morbidities listed above.

Other diseases or disorders associated with obesity are birth defects (maternal obesity associated with increased incidence of neural tube defects), carpal tunnel syndrome (CTS), chronic venous insufficiency (CVI), daytime sleepiness, deep vein thrombosis (DVT), end stage renal disease (ESRD), gout, heat disorders, impaired immune response, impaired respiratory function, infertility, liver disease, lower back pain, obstetric and gynecologic complications, pancreatititis, as well as abdominal hernias, acanthosis nigricans, endocrine abnormalities, chronic hypoxia and hypercapnia, dermatological effects, elephantitis, gastroesophageal reflux, heel spurs, lower extremity edema, mammegaly (causing considerable problems such as bra strap pain, skin damage, cervical pain, chronic odors and infections in the skin folds under the breasts, etc.), large anterior abdominal wall masses (abdominal panniculitis with frequent panniculitis, impeding walking, causing frequent infections, odors, clothing difficulties, low back pain), musculoskeletal disease, pseudo tumor cerebri (or benign intracranial hypertension), and sliding hiatil hernia.

The present disclosure relates to treating, prevention, ameliorating or alleviating conditions or disorders caused by, complicated by, or aggravated by a relatively high nutrient availability. By “condition or disorder which can be alleviated by reducing caloric (or nutrient) availability,” it is meant any condition or disorder in a subject that is either caused by, complicated by, or aggravated by a relatively high nutrient availability, or that can be alleviated by reducing nutrient availability, for example by decreasing food intake. Subjects who are insulin resistant, glucose intolerant, or have any form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes) can also benefit from this disclosure.

Such conditions or disorders are disorders associated with increased caloric intake, insulin resistance, or glucose intolerance and include, but are not limited to, obesity, diabetes, including type 2 diabetes, eating disorders, insulin-resistance syndromes, and Alzheimer\'s disease.