GB2505941A - Peptide analogues of glucagon for the treatment of obesity and diabetes - Google Patents

Abstract

Peptide analogues of glucagon are provided herein. Also provided herein are pharmaceutical compositions comprising said analogues, and methods of using said analogues for the treatment and/or prevention of conditions such as obesity and diabetes.

Description

NOVEL COMPOUNDS

FIELD OF TUE INVENTION

This invention relates to analogues of glucagon which are useful in treating diabetes and obesity. It also relates to use of the glucagon analogues as neuroprotective or cardioprotective agents.

BACKGROUND OF THE INVENTION

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 Unitcd Statcs arc 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 Mccl. 347:305, 2002; Massie, N EngL J.Mccl.

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. Recently it has been shown that considerable weight loss can effectively cure type 2 diabetes (Lim et al, Diabetologia June 2011).

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 proven to have serious adverse side effects, and have had to be withdrawn. 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 ofpatients. 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 as well as subjects who are of normal weight.

A number of derivatives of peptides deriving from the pro-glucagon molecule have been proposed for use in treatment of obesity and/or diabetes. Pro-glueagon is a precursor peptide of glucagon, as well as other hormones including oxyntomodulin (OXM) and GLPI (glueagon-like peptide 1). Glueagon is released in vivo when blood glucose levels fall low and has the activity of causing the liver to convert stored glyeogen into glucose which is released into the bloodstream raising blood glucose levels.

W02008/086086 (Indiana University Research and Technology Corporation) discloses certain glucagon peptides which have been modified by the incorporation of charged amino acids at the carboxy terminus of the peptide. The peptides are disclosed as having enhanced aqueous solubility at a pH ranging from about 5.5 to about 8.

Despite significant advances, the process of identifying substances useful as drugs remains a complex and, in many eases, unpredictable field. Compounds must possess a suitable balance of properties, for example in addition to having efficacy at the biological target of interest, they must have good in vim pharmacokinetic properties as well as low toxicity.

The present invention is based on the discovery that analogues of glucagon containing His residues at specified positions can be administered to a subject in order to cause an alteration in energy metabolism, so as to promote weight loss. In many case the analogues of the present invention have an improved pharmacokinetic profile, having a longer duration of action than native glucagon. In particular, analogues may have an improved pharmacokinetie profile characterised by a slower increase of plasma levels following administration, a lower Cmax and/or a more stable plateau. Such a pharmacokinetic profile may be associated with a decrease in side effects such as nausea, which are associated with a rapid onset of high plasma levels. Analogues of the invention may display improved potency and/or fewer side effects compared with native glucagon.

Increased potency at the glucagon receptor results in lower doses being required to decrease food intake, thereby decreasing the side effects associated with high doses.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an analogue of glucagon which is: a compound comprising an amino acid sequence represented by formula (T) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa'2-Xaa'3-Leu-Xaa15-Xaa'6- Xaa17 -Xaa1 8-Ala-Xaa20-Xaa21 -Phe-Xaa23-Xaa24-Trp-Leu-Leu-Asn-Xaa29-V (I) wherein V is selected from the group consisting of His, His-NH2, His-His, I-us-I-us-NI-I2, Gly-His, Gly-l-Iis-NH2, Lys-His, Lys-l-Iis-NH2, Gly-I-lis-His, Gly-His-His-NH2, His-His-His and His-His-His-NH2; Xaa'° is selected from the group consisting of Tyr and Leu; Xaa'2 is selected from the group consisting of Lys, His and Arg; Xaa'3 is selected from the group consisting of Tyr, Gin and His; Xaa'5 is selected from the group consisting of Asp and Glu; Xaa'6 is selected from the group consisting of Glu, Gin and Ser; Xaa" is selected from the group consisting of Arg, His and Lys; Xaa'8 is selected from the group consisting of Arg and Lys; Xaa2° is selected from the group consisting of His and Gin; Xaa2' is selected from the group consisting of Glu, His and Asp; Xaa23 is selected from the group consisting of Tie and Val; Xaa24 is selected from the group consisting of Gin and Giu; and Xaa2° is selected from the group consisting ofThr and Gly; wherein -NH2 represents a C-terminal amide group; or a derivative of the compound; or a salt of the compound or the derivative.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising an analogue of the invention together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

According to a further aspect of the invention, there is provided an analogue of the invention, or a pharmaccutical composition comprising an analogue of the invention, for use as a medicament. The analogue of the invention or pharmaceutical composition comprising the analogue of the invention finds use in the treatment of obesity and/or diabetes. The analogue or pharmaceutical composition is for use in increasing the energy expenditure of a subject, enhancing insulin release in a subject and/or improving carbohydrate metabolism in a subject and/or improving the lipid profile of a subject. The analogue or pharmaceutical composition also finds use as a cytoprotective agent, e.g. in preventing or treating neurodegeneration, providing neuroprotection and/or providing cardiac protection.

According to a further aspect of the invention, there is provided use of an analogue of the invention for the manufacture of a medicament for the treatment of obesity and/or diabetes. There is also provided use of an analogue for the manufacture of a medicament for increasing the energy expenditure ofa subject, enhancing insulin release in a subject, improving carbohydrate metabolism in a subject and/or improving the lipid profile of a subject. Thcrc is also providcd usc of an analogue for the manufacture of a medicament for providing eytoprotection.

According to a further aspect of the invention, there is provided a method of treating or preventing a disease or disorder or other non-desired physiological state in a subject comprising administration of a therapeutically effective amount of an analogue of the invention, or a pharmaceutical composition comprising the analogue of the invention.

There is also provided a method of treating obesity or diabetes in a subject in need thereof comprising administration of a therapeutically effective amount of an analogue or pharmaceutical composition. There is also provided a method of increasing the energy expenditure of a subject, enhancing insulin release in a subject, improving carbohydrate metabolism in a subject and/or improving the lipid profile of a subject, comprising administration of a therapeutically effective amount of an analogue or pharmaceutical composition. There is also provided a method of providing cytoprotection in a subject, comprising administration of a therapeutically effective amount of an analogue or pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequences of glucagon analogues of the invention. The peptide analogues in the table of Figure 1 are presented with the N-terminal residue at the left hand side of the table and the C-terminal residue at the right hand side of the table.

Analogues in the table containing the group "-Nl-12" have a C-terminal amide group (i.e. the C-terminal residue has a -C(O)NH2 group in place of a C-terminus carboxylic acid).

Figure 1 also shows data relating to cAMP signaling in cells over-expressing the human glueagon receptor or the human GLP-1 receptor following contact with glucagon analogues of the invention. Figure 1 also show summary food intake data for glucagon analogues of the invention.

Figures 2 to 11 show more detailed rat food intake and body weight change data for selected glucagon analogues of the invention compared with exendin-4.

Figures 12 to 21 show the results of rat pharmacokinetic studies with selected glucagon analogues of the invention.

DEFINITIONS

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided: Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. Thc term mammal includes both human and non-human mammals.

Similarly, the term "subject" includes both human and veterinary subjects.

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, amfcpramone (diethylpropion), phentermine, mazindol and phenylpropanolamine fenfluramine, dexfentluramine, and fluoxetine.

Body Mass Index (BMI): A mathematical formula for measuring body mass, also sometimes called Quetelets Index. BMI is calculated by dividing weight (in kg) by height2 (in meters2). The current standards for both men and women accepted as "normal are a BMI of 20-24.9 kg/m2. In one embodiment, a BMT of greater than 25 kglm2 can be used to identify an obese subject. Grade I obesity (which is sometimes referred to as being "overweight" rather than 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. I Cl/n. Nutr. 45:1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.

Cardioprotection refers to the protection of cardiac cells (and especially the myocardial cells) from apoptosis, necrotic cell death or degeneration (loss of function).

Cardioprotection is most often required following myocardial infarction, but may also be used in subjects suffcring from ischcmic hcart disease (for example angina) Conservative substitutions: The replacement of an amino acid residue by another, biologically similar residue in a polypeptide. The term "conservative variation" also includes the use of a substituted amino acid, i.e. an amino acid with one or more atoms replaced with another atom or group, in place of a parent amino acid provided that the polypeptide retains its activity or provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide Cytoprotection refers to the protection of cells from apoptosis, necrotic cell death or degeneration (loss of function).

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 f3 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.

Energy Metabolism: The body has to expend a certain amount of energy to maintain normal metabolism. In civilized man this is often set at about 2,800 Calories daily. If food consumption does not provide this, weight loss results. However, energy metabolism is also regulated and, for example, administration of glucagon is thought to increase the metabolic rate so that a greater food intake is required to achieve energy balance and maintain weight. Thus, if food intake is maintained at the usual level, but energy metabolism is increased, weight loss will result.

Food intake: The amount of food consumed by an individual. Food intake can be measured by volume or by weight. For example, food intake may be the total amount of food consumed by an individual. Or, food intake may be 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.

GLPI: Glucagon-like peptide 1 (GLPI) is derived from the transcription product of the proglucagon gene. The biologically active forms of GLP1 are truncated forms known as GLPI(737) and GLPIp3o)-C(O)NH2 /GLPl(7ro)-NI-l2 (i.e. the C-terminus has a C(O)NH2 group in place of a carboxylic acid). The sequence of human GLP1ç73o)-C(O)NH2 is His- Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Ly s-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-C(O)NH2.

Glucagon: Glucagon is a peptide derived from the proglucagon gene. It is a 29-amino acid polypeptide in humans and has the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp- Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-As n-Thr.

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 postsynaptie potential (IPSP). Although the threshold voltage of the cell is uncharged, a hyperpolarized cell requires a stronger excitatory stimulus to reach threshold.

S

Neuroprotection refers to the protection of neurons within the nervous system (preferably within the central nervous system) from apoptosis, necrotic cell death or degeneration (loss of function). Neuroprotective treatments, including those relating to various aspects of the present invention may be required following a brain injury (for example those following physical trauma or non-traumatic injury such as stroke, brain tumours, infection, poisoning, hypoxia, isehemia, encephalopathy or substance abuse). Neuroprotective treatments, including those relating to various aspects of the present invention may also be indicated in subjects having a chronic neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, Gehrig's disease or Huntington's disease.

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 ofan 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. For example, the Body Mass Index (BMI) may be used to assess obesity. In one commonly used convention, a BMI of 25.0 kg/rn2 to 29.9 kg/rn2 is overweight, while a BMI of3O kg/rn2 or greater is obese.

In another convention, waist circumfcrence is used to assess obesity. In this convention, in rnen a waist circumference of 102 cm or rnore is considered obese, while in women a waist circumference of 89cm 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 Madder disease, sleep apnca, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fat. 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. For example, an overweight individual is any individual who desires to decrease their weight. In one convention, an overweight individual is an individual with a BMI of 25.0 kg/rn2 to 29.9 kg/m2 PEGylated and PEGylation: the process of reacting a poly(alkylene glycofl, preferably an activated polyalkylene glycol) to form a covalent bond. A facilitator may be used, for example an amino acid, e.g. lysine. Although "PEGylation' is often carried out using p&y(ethylene gycol) or derivatives thercof such as mcthoxy p&y(ethyene glycol), the term is not limited herein to the use of methoxy poly(ethylene glycol) but also includes the use of any other useful poly(alkylene glycol), for example poly(propylene glycol).

p1: p1 is an abbreviation for isoelectric point. An alternative abbreviation sometimes used is IEP. It is the pH at which a particular molecule carries no net electric charge. At a pH below its p1 a protein or peptide carries a net positive charge. At a pH above its p1 a protein or peptide carries a net negative charge. Proteins and peptides can be separated according to their isoelcctric points using a technique called isoelcctric focussing which is an electrophoretic method that utilises a pH gradient contained within a polyacrylarnide gel.

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 intrayascular, intramuscular, subcutaneous, inhalation, oral, rectal, transdcrmal or intra-nasal administration.

Polvpcptide: 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 encompass any amino acid sequence and includc modified scqucnccs such as glycoprotcins. The term "polypcptide" covers 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 a fragment which exhibits at least one useful sequence in binding a receptor. The term "functional fragments ofapolypeptide" refers to all fragments ofapolypeptide that retain an activity of the polypeptide. Biologically functional peptides can also include fusion proteins, in which the pcptide of interest has bccn fused to anothcr peptide that does not decrease its desired activity.

Subcutaneous administration: Subcutaneous administration is administration of a substance to the subcutaneous layer of fat which is found between the dermis of the skin and the underlying tissue. Subcutaneous administration may be by an injection using a hypodcrmic ncedflc fitted, for cxample, to a syringc or a "pcn" type injcction dcvice. Other administration methods may be used for example microneedles. Injection with a hypodermic needle typically involves a dewee of pain on behalf of the recipient. Such pain may be masked by use of a local anaesthetic or analgesic. However, the usual method used to reduce the perceived pain of injections is to merely distract the subject immediately prior to and during the injection. Pain may be minimised by using a relatively small gauge hypodermic needle, by injecting a relatively small volume of substance and by avoiding excessively acidic or alkali compositions which may cause the subject to cxpcricnce a "stinging" sensation at thc injcction site. Compositions having a pH of between pH 4 and pH 10 are usually regarded as tolerably comfortable.

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 effective amount of a compound of the invention 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.

DETAILED DESCRIPTION

According to a first aspect of the invention, there is provided an analogue of glucagon which is: a compound comprising an amino acid sequence represented by formula (I) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa'3-Leu-Xaa'5-Xaa'6- Xaa17 -Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Asn-Xaa29-V (I) wherein V is selected from the group consisting of His, His-NH2, His-His, His-His-NH2, Gly-His, Gly-His-NH2, Lys-His, Lys-His-NH2, Gly-His-His, Gly-His-His-NH2, His-His-His and His-His-His-NH2; Xaa'° is selected from the group consisting ofTyr and Leu; Xaa'2 is selected from the group consisting of Lys, His and Arg; Xaa'3 is selected from the group consisting of Tyr, GIn and His; Xaa'5 is selected from the group consisting of Asp and Glu; Xaa'6 is selected from the group consisting of Glu, GIn and Ser; Xaa'7 is selected from the group consisting of Arg, His and Lys; Xaa'8 is selected from the group consisting of Arg and Lys; Xaa2° is selected from the group consisting of His and Gin; Xaa21 is selected from the group consisting of Glu, His and Asp; Xaa23 is selected from the group consisting of Tie and Val; Xaa24 is selected from the group consisting of Gin and Glu; and Xaa29 is selected from the group consisting of Thr and Gly; wherein -NI-I2 represents a C-terminal amide group; or a derivative of the compound; or a salt of the compound or the derivative.

The amino acid sequence of formula (I) above is shown with the N-terminus to the top left and the C-terminus to the bottom right.

Xaa'° is selected from the group consisting of Tyr and Leu. According to certain preferred embodiments, Xaa1° is Tyr. According to other embodiments Xaa1° is Leu.

Xaa'2 is selected from the group consisting of Lys, His and Arg. According to certain preferred embodiments Xaa'2 is Lys. According to other preferred embodiments Xaa'2 is His. According to other preferred embodiments Xaa12 is Arg.

Xaa'3 is selected from the group consisting of Tyr, GIn and His. According to certain preferred embodiments, Xaa'3 is Tyr. According to other preferred embodiments Xaa13 is His. According to other embodiments Xaa1 is Gin. According to certain preferred embodiments Xaa13 is selected from the group consisting of Tyr and His.

xaa'5 is selected from the group consisting of Asp and Glu. According to certain preferred embodiments, Xaa15 is Asp. According to other preferred embodiments Xaa15 is Glu.

Xaa'6 is selected from the group consisting of Glu, Gin and Ser. According to certain preferred embodiments, Xaa'6 is Scr. According to other embodiments, Xaa16 is GIn.

According to other embodiments, Xaa16 is Gln.

)(aa'' is selected from the group consisting of Arg, His and Lys. According to certain preferred embodiments, Xaa'7 is Arg. According to other preferred embodiments Xaa1' is Lys. According to other embodiments Xaa'7 is His. According to certain preferred embodiments Xaa17 is selected from the group consisting of Arg and Lys.

Xaa'8 is selected from the group consisting of Arg and Lys. According to certain preferred embodiments, Xaa is Arg. According to othcr preferred embodiments Xaa13 is Lys.

Xaa2° is selected from the group consisting of His and Gin. According to certain preferred embodiments, Xaa2° is His. According to other embodiments, Xaa2° is Gin.

Xaa2' is selected from the group consisting of Glu, His and Asp. According to certain preferred embodiments Xaa2' is Giu. According to other preferred embodiments Xaa2' is Asp. According to other embodiments Xaa2' is His. According to certain preferred embodiments Xaa2' is selected from the group consisting of Glu and Asp.

Xaa23 is selected from the group consisting of lie and Vai. According to certain preferred embodiments Xaa2 is Vai. According to other preferred embodiments Xaa23 is lie.

Xaa is selected from the group consisting of Gin and Giu. According to certain preferred embodiments Xaa24 is Gin. According to other preferred embodiments Xaa24 is Giu.

Xaa29 is selectcd from the group consisting of Thr and Giy. According to certain preferred embodiments Xaa29 is Thr. According to other preferred embodiments Xaa29 is Giy.

V is selected from the group consisting of His, His-N I12, His-His, His-His-N H2, Gly-His, (lily-His-NH2, Lys-His, Lys-His-NH2, Gly-His-His, Gly-His-His-NH2, His-His-His and His-His-His-NH2. According to certain preferred embodiments, V is His-His. According to other preferred embodiments, V is His-His-NH2. According to other preferred embodiments, V is Giy-His. According to other preferred embodiments, V is Giy-His-NH2. According to other embodiments, V is His. According to other embodiments, V is His-NH2. According to other embodiments, V is Lys-His. According to other embodiments, V is Lys-His-NH2. According to other embodiments, V is Giy-His-His.

According to other embodiments, V is Gly-His-His-NH2. According to other embodiments, V is His-His-His. According to other embodiments, V is His-His-His-NH2.

According to certain preferred embodiments, V is selected from the group consisting of His, His-NH2, His-His, His-His-NH2, Giy-His, and Giy-His-NH2.More preferably, V is selected from the group consisting of His-His, His-His-NH2, Gly-His and Gly-His-NH2. In certain embodiments, V is not His-NH2.

Where V is His-NH2, His-His-NH2 Lys-His-NH2 or Gly-His-NH2, the group -NH2 denotes the presence of a -C(O)NI-12 group at the C-terminus (in place of a carboxylic acid group).

In certain embodiments, the analogue is a compound which has a C-terminal amide group (i.e. -C(O)NH2, or a derivative of such a compound, or a salt of such a compound or such a derivative.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr and Xaa'6 is Ser. In another preferred group of glucagon analogues of the invention, Xaa1° is Tyr and Xaa2° is His. In another preferred group of glucagon analogues ofthe invention, Xaa'6 is 5cr and Xaa2° is His.

In onc preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa'6 is 5cr and Xaa2° is His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa16 is 5cr, and V is selected from the group consisting of His-His, His-His-NH2, Gly-His and Gly-His-NH2. In another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa2° is His, and V is selected from the group consisting of His-His, His-His-NH2, Gly-His and Gly-His-NH2. In another preferred group of glucagon analogues of the invention, Xaa'6 is 5cr, Xaa2° is His, and V is selected from the group consisting of His-His, His-His-NH2, Gly-His and Gly-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa16 is 5cr, Xaa2° is His, and V is selected from the group consisting of His-His, His-His-NH2, Gly-His and GIy-His-NI-12.

In one preferred group of glucagon analogues of the invention, Xaa13 is Tyr and/or Xaa' is Asp and/or Xaa'7 is Arg and/or Xaa'8 is Arg and/or Xaa2' is Glu and/or Xaa23 is \Tal. In one embodiment, Xaa13 is Tyr and Xaa15 is Asp. In another embodiment Xaa13 is Tyr and Xaa" is Arg. In another embodiment Xaa13 is Tyr and Xaa18 is Arg. In another embodiment Xaa1 is Tyr and Xaa2' is Glu. In another embodiment Xaa'3 is Tyr and Xaa23 is Va!. In another embodiment Xaa15 is Asp and Xaa'' is Arg. In another embodiment Xaa' is Asp and Xaa18 is Arg. In another embodiment Xaa15 is Asp and Xaa2' is Glu. In another embodiment Xaa' is Asp and Xaa23 is Val. In another embodiment Xaa'7 is Arg and Xaa18 is Arg. In another embodiment Xaa1' is Arg and Xaa21 is Glu. In another embodiment Xaa17 is Arg and Xaa2' is Va!. In another embodiment Xaa18 is Arg and Xaa2' is Glu. In another embodiment Xaa18 is Arg and Xaa23 is Val. In another embodiment Xaa21 is Glu and Xaa23 is Va].

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa'6 is Ser and Xaa2° is His. In another preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa16 is Ser and Xaa2° is His. In another preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa'2 is His, Xaa'6 is Ser and Xaa° is His. 10. 1

In one preferred group of glueagon analogues of the invention, Xaa is Tyr, Xaa is Tyr, Xaa'6 is Ser and Xaa2° is His. In another preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is His, Xaa'6 is Ser and Xaa2° is His.

In one preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa1 is Asp, Xaa16 is Ser and Xaa2° is His. In another preferred group of gueagon analogues of the invention, Xaa1° is Tyr, Xaa15 is Glu, Xaa16 is Ser and Xaa2° is His.

In one preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa16 is Ser, xaa17 is Arg and Xaa2° is His. In another preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa16 is Ser, Xaa17 is Lys and Xaa2° is His.

In one preferred group of glueagon analogues of the invention, XaaW is Tyr, Xaa16 is Ser, Xaa'8 is Arg and Xaa2° is His. In another preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa16 is 5cr, Xaa18 is Lys and Xaa2° is His.

bone preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'° is Ser, Xaa2° is His and Xaa2' is Glu. in another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa16 is Ser, Xaa2° is His and Xaa21 is Asp.

bone preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'6 is Set, Xaa2° is His and Xaa is Val. in another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, ç16 is Ser, Xaa2° is His and Xaa23 is ile.

In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'6 is Set, Xaa2° is His and Xaa24 is Gin. In another preferred group of giucagon analogues of the invention, XaaW is Tyr, Xaa'6 is Ser, Xa? is His and Xaa24 is (Mu.

bone preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'° is Set, Xaa2° is His and Xaa29 is Thr. In another preferred group of glucagon analogues of the invention, )(IO is Tyr, Xaa'6 is Ser, Xaa2° is His and Xaa is (My.

In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, is Asp, Xaa'6 is 5cr and Xaa2° is His. In another prcfcrrcd group of glucagon analogues of the invention, XaaW is Tyr, Xaa'3 is Tyr, Xaa'6 is Ser, Xaa'7 is Mg and Xad° is His. b another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, Xaa'6 is Ser, Xaa'8 is Arg and Xaa2° is His, in another preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa'3 is Tyr, Xaa16 is Set, Xaa2° is His and Xaa2' is (Mu. b another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, X&3 is Tyr, Xaa'6 is Set, Xaa2° is His and Xaa23 is Val. In another preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'5 is Asp, Xaa is Set, Xaa'7 is Arg and Xaa2° is His. In another preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa" I6 5)(18 is Mg and Xaa2° is His. In another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'5 is Asp, Xaa16 is Ser, Xaa2° is His and Xaa2' is Glu. in another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'5 is Asp, Xaa'6 is 5cr, Xaa2° is His and Xaz? is Val. In another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, 16 is Ser, Xaa'7 is Mg, Xaa'8 is Mg and X&° is His. In another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'6 is Set, Xaa'7 is Arg, Xaa2° is His and Xaa21 is Glu. In another preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'6 is Ser, Xaa'7 is Arg, Xaa2° is His and Xaa23 is Va]. In another preferred group ofg]ucagon analogues of the invention, Xaa'° is Tyr, Xaa16 is 5cr, Xaa18 is Arg, Xaa2° is His and Xaa2' is Glu. In anothcr prcfcrred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa16 is 5cr, Xaa18 is Arg, Xaa2° is His and Xaa23 is \Tal. In another preferred group of glueagon analogues of the invention, Xaa1° is Tyr, Xaa16 is Ser, Xaa2° is His, Xaa2' is Glu and Xaa23 is Val.

In one preferred group of g]ueagon analogues of the invention, Xaa1° is Tyr, Xaa16 is 5cr, Xaa2° is His, Xaa29 is Gly and V is selected from the group consisting of Gly-His and Gly-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa'3 is Tyr, Xaa15 is Asp, Xaa16 is Ser, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa2 is Val, Xaa24 is GIn, Xaa29 is Thr and V is His-His.

In onc preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa'2 is Arg, Xaa'3 is Tyr, Xaa15 is Asp, Xaa16 is 5cr, Xaa'7 is Arg, Xaa1 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val, Xaa24 is Gln, Xaa29 is Thr and V is His-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa'3 is His, Xaa15 is Asp, Xaa16 is Ser, Xaa'7 is Lys, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Va], Xaa24 is Gin, Xaa29 is Thr andY is His-His -NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa'3 is Tyr, Xaa15 is Asp, Xaa16 is Ser, Xaa'7 is Lys, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa2 is Val, Xaa24 is GIn, Xaa29 is Thr and V is Giy-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa'3 is Tyr, Xaa15 is Asp, Xaa16 is Ser, Xaa'7 is Arg, Xaa1 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val, Xaa24 is Gin, Xaa29 is Gly andY is Gly-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, XaaB is Tyr, Xaa'5 is Glu, Xaa'6 is Ser, Xaa'7 is Arg, Xaa18 is Lys, Xaa2° is His, Xaa21 is Glu, Xaa23 is lie, Xaa24 is Oh, Xaa29 is Thr and V is Gly-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa'3 is Tyr, Xaa15 is Asp, Xaa16 is Ser, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val, Xaa24 is Glu, Xaa29 is Thr and V is Gly-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, XaaB is Tyr, Xaa'5 is Asp, Xaa'6 is 5cr, Xaa' is Arg, Xaa'2 is Arg, Xaa2° is His, Xaa21 j Glu, Xaa2 is Val, Xaa24 is Glu, Xaa29 is Gly and V is Gly-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xa&2 is Lys, XaaB is Tyr, Xaa'5 is Asp, Xaa'6 is Ser, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Asp, Xaa23 is Val, Xaa24 is Glu, Xaa29 is Thr and V is His-His-NH2.

In one preferred group of giucagon analogues of the invention, Xaa1° is Tyr, Xa&2 is His, Xaa'3 is Tyr, Xaa15 is Asp, Xaa'6 is 5cr, Xaa" is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa2 is Val, Xaa24 is Gln, Xaa29 is Gly and V is Gly-His-NH2.

In one preferred group of giucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, IS -7 II 20 21 -2 Xaa is Asp, Xaa is Arg, Xaa is Arg, Xaa is His, Xaa is Gu and Xaa is Vat.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, 13-15-17-18-20-21-23 Xaa is Tyr, Xaa is Asp, Xaa is Arg, Xaa is Arg, Xaa is His, Xaa is Glu and Xaa is Val.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa'3 is Tyr, Xa&5 is Asp, Xaa17 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu and Xaa2 isVal.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa' is Asp, Xaa'6 is Glu, Xaa17 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Oh and Xaa23 is Va!.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa'5 is Asp, Xaa16 is Ser, Xaa17 is Arg, Xaa13 is Arg, Xaa2° is His, Xaa21 is Glu and Xaa23 is Val.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa' is Asp, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Gu, Xaa23 is Val and Xaa24 is Gln.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa'5 is Asp, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa2 is Va] and Xaa24 isGlu.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa' is Asp, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val and Xaa29 is Gly.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa' is Asp, Xaa'7 is Arg, Xaa12 is Arg, Xaa2° is His, Xaa2' is Gu, Xaa23 is Val and Xaa29 is Thr.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa'5 is Asp, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Va] and V is His-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa13 is Tyr, Xaa' is Asp, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val andY is Gly-His.

bone prekrred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa17 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is (flu, Xaa23 is Val and V is His.

bone prekrred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, Xaa15 is Asp, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val and V is His-MI2.

In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa17 is Arg, Xaa" is Arg, Xaa2° is His, Xaa2' is (flu, Xaa23 is Val and V is His-H2.

bone preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa17 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is (flu, Xaa23 is Val and V is Gly-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, is Asp, Xaa'6 is Scr, Xaa'7 is Mg, Xaa'8 is Mg, Xaa2° is His, Xaa2' is 0iu, Xaa24 is GIn and Xaar is VaL In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa16 is Glu, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa24 is Glu and Xaa is VaL bone preferred group of glucagon analogues of the invention, XaaW is Tyr, Xaa'2 is Lys, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa'6 is Scr, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val, Xaa24 is Gln, Xaa is Thr and v is His-His.

In one preferred group of glucagon analogues of the invention, Xaa'° is Tyr, Xaa'2 is Lys, Xaa'3 isTyr,Xaa'5 isAsp,Xaa6 is Scr,Xaa'7isArg,Xaa'8 isArg,Xaa2° isHis,Xaa2' is G1u,Xaa23isVa1,Xaa24isGln,XaaisG1yandVisG1y-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa' is Tyr, Xaa'5 is Asp, Xaa'6 is Glu, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Va! , Xaa24 is Glu, Xaa29 is Gly and V is Gly-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa'3 is Tyr, Xa&5 is Asp, Xaa16 is Ser, Xaa'7 is Arg, Xaa" is Arg, Xaa2° is His, Xaa21 is Glu, Xaa2 is Val, Xaa24 is Gin, Xaa29 is Thr and V is His-His.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa' is Tyr, Xaa'5 is Asp, Xaa'6 is Scr, Xaa' is Arg, Xaa'2 is Arg, Xaa2° is His, Xaa21 is Giu, Xaa2 is Val, Xaa24 is Gin, Xaa29 is Thr and V is His-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Arg, Xaa' is Tyr, Xaa'5 is Asp, Xaa'6 is Ser, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Giu, Xaa23 is Val, Xaa24 is Gin, Xaa29 is Thr and V is His-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, 15-16 17-18-20--21-Xaa is Tyr, Xaa is Asp, Xaa is Giu, Xaa is Arg, Xaa is Arg, Xaa is His, Xaa is Glu, Xaa2 is Val, Xaa24 is Glu, Xaa29 is Thr and V is His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xaa12 is Lys, Xaa' is Tyr, Xaa'5 is Asp, Xaa'6 is G!u, Xaa'7 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val, Xaa24 is Glu, Xaa29 is Giy and V is His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xa&2 is His, Xaa' is Tyr, Xaa'5 is Asp, Xaa'6 is Scr, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Giu, Xaa2 is Val, Xaa24 is Giu, Xaa29 is Giy and V is Gly-His-NH2.

In one preferred group of glucagon analogues of the invention, Xaa1° is Tyr, Xa&2 is His, Xaa'3 is Tyr, Xaa15 is Asp, Xaa'6 is Scr, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Giu, Xaa2 is Val, Xaa24 is Gin, Xaa29 is Giy and V is Gly-His-NH2.

According to certain embodiments, where Xaa'2 is Lys, Xaa'3 is not His. According to certain embodiments, where Xaa13 is His, Xaa17 is not His.

Glucagon analogues of thc invention include, but are not limited to, the compounds set out in Figure 1 or any of the Examples, or a derivative of such a compound, or a salt of such a compound or such a derivative.

The glucagon analogues of the invention have amino acid sequences that differ from the sequence of native glucagon in that they containing a His residue at position 30 and/or position 31. The glucagon analogues may comprise an amino acid sequence having additional changes compared with the native glueagon sequence. For example the glucagon analogues of the invention may contain 1,2,3,4, 5,6,7,8,9, 10, 11 or 12 amino acid residues that are substitutions of amino acid residues present in the native glucagon sequence. Prekrably the glucagon analogues of the invention contain up to 7 amino add residues that are substitutions of amino acid residues present in the native glucagon sequence, more preferably from 3 to 7 amino acid residues that arc substitutions of amino acid residues present in the native glucagon sequence.

According to certain preferred embodiments, the glucagon analogue is a compound consisting of an amino acid sequence represented by formula (1), or a derivative of the compound, or a salt of the compound or the derivative. The preferences for amino acid residues and combinations of amino acid residues set out above are also preferred for such glucagon analogues. In certain embodiments, the glucagon analogue is a compound consisting of an amino acid sequence represented by formula (I). In certain embodiments, the glucagon analogue is a derivative of such a compound. In certain embodiments, the glucagon analogue is a salt of such a compound or derivative.

Glucagon analogues of the invention may be pmduced by recombinant methods well known in the art or alternatively they may be produced by synthetic methods, again well knownintheart.

Derivatives A glucagon analogue of the invention may comprise the structure of formula (I) modified by well-known processes including amidation, glycosylation, carbamylation, acylation, for example acetylation, sulfation, phosphorylation, cyclization, I ipidization and PEGylation.

The structure of formula (I) may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

A glucagon analogue of the invention may be a fusion protein, whereby the structure of formula (I) is fused to another protein or polypeptide (the fusion partner) using recombinant methods known in the art. Alternatively, such a fusion protein may be synthetically synthesized by any known method. Such a fusion protein comprises the structure of formula (I). Any suitable peptide or protein can be used as the fusion partner (e.g., serum albumin, carbonic anhydrase, glutathione-S-transferase or thioredoxin, etc.).

Preferred fusion partners will not have an adverse biological activity in vivo. Such fusion proteins may be made by linking the carboxy-terminus of the fusion partner to the amino-terminus of the structure of formula (I) or vice versa. Optionally, a clcavable linker may be used to link the structure of formula (I) to the fusion partner. A resulting cleavable fusion protein may be cleaved in vivo such that an active form of a compound of the invention is released. Examples of such cleavable linkers include, but are not limited to, the linkers D-D-D-D-Y, G-P-R, A-G-G and H-P-F-H-L, which can be cleaved by enterokinase, thrombin, ubiquitin cleaving enzyme and renin, respectively. See, e.g., U.S. Patent No. 6,410,707, the contents of which are incorporated herein by reference.

A glucagon analogue of the invention may be a physiologically functional derivative of the structure of formula (I). The term "physiologically functional derivative" is used herein to denote a chemical derivative of a compound of formula (I) having the same physiological function as the corresponding unmodified compound of formula (I). For example, a physiologically functionally derivative may be convertible in the body to a compound of formula (I). According to the present invention, examples of physiologically functional derivatives include esters, amidcs, and carbamatcs; preferably esters and amidcs.

Pharmaceutically acceptable esters and amides of the compounds of the invention may comprise a C120 alkyl-, C220 alkenyl-, C540 aryl-, C510 ar-C120 alkyl-, or amino acid-ester or -amide attached at an appropriate site, for example at an acid group. Examples of suitable moieties are hydrophobic substituents with 4 to 26 carbon atoms, preferably 5 to 19 carbon atoms. Suitable lipid groups include fatty acids (e.g. lauroyl (C12F123), palmityl (C15H31), olcyl (C15H29) or stcaryl (C17H35)) and bile acids (e.g. cholatc or dcoxycholatc).

Methods for lipidization of sulthydryl-containing compounds with fatty acid derivatives are disclosed in U.S. Patent No. 5,936,092; U.S. Patent No. 6,093,692; and U.S. Patent No. 6,225,445, the contents of which are incorporated herein by reference. Fatty acid derivatives of a compound of the invention comprising a compound of the invention linked to fatty acid via a disulfidc linkage may be used for delivery ofa compound of the invention to neuronal cells and tissues. Lipidisation markedly increases the absorption of the compounds relative to the rate of absorption of the corresponding unlipidised compounds, as well as prolonging blood and tissue retention of the compounds.

Moreover, the disulfidc linkage in a lipidised derivative is relatively labile in the cells and thus facilitates intracellular release of the molecule from the fatty acid moieties. Suitable lipid-containing moictics arc hydrophobic substitucnts with 4 to 26 carbon atoms, preferably 5 to 19 carbon atoms. Suitable Suitable lipid groups include fatty acids (e.g. lauroyl (C12H23), palmityl (C15H31), olcyl (C15H29) or stcaryl (C17H35)) and bile acids (e.g. cholate or deoxycholate).

Cyclization methods include cyclization through the formation of a disulfide bridge and head-to-tail cyclization using a cyclization resin. Cyclizcd pcptidcs may have enhanced stability, including increased resistance to enzymatic degradation, as a result of their conformational constraints. Cyclization may in particular be expedient where the uncyclized peptide includes an N-terminal cysteine group. Suitable cyclized peptides include monomeric and dimcric head-to-tail cyclizcd structures. Cyclized pcptidcs may include one or more additional residues, especially an additional cysteine incorporated for the purpose of formation of a disulfide bond or a side chain incorporated for the purpose of resin-based cyclization.

A glucagon analogue of the invention may be a PEGylated structure of formula (I).

PEGylated compounds of the invention may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337, the contents of which are incorporated herein by reference).

Chemical moieties for derivitization of a compound of the invention may also be selected S from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol eopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. A polymer moiety for derivatisation of a compound of the invention may be of any molecular weight, and may be branched or unbranched. For ease in handling and manufacturing, the preferred molecular weight of a polyethylene glycol for derivatisation of a compound of the invention is from about I kDa to about 100 kDa, the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight. Polymers of other molecular weights may be used, depending on the desired therapeutic profile, for example the duration of sustained release desired, the effects, if any, on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog. For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

In certain embodiments, the glucagon analogue of the invention is not a derivative.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. Such complexes are known as "solvates". For example, a complex with water is known as a "hydrate". It will be understood by the skilled person that the invention also encompasses solvates of the compounds of formula (I), of derivatives of the compounds, and of salts of the compounds and derivatives.

Salts of compounds of formula (I) which are suitable for use in medicine are those wherein a counterion is pharmaceutically acceptable. However, salts having non-pharmaceutically acceptable counterions are within the scope of the present invention, for example, for use as intermediates in the preparation of the compounds of formula (I) and pharmaceutically acceptable salts and/or derivatives thereof S Suitable salts according to the invention include those formed with organic or inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed with hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycollic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzencsulfonic, and isethionic acids. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutical acceptable salts.

Pharmaceutically acceptable salts with bases include ammonium salts, ailcali metal salts, for example potassium and sodium salts, alkaline earth metal salts, for example calcium and magnesium salts, and salts with organic bases, for example dicyclohexylamine and N-mcthyl-D-glucominc.

Biological activity The glucagon analogues of the invention are active at the human glucagon receptor, and are glucagon receptor agonists. This may be assessed by, for example, an in vitro or cellular binding assay or by a reporter assay. Preferably, the glucagon analogues show binding to the human glucagon receptor with an affinity of at least 11200,000th, 1/20,OOO', 1/10000th 1/5000t11 1111000th or 1/400t1 of the affinity of human glucagon. More preferably, the analogues show affinity similar to that of human glucagon. Still more preferably, glucagon analogues of the invention show binding to the human glucagon-receptor with an affinity of at least 2-fold, 5-fold, 10-fold the binding affinity of human glucagon. Methods of assessing activity of analogues at the glucagon receptor are well known. For example, Thermo Scientific (Lafayette, CO, USA) market an in vitro glucagon receptor assay. The activity of the glucagon analogues of the invention at the glucagon receptor is preferably longer lasting in vivo than native glucagon.

Preferably, the glucagon analogues of the invention fulfil some or more preferably all, of the following criteria.

1) Sustained bioactivity at the human glucagon receptor resulting in enhancement of energy expenditure.

2) High solubility in aqueous solution at pH 5 to allow an effective dose to be administered in a low volume injection (thereby resulting in lower pain of injection). Solubility may be easily assessed by simple in vitro tests.

3) Long period of activity in vivo (as assessed in humans or an animal model) so as to permit injections no more frequently than daily and preferably no more than twicc, or morc prcfcraNy no more than once a wcck, whilst still producing acceptable therapeutic or cosmetic benefits.

4) Good weight loss (as assessed in human subjects or an animal model).

5) Low antigenicity in humans. This may be assessed in humans or animal models (in particular mice which have been experimentally reconstituted with a human immune system so as to mimic human antibody repertoire) or predicted using predictivc software such as that incorporating the "antigenic index" algorithm ((Janieson & Wolf (1 988) Comput. AppI. Biosci. 40): 181-6), or the PREDITOP algorithm (Pcllcqucr & Westhof, (1993) J. Mol. Graph. 1 1(3):204- 10, or using the methods of Kolaskar & Tongankar (1990) FEBS Leu.

10:276(1-2): 172-4, the contents of which are incorporated herein by reference).

In addition to having activity at the gucagon receptor, glucagon analogues of the invention may also have activity at the glucagon-like peptide 1 (GLP1) receptor. In other words, analogues of the invention may be GLP-1 receptor agonists as well as glucagon receptor agonists. GLP-1 is derived from the transcription product of the proglucagon gene. The biologically active forms of OLPI arc truncated forms known as GLP-1(?3?) and GLP-17 36)-NH2. GLP-1 is produced in vivo in the intestinal L cell in response to the presence of nutrients in the lumen of the gut. Once in the circulation, native GLP-1 has a half-life of only a few minutes in humans due to rapid degradation by the enzyme dipeptidyl peptidase. GLP-1 posscsscs a number of physiological functions including incrcasing insulin secretion from the pancreas in a glucose-dependent manner, decreasing glucagon secretion from the pallcreas, thhibiting gastric emptying and decreasing food intake by increasing satiety. Increased insulin secretion leads to a decrease in circulating glucose eon ccntration.

Zinc & solubility The glucagon analogues of the invention preferably have enhanced solubility at pH 5.

Enhanced solubility at pH 5 may be provided by incorporating histidine residues in the glucagon analogues of the invention. Histidine is unique among naturally occurring amino acids in That it is not charged at pH 7.4 (i.e. under physiological conditions in the circulation or subcutaneously following subcutaneous injection), but that it is fully charged at pH 5 since the p1 of the NH side chain group of histidine is about 6.0. The inclusion of histidine residues in the glucagon analogues of the invention therefore increases solubility at pH 5 which is a desirable feature. Histidine residues also bring an additional advantage in that when the analogue is injected subcutaneously, the solubility falls and this leads to subcutaneous precipitation of peptide. This is unexpected because in vitro zinc precipitation of His-containing peptidcs (as used for cxample in the purification of insulin) is typically slower and not expected to be sufficiently rapid in vivo to prevent dispersion of thc subcutaneous precipitate. The precipitate will resolubilise over time and this will produce an advantageous slow-release effect. The inclusion of histidine residues is especially advantageous wherein the glucagon analogue is to be formulated into a pharmaceutical composition containing zinc ions. This is because at pH 7.4 but not at pH zinc ions co-ordinate with histidine residues and result in a further reduction in solubility which can contribute to increased precipitation at a subcutaneous injection site, or which can contribute to increased stability of the precipitate. A zinc-containing precipitate will more gradually re-dissolve because the solubilisation is dependent on the zinc washing out of the injection site into the circulation and/or surrounding tissue fluid, increasing the longevity of the release into the circulation and decreasing the frequency of injections needed to sustain a useful biological effect.

The glucagon analogues of the invention contain a His residue at position 1, as found in native glucagon. In addition, the glucagon analogues of the invention contain His at positions 30 and/or 31, in contrast with native glucagon. . The glucagon analogues of the invention may also contain additional non-native histidine residues, at positions 12, 13, 17, and/or 21. Preferably, the glucagon analogues contain His at position 20, in contrast with native glueagon which contains Gin at that position. In certain embodiments, the glucagon analogues of the invention comprise 2,3,4,5 or 6 His residues (i.e. 1,2,3,4 or non-native His residues), preferably 3, 4 or S His residues (i.e. 2, 3 or 4 non-native His residues). Pharmaceutical compositions containing the glucagon analogues of the invention preferably contain zinc ions (preferably at a molar ratio of 1:4, 1:2, 1:1, 2:1 or 4:1 of zinc ions to glucagon analogue, or at a ratio which is a range between any two of the whole number ratios given immediately above).

According to certain embodiments of various aspects of the invention, especially embodiments relating to weight loss, obesity, carbohydrate metabolism and diabetes, glucagon analogues of the invention have one, several or all of the following features: A) Sufficient solubility between pH 4 and p1-IS to permit an effective dose to be administered in a volume of less than imI, less than 0.Sml or less than 0.3m1.

B) Activation of cAMP signaling in cells over-expressing the human glucagon receptor, C) One, several or all of the further ito 5 features listed above.

Conditions The invention also provides a glucagon analogue of the invention, or a pharmaceutical composition comprising the glucagon analogue of the invention, for use as a medicament.

The invention also provides a method of treating or preventing a disease or disorder or other non-desired physiological state in a subject comprising administration of a therapeutically effective amount of a glucagon analogue of the invention or of a pharmaceutical composition of the invention. Preferably the glucagon analogue or pharmaceutical composition is administered subcutaneously.

According to certain embodiments the disease or disorder or other non-desired physiological state is obesity or diabetes. Accordingly, the invention also provides a method for treating obesity or diabetes in a subject comprising administering to a subject a therapeutically effective amount of a glucagon analogue of the invention or of a pharmaceutical composition comprising the glucagon analogue of the invention.

According to certain embodiments the disease or disorder or other non-desired physiological state may be being the physiological state of being overweight.

The subject to whom the compound is administered may be overweight, for example, obese. Alternatively, or in addition, the subject may be diabetic, for example having insulin resistance or glucose intolerance, or both. The subject may have diabetes mellitus, for example, the subject may have Type IT diabetes. The subject may be overweight, for example, obese and have diabetes mellitus, for example, Type II diabetes.

In addition, or alternatively, the subject may have, or may be at risk of having, a disorder in which obesity or being overweight is a risk factor. Such disorders include, but are not limited to, cardiovascular disease, for example hypertension, atherosclerosis, congestive heart failure, and dyslipidcmia; strokc; gallbladder disease; ostcoarthritis; sleep apnca; reproductive disorders for example, polycystic ovarian syndrome; cancers, for example breast, prostate, colon, cndomctrial, kidney, and esophagus cancer; varicose veins; acanthosis nigricans; eczema; exercise intolerance; insulin resistance; hypertension hypercholesterolemia; cholithiasis; osteoarthritis; orthopedic injury; insulin resistance, for example, type 2 diabetes and syndrome X; and thromboembolic disease (see Kopelman, Nature404:635-43; Rissancn ctal., British Med. J 301, 835, 1990).

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

Other diseases or disorders associated with obesity are birth defects, maternal obesity being associated with increased incidence of neural tube defects, carpal tunnel syndrome (CTS); chronic venous insufficiency (CVI); daytime sleepiness; deep vein thrombosis (DYT); end stage renal disease (ESRD); gout; heat disorders; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecologic complications; pancreatitis; as well as abdominal hernias; acanthosis nigricans; endocrine abnormalities; chronic hypoxia and hypcrcapnia; dermatological effects; elephantitis; gastroesophageal reflux; heel spurs; lower extremity edema; mammegaly which causes 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, for example abdominal panniculitis with frequent panniculitis, impeding walking, causing frequent infections, odors, clothing difficulties, low back pain; musculoskclctal disease; pseudo tumor ccrcbri (or benign intracranial hypertension), and sliding hiatil hernia.

According to certain embodiments the disease or disorder or other non-desired physiological state may be being of a non-desired weight despite not being obese or overweight. The subject may be of normal weight (this includes but is not limited to subjects who were previously overweight or obese and who wish to prevent a retum to an unhealthy weight). A subject may be a subject who desires weight loss, for example female and male subjects who desire a change in their appearance. In some cases where the subject is of a normal weight, aspects of the invention may relate to cosmetic treatment rather than to therapeutic treatment.

The invention also provides a method of increasing the energy expenditure of a subject, enhancing insulin release in a subject, and!or improving carbohydrate metabolism in a subject, comprising administration of a therapeutically effective amount of a glucagon analogue of the invention, or of a pharmaceutical composition comprising the glucagon analogue of the invention. Such methods may relate to treating subjects having a pre-diabetic state such as insulin insensitivity or pre-diabetes.

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 one aspect, the method of the invention involves manipulation of the arcuate circuitry that alter food intake coordinately and reciprocally alter energy expenditure. Energy expenditure is a result of cdllular metabolism, protcin synthcsis, metabolic ratc, and calorie utilization. Thus, in this aspect of the invention, administration of a glucagon analogue of the invention results in increased energy expenditure, and decreased efficiency of calorie utilization.

The increase in energy expenditure may manifest as a lessening of the normal reduction in energy expenditure seen Ibilowing reduced food intake, or it may manifest as an absolute increase in energy expenditure for example by the promotion of increased physical activity levels or by an increase in the basal metabolic rate.

The invention also provides a method lbr improving a lipid profile in a subject comprising administration of a therapeutically effective amount of a glucagon analogue of the invention, or of a pharmaceutical composition comprising the glueagon analogue of the invention. The invention also provides a method for alleviating a condition or disorder that can be alleviated by reducing nutrient availability comprising administration of a therapeutically effective amount of a glucagon analogue of the invention, or of a pharmaceutical composition comprising the glucagon analogue of the invention.

A glucagon analogue of the invention may be used for weight control and treatment, for example reduction or prevention of obesity, in particular any one or more of the Slowing: preventing and reducing weight gain inducing and promoting weight loss; and reducing obesity as measured by the Body Mass Index. A glucagon analogue of the invention may be used in maintaining any one or more of a desired body weight, a desired Body Mass Index, a desired appearance and good health.

The present invention may also be used in treating, prevention, ameliorating or alleviating conditions or disorders caused by, complicated by, or aggravated by a relatively high nutrient availability. The term "condition or disorder which can be alleviated by reducing caloric (or nutrient) availability" is used herein to denote 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 arc insulin resistant, glucose intolerant, or have any form of diabetes mellitus, for example, type 1,2 or gestational diabetes, can also benefit from methods in accordance with the prcscnt invention.

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

As discussed above, glucagon analogues of the invention may also have activity against the GLP-1 receptor. Accordingly, the invention provides a method of reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, enhancing insulin release in a subject and/or improving carbohydrate tolerance in a subject, comprising administration of a therapeutically effective amount of an analogue of the invention, or of a pharmaceutical composition comprising the analogue.

J. Cereb. Blood Flow Metab. 2011 Apr13 (Teramoto Set al) discusses the use of both GLP-1 and exendin-4 to confer cardioprotection after myocardial infarction, and demonstrates that exendin-4 may be used to provide neuroprotection against cerebral ischemia-reperfiision injury. The study showed that mice receiving a flnsvenous injection of exendin-4, after a 60-minute focal cerebral ischemia showed significantly reduced infarct volume and improved functional deficit as wcfl as suppressed oxidative stress, inflammatory response, and cell death after reperfusion. The study provided evidence that the protective effect of exendin-4 is mediated through increased intracellular cAMP levels and suggested that Exendin-4 is potentially useful in the treatment of acute ischemic stroke.

Accordingly, the invention also provides a method of providing cytoprotection in a subject, such as providing cardiac protection, providing neuroprotection and/or treating or preventing neurodegeneration, comprising administration of a therapeutically effective amount of a glucagon analogue of the invention, or of a pharmaceutical composition comprising the glucagon analogue of the invention.

In certain embodiments the disease or disorder or other non-desired physiological state which the glueagon analogue may be used to treat or prevent is neurodegeneration. Such neurodegeneration maybe caused by apoptosis, necrosis or loss of function of neuronal cells, preferably in the CNS. Neurodegeneration treated or prevented may be that following a brain injury (for example following physical trauma or following a non-traumatic injury such a stroke, tumour, hypoxia, poisoning, infection, ischemia, eneephalopathy or substance abuse.). Alternatively or additionally, neurodegeneration may be prevented or treated in a subject having (or diagnosed as having a predisposition to) a neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, Gehrig's disease (Amyotrophie Lateral Sclerosis), Huntington's disease, Multiple Sclerosis, other demyelination related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS-associated dementia, other dementias, cerebral vasculitis, epilepsy, Tourette's syndrome, Guillain Bane Syndrome, Wilson's disease, Pick's disease, neuroinflammatory disorders, encephalitis, encephalomyelitis, meningitis, other central nervous system infections, prion diseases, cerebellar ataxias, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedrich's ataxia, ataxia teangiectasia, spinal dysmyotrophy, progressive supranuclear palsy, dystonia, muscle spasticity, tremor, retinitis pigmentosa, striatonigral degeneration, mitochondrial encephalomyopathies, neuronal ceroid lipofuscinosis. Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Gehrig's disease (Amyotrophic Lateral Sclerosis) and Huntington's disease. In such circumstances the treatment wodd be regarded as ncuroprotcctivc. According to certain preferred embodiments, the treatment is neuroprotective following cerebral ischemia or neuroprotective in a subject having a neurodegenerative disease or diagnosed as having a predisposition to a neurodegenerative disease.

According to other embodiments the disease or disorder or other non-desired physiological state is cardiac degeneration (in particular myocardial degeneration by apoptosis, necrosis or loss of function of myocardial cells), in which case the glucagon analogue or pharmaceutical composition provides cardiac protection. According to certain prcfencd embodiments that treatment is protective of myocardial function following myocardiac infarction.

The invention also provides a glucagon analogue of the invention, or a pharmaceutical composition comprising the glucagon analogue of the invention, for use in the treatment of obesity or diabetes.

The invention also provides a glucagon analogue of the invention or a pharmaceutical composition comprising the glucagon analogue of the invention for use in increasing energy expenditure of a subject, enhancing insulin release in a subject, improving carbohydrate tolerance in a subject and/or improving carbohydrate metabolism in a subject. Such use may relate to treating subjects having a pre-diabetic state such as insulin insensitivity or pre-diabctes.

The invention also provides a glucagon analogue of the invention or a pharmaceutical composition comprising the glucagon analogue of the invention for use in the reduction of appetite in a subject, use in the reduction of food intake in a subject, use in the reduction of calorie intake in a subject, use in enhancing insulin release in a subject, and/or use in improving carbohydrate tolerance in a subject.

The invention also provides a glucagon analogue of the invention, or a pharmaceutical composition comprising the glucagon analogue of the invention, for use as a cytoprotective agent (e.g. in treating or preventing neurodegeneration, providing neuroprotection and/or providing cardiac protection). For example, the glucagon analogue or pharmaceutical composition may be for usc in myocardial protection in a subject following myocardial infarction, or for use in neuroprotection in a subject following cerebral ischemia or stroke, or for use in neuroprotection in a subject having a chronic neurodegenerative disease.

Various features of neuroprotective or cardioprotective use of the glucagon analogue or pharmaceutical composition maybe as outlined above in relation to methods of the invention.

In the case of neuroprotection the subject may have experienced previously a brain injury, stroke or other event causing cerebral ischcmia. Alternatively, the subject may have or have been diagnosed with a predisposition to develop a chronic neurodegenerative disease.

In the case of cardioprotection the subject may have experienced previously an event causing myocardial ischemia such as a myocardial infarction and angina. According to some embodiments a glucagon analogue or pharmaceutical composition of the invention may be administered as soon as possible after the subject has experienced a suspected myocardial infarction. According to certain embodiments a glucagon analogue or pharmaceutical composition of thc invention may be administered as soon as possible after the subject has experienced as suspected stroke.

The invention also provides use of a glucagon analogue of the invention for the manufacture of a medicament for the treatment of obesity or diabetes, of a subject who may be as described above in reference to other aspects of the invention.

The invention also provides use of a glucagon analogue of the invention for the manufacture of a medicament for increasing energy expenditure in a subject, for enhancing insulin release in a subject, for improving carbohydrate tolerance in a subject and/or improving carbohydrate metabolism in a subject. Such use may relate to treating subjects with a pre-diabetic state such as insulin insensitivity or pre-diabetes.

The invention also provides use of a glucagon analogue of the invention for the manufacture of a medicament for the reduction of appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, enhancing insulin release in a subject, and/or use in improving carbohydrate tolerance in a subject.

The invention also provides usc of a glucagon analogue of the invention for the manufacture of a medicament for providing cytoprotection (e.g. preventing or treating neurodegeneration, providing neuroprotection and/or providing cardiac protection) of a subject who may be as described above in reference to other aspects of the invention.

According to certain embodiments the glucagon analogue or pharmaceutical composition is to be administered parentally. According to other embodiments the glucagon analogue or pharmaceutical composition is to be administered subcutaneously, intravenously, intramuscularly, intranasally, transdermally or sublingually. According to other embodiments the glucagon analogue or pharmaceutical composition is to be administered orally.

According to the present invention, a glucagon analogue of the invention is preferably used in the treatment of a human. However, while the glueagon analogues of the invention will typically be used to treat human subjects they may also be used to treat similar or identical conditions in other vertebrates for example other primates; farm animals for example swine, cattle and poulfry; sport animals for example horses; or companion animals for example dogs and eats.

Compositions While it is possible for the active ingredient to be administered alone, it is preferable for it to be present in a pharmaceutical formulation or composition. Accordingly, the invention provides a pharmaceutical formulation comprising a glucagon analogue of the invention together with a pharmaceutically acceptable excipient and optionally other therapeutic ingredients. According to certain preferred embodiments the pharmaceutical composition is present in a syringe or other administration device for subcutaneous administration to humans. According to certain prefened embodiments the composition has a pH of less than 5 prior to administration and the composition comprises zinc ions. Pharmaceutical compositions of the invention may take the form of a pharmaceutical formulation as described below.

The pharmaceutical formulations according to the invention include those suitable for oral, parcntcral (including subcutaneous, intradcrmal, intramuscular, intravenous, and intra-articular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators), rectal and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the canier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Formulations of the present invcntion suitable for oral administration may be prcscnted as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a foil-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation trcatises, c.g., Remington c Phannaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal fParenteraI Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988, the contents of which are incorporated herein by reference.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be preparcd by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a bindcr, lubricant, inert diluent, lubricating, surfacc active or dispersing agcnt.

Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingrcdient thercin. The prcscnt gucagon anaogucs can, for example, bc administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising the present glucagon analogues, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

The present glucagon analogues can also be administered liposomally.

Preferably, compositions according to the invention are suitable for subcutaneous administration, for cxamplc by injection. According to certain cmbodimcnts thc composition may contain metal ions, for example copper, iron, aluminium, zinc, nickel or cobalt ions. The presence of such ions may limit solubility and thus delay absorption into the circulatory system from the site of subcutaneous administration. In a particularly preferred embodiment, the composition contains zinc ions as described in more detail above.

Exemplary compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. The analogues of the invention can also be delivered through the oral cavity by sublingual and/or buccal administration. Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used.

Exemplary compositions include those formulating the present compound(s) with fast dissolving diluents such as mannitol, lactose, sucrose andior cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG). Such formulations can also include an excipient to aid mucosal adhesion such as hydroxypropyl cellulose (H PC), hydroxypropyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), malcic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g. Carbopol 934).

Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and s&utes which render the formulation isotonic with the blood of the intended recipient; aud aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to usc. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono-or diglycerides, and fatty acids, including olcic acid, or Cremaphor. An aqueous carrier may be, for example, an isotonic buffer solution at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, for example from 3.5 to 6.0, for example from 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers. The composition preferably does not include oxidizing agents and other compounds that are known to be deleterious to glueagon analogues of the invention and related molecules. Excipicnts that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Exemplary compositions for nasal aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art. Conveniently in compositions for nasal aerosol or inhalation administration the compound of the invention is delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoro-methanc, trichlorofluoromcthane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, for example lactose or starch. In one specific, non-limiting example, a compound of the invention is administered as an aerosol from a metered dose valve, through an aerosol adapter also known as an actuator. Optionally, a stabilizer is also included, and/or porous particles for deep lung delivery are included (e.g., see U.S. Patent No. 6,447,743).

Formulations for rectal administration may be presented as a retention enema or a suppository with the usual carriers such as cocoa buffer, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acaeia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerine or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).

Preferred unit dosage formulations are those containing an effective dose, as hereinbefore recited, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The glucagon analogues of the invention may also be suitably administered as sustained-release systems. Suitable examples of sustained-release systems of the invention include suitable polymeric materials, for example semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mierocapsules; suitable hydrophobic materials, for example as an emulsion in an acceptable oil; or ion exchange resins; and sparingly soluble derivatives of the compound of the invention, for example, a sparingly soluble salt.

Sustained-release systems may be administered orally; rectally; parenterally; intraeistemally; intravaginally; intraperitoneally; topically, for example as a powder, ointment, gel, drop or transdermal patch; bucally; or as an oral or nasal spray.

Preparations for administration can be suitably formulated to give controlled release of glucagon analogues of the invention. For example, the pharmaceutical compositions may be in the form of particles comprising one or more of biodegradable polymers, polysaccharidejellifying and/or bioadhesive polymers, amphiphilic polymers, agents capable of modifying the interface properties of particles of the glucagon analogues.

These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance, see U.S. Patent No. 5,700,486, the contents of which are incorporated by reference.

Controlled release of glucagon analogues of the invention may also be achieved by the use of pharmaceutical compositions comprising the glucagon analogue and zinc ions. As described above, at pH 7.4 but not at pH 5 zinc ions co-ordinate with histidine residues and result in increased precipitation at a subcutaneous injection site. A zinc-containing precipitate will more gradually re-dissolve because the solubilisation is dependent on the zinc washing out of the injection site into the circulation and/or surrounding tissue fluid, increasing the longevity of the release into the circulation. The use of a controlled release composition is prefened for indications such as the treatment of obesity and/or diabetes, where maximising the time period between injections is desirable. However, for indications such as providing neuroprotection or cardiac protection (e.g. following suspected myocardial infarction or stroke), where it is desired to achieve a therapeutic plasma concentration of the glucagon analogue in as short a time period as possible, an immediate release formulation will be preferred. In such eases, a dosage regime comprising administration of a dose of an immediate release formulation of the glucagon analogue (i.e. as soon as possible after suspected myocardial infarction or stroke) and subsequent administration of a dose of a controlled release formulation of the glucagon analogue may be preferred.

A glucagon analogue of the invention may be delivered by way of a pump (see Langer, supra; Sefton, CRC Crit Ref Biotned. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N Engi. J Med. 321:574, 1989) orby a continuous subcutaneous infusion, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by decreases in total body weight or ratio of fat to lean mass, or by other criteria for measuring control or prevention of obesity or prevention of obesity-related conditions, as are deemed appropriate by the practitioner. Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533, 1990) which is incorporated herein by reference. In another aspect of the disclosure, glucagon analogues of the invention are delivered by way of an implanted pump, described, for example, in U.S. Patent No. 6,436,091; U.S. Patent No. 5,939,380; U.S. Patent No. 5,993,414, the contents of which arc incorporatcd hcrcin by rcfcrcncc.

Implantabic drug infusion dcviccs arc uscd to providc paticnts with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Essentially such device maybe categorized as either active or passive. A glucagon analogue of the present invention may be formulated as a depot preparation. Such a long acting depot formulation can be administered by implantation, for example subcutaneously or intramuscularly; or by intramuscular injection. Thus, for example, the glucagon analogues can be formulated with suitablc polymcric or hydrophobic matcrials, for cxamplc as an emulsion in an acceptable oil; or ion exchange resins; or as a sparingly soluble derivatives, for example, as a sparingly soluble salt.

A therapeutically cffcctive amount of a glucagon analoguc of thc invcntion may bc administered as a single pulse dose, as a bolus dose, or as pulse doses administered over timc. Thus, in pulsc doscs, a bolus administration of a glucagon analogue of thc invcntion is provided, followed by a time period wherein no glucagon analogue of the invention is administered to thc subject, followcd by a second bolus administration. In specific, non-limiting examples, pulse doses of a glucagon analogue of the invention are administered during the course of a day, during the course of a week, or during the course of a month.

In ccrtain embodiments, a thcrapeutically effective amount of a glucagon analogue of thc invention is administered with a therapeutically effective amount of another agent. The glucagon analogue may be administered simultaneously with the further therapeutic agent, or it may be administered sequentially or separately. In certain embodiments, a glucagon analogue of thc invcntion is formulatcd and administcrcd with a furthcr thcrapcutic agcnt as a single dose.

In certain embodiments, the further therapeutic agent is an additional anti-diabetic, appetite supprcssant, a food-intakc-rcducing, plasma glucosc-lowcring or plasma lipid-altcring agent. Specific, non-limiting examples of an additional appetite suppressant include amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine, fenfluramine, dexfenfl uramine, phendimetrazine, benzphetamine, sibutramine, rimonabant, topiramate, fluoxetine, bupropion, zonisamide, naltrexone, orlistat and cetilistat. Specific, non-limiting examples of an additional anti-diabetic agent include metformin, phenformin, rosiglitazone, pioglitazone, troglitazone, repaglinide, nateglinide, tolbutamide, acetohexmaide, tolazamide, chiorpropamide, glipizide, glyburide, glimepiride, gliclazide, fibroblast growth factor 21, miglitol, acarbose, exenatide, pramlintide, vildagliptin and sitagliptin.

In alternative embodiments, the thrther therapeutic agent is an additional cardioprotective or neuroprotective agent. Specific, non-limiting, examples of additional cardioprotective agents include aspirin, N-aeetyleysteine, phenethylamines, eoenzyme Q10, vitamin E, vitamin C, L-camitine, carvedilol and dexrazoxane. Specific, non-limiting examples of neuroprotective agents include statins such as simvastatin, steroids such as progesterone, minocycline, resveratrol and vitamin E. Examples of agents used for the treatment of Parkinson's disease include antieholinergics, pramipexo Ic, bromoeriptine, levodopa, carbidopa, rasagiline, amantadine and ropinirole.

A glucagon analogue of the invention may be administered whenever the effect, e.g., appetite suppression, decreased food intake, increased energy expenditure or decreased caloric intake, is desired, or slightly before to whenever the effect is desired, such as, but not limited to about 10 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes, before the time the effect is desired.

The therapeutically effective amount of a glucagon analogue of the invention will be dependent on the molecule utilized, the subject being treated, the severity and type of the affliction, and the manner and route of administration. For example, a therapeutically effective amount of a glucagon analogue of the invention may vary from about 0.01 jig per kilogram (kg) body weight to about 1 g per kg body weight, for example about 0.1 jig to about 20 mg per kg body weight, for example about I jig to about 5 mg per kg body weight, or about 5 jig to about I mg per kg body weight.

In one embodiment of the invention, a glucagon analogue of the invention may be administered to a subject at from 4 to 1333 nmol per kg bodyweight, for example from 5 to 1000 nmol per kg bodyweight, for example at from 10 to 750 nmol per kg bodyweight, for example at from 20 to 500 nmol per kg bodyweight, in particular at from 30 to 240 nmol per kg bodyweight. For a 75 kg subject, such doses correspond to dosages of from 300 nmol to 100 limo!, for example from 375 nmol to 75 jimol, for example from 750 nmol to 56.25 umol, for example from 1.5 to 37.5 gmol, in particular from 2.25 to 18 tmol. The invention also contemplates dosages ranges bounded by any of the specific dosages mentioned herein.

In an alternative embodiment, a glucagon analogue of the invention may be administered to a subject at 0.5 to 135 picomole (pmol) per kg body weight, for example 5 to 100 picomole (pmol) per kg body weight, for example 10 to 90 picomole (pmol) per kg body weight, for example about 72 pmol per kg body weight. In onc specific, non-limiting example, a glucagon analogue of the invention is administered in a dose of about 1 nmol or more, 2 nmol or more, or 5 nmol or more. In this example, the dose of the glucagon analogue of thc invention is generally not morc than 100 nmol, for example, the dose is 90 nmols or less, 80 nmols or less, 70 nmols or less, 60 nmols or less, 50 nmols or less, 40 nmols or less, 30 nmols or less, 20 nmols or less, 10 nmols. For example, a dosage range may comprise any combination of any of the specified lower dose limits with any of the specified upper dose limits. Thus, examples of non-limiting dose ranges of glueagon analogues of the invention are within the range of from I to 100 nmols, from 2 to 90 mols, from 5 to 80 nmols.

In one specific, non-limiting example, from about ito about 50 nmol of a glucagon analogue of the invention is administered, for example about 2 to about 20 nmol, for example about 10 nmol is administered as a subcutaneous injection. The exact dose is readily determined by one of skill in the art based on the potency of the specific compound utilized, the route of delivery of the compound and the age, weight, sex and physiological condition of the subject.

The doses discussed above may be given, for example, once, twice, three-times or four-times a day or once or twice a week. Preferably a dose may be given no more frequently than once a week. Alternatively, they may be given once every 2, 3 or 4 days. According to certain embodiments they may be administered once shortly before each meal to be taken.

The invention is illustrated by the following non-limiting Examples.

S Materials and Methods: Animals Male C57BL/6 mice (Harlan) or male Wistar rats (Charles River Ltd, Margate, UK) were used for all animal experiments.

Peptkle Synthesis Peptides were made by a standard automated fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) method. Peptide synthesis was carried out on a tryclic amide linker resin. Amino acids were attached using the Fmoc strategy. Each amino acid was added sequentially from the C-to the N-termini. Peptide couplings were mediated by the reagent TBTU. Peptide cleavage from the resin was achieved with trifluoracetic acid in the presence of scavengers.

Peptides were purified by reverse phase HPLC. Full quality control was performed on all purified peptides and peptides were shown to be greater than 95% pure by HPLC in two buffer systems. Amino acid analysis following acid hydrolysis confirmed the amino acid composition. MALDI-MS showed the expected molecular ion.

Example I

Cellular Assays Cells (Chinese hamster ovary -hGCGr, or Human embryonic kidney -hGLP-l r) were plated at a density of 150000 cells/ml in 24 well plates. The cells were left for 18 hours, and were then serum starved for 1 hour by replacing with serum free media. The media was then replaced with that containing the example glucagon analogue at 12 concentrations in duplicate ranging from 0 up to 30 nM (1 analogue per 24 well plate).

Each plate was incubated for exactly 30 minutes. The incubation media was removed, and replaced with 120u1 of lysis buffer (0.IM HC1 O.5%Tritonx). l2Oulof sample (or a dilution thereof so as to hit ELISA standard curve) was added to an eppendorf tube, and was spun for 3 minutes at >5000g to remove cell debris. lOOul of sample was added to an ELISA plate (Direct cyclic AMP Enzyme Immunoassay Kit -Enzolifesciences). The ELISA assay was run as described in the manual.

Acute feeding studies in mice Mice were individually housed in IVC cages. Animals were randomised into treatment groups, with stratification by body weight. Mice were fasted overnight (16 hrs) prior to peptide or vehicle administration. All peptide solutions were prepared freshly, immediately prior to administration. The vehicle used for all studies was 5% v/v water and 95% v/v sodium choridc (0.9% w/v). Pcptidc and vehicle were administered by subcutaneous injection, with dosage corrected for bodyweight. The injection volume was p1. Vehicle or peptide was administered at 09:00 and animals were returned to their home cage with a known amount of food. Food intake was measured at 1, 4, 8 and 24 hours post injection. All statistics are calculated using a one-way ANOVA with Dunnett's post-test or one-way ANOVA with Bonferroni post-test.

Acute feeding studies in rats Rats were individually housed in PVC cages. Animals were randomiscd into treatment groups, with stratification by body weight. Rats were fasted overnight (24 hrs) prior to peptide or vehicle administration. All peptide solutions were prepared freshly, immediately prior to administration. The vehicle used for all studies was 5% v/v water and 95% v/v sodium choridc (0.9% w/v) with zinc chloride added at a 1:1 molar ratio to the peptide. Peptide and vehicle were administered by subcutaneous injection, with dosage corrected for bodyweight. The injection volume was 50 p1. Vehicle or peptide was administered at 09:00 and animals were returned to their home cage with a known amount offood. Food intake was measured at 1,4, 8, 24, 32, 48 and 72 hours post injection. All statistics are calculated using a one-way ANOVA with Dunnett's post-test.

Results Figure 1 shows a table providing amino acid sequences of glucagon analogues of the invention (each compound is identified by a G no., as well as an analogue no.). The table also provides the results of cellular assay experiments, and the results of experiments in which appetite suppressant effects in mice and rats were determined.

The column headed "hOCOr cAMP" shows signaling in human embryonic kidney cells or Chinese hamster ovary cells over-expressing human glucagon receptor following administration of the example glucagon analogues. The values provided in the column arc EC5O ratios relative to native glucagon (e.g. a value of 0.5 indicates that the concentration of the glucagon analogue required to stimulate 50% maximum release of cAMP is half the concentration of native glucagon that is required, and a value of 5 indicates that the concentration of the glucagon analogue required to stimulate 50% maximum release of cAMP is S times that of native glucagon).

The column headed "hGLP-lr cAMP" shows signaling in human embryonic kidney cells or chinese hamster ovary cells over-expressing human GLP1 receptor. The values provided in the column are EC5O ratios relative to native GLP-1 (e.g. a value of 0.5 indicates that the concentration of the glucagon analogue required to stimulate 50% maximum release of cAMP is half the concentration of native GLP-1 that is required, and a value of 5 indicates that the conccntration of the glucagon analogue required to stimulate 50% maximum release of cAMP is 5 times that of native GLP-1).

In the section of the table headed "Mouse food intake inhibition", the columns headed "0- 1", "0-4", "0-8", "4-8", "8-24" and "0-24" show the reduction in food intake relative to saline during the indicated time periods (in hours) since administration of the glucagon an&ogue. A positive value indicates that less food was consumed by mice to whom the glucagon analogue was administered compared with mice to whom saline was administered during the relevant time period (a value of 100 indicates that nothing was eaten). A negative value indicates that more food was consumed by mice to whom the glucagon analogue was administered compared with mice to whom saline was administered during the relevant time period (e.g. a value of -5 indicates that the glucagon analogue mice consumed 5% more food (in grams) than the saline mice). A value of 0 indicates that the same quantity of food was consumed by the glucagon analogue mice and the saline mice.

In the section of the table headed "Rat food intake inhibition", the columns headed "0-1", "0-4", "0-8", "4-8", "8-24", "24-32", "32-48", "48-72", "0-24", "0-48" and "0-72" show the reduction in food intake relative to saline during the indicated time periods (in hours) since administration of the glucagon analogue. A positive value indicates that less food was consumed by rats to whom the glucagon analogue was administered compared with rats to whom saline was administcrcd during the relevant time period (a value of 100 indicates that nothing was eaten). A negative value indicates that more food was consumed by rats to whom the glucagon analogue was administered compared with rats to whom saline was administered during the relevant time period (e.g. a value of -5 indicates that the glucagon analogue rats consumed 5% more food (in grams) than the saline mice). A value of 0 indicates that the same quantity of food was consumed by the gucagon anaoguc rats and the salinc rats.

Example 2 -Rat weight loss studies Male Wistar rats were individually housed in PVC cages. Animals were randomised into treatment groups, with stratification by body weight. Rats were injected with a) vehicle b) exendin-4 or c) a glucagon analogue of the invention in combination with exendin-4 at 16:00 and animals wcrc retumcd to their home cage. The vehiclc used for all studies was 5% v/v water and 95% v/v sodium chloride (0.9% w/v) with zinc chloride added at a 1:1 molar ratio to the pcptidc (glucagon analoguc). All peptide solutions were prepared freshly, immediately prior to administratioll. Peptide doses are stated in the figure legends.

Food intake (in grams) and change in body weight (in grams) were measured daily over 7 days. Peptide and vehicle were administered by subcutaneous injection, with dosage corrccted for bodyweight. The injection volumc was 50 pl. Thc rcsults of thc studies arc shown in Figures 2 to 11, which show cumulative food intake over 7 days for rats to which a glucagon analogue of the invention was administered (in conjunction with exendin-4), compared with cumulative food intake for rats to which saline or exendin-4 was administered. Figures 2 to 11 also show the change in body weight over 7 days for rats to which a glucagon analogue of the invention was administered (in conjunction with exendin-4), compared with results for saline or exendin-4.

Sincc the glucagon analogucs normally have no or minimal anorectic effect, both the exendin-4-treated and the combination-treated rats can be expected to lose the same amount of body weight through food intake inhibition. Accordingly, additional weight loss observed in the combination-treated rats can be attributed to effects of the glucagon analogue thmugh a mechanism independent of food intake.

Example 3-Rat pharmacoklnetle studIes The pharmacokinetics of glucagon analogues of the invention in rats over 3 day, 4day or 7 day time periods were evaluated. Rats were injected subcutaneously with a glucagon analogue. Each injection was of 20 gl total volume/rat containing 1 mg peptide and 1 zinc ion (as ZnCI2) per peptide molecule. Blood was collected at the indicated time points, and the concentration of the glucagon analogue was determined. Peptide levels were assessed radioinimunoassay using R2641 glucagon antibody. The results are presented in Figures 12 to 21, and show that glucagon analogues of the invention can achieve good in vivo pharmacokinetic proffles over extended periods of time.

Claims (33)

1. CLAIMSAn analogue of glucagon which is: a compound comprising an amino acid scqucncc rcprcscntcd by formula (I) His-Ser-GIn-GIy-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Leu-Xaa15-Xaa'6- Xaa17 -Xaa1 8-Ala-Xaa20-Xaa21 -Phe-Xaa23-Xaa24-Trp-Leu-Leu-Asn-Xaa29-V (I) wherein V is selected from the group consisting of His, His-NH2, His-His, His-His-NH2, Gly-His, Gly-His-NH2, Lys-His, Lys-His-NH2, Gly-His-His, Gly-His-His-NH2, His-His-His and His-His-His-NH2; Xaa'° is selected from the group consisting ofTyr and Leu; Xaa'2 is selected from the group consisting of Lys, His and Arg; Xaa'3 is selected from the group consisting of Tyr, GIn and His; Xaa1 is selected from the group consisting of Asp and Glu; Xaa'6 is selected from the group consisting of Glu, GIn and Ser; Xaa'7 is selected from the group consisting of Arg, His and Lys; Xaa'8 is selected from the group consisting of Arg and Lys; Xaa2° is selected from the group consisting of His and Gin; Xaa2' is selected from the group consisting ofGlu, His and Asp; Xaa23 is selected from the group consisting of Tie and Val; Xaa24 is selected from the group consisting of Gin and Glu; and Xaa29 is selected from the group consisting of Thr and Gly; wherein -NH2 represents a C-terminal amide group; or a derivative of the compound; or a sait of the compound or the derivative.
2. 2. An analogue as claimed in claim 1, wherein Xaa2° is His.
3. 3. An analogue as claimed in claim 2, wherein Xaa1° is Tyr and Xaa'6 is Ser.
4. 4. An analogue as claimed in any one of claims I to 3, wherein V is selected from the group consisting of His-His, His-His-NH2, Gly-His and Gly-His-NH2.
5. 5. An analogue as claimed in anyone of claims I to 4, wherein Xaa1 is Tyr and/or Xaa15 is Asp and/or Xaa17 is Arg and/or Xaa18 is Arg and/or Xaa21 is Glu and/or Xaa23 is Va!
6. 6. An analogue as claimed in any one of claims I to 5, wherein the compound consists of an amino acid sequence represented by formula (1).
7. 7. An analogue as claimed in any one of claims 1 to 6, wherein i) Xaa1° is Tyr, Xaa'2 is Lys, Xaa' is Tyr, Xaa' is Asp, Xaa'6 is Ser, Xaa1' is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val, Xaa24 is GIn, Xaa29 is Tlir and V is His-His; ii) Xaa1° is Tyr, Xaa12 is Arg, Xaa'3 is Tyr, Xaa15 is Asp, Xaa'6 is Ser, Xaa" is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa21 is Glu, Xaa23 is Val, Xaa24 is GIn, Xaa29 is Tlir and V is His-His-NH2; iii) Xaa'° is Tyr, Xaa'2 is Arg, Xaa'3 is His, Xaa'5 is Asp, Xaa16 is 5cr, Xaa17 is Lys, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Va!, Xaa24 is Gln, Xaa29 is Thr and V is His-His -NH2; iv) Xaa1° is Tyr, Xaa12 is Arg, Xaa13 is Tyr, Xaa' is Asp, Xaa16 is 5cr, Xaa17 is Lys, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val, Xaa24 is G!n, Xaa29 is Thr and V is Gly-His; v) Xaa1° is Tyr, Xaa12 is Lys, Xaa13 is Tyr, Xaa15 is Asp, Xaa'6 is Ser, Xaa'7 is Aig, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val, Xaa24 is Gin, Xaa29 is Gly and V is Gly-His; vi) Xaa'° is Tyr, Xaa'2 is Lys, Xaa" is Tyr, Xaa'5 is Glu, Xaa16 is Ser, Xaa'7 is Arg, Xaa'8 is Lys, Xaa2° is His, Xaa21 is Gin, Xaa23 is lie, Xaa24 is Glu, Xaa29 is Thr and V is Gly-His; vii) Xaa'° is Tyr, Xaa12 is Lys, Xaa13 is Tyr, Xaa15 is Asp, Xaa16 is 5cr, Xaa'7 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Va!, Xaa24 is Glu, Xaa29 is Thr and V is Gly-His; viii) Xaa1° is Tyr, Xaa12 is Lys, Xaa'3 is Tyr, Xaa' is Asp, Xaa'6 is Ser, Xaa17 is Arg, Xaa'8 is Arg, Xaa2° is His, Xaa2' is Gin, Xaa23 is Vai, Xaa24 is Gin, Xaa29 is Giy and V is Gly-His-N1-12; ix) Xaa'° is Tyr, Xaa'2 is Lys, Xaa'3 is Tyr, Xaa" is Asp, Xaa'6 is Ser, Xaa17 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Asp, Xaa23 is Va!, Xaa24 is Glu, Xaa29 is Thr and V is His-His-NH2; x) Xaa1° is Tyr, Xaa12 is His, Xaa'3 is Tyr, Xaa'5 is Asp, Xaa'6 is 5cr, Xaa17 is Arg, Xaa18 is Arg, Xaa2° is His, Xaa2' is Glu, Xaa23 is Val, Xaa24 is Gin, Xaa29 is Gly and V is Gly-His-NH2;
8. 8. An analogue as claimed in any one of ciaims 1 to 7 which is a derivative that has been modified by one or more processes seiected from amidation, glycosyiation, carbamylation, acylation, sulfation, phosphorylation, cyclization, !ipidization, pegyiation and fusion to another peptide or protein to form a fusion protein.
9. 9. An analogue as claimed in any one of ciaims 1 to 7 which is not a derivative.
10. 10. An analogue as ciaimed in claim 1, which is any one of the compounds hstcd in Figure 1.
11. 11. An analogue as claimed in any one of claims I to 10 together with a further therapeutic agent, for simultaneous, sequential or separate administration.
12. 12. A pharmaceutical composition comprising an analogue as ciaimed in any one of claims 1 to 11 together with a pharmaceuticaily acceptable carrier and optionaily other therapeutic ingredients.
13. 13. A pharmaceutical composition as claimed in claim 12, present in a syringe or other administration device for subcutaneous administration to humans.
14. 14. A pharmaceutical composition as ciaimed in claim 12 or claim 13 wherein the composition has a pH of less than 5 prior to administration and wherein the composition comprises zinc ions.
15. 15. An analogue as claimed in any one of claims I to II, or a pharmaceutical composition as claimed in any one of claims 12 to 14, for use as a medicament.
16. 16. A method of treating or preventing a disease or disorder or other non-desired physiological state in a subject comprising administration of a therapeutically effective amount of an analogue as claimed in any one of claims Ito 11, or a pharmaceutical composition as claimed in any one of claims 12 to 14.
17. 17. An analogue as claimed in any one of claims I to II or a pharmaceutical composition as claimed in any one of claims 12 to 14 for use in the treatment of obesity or diabetes.
18. 18. An analogue as claimed in any one of claims Ito 11 or a pharmaceutical composition as claimed in any one of claims 12 to 14 for usc in increasing the energy expenditure of a subject, enhancing insulin release in a subject, improving carbohydrate metabolism in a subject and/or for improving the lipid profile of a subject.
19. 19. An analogue as claimed in any one of claims 1 to 11, or a pharmaceutical composition as claimed in any one of claims 12 to 14 for use in the reduction of appetite in a subject, use in the reduction of food intake in a subject, use in the reduction of calorie intake in a subject, use in enhancing insulin release in a subject, and/or use in improving carbohydrate tolerance in a subject.
20. 20. An analogue as claimed in any one of claims 1 to 11 or a pharmaceutical composition as claimed in one of claims 12 to 14 for use as a cytoprotective agent.
21. 21. A method of treating obesity or diabetes in a subject in need thereof comprising administration of a therapeutically effective amount of an analogue as claimed in any one of claims I to 11, ora pharmaceutical composition as claimed in any of claims 12 to 14.
22. 22. A method of increasing the energy expenditure of a subject, enhancing insulin release in a subject, improving carbohydrate metabolism in a subject and/or improving the lipid profile of a subject, comprising administration of a therapeutically effective amount of an analogue as claimed in any one of claims Ito 11, or a pharmaceutical composition as claimed in any of claims 12 to 14.
23. 23. A mcthod of rcducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, enhancing insulin release in a subject and/or improving carbohydrate tolerance in a subject, comprising administration of a therapeutically effective amount of an analogue as claimed in any one of claims Ito 11, or a pharmaceutical composition as claimed in any of claims 12 to 14.
24. 24. A method ofproviding eytoprotection in a subject, comprising administration of a therapeutically effective amount of an analogue as claimed in any one of claims Ito 11, or a pharmaceutical composition as claimed in any of claims 12 to 14.
25. 25. Use ofan analogue as claimed in any one of claims I to 11 for the manufacture ofa medicament for the treatment of obesity or diabetes.
26. 26. Use ofan analogue as claimed in any one of claims I to 11 for the manufacture ofa mcdicamcnt for increasing the energy expenditure of a subject, enhancing insulin release in a subject, improving carbohydrate metabolism in a subject and/or improving the lipid profile of a subject.
27. 27. Use ofan analogue as claimed in any one of claims I to 11 tbr the manufacture ofa medicament for the reduction of appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, enhancing insulin release in a subject, and/or use in improving carbohydrate tolerance in a subject.
28. 28. Use of an analogue as claimed in any one of claims Ito 11 for the manufacture of a medicament for providing cytoprotection in a subject.
29. 29. An analogue or pharmaceutical composition as claimed in any one of claims 17 to 19, a method as claimed in any one of claims 21 to 23, or a use as claimed in any one of claims 25 to 27, wherein the subject is overweight and/or obese and/or diabetic.
30. 30. An analogue or pharmaceutical composition for use as claimed in claim 20, a method as claimed in claim 24, or a use as claimed in claim 28, wherein the analogue or composition is for treating or preventing neurodegencration, providing neuroprotect ion and/or providing cardiac protection.
31. 31. An analogue or pharmaceutical composition for use, a method or use as claimed in claim 30, wherein the analogue or pharmaceutical composition is for providing cardiac protection in a subject following a myocardial infarction.
32. 32. An analogue or pharmaceutical composition for use, a method or use as claimed in claim 30, wherein the analogue or pharmaceutical composition is for providing neuroprotection in a subject having or diagnosed as being at risk of a chronic neurodegenerative disease.
33. 33. An analogue or pharmaceutical composition for use, a method or use as claimed in claim 32, wherein the chronic ncurodegencrativc discasc is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Gehrig's disease (Amyotrophic Lateral Sclerosis), Huntington's disease, Multiple Sclerosis, other demyclination related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS-associated dementia, other dementias, cerebral vasculitis, epilepsy, Tourette's syndrome, Gui I lain Bane Syndrome, Wilson's disease, Pick's disease, neuroinflammatory disorders, encephalitis, cnccphalomyclitis, meningitis, other central nervous system infections, prion diseases, cerebellar ataxias, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedrich's ataxia, ataxia teangiectasia, spinal dysmyotrophy, progressive supranuclear palsy, dystonia, muscle spasticity, tremor, retinitis pigmentosa, striatonigral degeneration, mitochondrial cncephalomyopathies and nouronal ceroid lipof'uscinosis.