WO2017178829A1

WO2017178829A1 - Peptide analogues

Info

Publication number: WO2017178829A1
Application number: PCT/GB2017/051045
Authority: WO
Inventors: Stephen Robert Bloom; Benjamin John JONES
Original assignee: Imperial Innovations Limited
Priority date: 2016-04-15
Filing date: 2017-04-13
Publication date: 2017-10-19

Abstract

Peptide hormone analogues of formulae (I) to (IV) are provided comprising amino acid substitutions relative to the native sequences at one or more of positions 1 to 5. Also provided herein are pharmaceutical compositions comprising said analogues, and methods of using said analogues for the treatment of conditions such as obesity and diabetes.

Description

PEPTIDE ANALOGUES

FIELD OF THE INVENTION

This invention relates to peptide hormones analogues of GLP-1, exendin-4, oxyntomodulin and glucagon, which are useful in treating disorders such as diabetes and obesity.

BACKGROUND TO THE INVENTION

According to the National Health and Nutrition Examination Survey (NHANES, 2009-2010), 33.0% of adults in the United States aged 20 and over are overweight, 35.7% are obese, and 6.3% are extremely obese. In addition, a large percentage of children in the United States are overweight or obese.

The cause of obesity is complex and multi-factorial. Increasing evidence suggests that obesity is not a simple problem of self-control but is a complex disorder involving appetite regulation and energy metabolism. 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).

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 (type 1 diabetes) and non-insulin dependent diabetes mellitus (type 2 diabetes). Diabetes type I, or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of β cells, which results in insufficient levels of endogenous insulin. Diabetes type 2, or non-insulin dependent diabetes, results from a defect in both the body's sensitivity to insulin, and a relative deficiency in insulin production. According to the National Diabetes Statistics Report, 2014 around 28.9 million adults in the United States aged 20 and over have diabetes (2009-2012 National Health and Nutrition Examination Survey estimates applied to 2012 U.S. Census data). In adults 90 to 95% of the diabetes is type 2 diabetes.

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

A number of approaches to the development of agents useful in effecting weight loss have involved gastrointestinal peptide hormones and their analogues. For example, derivatives of peptides deriving from the preproglucagon molecule have been proposed for use in treatment of obesity and/or diabetes. Preproglucagon is a precursor peptide of glucagon, as well as other hormones including glucagon-like peptide 1 (GLP-1), oxyntomodulin (OXM). Despite significant advances, the process of identifying substances useful as drugs remains a complex and, in many cases, unpredictable field. In order to be useful as therapeutic agents, compounds must possess a suitable range of properties. For example, in addition to having efficacy at the biological target of interest, compounds must have good in vivo pharmacokinetic properties, low toxicity, and low side effects, as well as having appropriate physical properties (e.g. solubility).

In the field of peptide therapeutics, native peptides or analogues thereof often suffer from poor pharmacokinetic properties. Research has led to the identification of peptide therapeutics having improved pharmacokinetic properties. WO 2013/004983 peptides that are both a GLP-1 receptor agonist and a glucagon receptor agonist and teaches that such compounds can reduce appetite and therefore food intake and also change energy metabolism so as to promote weight loss. WO

2014/041375 discloses peptide analogues of glucagon and peptide analogues of GLP-1 containing His residues at specified positions and teaches that the analogues exhibit improved pharmacokinetic properties (e.g. longer duration of action) compared with native GLP-1. WO2015/132599 discloses glucagon analogues and a GLP-1 analogues, having a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said residues being His, and teaches that such compounds exhibit potent and prolonged duration of action in vivo following subcutaneous administration.

The peptide hormones exendin-4 (Ex-4), glucagon-like peptide 1 (GLP-1), glucagon and

oxyntomodulin (OXM) are each GLP-1 receptor (GLP-1 R) agonists. Binding of Ex-4, GLP-1, glucagon or oxyntomodulin to the GLP-1R results in recruitment of both G proteins and β-arrestins, which have been implicated in signaling to divergent downstream biological events including potentiation of insulin secretion and reduction in β-cell apoptosis. Preferential signaling through one of those pathways is known as "ligand bias"; and an analogue that preferentially signals through one of those pathways may be referred to as "biased". Ligand bias for Ex-4 analogues at the GLP-IR has been previously reported for the compound P5, a GLP-IR G-protein-biased agonist (Zhang, H., et al, Nature Communications, 2005, 6:8919). P5 comprises 7 amino acids fused to the N terminus of Exendin(9 - 39). The N terminus 7 amino acid sequence of P5, as well as the sequences of the other analogues fused to Exendin 9 - 39 found to have ligand bias in Zhang, are unrelated to the N terminus of Ex4. It has been found that oxyntomodulin itself, which is a full agonist at GLP-IR, may be ligand biased at GLP-IR for the G-protein-dependent pathway (Jorgensen, R., et al, J Pharamcol Exp Ther, 2007, 322(1), 148-54).

There remains a need for further compounds which are effective as therapeutic agents; in particular having potent biological activity combined with improved pharmacokinetic properties and/or fewer unwanted side effects.

The present invention is based on the discovery that a single or multiple amino acid substitutions at positions 1 to 5 of certain GLP-1 receptor agonists can result in significant changes to the ligand biasing of those agonists. Furthermore, the inventors have found that peptide hormone analogues with modified ligand biasing have therapeutically useful characteristics, such as improved pharmacokinetic properties (e.g. longer duration of action in terms of insulin release and/or glycemic control), decreased insulin resistance during chronic treatment, improved long-term efficacy, increased potency, and/or fewer side effects compared with native peptide.

SUMMARY OF THE INVENTION

The present invention provides a peptide hormone analogue, or a derivative of the peptide hormone analogue, or a salt of the compound or the derivative, which is: (i) an analogue of Exendin-4 which is a compound of formula (I):

Xaa^Xaa^Xaa^Xaa^Xaa^Phe-Thr-Ser-Asp-Leu-Ser-Lys-XaaP-Xaa^Glu-Glu-Glu-Ala-Val-Arg- Leu-Phe-Ile-Glu-T -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser

(I)

wherein

Xaa is selected from the group consisting of Phe, His, D-His, Asn, Gin, Tyr, and D-Tyr; Xaa² is selected from the group consisting of Gly, Ala, an a-aminoisobutyric acid residue (AIB), Ser, D-Ser, Thr and Pro;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and Xaa⁵ is selected from the group consisting of Thr and Ser;

Xaa^p is selected from the group consisting of Gin and Tyr,

Xaa^Y is selected from the group consisting of Met and Leu; or a variant thereof having up to three conservative amino acid substitutions at any one of positions 6 to 39;

(ii) an analogue of GLP-1 which is a compound of formula (II): Xaa^Xaa'-Xaa'-Xaa'-Xaa'-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(II)

wherein

Xaa¹ is selected from the group consisting of Phe, His, D-His, Asn, Gin, Tyr, and D-Tyr; Xaa² is selected from the group consisting of Ala, an a-aminoisobutyric acid residue (AIB),

Ser, D-Ser, Gly, Thr and Pro;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and

Xaa⁵ is selected from the group consisting of Thr and Ser;

Xaa" is absent or Gly (preferably Xaa^p is absent); or a variant thereof having up to three conservative amino acid substitutions at any one of positions 6 to 31 when Xaa" is present, or at any one of positions 6 to 30 when Xaa" is absent;

(iii) an analogue of oxyntomodulin which is a compound of formula (III):

Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp- Phe-Val-Gln-T -Leu-Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala

(III)

wherein

Xaa¹ is selected from the group consisting of Phe, His, D-His, Asn, Gin, Tyr, and D-Tyr; Xaa² is selected from the group consisting of Ala, an a-aminoisobutyric acid residue (AIB), Ser, D-Ser, Gly, Thr and Pro;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and

Lys;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and Xaa⁵ is selected from the group consisting of Thr and Ser; or a variant thereof having up to three conservative amino acid substitutions at any one of positions 6 to 37;

(iv) an analogue of glucagon which is a compound of formula (IV)

Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸- Ala-Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-T -Leu-Leu-Asn-Xaa²⁹-V

(IV) wherein

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and

Xaa⁵ is selected from the group consisting of Thr and Ser;

Xaa¹⁰ is selected from the \ group consisting i of Tyr and Leu;

Xaa¹² is selected from the \ group consisting i of Lys, His and Arg;

Xaa¹³ is selected from the \ group consisting i of Tyr, Gin and His;

Xaa¹⁵ is selected from the ; group consisting ; of Asp and Glu;

Xaa¹⁶ is selected from the ; group consisting ; of Glu, Gin and Ser;

Xaa¹⁷ is selected from the ; group consisting ; of Arg, His and Lys;

Xaa¹⁸ is selected from the \ group consisting i of Arg and Lys;

Xaa²⁰ is selected from the \ group consisting i of His and Gin;

Xaa²¹ is selected from the \ group consisting i of Glu, His and Asp;

Xaa²³ is selected from the ; group consisting ; of lie and Val;

Xaa²⁴ is selected from the \ group consisting ; of Gin and Glu;

Xaa²⁹ is selected from the \ group consisting i of Thr and Gly;

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, His-His-His-NH₂, and a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues; or V is absent; with the proviso that when the peptide hormone analogue is:

(i) an analogue of Exendin-4 which is a compound of formula (I), Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵ is not His-Gly-Glu-Gly-Thr;

(ii) an analogue of GLP-1 which is a compound of formula (II), Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵ is not His- Ala-Glu-Gly-Thr;

(iii) an analogue of oxyntomodulin which is a compound of formula (III), Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵ is not His-Ser-Gln-Gly-Thr; and

(iv) an analogue of glucagon which is a compound of formula (IV), Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵ is not His-Ser-Gln-Gly-Thr or His-AIB-Gln-Gly-Thr.

The present inventors have identified peptide hormone analogues having one or more amino acid substitutions at any of positions 1 to 5 that possess therapeutically useful characteristics in terms of advantageous biological and/or pharmacokinetic properties. The therapeutically useful characteristics are due to the compounds having ligand bias for the GLP-1 receptor G-protein-dependent pathway (i.e. the GLP-1 receptor pathway dependent on generation of cAMP) or having ligand bias for the GLP-1 receptor G-protein-independent pathway (i.e. the GLP-1 receptor pathway dependent on analogue-induced -arrestin-2 ( ARR2) recruitment). For example, the present inventors have surprisingly found that even single N-terminal substitutions to can lead to 10- to 20-fold bias towards cAMP and away from ARR2 recruitment.

Furthermore, the present inventors surprisingly found that analogues of the invention with ligand bias for GLP-1 receptor G-protein-dependent pathway are excellent insulin secretagogues in vitro and can reduce glucose levels in vivo. For example, analogues that were biased for the G-protein-dependent pathway compared to the native peptide were approximately twice as insulinotropic when

administered to β-cells over 24 hours, even though the analogues had a lower potency for cAMP compared to the native peptide. The increased insulinotropic effect was also associated with a 30% reduction in GLP-1R desensitization. In vivo analogues of the invention have been shown to significantly lower glucose level in mice compared to the native peptide hormone.

The GLP-1R undergoes rapid agonist-mediated endocytosis. The present inventors have also found that exendin analogues of the invention which have lesser propensity for GLP-1 R internalization and recycling, i.e. which retain the GLP-1 R at the plasma membrane, produce greater insulin release and glucose lowering in vitro and in vivo, resulting in substantially improved responses compared to the native peptide (i.e. exendin-4). The present inventors have also found that residence time at the GLP-IR of an exendin analogue of the invention is associated with propensity for GLP-IR internalization. Longer residence time of the analogue at the GLP-IR receptor is associated with a greater degree of GLP-IR internalization. Without wishing to be bound by any theory, the inventors consider that residence time of peptide analogues at the GLP-IR is associated with the degree of internalization of the GLP-IR, the degree of recycling of the receptor, and the degree of ligand bias for the GLP-1 receptor G-protein-dependent pathway (i.e. the GLP-1 receptor pathway dependent on generation of cAMP) vs the GLP-1 receptor G-protein-independent pathway (i.e. the GLP-1 receptor pathway dependent on analogue-induced β- arrestin-2 ( ARR2) recruitment).

Improvement in glycaemia with existing GLP-1 based agents is limited by dose-dependent nausea. It has further been found that the glycemic benefits provided by analogues of the invention occur without alterations to appetite suppression or aversive behavior indicative of nausea, indicating that such benefits may be achieved without concomitant increase in nausea.

The present invention further provides a peptide hormone of the invention together with a further therapeutic agent, for simultaneous, sequential or separate administration. The present invention also provides a pharmaceutical composition comprising a peptide hormone analogue of the invention together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

The invention also provides a peptide hormone analogue of the invention, or a pharmaceutical composition of such an analogue, for use as a medicament. The peptide hormone analogues of the invention or pharmaceutical composition comprising a peptide hormone analogue of the invention finds use in the prevention or treatment of obesity and/or diabetes, for use in the prevention or treatment of obesity related diseases and/or diabetes related diseases, increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject; restoring insulin responsiveness in a subject, for providing long- term glycemic control in a subject and/or for use as a cytoprotective agent. The peptide hormone analogues of the invention or pharmaceutical composition comprising a peptide hormone analogue of the invention also find use in preventing or reducing nausea associated with GLP-1 agonist therapy, and find use in the prevention or treatment of diabetes or a diabetes-related disorder whilst reducing or preventing the incidence of nausea. 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 peptide hormone analogue of the invention. There is also provided a method of treating or preventing obesity and/or diabetes, obesity related diseases and/or diabetes related diseases, increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject; restoring insulin responsiveness in a subject, for providing long-term glycemic control in a subject and/or for use as a cytoprotective agent, comprising administration of a therapeutically effective amount of a peptide hormone analogue of the invention, or a pharmaceutical composition of such a analogue. The invention also provides a method of preventing or reducing nausea associated with GLP-1 agonist therapy, and a method of preventing or treating diabetes or a diabetes-related disorder whilst reducing or preventing the incidence of nausea, the method comprising administration of a therapeutically effective amount of a peptide hormone analogue of the invention, or a pharmaceutical composition of such an analogue.

The invention further provides the use of a peptide hormone analogue of the invention for the manufacture of a medicament for the prevention or treatment of obesity and/or diabetes, obesity related diseases and/or diabetes related diseases, increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de- sensitization in a subject; restoring insulin responsiveness in a subject, for providing long-term glycemic control in a subject and/or for use as a cytoprotective agent. The invention further provides the use of a peptide hormone analogue of the invention for the manufacture of a medicament for preventing or reducing nausea associated with GLP-1 agonist therapy, or for preventing or treating diabetes or a diabetes-related disorder whilst reducing or preventing the incidence of nausea.

There is also provided a method of causing weight loss or preventing weight gain in a subject for cosmetic purposes comprising administration of an effective amount of a peptide hormone analogue of the invention, or a composition of such an analogue. The present invention further provides use of a peptide hormone analogue of the invention in labelled form as a diagnostic agent for the diagnosis of conditions associated with a disease or disorder associated with the GLP-1 receptor. The invention also provides use of a peptide hormone analogue of the invention or a labelled form of such a peptide hormone analogue as a reference compound in a method of identifying ligands for the identifying ligands for the GLP-1 receptor, ligands for the GLP- 1 receptor that are biased for the GLP-1 receptor G-protein-dependent pathway, or ligands for the GLP-1 receptor that are biased for the GLP-1 receptor G-protein-independent pathway.

Analogues of the invention also find use as agents for targeting β-cells, e.g. for imaging purposes. Analogues of the invention which have greater propensity for GLP-1 R internalization, find particular use in such applications. For example, analogues of the invention comprising an appropriate label, e.g. a radioactive isotope atom such as H or other detectable isotope atom may be used for imaging purposes. Such labelled analogues may be produced by conventional methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a table showing the sequences of the analogues of the invention (analogues 1 to 42 and reference compounds Ex-4, GLP-1 and G1950.

Figure 2 shows the results of ligand biasing experiments for analogues of the invention including delta delta log(tau/KA) relative to GLP-1 and delta delta log(tau/KA) relative to exendin-4. In this case, bias was calculated using method 1A as described in "Materials and Methods".

Figure 3 shows the results of ligand biasing experiments for analogues of the invention including delta log(tau/KA), cAMP*, delta log(tau/KA), bARR2**, delta delta log(tau/KA) normalised to GLP-1 (* agonist transduction ratio, normalized to GLP-1; ** agonist bARR2 transduction ratio, normalized to GLP-1). The table of Figure 3 also shows the calculated delta delta log(tau/KA) of analogues of the invention relative to Ex-4 and of analogues of the invention relative to the reference compound

G1950. In this case, bias was calculated using method IB as described in "Materials and Methods".

Figure 4 shows a dose response curve for cAMP response after 30 minute stimulation with Ex-4, Ex-4 dHisl or Ex-4 Phe l in CHO-GLP-1R cells, n=6.

Figure 5 shows a dose response curve for ARR2 recruitment after 90 minute simulation with Ex-4, Ex-4 dHisl or Ex-4 Phe l in CHO-GLP-1R cells, n=6. Also shown is the inhibitory effect of Ex-4 dHisl and Ex-4 Phe 1 on recruitment of ARR2 by a fixed concentration of Ex-4. Figure 6 shows the pathway bias (log scale) of Ex-4 dHisl and Ex-4 Phe l relative to Ex-4 using the data from Figures 4 and 5. 95% CI indicated. In this case, pathway bias was calculated using "method 2" as described in "Materials and Methods) Figure 7 shows insulin secretion response in adherent INS-1 832/3 cells after 24 h incubation at 1 ImM glucose (Gl 1), n=6. Ex-4, Ex-4 dHisl or Ex-4 Phe l administered at ΙΟΟηΜ. The response expressed as insulin stimulation index (ISI) relative to Gl 1. (*p<0.05, **p<0.01 vs Ex-4 using randomized block ANOVA + Friedman test.)

Figure 8 shows cAMP response in INS-1 832/3 cells to ΙΟηΜ GLP-1 after prior exposure to ΙΟΟηΜ Ex-4, Ex-4 dHisl or Ex-4 Phe l or vehicle for 20 minutes (****p<0.001 vs Ex-4 using randomized block ANOVA + Sidak test.)

Figure 9 shows change in blood glucose level (mM) over 24 hours in female C57BL/6J mice with obesity-induced dysglycaemia after administration of lOug/kg of Ex-4, Ex-4 dHisl, or vehicle (0.9% NaCl). Figure 10 shows the glucose AUC (mM.hr) calculated from the data in Figure 9 for Ex-4, Ex-4 dHisl, and vehicle (0.9% NaCl) (*p<0.05, ** p<0.01, *** p<0.001 by one way ANOVA + Tukey test).

Figure 11 shows change in blood glucose level (mM) over 24 hours in female C57BL/6J mice with obesity-induced dysglycaemia after administration of lOug/kg of Ex-4, Ex-4 Phel, or vehicle (0.9% NaCl).

Figure 12 shows the glucose AUC (mM.hr) calculated from the data in Figure 11 for Ex-4, Ex-4 Phel, and vehicle (0.9% NaCl) (*p<0.05, ** p<0.01, *** pO.001 by one way ANOVA + Tukey test). Figure 13 shows dose response curves for cAMP response after 90 minute stimulation with Ex-4 or analogues of the invention in CHO-GLP-1R cells, n=5

Figure 14 shows dose response curves for ARR2 recruitment after 90 minute simulation with Ex-4 or analogues of the invention in CHO-GLP-1R cells, n=5

Figure 15 shows dose response curves for cAMP response after 90 minute stimulation with GLP-1 or analogues of the invention in CHO-GLP-1R cells, n=5

Figure 16 shows dose response curves for ARR2 recruitment after 90 minute simulation with GLP-1 or analogues of the invention in CHO-GLP-1R cells, n=5

Figure 17 shows competitive antagonism by exendin-phel against GLP-1 -induced (a) β-arrestin-l recruitment, n=5, and (b) -arrestin-2 recruitment, n=6, in Pathhunter GLP-1R cells. Ex-4 Phel and GLP-1 (150 nM) applied simultaneously for 90 min. Data indicated as mean ± SEM with 4-parameter logistic fit shown. Figure 18 shows screening results for exendin-4 and analogues of the invention: cAMP response measured in Pathhunter GLP-1R -arrestin-2 cells, 30 min incubation, quantified as pEC50 (negative logarithm of EC50), n=3-6; β-arrestin-l recruitment in Pathhunter GLP-1R β-arrestin-l cells, 1 μΜ agonist incubation for 90 min, normalized to 1 μΜ GLP-1 response, n=3; -arrestin-2 response measured similarly in Pathhunter GLP-1R β-13 arrestin-2 cells, n=3.

Figure 19 shows screening results for exendin-4 and analogues of the invention: GLP-1R

internalization measured by cell-surface ELISA in Pathhunter GLP-1 R -arrestin-2 cells, 1 μΜ agonist incubation for 90 min, quantified as % loss of cell surface receptor vs. vehicle control, n=3; agonist residence time measured in CHO-SNAP-GLP-1R cells by competitive association kinetics (Motulsky, H. J. & Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1-9 (1984)) using agonist in competition with 50 nM exendin-4- FITC, n=4.

Figure 20 shows the % recycling of internalized GLP-1 R following incubation with an analogue of the invention in CHO-SNAP-GLP-1R cells. After 60 minute prior exposure to the indicated analogue (100 nM) of the invention to induce internalization, analogue was removed and cells washed, and recycling allowed to proceed for 60 minutes. Cell surface SNAP-GLP-1R was then detected, allowing recycling to be determined as a percentage of that originally internalized. Figure 21 shows residual human islet surface GLP-1R labeled with exendin-4-FITC after overnight treatment with Gl 1 ± agonist, representative image from n=3 donors; scale bars, 10 um.

Figure 22 shows Ca2+ responses in Fluo-2 -loaded human islets to acute stimulation with exendin-4 or Ex-4 Phel at Gl 1, n=2 donors, 13-21 islets per condition analyzed. Agonists applied at 100 nM.

Figure 23 shows area under the curve (AUC) determined from Figure 21 relative to individual islet baselines, two-tailed unpaired t-test. Agonists applied at 100 nM. Data expressed as mean ± SEM, or as individual data points, * p<0.05, ** p<0.01, *** pO.001, by statistical test indicated above. Figure 24 shows Ca2+ responses in Fluo-2 -loaded human islets to exendin-4 after overnight pre- treatment with Ex-4 or Ex-4 Phel, n=2 donors, 13-21 islets per condition analyzed. Agonists applied at 100 nM. Figure 25 shows area under the curve (AUC) determined from Figure 23 relative to individual islet baselines, two-tailed unpaired t-test. Agonists applied at 100 nM. Data expressed as mean ± SEM, or as individual data points, * p<0.05, ** p<0.01, *** p<0.001, by statistical test indicated above.

Figure 26 shows screening results for exendin-4 and analogues of the invention: insulin secretion index relative to 1 ImM glucose alone, measured in INS-1 832/3 cells after overnight (16 h) incubation with an analogue of the invention at ΙΟΟηΜ concentration, n=5. Figure 27 shows dose response for agonist-induced (Ex4, Ex-4 Phel, Ex-4 Asp3) insulin release during overnight (16 h) stimulation in INS-1 832/3 cells (n=5), expressed as insulin stimulation index (ISI) relative to the response to 11 mM glucose (Gl 1) alone.

Figure 28 shows agonist-induced (Ex-4, Ex-4 Phel) insulin release during overnight (16 h) stimulation in intact human islets (HI, n=l 1 donors), expressed as insulin stimulation index (ISI) relative to the response to 11 mM glucose (Gl 1) alone.

Figure 29 shows change in blood glucose level (mM) over 8 hours following administration of 0.24 or 2.4 nmol/kg Exendin-4 (Ex-4), Ex-4 Phel or vehicle (0.9% saline) in male obese C57B1/6J mice fed a high fat, high sucrose diet for 4 months

Figure 30 shows the change in glucose AUC (mM.hr) over 8 hours, calculated from the data in Figure 29 for Ex-4, Ex-4 Phel, and vehicle, using the t = 0 glucose as the baseline for AUC calculation. Figure 31 shows blood glucose during IPGTT (2 mg/kg glucose) performed in HFHS mice at indicated time points after intraperitoneal (IP) injection of agonist (Ex-4 Phel = light blue bottom line; Ex-4 = dark blue middle line; vehicle = black top line) (2.4 nmol/kg), n=10/group except vehicle group at 4/8 h (n=9), two-way repeat measures ANOVA with Tukey's test, with significance vs. exendin-4 shown.

Figure 32 shows AUC determined from studies of Figure 30, relative to baseline glucose at t=0, two- way ANOVA with Dunnett's test vs. 0 h.

Figure 33 shows plasma insulin before and 10 min after IP administration of glucose (2 g/kg) in HFHS mice, at indicated time point after IP injection of agonist (2.4 nmol/kg), n=10/group except vehicle 0 h (n=8) and 4 h vehicle/exendin-4 (n=9), two-way repeat measures ANOVA with Sidak's test. Figure 34 shows cumulative food intake in fasted HFHS mice after IP injection of agonist (Ex-4 Phel = bottom light blue line; Ex-4 = bottom light blue line; vehicle = top black line) (2.4 nmol/kg), n=8/group.

Figure 35 shows blood glucose during IPGTT (2 g/kg) in HFHS mice 8 h after IP injection of agonist (Ex-4 Phel = bottom light blue line; Ex-4 = middle dark blue line; vehicle = top black line) (0.24 nmol/kg), n=8 per group, two-way repeated-measures ANOVA with Tukey's test, significance shown for Ex-4 Phel vs. exendin-4.

Figure 36 shows AUC calculated from Figure 33, relative to baseline glucose at t=0, one-way ANOVA with Tukey's test.

Figure 37 shows cumulative food intake in fasted HFHS mice after IP injection of agonist (Ex-4 Phel = bottom light blue line; Ex-4 = bottom dark blue line; vehicle = top black line) (0.24 nmol/kg), n=8 per group.

Figure 38 shows blood glucose during IPGTT (2 g/kg) in lean, chow-fed male C57B1/6 mice performed 8 h after IP injection of indicated agonist (2.4 nmol/kg), n=4 per group.

Figure 39 shows the results of a conditioned taste aversion experiment to assess nausea, showing the lack of difference in aversive affect between Ex-4 and Ex-4 Phel (0.24 nmol/kg). Mice (n=8/group) were conditioned to associate the indicated treatment, delivered i.p., with Kool-Aid consumption (cherry flavour), prior to being given free choice of Kool-Aid versus water for one hour. Preference, calculated as ratio of weight of each drink consumed, is indicated in the chart. LiCl, known to induce transient nausea in rodents, used as positive control.

Figure 40 shows the results of a conditioned taste aversion experiment to assess nausea, showing the lack of difference in aversive affect between Ex-4 and Ex-4 Phel (2.4 nmol/kg). Mice (n=8/group) were conditioned to associate the indicated treatment, delivered i.p., with Kool-Aid consumption

(grape flavour), prior to being given free choice of Kool-Aid versus water for one hour. Preference, calculated as ratio of weight of each drink consumed, is indicated in the chart. LiCl, known to induce transient nausea in rodents, used as positive control. Figure 41 shows blood glucose during IPGTT performed after 14 days of continuous agonist administration (Ex-4 Phel = bottom light blue line; Ex-4 = dark blue middle line; vehicle = top black line) (0.24 nmol/kg/day) in HFHS mice, n=10/group, two-way repeat measures ANOVA with Tukey's test, with significance for Ex-4 Phel vs. exendin-4 indicated.

Figure 42 shows AUC calculated from Figure 41, relative to baseline glucose at t=0, one-way ANOVA with Tukey's test.

Figure 43 shows fasting blood glucose in HFHS mice treated with continuous SC agonist (0.24 nmol/kg/day) for 16 days, n=10/group, one-way ANOVA with Tukey's test. Data expressed as mean ± SEM. *p<0.05, ** p<0.01, *** pO.001, by statistical test defined in the text.

Figure 44 shows cumulative food intake with continuous subcutaneous (SC) administration of agonist (eEx-4 Phel; Ex-4; Ex-4 Asp3) (0.24 nmol/kg/day), n=10/group.

Figure 45 shows body weight change with continuous subcutaneous (SC) administration of agonist (Ex-4 = dark blue bottom line; vehicle = black middle line; Ex-4 Phel = light blue top line) (0.24 nmol/kg/day), n=10/group.

Figure 46 shows histologically determined steatohepatitis, quantified as non-alcoholic activity score (NAS), after 16 days agonist administration (0.24 nmol/kg/day), n=10/group, Kruskal-Wallis with Dunn's test.

Figure 47 shows in vivo plasma concentrations of exendin-4 and Ex-4 Phel (24 nmol/kg i.p.) at 4 and 8 hrs following administration to HFHS-fed male C57BL/6 mice. 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. The 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, amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine fenfluramine, dexfenfluramine, fluoxetine, rimonabant, and sibutramine.

Body Mass Index (BMI): A mathematical formula for measuring body mass, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height² (in 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 BMI of greater than 25 kg/m² 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. J Clin. Nutr. 45: 1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.

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 suffering from ischemic heart 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. Typical but not limiting conservative substitutions are the replacements, for one another, among the aliphatic amino acids Ala, Val, Leu and lie; interchange of hydroxyl -containing residues Ser and Thr, interchange of the acidic residues Asp and Glu, interchange between the amide- containing residues Asn and Gin, interchange of the basic residues Lys and Arg, interchange of the aromatic residues Phe and Tyr, and interchange of the small-sized amino acids Ala, Ser, Thr, Met and Gly. Additional conservative substitutions include the replacement of an amino acid by another of similar spatial or steric configuration, for example the interchange of Asn for Asp, or Gin for Glu.

Table 1 : Non-limiting examples of conservative amino acid substitutions

Original Residue Conservative Substitutions

Ala Gly, Val, Leu, lie, Ser, Thr, Met

Arg Lys

Asn Asp, Gin, His

Asp Glu, Asn

Cys Ser

Gin Asn, His, Lys, Glu Glu Asp, Gin

Gly Ala, Ser, Thr, Met

His Asn, Gin

lie Ala, Leu, Val, Met

Leu Ala, lie, Val, Met,

Lys Arg

Met Leu, He, Ala, Ser, Thr, Gly

Phe Leu, Tyr, Trp

Ser Thr, Cys, Ala, Met, Gly

Thr Ser, Ala, Ser, Met, Gly

Trp Tyr, Phe

Tyr Trp; Phe

Val Ala, lie, Leu Non-conservative substitutions: The replacement, in a polypeptide, of an amino acid residue by another residue which is not biologically similar. For example, the replacement of an amino acid residue with another residue that has a substantially different charge, a substantially different hydrophobicity or a substantially different spatial or steric configuration. 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 (type 1 diabetes) and non-insulin dependent diabetes mellitus (type 2 diabetes) which differ in etiology, pathology, genetics, age of onset, and treatment. The two major forms of diabetes are both characterized by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis. Diabetes type I, or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of β cells, which results in insufficient levels of endogenous insulin. Diabetes type 2, 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. Exendin-4 (Ex-4): Ex-4 is a 39 amino acid peptide agonist of the glucagon-like peptide 1 receptor (GLP-1 R) and is found in the saliva of the Gilamonster. It is a stable glucagon-like peptide- 1 (GLP-1) mimetic, having similar functional properties to native GLP-1, and is an agonist of the GLP-1 receptor. It is an insulin secretagogue, with gluco-regulatory effects. Exenatide, a synthetic version of Ex-4, was approved by the FDA in 2005 for patients with type 2 diabetes mellitus who have not achieved adequate glycemic control on other medication.

The sequence of Ex-4 is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu- Ala-Val-Arg-Leu-Phe-Ile-Glu-T -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.

An "analogue of Ex-4" according to the present invention does not have the same sequence as Ex-4. It has at least one different amino acid at positions 1, 2, 3, 4 and/or 5. It may have other conservative amino acid substitutions at any one of positions 6 to 30. An analogue of Exendin-4 according to the present invention must retain at least some of the same activity as Exendin-4, for example the ability to reduce appetite and/or the ability to increase insulin levels and/or reduce glucose levels in a subject. Where an analogue of the invention is referred to by the name, for example, Ex-4 Phe l, this represents a peptide having an amino acid sequence analogous to that of exendin-4 but in which the amino acid residue at position 1 of exendin-4 has been substituted for Phe (phenylalanine).

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.

GLP-1: Glucagon-like peptide 1 (GLP-1) is derived from the transcription product of the proglucagon gene. 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 possesses a number of physiological functions including increasing insulin secretion from the pancreas in a glucose- dependent manner, decreasing glucagon secretion from the pancreas, inhibiting gastric emptying and decreasing food intake by increasing satiety. Increased insulin secretion leads to a decrease in circulating glucose concentration. The biologically active forms of GLP-1 are truncated forms known as GLP-l(7-37) and GLP-1 (7-36)-NH2 (i.e. the C-terminus has a -CONH2 group in place of a carboxylic acid). Peptide analogues of GLP-1 useful in treating metabolic disorders are disclosed in, for example, WO2013/004983.

The sequence of human GLP-l(7-37) is His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu- Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Gly.

The sequence of human GLP-l(7-36₎-NH2 is His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-T -Leu-Val-Lys-Gly-Arg-NH2.

An "analogue of GLP-1" according to the present invention does not have the same sequence as GLP- 1 (GLP- 1(7-37) or GLP-l(7-36)-NH2: it has at least one different amino acid at positions 1, 2, 3, 4 and/or

5. It may have other conservative amino acid substitutions at any one of positions 6 to 30 or 6 to 31.

An analogue of GLP-1 according to the present invention must retain at least some of the same activity as native GLP-1, for example the ability to reduce appetite and/or the ability to increase insulin levels and/or reduce glucose levels in a subject. Where an analogue of the invention is referred to by the name, for example, GLP- 1 Phe 1 , this represents a peptide having an amino acid sequence analogous to that of GLP-l(7-36)-NH2 but in which the amino acid residue at position 1 of exendin-4 has been substituted for Phe (phenylalanine).

Glucagon: Glucagon is a 29 amino acid peptide derived from the proglucagon gene. Glucagon is released in vivo when blood glucose levels fall low and has the activity of causing the liver to convert stored glycogen into glucose which is released into the bloodstream raising blood glucose levels. In humans, glucagon 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-Asn-Thr.

An "analogue of glucagon" according to the present invention must retain at least some of the same activity as native glucagon, for example the ability to reduce appetite and/or the ability to increase insulin levels and/or reduce glucose levels in a subject. Ligand bias: Binding of Ex-4, GLP-1, glucagon or oxyntomodulin to the GLP-1R results in recruitment of both G proteins and β-arrestins, which have been implicated in signaling to divergent downstream biological events including potentiation of insulin secretion and reduction in β-cell apoptosis. Preferential signaling through one of those pathways is known as "ligand bias"; and an analogue that preferentially signals through one of those pathways may be referred to as "biased".

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

In animals, the caloric and nutrient requirements vary depending on the species and size of the animal. For example, in cats, the total caloric intake per pound, as well as the percent distribution of protein, carbohydrate and fat varies with the age of the cat and the reproductive state. A general guideline for cats, however, is 40 cal/lb/day (18.2 cal/kg/day). About 30% to about 40% should be protein, about 7% to about 10% should be from carbohydrate, and about 50% to about 62.5% should be derived from fat intake. One of skill in the art can readily identify the normal daily diet of an individual of any species. Obesity: A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102:E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure. For example, the Body Mass Index (BMI) may be used to assess obesity. In one commonly used convention, a BMI of 25.0 kg/m² to 29.9 kg/m² is overweight, while a BMI of 30 kg/m² or greater is obese.

In another convention, waist circumference is used to assess obesity. In this convention, in men a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese. Strong evidence shows that obesity affects both the morbidity and mortality of individuals. For example, an obese individual is at increased risk for heart disease, non- insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001). Overweight: An individual who weighs more than their ideal body weight. An overweight individual can be obese, but is not necessarily obese. 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/m² to 29.9 kg/m². Oxyntomodulin (OXM): Oxyntomodulin is a 37 amino acid peptide member of the glucagon superfamily comprising the entire 29 amino acid sequence of glucagon, with an eight amino acid carboxy terminal (C-terminal) extension, resulting from the tissue-specific processing of the pre-pro- glucagon precursor in the brain and gut. OXM is known to bind both the GLP-1 receptor and the glucagon receptor. The human OXM sequence (which is the same as the rat and hamster) is as follows: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- Asp-Phe-Val-Gln-T -Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala.

An "analogue of OXM" according to the present invention does not have the same sequence as OXM: it has at least one different amino acid at positions 1, 2, 3, 4 and/or 5. It may have other conservative amino acid substitutions at any one of positions 6 to 37. An analogue of OXM according to the present invention must retain at least some of the same activity as native OXM, for example the ability to reduce appetite and/or the ability to increase insulin levels and/or reduce glucose levels in a subject.

PEGylated and PEGylation: the process of reacting a poly(alkylene glycol), preferably an activated poly(alkylene 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 poly(ethylene glycol) or derivatives thereof, such as methoxy poly(ethylene 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).

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

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms "polypeptide" or "protein" as used herein encompass any amino acid sequence and include modified sequences such as

glycoproteins. The term "polypeptide" 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 of a polypeptide" refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional peptides can also include fusion proteins, in which the peptide of interest has been fused to another peptide that does not decrease its desired activity.

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 hypodermic needle fitted, for example, to a syringe or a "pen" type injection device. Other administration methods may be used for example microneedles. Injection with a hypodermic needle typically involves a degree 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 experience a "stinging" sensation at the injection 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.

ABBREVIATIONS

AUC Area under curve

BETP 2-(Ethylsulfinyl)-4- [3 -(phenylmethoxy)phenyl] -6-(trifluoromethyl)-pyrimidine cAMP Cyclic adenosine monophosphate

CHO Chinese hamster ovary

CI Confidence intervals

DERET Diffusion-enhanced resonance energy transfer

EC50 Effective half-maximal concentration

ER Endoplasmic reticulum

ex(9-39) Exendin(9-39)

FACS Fluorescence-activated cell sorting

FITC Fluorescein isothiocyanate

GLP-1 Glucagon-like peptide- 1

GLP-1R Glucagon-like peptide- 1 receptor

HFHS High fat, high sucrose

HTRF Homogenous time-resolved fluorescence

IBMX 3 -isobutyl- 1 -methylxanthine

IPGTT Intraperitoneal glucose tolerance test MesNa 2-sulfanylethanesulfonate

pEC50 Negative logarithm of EC50

RNAi RNA interference

SC Subcutaneous

SEM Standard error of the mean

T2D Type 2 diabetes

TR-FRET Time-resolved Forster resonance energy transfer

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

DETAILED DESCRIPTION OF THE INVENTION

The analogues of the invention are peptide hormone analogues. The peptide hormone analogues of the present invention are agonists of the GLP-1 receptor (also referred to as GLP-1 receptor (GLP-1 R) agonists). Certain analogues of the invention are gastrointestinal peptide hormone analogues. The gastrointestinal hormones (or gut hormones) constitute a group of hormones secreted by

enteroendocrine cells in the stomach, pancreas, and small intestine that control various functions of the digestive organs, and include GLP-1, OXM and glucagon. Peptide hormone analogues of the present invention also include analogues of Ex-4, which is a gastrointestinal peptide hormone mimetic.

Analogues of the invention are biased for a particular GLP-1 R pathway compared to GLP-1 and/or the native peptide from which they are derived (for example, biased for the G-protein-dependent pathway via generation of cAMP; or biased for the G-protein independent pathway via analogue- induced -arrestin-2 ( ARR2) recruitment). Other preferred compounds of the invention are biased for a particular pathway (for example compared to the native hormone from which they are derived) combined with having good potency at the GLP-1 receptor. In certain embodiments, preferred analogues of the invention are biased for the G-protein-dependent pathway compared to GLP-1 and/or the native peptide from which they are derived. It has been found that analogues of the invention which have lesser propensity for GLP-IR internalization and recycling, i.e. which retain the GLP-IR at the plasma membrane, which have short residence times at the GLP-IR, and/or which have low levels of β-arrestin 1 and/or β-arrestin 2 recruitment (e.g. compared with exendin-4) produce greater insulin release and glucose lowering effects in vitro and in vivo. Thus, in some embodiments, the analogues of the invention have one or more of the following properties: low levels of GLP-IR internalization upon binding to GLP-IR, short residence time upon binding to GLP-IR, and/or low levels of β-arrestin 1 and/or β-arrestin 2 recruitment. In some embodiments, the analogues of the invention possess low GLP-IR internalization properties, for example on binding less than 60%, less than 50% or less than 40% of the GLP-1R may be internalized, for example using a cell-surface ELISA assay, e.g. as described below. In some embodiments, the analogues of the invention have short average (e.g. mean) residence time upon binding to GLP-1R, for example the average residence time may be less than 25 minutes, less than 20 minutes, or less than 15 minutes, for example when assayed by assessing binding of FITC -agonist to labeled SNAP-GLP-1R by TR-FRET, e.g. as described below. In some embodiments, the analogues of the invention possess low β-arrestin 1 recruitment properties, for example where administration of the analogue leads to less than 80%, less than 60%, less than 40% or less than 30% of the levels of β-arrestin 1 recruitment compared with administration of an equimolar amount of exendin-4, for example when assayed in CHO-GLP-1R β-arrestin 1 cell line, e.g. as described below. In some embodiments, the analogues of the invention possess low β-arrestin 2 recruitment properties, for example where administration of the analogue leads to less than 80%, less than 60%, less than 40% or less than 30% of the levels of β-arrestin 2 recruitment compared with administration of an equimolar amount of exendin-4, for example when assayed in CHO-GLP-1R β- arrestin 2 cell line, e.g. as described below.

The amino acid sequences described here (for example the sequences of formula (I), (II), (III), (IV) and (X) are shown with the N-terminus to the top left. Unless indicated otherwise (e.g. D-His), the amino acid residues in the sequence disclosed herein (for example the sequences of formula (I), (II), (III), (IV)) are L-amino acids. The C-terminus of an analogue of the invention may be an

unfunctionalised acid group, i.e. CO2H, or may be in salt form or derivatised as described elsewhere herein.

The present invention envisages the combination of the various embodiments and aspects of the invention described herein, unless stated otherwise.

In one aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Gin, Tyr, and D-Tyr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is His. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His, Phe, Gin, Tyr, and D-Tyr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His, Asn, Tyr, and D-Tyr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II),

(III) or (IV), and Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Tyr, and D-Tyr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His and Asn. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His and Phe. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is selected from the group consisting of D-His and D-Tyr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is D-His. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa¹ is Phe. In one aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa² is selected from the group consisting of Ala, Ser, D-Ser, Gly, Thr and Pro. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa² is selected from the group consisting of Ala, Ser, and Gly. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa² is selected from the group consisting of AIB, D- Ser, Thr and Pro. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa² is selected from the group consisting of D-Ser, Thr and Pro. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa² is D-Ser.

In one aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa³ is selected from the group consisting of D-Gln, His, Asp, Ala, Tyr, Leu and Lys. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I) or (II), and Xaa³ is selected from the group consisting of Glu, D-Gln, His, Ala, Tyr, Leu and Lys. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (III) or (IV), and Xaa³ is selected from the group consisting of Gin, D-Gln, His, Ala, Tyr, Leu and Lys. In another aspect of the invention, the peptide hormone analogue of the invention is an analogue of formula (I), (II), (III) or (IV), and Xaa³ is selected from the group consisting of D-Gln, His, Ala, Tyr, Leu and Lys. In another aspect of the invention, the peptide hormone analogue of the invention is an analogue of formula (I), (II), (III) or (IV), and Xaa³ is selected from the group consisting of Gin, Glu and Asp. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I) or (II) and Xaa³ is Asp. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I) or (II) and Xaa³ is Glu. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (III) or (IV) and Xaa³ is Gin. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa³ is D-Gln. In one aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa⁴ is selected from the group consisting of Gly, Leu, Ala and Ser. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa⁴ is selected from the group consisting of Leu, Ala and Ser. In a preferred aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa⁴ is Gly.

In one aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa⁵ is Thr. In another aspect of the invention, the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and Xaa⁵ is Ser.

In one preferred aspect of the invention (for example for peptide hormone analogues biased for the GLP-1 receptor G-protein-dependent pathway), analogue is (i) an analogue of Exendin-4 which is a compound of formula (I) and Xaa² is Gly; (ii) an analogue of GLP-1 which is a compound of formula (II) and Xaa² is Ala; (iii) an analogue of oxyntomodulin which is a compound of formula (III) and Xaa² is Ser; or (iv) an analogue of glucagon which is a compound of formula (IV) and Xaa² is Ser. Even more preferably, additionally, Xaa⁵ is Thr, or Xaa⁴ is Gly, or Xaa⁵ is Thr and Xaa⁴ is Gly. In a further embodiment, additionally Xaa¹ is His or Xaa³ is Glu when the analogue is a compound of formula (I) or (II) or Xaa³ is Gin when the analogue is a compound of formula (III) or (IV).

In especially preferred embodiments of the invention, the amino acid at Xaa¹, Xaa² and/or Xaa³ is a D- amino acid, for example: Xaa¹ is selected from the group consisting of D-His and D-Tyr (preferably D- His); and/or Xaa² is D-Ser; and/or Xaa³ D-Gln.

In one embodiment (for example for peptide hormone analogues biased for the GLP-1 receptor G- protein-dependent pathway), the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D-Tyr; Xaa² is selected from the group consisting of Ala, Ser, and Gly;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, Gin, D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala and Ser, and more preferably Gly); and

Xaa⁵ is selected from the group consisting of Thr and Ser. In one embodiment (for example for peptide hormone analogues biased for the GLP-1 receptor G- protein-dependent pathway), the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and

Xaa⁵ is Thr.

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Tyr, and D-Tyr

(preferably His, D-His, Phe, Asn and Tyr, more preferably His, D-His and Phe);

Xaa² is selected from the group consisting of Ala, Ser, and Gly;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly,

Leu, Ala and Ser, more preferably Gly); and

Xaa⁵ is Thr.

Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Gin, Tyr, and D-Tyr

(preferably D-His, Phe, Asn and Tyr, more preferably D-His and Phe);

Xaa² is selected from the group consisting of Ala, Ser, and Gly;

Lys (preferably Glu, Gin, D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala, and Ser, more preferably Gly); and

Xaa⁵ is Thr. In one embodiment (for example for peptide hormone analogues biased for the GLP-1 receptor G- protein-dependent pathway), the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and

Xaa³ is selected from the group consisting of D-Gln, His, Asp, Ala, Tyr, Leu and Lys

(preferably D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala and Ser, more preferably Gly); and

Xaa⁵ is Thr.

(preferably His, D-His, Phe, Asn and Tyr, more preferably His, D-His and Phe);

Xaa² is selected from the group consisting of Ala, Ser, and Gly;

Xaa³ is selected from the group consisting of D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys);

Leu, Ala and Ser, more preferably Gly); and

Xaa⁵ is Thr.

(preferably D-His, Phe, Asn and Tyr, more preferably D-His and Phe);

Xaa² is selected from the group consisting of Ala, Ser, and Gly;

(preferably D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa¹ is selected from the group consisting of His, D-His, and D-Tyr;

Xaa² is selected from the group consisting of Ala, Gly and Ser;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr,

Leu and Lys (preferably Glu, Gin, D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably

Gly, Leu, Ala and Ser, more preferably Gly); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Tyr, and D-Tyr;

Xaa² is selected from the group consisting of Ala, Gly and Ser;

Xaa³ is selected from the group consisting of D-Gln;

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

Xaa¹ is selected from the group consisting of D-His and D-Tyr;

Xaa² is selected from the group consisting of Ala, Gly and Ser;

Leu and Lys (preferably D-Gln);

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

Xaa¹ is D-His and D-Tyr;

Xaa² is D-Ser; Xaa³ is selected from the group consisting of Glu, Gin, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys, more preferably D-Gln);

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

In one embodiment (for example for peptide hormone analogues biased for the GLP-1 receptor G- protein-independent pathway), the peptide hormone analogue of the invention is a compound of formula (I), (II), (III) or (IV), and

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D-

Tyr;

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala, Ser, D-Ser, Gly, Thr and Pro);

Xaa³ is selected from the group consisting of Glu, Gin, and Asp (for example Glu and Gin);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and Xaa⁵ is selected from the group consisting of Thr and Ser.

Xaa¹ is selected from the group consisting of His, Phe, Gin, and D-Tyr (preferably

His or Gin, more preferably His);

Xaa¹ is selected from the group consisting of His, Phe, Gin, and D-Tyr (preferably His or Gin, more preferably His);

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro

(preferably Ala, Ser, D-Ser, Gly, Thr and Pro); Xaa³ is selected from the group consisting of Glu, Gin, and Asp (for example Glu and Gin);

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser.

Xaa² is selected from the group consisting of Ala, Ser, D-Ser, Gly, Thr and Pro

(preferably D-Ser, Thr and Pro);

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser.

His or Gin, more preferably His);

Xaa² is D-Ser;

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser.

(i) an analogue of Exendin-4 which is a compound of formula (I):

In one preferred embodiment of the invention the peptide hormone analogue is an analogue of Exendin-4 which is a compound of formula (I) as defined above.

For a compound of formula (I), preferably Xaa^p is Gin, and preferably Xaa^Y is Met. Most preferably Xaa^p is Gin and Xaa^Yis Met. Preferably the C-terminal of the Ex-4 analogue is amidated.

An analogue of Ex-4 which is a compound of formula (I) has 0, 1, 2 or 3 conservative substitutions at positions 6 to 39, preferably 0, 1 or 2, more preferably 0 or 1 and most preferably 0. Certain preferred Ex-4 analogues of the invention contain at least 79% sequence homology with Ex-4, for example from 79-97%. Some particularly preferred analogues of the invention have between 75 and 97% sequence homology with Ex-4, for example 80 to 97%, 85 to 97%, 90 to 97%, or 93 to 97%. Some preferred analogues of the invention contain greater than 70% sequence homology with Ex-4: for example 79%, 82%, 85%, 87%, 90%, 92%, 95% or 97% sequence homology. In some embodiments, an analogue contains an amino acid sequence that corresponds to the amino acid sequence of Ex-4 except that the analogue contains from 1 to 8 amino acid substitutions, preferably the analogue contains from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 6, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 8, from 5 to 7, or from 5 to 6, amino acid substitutions from the amino acid sequence of Ex-4 (for example 1, 2, 3, 4, 5, 6, 7 or 8).

In one particularly preferred embodiment the analogue of Ex-4 which is a compound of formula (I) is not a variant. In another particularly preferred embodiment the analogue of Ex-4 which is a compound of formula (I) is not a derivative.

In one particularly preferred embodiment, the Ex-4 analogue is a compound consisting of an amino acid sequence represented by formula (I) or a variant having up to three conservative amino acid substitutions at any one of positions 6 to 39, for example 0, 1, 2 or 3 conservative amino acid substitutions; or a derivative of the analogue or variant thereof, or a salt of the compound, variant thereof or the derivative of the compound or variant. The preferences for amino acid residues and combinations of amino acid residues set out above are also preferred for such Ex-4 analogues. In one particularly preferred embodiment the Ex-4 analogue is a compound consisting of an amino acid sequence represented by formula (I), or a salt of the analogue.

In embodiments of the invention where the peptide hormone analogue is an analogue of Ex-4 which is a compound of formula (I), preferably:

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Phe);

(preferably Gly);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln, His, Ala, Tyr, Leu and Lys or Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example Asp); Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB, (preferably Gly, Leu, Ala and Ser); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr). In another preferred embodiment of a compound of formula (I):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, most preferably D-His and Phe); Xaa² is Gly;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln, His, Ala, Tyr, Leu and Lys or Glu, D-Gln, His,

Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example Asp);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Phe); Xaa² is Gly;

Xaa³ is Glu;

Xaa⁵ is Thr. In another preferred embodiment of a compound of formula (I):

Xaa¹ is His;

Xaa² is Gly;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln, His, Ala, Tyr, Leu and Lys, or Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example Asp);

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I): Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, most preferably D-His and Phe); Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala, Ser, D-Ser, Gly, Thr and Pro, more preferably Ser, D-Ser, Gly, Thr and Pro);

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I):

Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Gin, Tyr, and D-Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, most preferably D-His and Phe); Xaa² is Gly;

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I):

Xaa¹ is His;

Xaa² is Gly;

Xaa³ is selected from the group consisting of Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I):

Xaa¹ is His;

Xaa² is Gly;

Xaa³ is Glu;

Xaa⁴ is selected from the group consisting of Leu, Ala, Ser and AIB (preferably Gly,

Leu, Ala and Ser); and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (I):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably Gin, D-Tyr and His, more preferably Gin and His); Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably AIB and Ala);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, Asp and Glu);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB, (preferably

Gly); and

Xaa⁵ is selected from the group consisting of Thr and Ser.

In another preferred aspect of the invention, the peptide hormone analogue is an analogue of Ex-4 which is a compound selected from the group consisting of:

formula (la):

Xaa¹-Gly-Xaa -Xaa⁴-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(la);

formula (lb):

Xaa^Xaa'-Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(lb)

formula (Ic):

Xaa^Gly-Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ic)

formula (Id):

Xaa^Gly-Glu-Xaa'-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Id)

formula (le):

Xaa^Xaa'-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(le)

formula (If):

Xaa^Gly-Glu-Gly-Thr -Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(If)

formula (Ig):

His- Xaa²-Glu-Gly-Thr -Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a (Ig)

formula (Ih):

His-Gly- Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ih)

formula (Ij):

His-Gly-Glu- Xaa⁴-Thr Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ij)

and formula (Ik) :

His-Gly-Glu-Gly- Xaa^Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ik) wherein the definitions of Xaa¹, Xaa², Xaa³, Xaa⁴, Xaa⁵ are as for a compound of formula (I) as described above, and with the proviso that the sequence at positions 1 to 5 is not His-Gly-Glu -Gly- Thr.

In one preferred embodiment of the compound of formula (I), Xaa² is Gly, Xaa³ is Glu, Xaa⁴ is Gly, Xaa⁵ is Thr, Xaa^p is Gin and Xaa^Y is Met. In that embodiment, more preferably Xaa¹ is D-His, Asn, Gin, Tyr, Phe or D-Tyr,

In one preferred embodiment of the compound of formula (I), Xaa¹ is His, Xaa² is Gly, Xaa⁴ is Gly, Xaa⁵ is Thr, Xaa^p is Gin and Xaa^Y is Met. In that embodiment, more preferably Xaa³ is D-Gln.

In one particularly preferred embodiment, the Ex-4 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 11 to 19 or 19a, or a derivative of the compound, or a salt of the compound or the derivative. The amino acid sequences of analogue nos. 11 to 19 and 19a are provided in the Table of Figure 1. In one preferred embodiment the Ex-4 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 11 to 19 or 19a, or a salt of the compound. In one preferred embodiment the Ex-4 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 11 to 19 or 19a.

(ii) an analogue of GLP-1 which is a compound of formula (II):

In one preferred embodiment of the invention the peptide hormone analogue is an analogue of GLP-1 which is a compound of formula (II), as defined above. Preferably, in the compound of formula (II), Xaa^p is absent. More preferably, Xaa^p is absent and the C-terminal of the GLP-1 analogue is amidated.

An analogue of GLP-1 which is a compound of formula (II) has 0, 1, 2 or 3 conservative substitutions at positions 6 to 30 or 31, preferably 0, 1 or 2, more preferably 0 or 1 and most preferably 0.

Certain preferred GLP-1 analogues of the invention contain at least 70% sequence homology with native GLP-1, for example from 70-97%. Some particularly preferred analogues of the invention have between 73 and 97% sequence homology with GLP-1 (7-36)-NH2, for example 80 to 97%, 85 to 97%, 90 to 97%, or 93 to 97%. Some preferred analogues of the invention contain greater than 70% sequence homology with native GLP-1 : for example 73%, 76%, 80%, 83%, 86%, 90%, 93% or 97% sequence homology. In some embodiments, an analogue contains an amino acid sequence that corresponds to the amino acid sequence of native GLP-1 (e.g. GLP-1 (7-36ΓΝΗ2) except that the analogue contains from 1 to 8 amino acid substitutions, preferably the analogue contains from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 6, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 8, from 5 to 7, or from 5 to 6, amino acid substitutions from the amino acid sequence of native GLP-1 (for example 1, 2, 3, 4, 5, 6, 7 or 8).

In one particularly preferred embodiment the analogue of GLP-1 which is a compound of formula (II) is not a variant. In another particularly preferred embodiment the analogue of GLP-1 which is a compound of formula (II) is not a derivative. In one particularly preferred embodiment, the GLP-1 analogue is a compound consisting of an amino acid sequence represented by formula (II), or a variant having up to three conservative amino acid substitutions at any one of positions 6 to 31 where Xaa" is present or at any one of positions 6 to 30 where Xaa" is absent, for example 0, 1, 2 or 3 conservative amino acid substitutions; or a derivative of the analogue or variant thereof, or a salt of the compound, variant thereof or the derivative of the compound or variant. The preferences for amino acid residues and combinations of amino acid residues set out above are also preferred for such GLP-1 analogues. In one particularly preferred embodiment the GLP-1 analogue is a compound consisting of an amino acid sequence represented by formula (II), or a salt of the analogue.

In embodiments of the invention where the peptide hormone analogue is an analogue of GLP-1 which is a compound of formula (II), preferably: Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, more preferably D-His and Phe);

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln, His, Ala, Tyr, Leu and Lys or Glu, D-Gln, His, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala and Ser); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

In another preferred embodiment of a compound of formula (II):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, most preferably D-His and

Phe);

Xaa² is Ala;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln, His, Ala, Tyr, Leu and Lys, more preferably D- Gin, His, Ala, Tyr, Leu and Lys);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, more preferably D-His and Phe);

Xaa² is Ala;

Xaa³ is Glu;

Gly, Leu, Ala and Ser, more preferably Gly); and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is His;

Xaa² is Ala; Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln, His, Ala, Tyr, Leu and Lys or Glu, D-Gln, His, Ala, Tyr, Leu and Lys);

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, more preferably D-His and

Phe);

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala, Ser, D-Ser, Gly, Thr and Pro or, more preferably Ser, D-Ser, Gly, Thr and Pro);

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Gin, Tyr, and D-Tyr (preferably D-His, Phe, Asn, Gin, Tyr, and D-Tyr, most preferably D-His and Phe); Xaa² is Ala;

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is His;

Xaa² is Ala;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is His;

Xaa² is Ala; Xaa³ is Glu;

Xaa⁴ is selected from the group consisting of Leu, Ala, Ser and AIB, (preferably Gly, Leu, Ala and Ser); and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (II):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably Gin, D-Tyr and His, more preferably His);

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro; Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr,

Leu and Lys (preferably Gin, Asp or Glu, more preferably Glu and Asp);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB, (preferably Gly); and

Xaa⁵ is selected from the group consisting of Thr and Ser.

In another preferred aspect of the invention, the peptide hormone analogue is an analogue of GLP-1 which is a compound selected from the group consisting of:

formula (Ila):

Xaa¹-Ala-Xaa -Xaa⁴-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ila);

formula (lib):

(lib)

formula (lie):

Xaa^Ala-Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(lie)

formula (lid):

Xaa^Ala-Glu-Xaa'-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(lid)

formula (He):

(He) formula (Ilf):

Xaa^Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Ilf)

formula (Ilg):

His-Xaa'-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Ilg)

formula (Ilh) :

His-Ala- Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Ilh)

formula (Ilj):

His-Ala-Glu-Xaa⁴-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Ilj); and

formula (Ilk):

His-Ala-Glu-Gly-Xaa⁵-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Tφ-Leu-Val-Lys-Gly-Arg-Xaa^α

(Ilk) wherein the definitions of Xaa¹, Xaa², Xaa³, Xaa⁴, Xaa⁵ are as for a compound of formula (II) as described above, and with the proviso that the sequence at positions 1 to 5 is not His-Ala-Glu-Gly- Thr.

In one particularly preferred embodiment, the GLP-1 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 1 to 10, or a derivative of the compound, or a salt of the compound or the derivative. The amino acid sequences of analogue nos. 1 to 10 are provided in the Table of Figure 1. In one preferred embodiment the GLP-1 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 1 to 10, or a salt of the compound. In one preferred embodiment the GLP-1 analogue is a compound consisting of the amino acid sequence of any of analogue nos. 1 to 10.

(iii) an analogue of oxyntomodulin which is a compound of formula (III):

In one preferred embodiment of the invention the peptide hormone analogue is an analogue of oxyntomodulin which is a compound of formula (III) as defined above. An analogue of OXM which is a compound of formula (III) has 0, 1, 2 or 3 conservative substitutions at positions 6 to 37, preferably 0, 1 or 2, more preferably 0 or 1 and most preferably 0.

Certain preferred OXM analogues of the invention contain at least 78% sequence homology with native OXM, for example from 75-97%. Some particularly preferred analogues of the invention have between 75 and 97% sequence homology with native OXM, for example 80 to 97%, 85 to 97%, 90 to 97%, or 93 to 97%. Some preferred analogues of the invention contain greater than 70% sequence homology with native OXM: for example 78%, 81%, 84%, 86%, 89%, 92%, 95% or 97% sequence homology. In some embodiments, an analogue contains an amino acid sequence that corresponds to the amino acid sequence of native OXM except that the analogue contains from 1 to 8 amino acid substitutions, preferably the analogue contains from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 6, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 8, from 5 to 7, or from 5 to 6, amino acid modifications from the amino acid sequence of native OXM (for example 1, 2, 3, 4, 5, 6, 7 or 8).

In one particularly preferred embodiment the analogue of OXM which is a compound of formula (III) is not a variant. In another particularly preferred embodiment the analogue of OXM which is a compound of formula (III) is not a derivative.

In one particularly preferred embodiment, the OXM analogue is a compound consisting of an amino acid sequence represented by formula (III), or a variant having up to three conservative amino acid substitutions at any one of positions 6 to 37, for example 0, 1, 2 or 3 conservative amino acid substitutions; or a derivative of the analogue or variant thereof, or a salt of the compound, variant thereof or the derivative of the compound or variant. The preferences for amino acid residues and combinations of amino acid residues set out above are also preferred for such OXM analogues. In one particularly preferred embodiment the OXM analogue is a compound consisting of an amino acid sequence represented by formula (III), or a salt of the analogue. In embodiments of the invention where the peptide hormone analogue is an analogue of OXM which is a compound of formula (III), preferably:

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Asn); Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ser);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys or D-Gln, His, Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example D-Gln or His);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB, (preferably Gly, Leu, Ala, Ser); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

In another preferred embodiment of a compound of formula (III):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Asn); Xaa² is Ser;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Asn);

Xaa² is Ser;

Xaa³ is Gin;

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa¹ is His;

Xaa² is Ser;

Leu and Lys (preferably Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys or D-Gln, His, Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example D-Gln or His);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala, and Ser); and

Xaa⁵ is Thr. In another preferred embodiment of a compound of formula (III):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Asn); Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala, Ser, D-Ser, Gly, Thr and Pro, more preferably Ala, D-Ser, Gly, Thr and Pro);

Xaa³ is Gin;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa³ is Gin;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is selected from the group consisting of Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example D-Gln or His) Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is Gin;

Xaa⁴ is selected from the group consisting of Leu, Ala, Ser and AIB, (preferably Gly, Leu, Ala and Ser).

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (III):

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably His, Gin, Phe and D-Tyr, more preferably His, Gin and D-Tyr); Xaa² is selected from the group consisting of AIB, Ser, D-Ser, Gly, Thr and Pro (preferably D-Ser, Gly, Thr and Pro);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, Asp or Glu, more preferably Glu);

Gly); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Ser).

In another preferred aspect of the invention, the peptide hormone analogue is an analogue of OXM which is a compound selected from the group consisting of:

formula (Ilia):

Xaa¹-Ser-Xaa -Xaa⁴-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Ilia);

formula (Illb):

(Illb)

formula (IIIc):

Xaa^Ser-Xaa'-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(IIIc)

formula (Hid):

Xaa¹-Ser-Gln-Xaa⁴-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Hid)

formula (Hie):

Xaa^Xaa'-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(Hie)

formula (Illf):

Xaa^Ser-Gln-Gly-Thr -Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

( f)

formula (Illg):

His- Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a (nig)

formula (Illh):

His-Ser- Xaa -Gly-Thr -Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Illh)

formula (IHj):

His-Ser-Gln- Xaa⁴-Thr Phe-TTir-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-T -Leu-Val-Lys-Gly-Arg-Xaa^α

(Illj)

and formula (Illk):

His-Ser-Gln-Gly- Xaa⁵-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(ink) wherein the definitions of Xaa¹, Xaa², Xaa³, Xaa⁴, Xaa⁵ are as for a compound of formula (II) described above, and^wlth the proviso that the sequence at positions 1 to 5 is not His-Ser-Gln-Gly-Thr.

(iv) an analogue of glucagon which is a compound of formula (IV)

In one preferred embodiment of the invention the peptide hormone analogue is an analogue of glucagon which is a compound of formula (IV) as defined above.

In one particularly preferred embodiment the analogue of glucagon which is a compound of formula (IV) is not a derivative. In one particularly preferred embodiment, the glucagon analogue is a compound consisting of an amino acid sequence represented by formula (IV), 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 one particularly preferred embodiment the glucagon analogue is a compound consisting of an amino acid sequence represented by formula (IV), or a salt of the analogue.

In embodiments of the invention where the peptide hormone analogue is an analogue of glucagon which is a compound of formula (IV), preferably:

(preferably Ser); Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys or D-Gln, His, Asp, Ala, Tyr, Leu and Lys, more preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example D-Gln or His);

Gly, Leu, Ala, Ser); and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Thr).

In another preferred embodiment of a compound of formula (IV):

Tyr (preferably D-His, Phe, Asn, Tyr, and D-Tyr, more preferably D-His and Asn); Xaa² is Ser;

Xaa⁴ is Gly; and

Xaa⁵ is Thr. In another preferred embodiment of a compound of formula (IV):

Xaa³ is Gin;

Gly, Leu, Ala and Ser); and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV):

Xaa¹ is His;

Xaa² is Ser;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB (preferably Gly, Leu, Ala, and Ser); and Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV):

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ala, Ser, D-Ser, Gly, Thr and Pro, more preferably Ala, D-Ser, Gly, Thr and Pro);

Xaa³ is Gin;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV):

Xaa² is Ser;

Xaa³ is Gin;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV):

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is selected from the group consisting of Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys, for example D-Gln or His)

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV):

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is Gin;

Xaa⁵ is Thr.

In another preferred embodiment of a compound of formula (IV): Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably His, Gin, Phe and D-Tyr, more preferably His, Gin and D-Tyr); Xaa² is selected from the group consisting of AIB, Ser, D-Ser, Gly, Thr and Pro (preferably D-Ser, Gly, Thr and Pro);

Leu and Lys (preferably Gin, Asp or Glu, more preferably Glu and Asp);

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Ser).

In another preferred aspect of the invention, the peptide hormone analogue is an analogue of glucagon which is a compound selected from the group consisting of:

formula (IVa):

Xaa¹-Ser-Xaa -Xaa⁴-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸- Ala-Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVa);

formula (IVb):

Xaa¹-Xaa²-Xaa -Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVb)

formula (IVc):

Xaa¹-Ser-Xaa -Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVc)

formula (IVd):

Xaa¹-Ser-Gln-Xaa⁴-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVd)

formula (IVe):

Xaa¹-Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVe)

formula (I Vf):

Xaa¹-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVf) formula (IVg):

His- Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-T -Leu-Leu-Asn-Xaa²⁹-V

(IVg)

formula (IVh):

His-Ser- Xaa -Gly-Thr -Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVh)

formula (IVj):

His-Ser-Gln- Xaa⁴-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-T -Leu-Leu-Asn-Xaa²⁹-V (IVj) and formula (IVk):

His-Ser-Gln-Gly- Xaa⁵-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹ -Leu-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Ala- Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-Trp-Leu-Leu-Asn-Xaa²⁹-V

(IVk) wherein the definitions of Xaa¹, Xaa², Xaa³, Xaa⁴, Xaa⁵ are as for a compound of formula (IV) as described above, and^wlth the proviso that the sequence at positions 1 to 5 is not His-Ser-Gln-Gly-Thr or His-AIB-Gln-Gly-Thr.

In formula (IV), 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, His-His-His-NH₂, and a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues; or V is absent.

In embodiments where V is absent, Xaa²⁹ may be derivatised, for example Xaa²⁹ may be amidated.

In preferred embodiments of the invention, V is present and 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, His-His-His-NH₂, and a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues. More preferably, V is a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues. In one preferred embodiment of the peptide hormone analogue of formula (IV), V has the formula (X) (X)

wherein Xaa¹ is Gly or His;

Xaa¹¹ is absent or selected from the group consisting of Gly and His;

Xaa¹¹¹ is absent or selected from the group consisting of Gly and His;

Xaa^lv is absent or selected from the group consisting of Gly and His;

Xaa^v is absent or His;

Xaa" is absent or His;

Xaa^vu is absent or His;

Xaa^vm is absent or is selected from the group consisting of Ala, Glu, Gly, Gin and Ser;

Xaa^lx is absent or is selected from the group consisting of Ala, Glu, Gly, Gin and Ser; Xaa^x is absent or is selected from the group consisting of Ala, Glu, Gly, Gin and Ser; and wherein the C-terminal residue may optionally terminate in a -CONH2 group in place of a carboxylic acid group. Unless indicated otherwise, the amino acid residues in the sequence of formula (X) are L- amino acids.

In one preferred embodiment V has the formula (II) and one of Xaa¹, Xaa¹¹ and Xaa¹¹¹ is Gly; more preferably one of Xaa¹ and Xaa¹¹ is Gly.

In one preferred embodiment V has the formula (II), Xaa¹ is Gly and Xaa¹¹ and Xaa¹¹¹ are each independently absent or His.

In one preferred embodiment V has the formula (II), Xaa¹ is His, Xaa¹¹ is Gly and Xaa¹¹¹ is His or absent.

In one preferred embodiment V has the formula (II) and V contains from 3 to 6 His residues, more preferably 3, 4 or 5 His residues.

In one preferred embodiment V has the formula (II) and V contains from 0 to 4 non-His residues; more preferably 0, 1, 2 or 3 non-His residues.

In one preferred embodiment V has the formula (II) and contains 3 His residues and 1, 2 or 3 non-His residues, or V has the formula (II) and contains 4 or 5 His residues and contains 0, 1, 2 or 3 non-His residues.

In one preferred embodiment V is selected from the group consisting of His, His-NH₂, His-His, His- H1S-NH2, Gly-His, Gly-His-NH₂ , Lys-His, Lys-His-NH₂, Gly-His-His, Gly-His-His-NH₂, His-His- His, His-His-His-NH₂, Gly-His-His-His-His-Ala-NH₂, Gly-His-His-His-His-Gln-NH₂, Gly-His-His- His-His-His-Glu-NH₂, Gly-His-His-His-His-Ser-NH₂, Gly-His-His-His-His-Gln-Gln-NH₂, His-His- His-His-Gly, His-His-His-His-Ala-Gly, Gly-His-His-His-NH₂, His-Gly-His-His-NH₂, His-His-His- His-NH₂, His-His-His-His-His-Gln-NH₂, His-His-His-Gly and His-His-His-His-His-Gly. Most preferably V is His-His-His-His-His-Gln-NH2; and more preferably Gly-His-His-His-His-Ala-NH2, Gly-His-His-His-His-Gln-NH₂, Gly-His-His-His-His-His-Glu-NH₂, Gly-His-His-His-His-Ser-NH₂, Gly-His-His-His-His-Gln-Gln-NH₂, His-His-His-His-Gly, His-His-His-His-Ala-Gly, Gly-His-His- H1S-NH2, His-Gly-His-His-NH₂, His-His-His-His-NH₂, His-His-His-His-His-Gln-NH₂, His-His-His- Gly and His-His-His-His-His-Gly. Most preferably V is His-His-His-His-His-Gln-NH2

For the avoidance of doubt, references to V groups containing a C-terminal -Ν¾ group will be understood as referring to peptides in which the carboxylic acid group of the C-terminal amino acid residue is replaced by a -CONH2 group. For example, where V has the sequence His-His-His-His- His-Gln-NH2, the C-terminal Gin residue has a-CONH2 group in place of the carboxylic acid (i.e. - CO2H) group.

In one preferred embodiment for a compound of formula (IV), Xaa¹⁰ is Tyr, Xaa²⁰ is His, and V has the formula (X); more preferably V has the formula (X) and contains 3 His residues and 1, 2 or 3 non- His residues, or V has the formula (X) and contains 4 or 5 His residues and 0, 1, 2 or 3 non-His residues.

In one particularly preferred embodiment, in the compound of formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, and V has the formula (X); more preferably V has the formula (X) and contains 3 His residues and 1, 2 or 3 non-His residues, or V has the formula (X) and contains 4 or 5 His residues and 0, 1, 2 or 3 non-His residues.

In one particularly preferred embodiment, in the compound of formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin (preferably Glu), Xaa¹⁷ is Lys or Arg (preferably Lys), Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie; and 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, His-His-His-NH₂, Gly-His-His-His-His-Ala-NH₂, Gly- His-His-His-His-Gln-NH₂, Gly-His-His-His-His-His-Glu-NH₂, Gly-His-His-His-His-Ser-NH₂, Gly- His-His-His-His-Gln-Gln-NH₂, His-His-His-His-Gly, His-His-His-His-Ala-Gly, Gly-His-His-His- NH₂, His-Gly-His-His-NH₂, His-His-His-His-NH₂, His-His-His-His-His-Gln-NH₂, His-His-His-Gly and His-His-His-His-His-Gly (more preferably , Gly-His-His-His-His-Ala-NH2, Gly-His-His-His-His- Gln-NH₂, Gly-His-His-His-His-His-Glu-NH₂, Gly-His-His-His-His-Ser-NH₂, Gly-His-His-His-His- Gln-Gln-NH₂, His-His-His-His-Gly, His-His-His-His-Ala-Gly, Gly-His-His-His-NH₂, His-Gly-His- H1S-NH2, His-His-His-His-NH₂, His-His-His-His-His-Gln-NH₂, His-His-His-Gly and His-His-His- His-His-Gly). In formula (IV), Xaa is selected from the group consisting of Tyr and Leu. In one preferred embodiment Xaa¹⁰ is Tyr. In one embodiment Xaa¹⁰ is Leu.

In formula (IV), Xaa¹² is selected from the group consisting of Lys, His and Arg. In one preferred embodiment Xaa¹² is Lys. In one embodiment Xaa¹² is His. In one embodiment Xaa¹² is Arg.

In formula (IV), Xaa¹³ is selected from the group consisting of Tyr, Gin and His. In one preferred embodiment Xaa¹³ is Tyr. In one embodiment Xaa¹³ is His. In one embodiment Xaa¹³ is Gin. In one embodiment Xaa¹³ is selected from the group consisting of Tyr and His.

In formula (IV), Xaa¹⁵ is selected from the group consisting of Asp and Glu. In one preferred embodiment Xaa¹⁵ is Asp. In one embodiment Xaa¹⁵ is Glu.

In formula (IV), Xaa¹⁶ is selected from the group consisting of Glu, Gin and Ser. In one embodiment

Xaa¹⁶ is Ser. In one preferred embodiment Xaa¹⁶ is Glu. In one preferred embodiment Xaa¹⁶ is Gin.

In one preferred embodiment Xaa¹⁶ is selected from the group consisting of Glu and Gin.

In formula (IV), Xaa¹⁷ is selected from the group consisting of Arg, His and Lys. In one preferred embodiment Xaa¹⁷ is Arg. In one preferred embodiment Xaa¹⁷ is Lys. In one embodiment Xaa¹⁷ is His. In one preferred embodiment Xaa¹⁷ is selected from the group consisting of Arg and Lys.

In formula (IV) Xaa¹⁸ is selected from the group consisting of Arg and Lys. In one preferred embodiment Xaa¹⁸ is Arg. In one embodiment Xaa¹⁸ is Lys.

In formula (IV), Xaa²⁰ is selected from the group consisting of His and Gin. In one preferred embodiment Xaa²⁰ is His. In one embodiment Xaa²⁰ is Gin.

In formula (IV), Xaa²¹ is selected from the group consisting of Glu, His and Asp. In one preferred embodiment Xaa²¹ is Glu. In one embodiment Xaa²¹ is Asp. In one embodiment Xaa²¹ is His. In one embodiment Xaa²¹ is selected from the group consisting of Glu and Asp.

In formula (IV), Xaa²³ is selected from the group consisting of lie and Val. In one embodiment Xaa²³ is Val. In one preferred embodiment Xaa²³ is lie.

In formula (IV), Xaa²⁴ is selected from the group consisting of Gin and Glu. In one preferred embodiment Xaa²⁴ is Gin. In one preferred embodiment Xaa²⁴ is Glu.

In formula (IV), Xaa²⁹ is selected from the group consisting of Thr and Gly. In one preferred embodiment Xaa²⁹ is Thr. In one preferred embodiment Xaa²⁹ is Gly. In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr and Xaa¹⁶ is Ser. In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr and Xaa²⁰ is His. In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁶ is Ser and Xaa²⁰ is His. In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser and Xaa^2C is His.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹³ 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 Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹³ is Tyr and Xaa¹⁵ is Asp. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹³ is Tyr and Xaa¹⁷ is Arg. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹³ is Tyr and Xaa¹⁸ is Arg. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹³ is Tyr and Xaa²¹ is Glu. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹³ is Tyr and Xaa²³ is Val. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁵ is Asp and Xaa¹⁷ is Arg. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁵ is Asp and Xaa¹⁸ is Arg. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁵ is Asp and Xaa²¹ is Glu. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁵ is Asp and Xaa²³ is Val. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁷ is Arg and Xaa¹⁸ is Arg. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁷ is Arg and Xaa²¹ is Glu. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁷ is Arg and Xaa²³ is Val. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁸ is Arg and Xaa²¹ is Glu. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁸ is Arg and Xaa²³ is Val. In one preferred embodiment, the analog *ue has the formula (IV), Xaa²¹ is Glu and Xaa²³ is Val. In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Arg, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is His, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is His, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Glu, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analog me has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analog me has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁷ is Lys and Xaa²⁰ is His.

In one preferred embodiment, the analog *ue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁸ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analot me has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁸ is Lys and Xaa²⁰ is His. In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²¹ is Glu.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²¹ is Asp.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²³ is lie.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²⁴ is Gin.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²⁴ is Glu.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²⁹ is Thr.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²⁹ is Gly.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser and Xaa²⁰ is His.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁸ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²¹ is Glu.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula TV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa¹⁸ is Arg and Xaa²⁰ is His.

In one preferred embodiment, the analogue has the formula TV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²¹ is Glu.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa²⁰ is His and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula ;iV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg and Xaa²⁰ is His. In one preferred embodiment, the analogue has the formula (IV), Xaa is Tyr, Xaa is Ser, Xaa is Arg, Xaa²⁰ is His and Xaa²¹ is Glu.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg, Xaa²⁰ is His and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁸ is Arg, Xaa²⁰ is His and Xaa²¹ is Glu.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa¹⁸ is Arg, Xaa²⁰ is His and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹⁶ is Ser, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Arg, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu and Xaa²³ is Val.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is Val and Xaa²⁴ is Gin.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is Val and Xaa²⁴ is Glu.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is Val and Xaa²⁹ is Gly.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is Val and Xaa²⁹ is Thr.

In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Ser, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²⁴ is Gin and Xaa²³ is Val. In one preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²⁴ is Glu and Xaa²³ is Val. In one particularly preferred embodiment, the analogue has the formula (IV), Xaa is Tyr, Xaa is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, and Xaa²⁴ is Glu.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, and Xaa²⁴ is Gin.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, and Xaa²⁹ is Gly.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie. In one particularly preferred embodiment, the analogue has the formula (IV), Xaa is Tyr, Xaa is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Glu.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Gin.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Glu.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Gly.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Glu.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Gin.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Glu.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁴ is Gin.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Thr. In one particularly preferred embodiment, the analogue has the formula (IV), Xaa is Tyr, Xaa is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Gly.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie and Xaa²⁹ is Gly.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, Xaa²⁴ is Glu and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, Xaa²⁴ is Gin and Xaa²⁹ is Thr.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, Xaa²⁴ is Glu and Xaa²⁹ is Gly.

In one particularly preferred embodiment, the analogue has the formula (IV), Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin, Xaa¹⁷ is Lys or Arg, Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, Xaa²³ is lie, Xaa²⁴ is Gin and Xaa²⁹ is Gly.

In one embodiment, where the analogue has the formula (IV) and Xaa¹² is Lys, Xaa¹³ is not His. In one embodiment, where the analogue has the formula (IV) and Xaa¹³ is His, Xaa¹⁷ is not His.

In one particularly preferred embodiment, the glucagon analogue is a compound consisting of the amino acid sequence of any of analogue nos. 20 to 37, or a derivative of the compound, or a salt of the compound or the derivative. The amino acid sequences of analogue nos. 20 to 37 are provided in the Table of Figure 1. In one preferred embodiment the glucagon analogue is a compound consisting of the amino acid sequence of any of analogue nos. 20 to 37, or a salt of the compound. In one preferred embodiment the glucagon analogue is a compound consisting of the amino acid sequence of any of analogue nos. 20 to 37.

The GLP-1 analogues, Ex-4 analogues, OXM analogues and glucagon analogues of the present invention may be produced by recombinant methods well-known in the art or alternatively they may be produced by synthetic methods, again well-known in the art. The present invention also provides a peptide hormone analogue that is biased for the GLP-1 receptor G-protein-dependent pathway compared to the GLP-1 receptor G-protein-independent pathway. Such a peptide hormone analogue may be a GLP-1 analogue, OXM analogue, Ex-4 analogue or glucagon analogue having from 1 to 5 amino acid substitutions at positions 1 to 5 of the analogue (for example 1, 2, 3, 4 or 5, amino acid substitutions at positions 1 to 5 of the analogue, preferably 1, 2 or 3, and more preferably 1 or 2). Such a peptide hormone analogue may be a GLP-1 analogue, OXM analogue, Ex-4 analogue or glucagon analogue as described above.

The present invention also provides a peptide hormone analogue that is biased for the GLP-1 receptor G-protein-independent pathway compared to the GLP- 1 receptor G-protein-independent pathway. Such a peptide hormone analogue may be a GLP-1 analogue, OXM analogue, Ex-4 analogue or glucagon analogue having from 1 to 5 amino acid substitutions at positions 1 to 5 of the analogue (for example 1, 2, 3, 4 or 5, amino acid substitutions at positions 1 to 5 of the analogue, preferably 1, 2 or 3, and more preferably 1 or 2). Such a peptide hormone analogue may be a GLP-1 analogue, OXM analogue, Ex-4 analogue or glucagon analogue as described above.

For example, the peptide hormone analogue of the present invention may be

(i) an analogue of Exendin-4 which is a compound of formula (I) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to the G- protein-independent pathway; or (/^') an analogue of Exendin-4 which is a compound of formula (I) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein- independent pathway compared to the G-protein-dependent pathway; or (ii) an analogue of GLP-1 which is a compound of formula (II) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to the G- protein-independent pathway; or (/^'/^') an analogue of GLP-1 which is a compound of formula (II) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein- independent pathway compared to the G-protein-dependent pathway;

(iii) an analogue of oxyntomodulin which is a compound of formula (III) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to the G-protein-independent pathway; (in) an analogue of oxyntomodulin which is a compound of formula (III) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to the G-protein-dependent pathway; (iv) an analogue of glucagon which is a compound of formula (IV) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to the G-protein-independent pathway; or (iv) an analogue of glucagon which is a compound of formula (IV) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to the G-protein-dependent pathway.

Additionally, or alternatively, the peptide hormone analogue of the present invention may be

(i) an analogue of Exendin-4 which is a compound of formula (I) as described above: and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to GLP-1 and/or Exendin-4; or (/^') an analogue of Exendin-4 which is a compound of formula (I) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to GLP-1 and/or Exendin-4; or (ii) an analogue of GLP-1 which is a compound of formula (II) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to GLP- 1; or (/^'/^') an analogue of GLP-1 which is a compound of formula (II) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to GLP-1;

(iii) an analogue of oxyntomodulin which is a compound of formula (III) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to GLP-1 and/or oxyntomodulin; (Hi) an analogue of oxyntomodulin which is a compound of formula (III) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G- protein-independent pathway compared to GLP-1 and/or oxyntomodulin;

(iv) an analogue of glucagon which is a compound of formula (IV) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to GLP-1 and/or glucagon (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues); or (iv) an analogue of glucagon which is a compound of formula (IV) as described above, and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to GLP-1 and/or glucagon (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues).

A person skilled in the art can determine whether a compound is biased for the G-protein-dependent pathway or GLP-1 receptor G-protein-independent pathway, for example by measuring analogue- induced β-arrestin recruitment (for example βΑΡνΡν2 and/or ARR2 recruitment) and cyclic AMP (cAMP) generation in Pathhunter CHO-K1-GLP-1R cells and these data used to calculate pathway- specific relative activities. Derivatives

A peptide hormone analogue of the invention may be a derivative which comprises the structure of formula (I), (II), (III) or (IV) modified by well-known processes including amidation (for example amidation of the C-terminal amino acid), glycosylation, carbamylation, acylation, for example acetylation, sulfation, phosphorylation, cyclization, lipidization and pegylation and fusion to another peptide or protein to form a fusion protein. Preferred modifications of the analogues of the present invention are amidation (for example amidation of the C-terminal amino acid), lipidization, pegylation and fusion to another peptide or protein to form a fusion protein. The structure of formula (I), (II), (III) or (IV) 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 peptide hormone analogue of the invention may be a fusion protein, whereby the structure of formula (I), (II), (III) or (IV) 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), (II), (III) or (IV). 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 C- terminus of the fusion partner to the amino-terminus of the structure of formula, (I) (II), (III) or (IV); or vice versa. Optionally, a cleavable linker may be used to link the structure of formula (I), (II), (III) or (IV) to the fusion partner. A resulting cleavable fusion protein may be cleaved in vivo such that an active form of an analogue 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 peptide hormone analogue of the invention may be a physiologically functional derivative of the structure of formula (I), (II), (III) or (IV). The term "physiologically functional derivative" is used herein to denote a chemical derivative of an compound of formula (I), (II), (III) or (IV) having the same physiological function as the corresponding unmodified compound of formula (I), (II), (III) or (IV). For example, a physiologically functionally derivative may be convertible in the body to a compound of formula (I), (II), (III) or (IV). According to the present invention, examples of physiologically functional derivatives include esters, amides, and carbamates; preferably esters and amides.

Pharmaceutically acceptable esters and amides of the analogues of the invention may comprise a Ci-20 alkyl-, C2-20 alkenyl-, C5-10 aryl-, C5-10 ar-Ci-20 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 (C12H23), palmityl (C15H31), oleyl (C15H29) or stearyl (C17H35)) and bile acids (e.g. cholate or deoxycholate).

Methods for lipidization of sulfhydryl-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 an analogue of the invention comprising an analogue of the invention linked to fatty acid via a disulfide linkage may be used for delivery of an analogue of the invention to neuronal cells and tissues. Lipidisation markedly increases the absorption of the analogues relative to the rate of absorption of the corresponding unlipidised analogues, as well as prolonging blood and tissue retention of the analogues. Moreover, the disulfide 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 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 (C12H23), palmityl (C15H31), oleyl (C15H29) or stearyl (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. Cyclized peptides 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 dimeric head-to-tail cyclized structures. Cyclized peptides 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 peptide hormone analogue of the invention may be a PEGylated structure of formula (I), (II), (III) or (IV). PEGylated analogues 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 derivatization of a peptide hormone analogue of the invention may also be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. A polymer moiety for derivatisation of an analogue 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 an analogue of the invention is from about 1 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.

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 peptide hormone analogues of formula (I), formula (II), formula (III) or formula (IV); of derivatives of the analogues, and of salts of the analogues and derivatives.

Salts of analogues of formula (I), (II), (III) or (IV) 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 analogues of formula (I), (II), (III) or (IV) and

pharmaceutically acceptable salts and/or derivatives thereof.

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, glycolic, salicylic, oxaloacetic, methanesulfonic, ethane sulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, and isethionic acids. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the analogues of the invention and their pharmaceutical acceptable salts. Pharmaceutically acceptable salts with bases include ammonium salts, alkali 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-methyl-D- glucomine.

Biological Activity

Peptide hormone analogues of the invention have activity at the human GLP-1 receptor and can be considered GLP-1 receptor agonists. This may be assessed by, for example, an in vitro or cellular binding assay or by a reporter assay.

Preferred peptide hormone analogues of the invention exhibit an activity at the human GLP-1 receptor that is at least l/50^th that of human GLP-1, preferably an activity which is at least l/20^th, l/10^th, l/5^th, l/3^rd or ½ that of the corresponding native peptide (e.g. Ex-4 for Ex-4 analogues; OXM for OXM analogues; glucagon (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues), and/or human GLP-1. Especially preferred peptide hormone analogues of the invention exhibit an activity at the human GLP-1 receptor that is at least equivalent to that of that of the corresponding native peptide, and/or human GLP-1. Methods of assessing activity at the GLP-1 receptor are well known.

Peptides active at the GLP-1 receptor signal through the G-protein-dependent pathway, which is dependent on generation of cAMP; and the G-protein-independent pathway, which is dependent on analogue-induced -arrestin-2 ( ARR2) and β-arrestin-l (βΑΡνΡνΙ) recruitment. A native peptide may be biased for one of those pathways over the other. For example, OXM is more biased at GLP-1 R for the G-protein-dependent pathway than the G-protein-independent pathway. Peptide hormone analogues of the invention are more biased (either for the G-protein-dependent pathway or the G- protein-independent pathway) than the corresponding native peptide.

As well as being GLP-1 receptor agonists, peptide hormone analogues of the invention are more biased (either for the G-protein-dependent pathway or the G-protein-independent pathway) than the corresponding native peptide. For example, the analogues of the invention are be biased for either pathway compared to Ex-4 for Ex-4 analogues; OXM for OXM analogues; glucagon (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues. Additionally, or alternatively, the peptide hormone analogues of the invention may be biased for either pathway compared to GLP- 1. Particularly preferred analogues of the invention are biased at the GLP-1R for the G-protein- dependent pathway (for example compared to the native peptide and/or GLP-1). For example, preferred compounds of the invention are at least 1.1 -fold biased towards cAMP and away from ARR2 recruitment; preferably at least 1.2-fold biased towards cAMP and away from βΑΡνΡν2 recruitment; preferably at least 1.3-fold biased towards cAMP and away from βΑΡνΡν2 recruitment; preferably at least 2-fold biased towards cAMP and away from βΑΡνΡν2 recruitment; preferably at least 1.5-fold biased towards cAMP and away from βΑΡνΡν2 recruitment; preferably at least 1.7-fold biased towards cAMP and away from βΑΡνΡν2 recruitment; more preferably at least 3 -fold biased towards cAMP and away from βΑΡνΡν2 recruitment, more preferably at least 5 -fold biased towards cAMP and away from βΑΡνΡν2 recruitment and even more preferably at least 10-fold biased towards cAMP and away from βΑΡνΡν2 recruitment compared to native peptide and/or GLP-1. The corresponding native peptide is the peptide that the compound is an analogue of (e.g. Ex-4 for Ex-4 analogues; OXM for OXM analogues; glucagon (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues). Methods of assessing ligand bias are well known. For example, Christopoulos, et al, ACS Chem Neurosci, 2012, Vol , pages 193 - 203, and Herenbrink, et al, Nature

Communications, 2016, Vol 7, Article number: 10842. A specific method is described herein below.

Preferred peptide hormone analogues of the invention have a more sustained effect on insulin secretion than GLP-1 and/or the corresponding native peptide (e.g. Ex-4 for Ex-4 analogues; OXM for OXM analogues; glucagon, (or a glucagon analogue not within the scope of the present invention having Xaa¹ = His, Xaa² = Ser; Xaa³ = Gin; Xaa⁴ = Gly and Xaa⁵ = Thr) for glucagon analogues).

Preferred analogues of the invention reduce glucose levels in vivo (as assessed in humans or an animal model). Preferred analogues have an effect on blood glucose level and/or a subject's glycemic control which is at least as strong as GLP-1, Ex-4 and/or glucagon. For especially preferred analogues, that effect is more sustained compared to GLP-1, Ex-4 and/or glucagon.

Preferred peptide hormone analogues of the invention fulfil some, or more preferably all, of the following criteria.

1) Biased at the human GLP-1 receptor for the generation of cAMP over recruitment of βΑΡΡ2.

2) Lead to a reduction in GLP-1 R desensitisation (for example compared to GLP-1 and/or Ex-4 and/or glucagon)

3) Are more insulinotropic than GLP-1 and/or Ex-4 and/or glucagon when administered to β-cells over 24 hour 4) Have sustained bioactivity at the human GLP-1 receptor resulting in enhancement of insulin release.

5) Have sustained bioactivity at the human GLP-1 receptor resulting in enhancement of glycemic control.

6) Control glucose levels in vivo (as assessed in humans or an animal model)

7) Have a long period of activity in vivo (as assessed in humans or an animal model) so as to permit injections of an analogue of the invention no more frequently than daily and preferably no more than twice, or more preferably no more than once a week, whilst still producing acceptable therapeutic or cosmetic benefits.

8) Good control of the cause and/or symptoms of diabetes (for example improving insulin release in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject and/or providing long-term glycemic control in a subject), especially type 2 diabetes, (as assessed in human subjects or an animal model).

9) Good weight loss or appetite suppression (as assessed in human subjects or an animal model).

Conditions

The invention also provides a peptide hormone analogue of the invention, or a pharmaceutical composition comprising the peptide hormone 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 peptide hormone analogue of formula (I), (II), (III) or (IV) of the invention or of a pharmaceutical composition comprising an compound of formula (I), (II), (III) or (IV) of the invention. Preferably the peptide hormone analogue or pharmaceutical composition is administered subcutaneously.

In a preferred embodiment of the invention, the disease or disorder or other non-desired physiological state is obesity or diabetes, for example type 2 diabetes, type 1 diabetes (especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function). Accordingly, the invention also provides a method for treating obesity or diabetes in a subject comprising administering to the subject a therapeutically effective amount of a peptide hormone analogue of the invention or of a pharmaceutical composition comprising the peptide hormone analogue of the invention; and preferably, a method for treating type 2 diabetes or type 1 diabetes (especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function) in a subject comprising administering to the subject a therapeutically effective amount of a peptide hormone analogue of the invention or of a pharmaceutical composition comprising the peptide hormone analogue of the invention.

In a preferred embodiment of the invention, the disease or disorder or other non-desired physiological state is obesity related diseases and/or diabetes related diseases (for example diabetic peripheral neuropathy, diabetic retinopathy and other forms of diabetic eye disease, diabetic nephropathy, fatty liver disease, non-alcoholic steatosis, non-alcoholic steatohepatitis, obstructive sleep apnoea and/or polycystic ovarian syndrome). Further examples of obesity related diseases and/or diabetes are described herein below.

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

In an especially preferred embodiment, the subject to whom the peptide hormone analogue is administered 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 2 diabetes or type 1 diabetes, especially type 2 diabetes (for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function). Alternatively, or in addition, the subject may be overweight, for example, obese. For example, the subject may be overweight, for example, obese, and have diabetes mellitus, for example, type 2 diabetes or type 1 diabetes, especially type 2 diabetes (for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function).

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 dyslipidemia; stroke; gallbladder disease; osteoarthritis; sleep apnea; reproductive disorders for example, polycystic ovarian syndrome; cancers, for example breast, prostate, colon, endometrial, 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; thromboembolic disease (see Kopelman, Nature 404:635-43; Rissanen et al., British Med. J. 301, 835, 1990); fatty liver disease; non-alcoholic steatosis; non-alcoholic steatohepatitis; and obstructive sleep apnoea. For example, the subject may one or more of the following disorders polycystic ovarian syndrome, fatty liver disease; non-alcoholic steatosis; non-alcoholic steatohepatitis; obstructive sleep apnoea; and polycystic ovarian syndrome

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 (DVT); end stage renal disease (ESRD); gout; heat disorders; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecologic complications; pancreatitis; as well as abdominal hernias; acanthosis nigricans; endocrine abnormalities; chronic hypoxia and hypercapnia; 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; musculoskeletal disease; pseudo tumor cerebri (or benign intracranial hypertension), and sliding hiatil hernia.

In addition, or alternatively, the subject may have, or may be at risk of having, a disorder in which diabetes is a risk factor. Such disorders include, diabetic peripheral neuropathy, diabetic retinopathy and other forms of diabetic eye disease, and diabetic nephropathy.

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 return 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.

According to certain embodiments the disease or disorder or other non-desired physiological state may be having a pre-diabetic state such as insulin insensitivity or pre-diabetes. The invention also provides a method of improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de- sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, improving carbohydrate metabolism in a subject, and/or improving carbohydrate tolerance in a subject, increasing the energy expenditure of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, comprising administration of a therapeutically effective amount of a peptide hormone analogue of the invention, or of a

pharmaceutical composition comprising the peptide hormone analogue of the invention. Such methods may relate to treating subjects having a pre-diabetic state such as insulin insensitivity or pre- diabetes. Preferably the invention provides a method of improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, improving carbohydrate metabolism in a subject, and/or improving carbohydrate tolerance in a subject comprising administration of a therapeutically effective amount of a peptide hormone analogue of the invention, or of a pharmaceutical composition comprising the peptide hormone analogue of the invention.

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 cellular metabolism, protein synthesis, metabolic rate, and calorie utilization. Thus, in this aspect of the invention, administration of a peptide hormone 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 following 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 for improving a lipid profile in a subject comprising administration of a therapeutically effective amount of a peptide hormone analogue of the invention, or of a pharmaceutical composition comprising the peptide hormone 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 peptide hormone analogue of the invention, or of a pharmaceutical composition comprising the peptide hormone analogue of the invention.

A peptide hormone 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 following: preventing and reducing weight gain; inducing and promoting weight loss; and reducing obesity as measured by the Body Mass Index. A peptide hormone 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 are insulin resistant, glucose intolerant, or have any form of diabetes mellitus, for example, type 1, 2 or gestational diabetes (especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function), can benefit from methods in accordance with the present 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 and type 1 diabetes (especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function), eating disorders, insulin-resistance syndromes, and Alzheimer's disease.

J. Cereb. Blood Flow Metab. 2011 Apr 13 (Teramoto S et 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-reperfusion injury. The study showed that mice receiving a transvenous injection of exendin-4, after a 60-minute focal cerebral ischemia showed significantly reduced infarct volume and improved functional deficit as well 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 peptide hormone analogue of the invention, or of a pharmaceutical composition comprising the peptide hormone analogue of the invention. In certain embodiments the disease or disorder or other non-desired physiological state which the peptide hormone analogue may be used to treat or prevent is neurodegeneration. Such

neurodegeneration may be 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, encephalopathy 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 (Amyotrophic 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 Barre 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 would be regarded as neuroprotective. 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 peptide hormone analogue or pharmaceutical composition comprising the peptide hormone analogue provides cardiac protection. According to certain preferred embodiments that treatment is protective of myocardial function following myocardiac infarction. In a preferred aspect, the present invention also provides a peptide hormone of the invention, or a pharmaceutical composition comprising the peptide hormone of the invention, for use in the treatment of obesity or diabetes, for example type 2 diabetes or type 1 diabetes, especially type 2 diabetes (for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function). In a more preferred aspect, the present invention provides a peptide hormone of the invention, or a pharmaceutical composition comprising the peptide hormone of the invention, for use in the treatment of diabetes, for example type 2 diabetes or type 1 diabetes, especially type 2 diabetes (for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function).

The invention also provides a peptide hormone analogue of the invention or a pharmaceutical composition comprising the peptide hormone analogue of the invention for use in increasing energy expenditure of a subject, improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, improving carbohydrate tolerance in a subject and/or improving carbohydrate metabolism in a subject (preferably for use improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control 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-diabetes.

The invention also provides a peptide hormone analogue of the invention or a pharmaceutical composition comprising the peptide hormone 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 improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, and/or use in improving carbohydrate tolerance in a subject. Such use may relate to treating subjects having a pre-diabetic state such as insulin insensitivity or pre-diabetes.

The invention also provides a peptide hormone analogue of the invention, or a pharmaceutical composition comprising the peptide hormone 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 peptide hormone analogue or pharmaceutical composition may be for use 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 peptide hormone analogue or pharmaceutical composition may be 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 ischemia. 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 peptide hormone analogue or pharmaceutical composition comprising the peptide hormone analogue of the invention may be administered as soon as possible after the subject has experienced a suspected myocardial infarction. According to certain embodiments a peptide hormone analogue or pharmaceutical composition comprising the peptide hormone analogue of the invention may be administered as soon as possible after the subject has experienced as suspected stroke.

In another preferred aspect, the invention also provides use of a peptide hormone analogue of the invention for the manufacture of a medicament for the treatment of obesity or diabetes, for example type 2 diabetes or type 1 diabetes (especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function), of a subject, who may be as described above in reference to other aspects of the invention. In a more preferred aspect, the invention provides use of a peptide hormone analogue of the invention for the manufacture of a medicament for the treatment of diabetes, for example type 2 diabetes or type 1 diabetes

(especially type 2 diabetes, for example type 2 diabetes in subjects with insulin resistance and/or type 2 diabetes in subjects with reduced beta cell function), of a subject, who may be as described above in reference to other aspects of the invention.

The invention also provides use of a peptide hormone analogue of the invention for the manufacture of a medicament for increasing energy expenditure in a subject, for improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, for improving carbohydrate tolerance in a subject and/or improving carbohydrate metabolism in a subject (preferably for improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control 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 peptide hormone analogue of the invention for the manufacture of a medicament for improving insulin release in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject, restoring insulin responsiveness in a subject, providing long-term glycemic control in a subject, use in improving carbohydrate tolerance in a subject, the reduction of appetite in a subject, reducing food intake in a subject, and/or reducing calorie intake in a subject.

The invention also provides use of a peptide hormone 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 peptide hormone analogue or pharmaceutical composition is to be administered parentally. According to other embodiments the peptide hormone analogue or pharmaceutical composition is to be administered subcutaneously, intravenously, intramuscularly, intranasally, transdermally or sublingually. According to other embodiments the peptide hormone analogue or pharmaceutical composition is to be administered orally. In one preferred embodiment the peptide hormone analogue or pharmaceutical composition is administered subcutaneously.

According to the present invention, a peptide hormone analogue of the invention is preferably used in the treatment of a human. However, while the peptide hormone 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 poultry; sport animals for example horses; or companion animals for example dogs and cats.

Compositions

While it is possible for the peptide hormone analogue of the present invention 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 peptide hormone 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 preferred embodiments the composition has a pH of less than 5 prior to administration and the composition comprises zinc ions. In such an embodiment the peptide hormone analogue is preferably an analogue of glucagon which is a compound of formula (IV).

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, parenteral (including subcutaneous, intradermal, 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 carrier 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 invention suitable for oral administration may be presented 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 nonaqueous 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 treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral 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 prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. 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 ingredient therein. The present peptide hormone analogues can, for example, be 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 peptide hormone analogues, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. The present peptide hormone analogues can also be administered liposomally.

Preferably, compositions according to the invention are suitable for subcutaneous administration, for example by injection. According to certain embodiments the 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 (preferably at a molar ratio of 1:4, 1 :2, 1 : 1, 2: 1 or 4: 1 of zinc ions to peptide hormone analogue, or at a ratio which is a range between any two of the whole number ratios given immediately above). In such an embodiment the peptide hormone analogue is preferably an analogue of glucagon which is a compound of formula (IV) . In another preferred embodiment, the composition has a pH of less than 5 prior to administration and the composition contains zinc ions. In such an embodiment the peptide hormone analogue is preferably an analogue of glucagon which is a compound of formula (IV).

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 peptide hormone analogue 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 and/or 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 (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic 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 solutes which render the formulation isotonic with the blood of the intended recipient; and 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 use. 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 oleic 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 peptide hormone analogues of the invention and related molecules. Excipients 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-methane,

trichlorofluoromethane, 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 butter, 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 acacia 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 peptide hormone 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 microcapsules; 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; intracisternally; 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 peptide hormone analogues of the invention. For example, the pharmaceutical compositions may be in the form of particles comprising one or more of biodegradable polymers, polysaccharide jellifying and/or bioadhesive polymers, amphiphilic polymers, agents capable of modifying the interface properties of particles of the peptide hormone 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 peptide hormone analogues of the invention may also be achieved by the use of pharmaceutical compositions comprising the peptide hormone analogue and zinc ions. Preferably, in such an embodiment, the peptide hormone analogue is an analogue of glucagon which is a compound of formula (IV). 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 preferred for indications such as the treatment of obesity and/or diabetes, where maximising the time period between injections is desirable. For indications where it is desired to achieve a therapeutic plasma concentration of the peptide hormone analogue in as short a time period as possible, an immediate release formulation will be preferred. In such cases, a dosage regime comprising administration of a dose of an immediate release formulation of the peptide hormone analogue and subsequent administration of a dose of a controlled release formulation of the peptide hormone analogue may be preferred.

A peptide hormone analogue of the invention may be delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al, N. Engl. J. Med. 321 :574, 1989) or by 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, peptide hormone 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 are incorporated herein by reference.

Implantable drug infusion devices are used to provide patients with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Essentially such device may be categorized as either active or passive. A peptide hormone 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 peptide hormone analogues can be formulated with suitable polymeric or hydrophobic materials, for example 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 effective amount of a peptide hormone analogue of the invention may be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. Thus, in pulse doses, a bolus administration of a peptide hormone analogue of the invention is provided, followed by a time period wherein no peptide hormone analogue of the invention is administered to the subject, followed by a second bolus administration. In specific, non-limiting examples, pulse doses of a peptide hormone 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 certain embodiments, a therapeutically effective amount of a peptide hormone analogue of the invention is administered with a therapeutically effective amount of a further agent. The peptide hormone analogue may be administered simultaneously with the further therapeutic agent, or it may be administered sequentially or separately. Accordingly, the invention provides a peptide hormone analogue of the invention for use as a medicament, wherein the peptide hormone analogue is for use with a therapeutically effective amount of a further therapeutic agent (e.g. for administration simultaneously, sequentially or separately). In certain embodiments, a peptide hormone analogue of the invention is formulated and administered with a further therapeutic agent as a single dose.

In certain embodiments, the further therapeutic agent is an additional anti-diabetic, appetite suppressant, a food-intake-reducing, plasma glucose-lowering or plasma lipid-altering agent. Specific, non-limiting examples of an additional appetite suppressant include amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine, fenfluramine, dexfenfluramine, 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, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride, gliclazide, fibroblast growth factor 21, miglitol, acarbose, exenatide, pramlintide, vildagliptin, sitagliptin, and dapagliflozin, canagliflozin, emaglifozin.

In alternative embodiments, the further therapeutic agent is an additional cardioprotective or neuroprotective agent. Specific, non-limiting, examples of additional cardioprotective agents include aspirin, N-acetylcysteine, phenethylamines, coenzyme Q10, vitamin E, vitamin C, L-carnitine, 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 anticholinergics, pramipexole, bromocriptine, levodopa, carbidopa, rasagiline, amantadine and ropinirole. A peptide hormone 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 peptide hormone 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 peptide hormone analogue of the invention may vary from about 0.01 μg per kilogram (kg) body weight to about 1 g per kg body weight, for example about 0.1 μg to about 20 mg per kg body weight, for example about 1 μg to about 5 mg per kg body weight, or about 5 μg to about 1 mg per kg body weight. In one embodiment of the invention, a peptide hormone 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 μιηοΐ, for example from 375 nmol to 75 μιηοΐ, for example from 750 nmol to 56.25 μιηοΐ, for example from 1.5 to 37.5 μιηοΐ, in particular from 2.25 to 18 μιηοΐ. The invention also contemplates dosages ranges bounded by any of the specific dosages mentioned herein.

In an alternative embodiment, a peptide hormone 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 one specific, non-limiting example, a peptide hormone 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 peptide hormone analogue of the invention is generally not more 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 peptide hormone analogues of the invention are within the range of from 1 to 100 nmols, from 2 to 90 mols, from 5 to 80 nmols. In one specific, non-limiting example, from about 1 to about 50 nmol of a peptide hormone 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 present invention also provides a peptide hormone analogue of the invention in labelled form as a diagnostic agent for the diagnosis of conditions associated with a disease or disorder associated with the GLP-1 receptor, for example as a diagnostic agent for the diagnosis of conditions selected from the group consisting of obesity, diabetes, a disease or disorder associated with the GLP-1 receptor G- protein-dependent pathway, and a disease or disorder associated with the GLP-1 receptor G-protein- independent pathway.

The present invention further provides a peptide hormone analogue of the invention in labelled form of such a peptide hormone analogue as a reference compound in a method of identifying ligands for the GLP-1 receptor, for example ligands for the GLP-1 receptor that are biased for the GLP-1 receptor G-protein-dependent pathway, or as a reference compound in a method of identifying ligands for the GLP-1 receptor that are biased for the GLP-1 receptor G-protein-independent pathway. Materials and Methods:

Peptides

Analogues of the invention were purchased as custom peptides from Insight Biotechnology. Peptides were synthesized by a standard automated fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) method. Peptide synthesis was carried out on a tricyclic 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.

Exendin-4, exendin (9-39) and GLP-1 (7-36)NH2 are obtainable from Bachem.

Cell culture and generation of stable cell lines

Pathhunter CHO-GLP-1R -arrestin-l/-2 reporter cell lines (DiscoverX) were maintained in Culture Medium 6 (DiscoverX). INS-1 832/3 were maintained as previously described (Hohmeier, H. E. et al. Diabetes 49, 424-430 (2000)). Monoclonal stable CHO-SNAP-GLP-1R cells were generated by transfecting pSNAP-GLP-lR (Cisbio) into wild-type CHO-K1 (ECACC), G418 (1 mg/ml) selection, and single-cell sorting by fluorescence-activated cell sorting (FACS) following SNAP-Surface 488 (New England Biolabs) labeling, and maintained in RPMI-1640 + 0.5 mg/ml G418. Mycoplasma testing was performed yearly. Human islet isolation and culture

Human islet studies were approved by the National Research Ethics Committee London (REC 07/H0711/114). Islets were obtained from normoglycemic donors at isolation centers in Geneva, Pisa, Edmonton41, Milan and Oxford according to local ethics rules (including next-of-kin consent) and isolation techniques, and cultured as previously described (Hodson, D. J. et al. J. Clin Invest 123, 4182-4194 (2013)). Experiments were performed with randomly allocated, size-matched islets. cAMP assays

Cyclic AMP accumulation was determined by HTRF (cAMP Dynamic 2, Cisbio) from experiments at 37°C. CHO cells were stimulated in serum-free DMEM without phosphodiesterase inhibitors. For dose-responses, a full GLP-1 curve was included to establish the assay Emax. Curve fitting to a 4- parameter logistic fit was performed using GraphPad Prism 6.0. β-arrestin recruitment assay

Pathhunter CHO-GLP-1R β-arrestin-l and -2 reporter cell lines were used as per manufacturer's instructions, with 90 min stimulation. A full GLP-1 dose-response was included to establish the assay Emax.

Quantitation of bias

Bias between cAMP and -arrestin-2 responses was determined using modified versions of the operational model of agonism.

METHOD 1A and IB: Concentration response data was fitted using GraphPad Prism with previously described equations (van der Westhuizen et al, Mol. Pharmacol. 85, 492-509 (2014); Kenakin, T. et al, ACS Chem Neurosci 3, 193-203 (2012)) to derive transduction ratio ( /K_A) values for each agonist in each pathway. Log(r/¾) values for each agonist were normalized by subtracting log(r/¾) for the reference agonist (exendin-4, GLP-1, or G1950) in each pathway, giving

Alog(r/¾). To determine bias between two pathways, Alog(r/¾) values from one were subtracted from the other, yielding AAlog(r/¾). In METHOD IB (in particular for results shown in figure 3), these calculations were performed with data derived from 30 minute incubations for cAMP, 90 minutes for -arrestin-2. Further, as these experiments were performed during the screening phase of the invention, comparisons with control peptides (e.g. G1950) were made with results from non- matched experiments. METHOD 1A: Subsequently, the method was refined to standardize the incubation time for both pathways, with all analogue treatments performed in parallel in each pathway to reduce variability (especially as in figure 2).

METHOD 2: For compounds which are weak partial agonists for arrestin recruitment, the precision of bias estimate can be improved by incorporating data from a competitive experiment where ascending doses of the test compound inhibit the activity of the reference agonist, as described in Stahl, et al, Mol Pharmacology, 2015, Vol. 87(5), pages 866 - 77. This experiment was performed as described above for the beta arrestin recruitment assay, with test compound also incubated in the presence of 90nM of exendin-4. Thus, stimulatory and inhibitory dose response curves were generated, and relative activity for the beta arrestin pathway then calculated as described by Stahl et al. The results of this experiment for analogues Ex-4 dHisl and Ex-4 Phe l are shown in Figures 4 to 6. Figure 4 shows the concentration-dose response data for analogues Ex-4 dHis 1 and Ex-4 Phe 1 and Ex-4 for cAMP in graphical form; Figure 5 shows the concentration-dose response data for analogues Ex-4 dHisl and Ex-4 Phel and Ex-4 for bARR2 in graphical form.

Cell surface ELISA assay

Pathhunter CHO-GLP-1R cells were treated with agonist for 90 min at 37°C, then placed on ice to arrest further receptor endocytosis before fixation. A cell surface ELISA was used to detect remaining surface GLP-lR with monoclonal anti-human GLP-1R antibody44 (Mab 3F52, Developmental Studies Hybridoma Bank) and HRP -conjugated rabbit anti-mouse secondary (Abeam). TMB substrate was added and absorbance read at 450 nm after 1 M HCl addition. Surface expression was determined as absorbance from peptide- vs. control-treated wells.

GLP-1R recycling assay

To measure GLP-1 R recycling, two plates of CHO-SNAP-GLP-1R cells were exposed to indicated agonist (100 nM) or vehicle for 60 min to induce internalisation followed by thorough washing to remove extracellular agonist. At this point, one plate ("internalisation plate") was placed in the cold room to arrest further GLP-1R trafficking. The other plate ("recycling plate") remained at 37°C for a further 60 minutes to allow GLP-lR to recycle back to the plasma membrane. Recycling was performed in the presence of exendin(9-39) (10 μΜ) to prevent rebinding. After further washing, both "internalisation" and "recycling" plates were labelled with the time-resolved fluorescence cell- impermeating probe Lumi4-Tb (40 μΜ) in the cold for 60 minutes, followed by washing, allowing quantification of surface SNAP-GLP-1R in each well. Agonist-specific GLP-1R recycling rates were determined by comparing the residual surface SNAP-GLP-1R in "recycling" vs "internalisation" plates to calculate the % of internalized receptor that had been recycled in the 60 minute recycling period. Competitive association kinetic binding experiment to determine agonist residence time

Binding of FITC -agonist to labeled SNAP-GLP-1R was measured in real time by time-resolved Forster resonance energy transfer (TR-FRET). CHO-SNAP-GLP-1R cells were labeled with 40 μΜ Lumi4-Tb and, to prevent agonist-induced GLP-1R internalisation during the binding experiment, cells were treated with metabolic inhibitors (10 mM NaN3, 20 mM 2-deoxyglucose) (Widmann, C. at al, Biochem J 310 ( Pt 1), 203-214 (1995)) . On simultaneous addition of five concentrations of unlabeled agonist in conjunction with 50 nM exendin-4-FITC, TR-FRET was serially read with a Flexstation 3 plate reader (Molecular Devices) with the following parameters: excitation 340 nm, emission 520 nm (cut-off 495 nm) and 620 nm (cut-off 570 nm), 50 delay time, 350 μβ integration time. . The dissociation rate constant koff was calculated as previously described (Motulsky, H. J. & Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1-9 (1984)) using GraphPad Prism. Residence time = 1/koff.

Human islet histology

Human islets were incubated overnight at 37°C with medium containing 11 mM glucose ± 100 nM agonist prior to transfer to 4°C to arrest endocytosis and labeling for 1 h with 1 μΜ exendin-FITC at 4°C. Islets were fixed with 4% PFA for 20 min, followed by dehydration with 70% ethanol before suspension in 4% agarose in order to form small agarose plugs. Once cooled to room temperature, plugs were placed in processing cassettes (Histosette II®; Simport) and dehydrated through a serial ethanol gradient (70, 90, and 100%) and HistoChoice clearing agent (Sigma) before embedding in paraffin blocks using the Histoembedder station (Leica). 1 μιη sections were cut with a Leica Jung RM2035 microtome and placed on poly-lysine-coated microscope slides before de-waxing in HistoChoice clearing agent and rehydration in ethanol and water. Sections were stained for FITC with a rabbit polyclonal anti-FITC antibody (711900, Thermo Fisher Scientific) and secondary Alexa Fluor 488 antibody (Life Technologies) plus a guinea pig anti-human insulin antibody (A0564, Dako) and secondary Alexa Fluor 546 antibody (Life Technologies) prior to mounting and imaging by confocal microscopy. Insulin secretion assays

In each case, samples were obtained for secreted and total insulin, and analyzed by HTRF (Cisbio). Percentage release was calculated, and agonist-stimulated results expressed relative to 11 mM glucose-alone results as insulin stimulation index (ISI). INS-1 832/3 cells were exposed to agonist (100 nM) in complete RPMI + 11 mM glucose after a prior 24 h period in low glucose (3 mM) medium; initial experiments were performed with cells adherent to the plate (figure 7), subsequent experiments were performed with suspension cells (figure 25). For human islets, overnight incubations were performed in complete RPMI with 11 mM glucose ± 100 nM agonist.

Human islet calcium imaging

Ca2+ imaging was performed as previously described (Hodson, D. J. et al., J Clin Invest 123, 4182- 4194 (2013); Hodson, D. J. et al., Mol. Endocrinol. 28, 860-871 (2014)). Briefly, islets were incubated for 1 h with Fluo-2 (10 μΜ) in HEPES-bicarbonate buffer containing 11 mM glucose. For homologous desensitization measurements, islets were pre-incubated overnight with 100 nM exendin- 4 or Ex-4 Phel . Fluorescent signals were normalized using the function F/Fbaseline where F is fluorescence at a given time-point and Fbaseline is average fluorescence intensity between 2 and 4 min. Animal studies

All animal procedures were approved by the British Home Office under the UK Animal (Scientific Procedures) Act 1986 (Project Licence 70/7596). C57BL/6J mice (8-10 weeks, Charles River) were maintained under controlled temperature (21-23°C) and light-dark cycles (12: 12 hour light-dark schedule, lights on at 0700). Ad libitum access to water and normal chow (RM1, Special Diet Services) or a HFHS diabetogenic diet (AIN-76A, TestDiet) was provided unless otherwise stated. HFHS animals were initially group housed (5 per cage) for >4 months before transfer to single cages for experimental procedures. Treatments were randomly allocated according to body weight. Group sizes of 8-10 were deemed adequate to detect treatment-related differences as per initial dose-finding glycaemia study. During experiments, one researcher was aware of treatment allocation but others were blinded. No animals were excluded from the analysis.

Dose-finding glycaemia study

Mice (either female high fat died-induced obese C57BL/6, or male high fat / high sucrose [HFHS] diet-induced obese C57BL/6) were fasted for two hours before IP injection of 50 μΐ agonist or vehicle (0.9% NaCl). Blood samples were obtained at indicated time-points, and blood glucose measured using a Contour glucose meter (Bayer). Intra-peritoneal glucose tolerance tests

Mice were fasted overnight before the procedure (HFHS mice) or the morning of the procedure (lean mice). Agonist was administered by IP injection. D-glucose (2 g/kg) was injected IP immediately, 4 h, or 8 h after agonist. Tail vein samples were obtained for immediate glucose measurement as above, or into lithium heparin-coated microvette tubes for plasma insulin measurement using a mouse insulin- specific HTRF assay (Cisbio).

Pharmacokinetic study

Plasma agonist concentration after a single IP injection (24 nmol/kg) was measured using an ELISA (Phoenix Pharmaceuticals) specific for the C-terminus of exendin-4, a peptide region that does not differ between agonists, hence detecting exendin-4 and Ex-4 Phe 1 equally (confirmed by analysis of known concentrations of each agonist).

Acute food intake study

Mice were fasted overnight and access to their normal diet was returned after IP agonist injection with food intake monitored by weight.

Chronic administration study

Subcutaneous osmotic minipumps (ALZET model 2004, Charles River) filled with agonist or vehicle (0.9% NaCl) to ensure delivery of a weight-adjusted dose of 0.24 nmol/kg/day were inserted under gas anesthesia. Mice and diet were weighed day 1 post surgery and body and food weight were measured at indicated intervals. IPGTTs were performed on day 14. Mice were sacrificed by decapitation in the fasting state. Liver histology

Liver tissue was fixed in 4% PFA followed by dehydration in 70% ethanol. Heaematoxylin- and eosin-stained liver sections were scored by a histopathologist blinded to treatment allocation using the Nonalcoholic Activity Score (Kleiner, D. E. et al., Hepatology 41, 1313-1321 (2005)) with fat scored 0-3, ballooning 0-2 and lobular inflammation 0-2.

Statistical testing

GraphPad Prism 6.0 was used for all analyses. Curve fitting and bias calculation were performed as described. For in vitro experiments, intra-experimental replicate mean was treated as a single experimental replicate. ANOVA or two-tailed t-tests were performed throughout, according to number of treatments compared. Data were visually confirmed as approximately normally distributed, allowing parametric analyses (robust to minor deviations from normality (Krzywinski, M., & Altman, N., Nat Methods. 11, 215-216 (2014))). Where n<5, individual data points are depicted, except for time-course experiments (for clarity). Randomized block (ANOVA) or paired (t-test) analyses were used in matched design experiments. Two-way ANOVA was used for experiments including multiple time-points. For ANOVA, post hoc tests were performed according to the primary question of the experiment: Dunnett's test for specifically comparing Ex-4 Phel and Ex-4 Asp3 responses with exendin-4, and Tukey's test for differences between all groups. Other post hoc tests are indicated in the figure legends.

The table of Figure 1 shows the amino acid sequences of the example analogues of the invention (each compound is identified by an analogue no., and certain compounds are also identified with a G. no). The sequences of Ex-4, GLP-1 and G1950, which are not analogues of the present invention, are also provided in that table.

Example 1 - Measurement of GLP-1 Receptor Ligand Bias In addition to G proteins, activated GLP-IR recruits β-arrestins (Jorgensen, R. et al., Mol. Endocrinol. 19, 812-823 (2005)). Arrestin recruitment is classically a signal-terminating event as it promotes GPCR de sensitization via endocytosis into clathrin-coated pits (Goodman, O. B. et al., Nature 383, 447-450 (1996)). However, β-arrestins also mediate non canonical signaling, which for the GLP-IR is linked to insulin secretion and inhibition of beta cell apoptosis (Sonoda, N. et al., Proc Natl Acad Sci USA 105, 6614-6619 (2008); Quoyer, J. et al., J Biol Chem 285, 1989-2002 (2010)). Therefore, it was decided to measure arrestin and cyclic AMP responses to analogues of the invention to determine whether analogues exhibited evidence of signaling bias between these pathways.

For analogues of the invention, analogue-induced -arrestin-2 ( ARR2) recruitment and cyclic AMP (cAMP) generation were tested in Pathhunter CHO-K1-GLP-1R cells and these data used to calculate pathway-specific relative activities and pathway bias for each analogue. The tables of Figures 2 and 3, and Figure 6 provide the experimentally derived bias factors, also termed "normalized transduction ratios", delta delta log(tau/KA) for analogues of the invention, determined relative to Ex-4, GLP-1 and G1950.

In Figure 2, the bias (calculated using method IB) for each pathway relative to GLP-1 and exendin-4 is shown ("delta delta log(tau/KA") for certain analogues. For the GLP-1 analogues (nos. 1-9) and exendin-4 analogues (nos. 12-22) the calculated bias is provided in the tables of Figure 2 relative to Ex-4 and GLP-1. The dose-response experiments used to calculate these bias factors are shown in figures 13 - 16. Figure 13 shows the concentration-dose response data for exendin-4 analogues and Ex-4 for cAMP in graphical form. Figure 14 shows the concentration-dose response data for exendin- 4 analogues and Ex-4 for bARR2 in graphical form. Figure 15 shows the concentration-dose response data for GLP-1 analogues and GLP-1 for cAMP in graphical form; Figure 16 shows the

concentration-dose response data for GLP-1 analogues and GLP-1 for bARR2 in graphical form. In Figure 3, the normalized transduction ratio/bias (calculated using method 1A) for each agonist in the cAMP and bARR2 pathways is shown in the table ("delta log(tau KA), cAMP" and "delta log(tau/KA), bARR2").

In Figure 6, separately performed bias calculations for Ex-4 Phel and Ex-4 dHisl are shown. These calculations were performed using an alternative methodology (Method 2) reported to improve precision of bias estimation for agonists which are weak partial agonists for a particular pathway, as is the case for the bARR2 response for these two analogues. The dose-response data used for these calculations are shown in Figures 4 and 5. Figure 5 in particular shows the inhibitory effect of both Ex-4 dHisl and Ex-4 Phel on bARR2 recruitment induced by exendin-4, which is used in this calculation. Similar inhibitory effects of Ex-4 Phel on recruitment of bARRl and bARR2 by GLP-1 are shown in Figure 17.

Additionally, it was found that β-arrestin-l and -2 responses at a single maximal dose (1 μΜ) with different analogues of exendin-4 were highly correlated (Figure 18), indicating that bias between bARRl and cAMP is likely to mirror that of bARR2 and cAMP, the latter being more extensively investigated.

In summary, these experiments identify several analogues of Exendin-4, GLP-1, and G1950, which exhibit bias between cAMP and bARR2 signalling.

Example 2 - Effect of agonist binding kinetics on control of GLP-1 receptor trafficking

It was hypothesized that analogues of the invention with amino acid substitutions close to the N- terminus might also differ in binding kinetics at the GLP-1 R and in how they modulate receptor trafficking. Figure 19 shows screening results for analogues of the invention: GLP-IR internalization was measured by cell-surface ELISA in Pathhunter GLP-IR -arrestin-2 cells, following 1 μΜ agonist incubation for 90 min, quantified as % loss of cell surface receptor vs. vehicle control, n=3. The agonist residence time was measured in CHO-SNAP-GLP-1R cells by competitive association kinetics (Motulsky, H. J. & Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1-9 (1984)) using agonist in competition with 50 nM exendin-4-FITC, n=4. From the table of Figure 19 it can be seen that longer residence time at the GLP-IR correlates with a greater degree of internalization of the GLP-IR. Further experiments were performed to determine agonist-specific differences in GLP-IR recycling back to the plasma membrane after removal of stimulatory concentrations of extracellular agonist. These results indicated that not only did exendin-4 analogues with N-terminal substitutions lead to different rates of GLP-IR internalisation, but also, once internalized, different agonist treatment led to differences in rates of GLP-IR recycling (see table of Figure 20). Notably, agonists with short residence times and reduced internalisation, including Ex-4 dHisl, Ex-4 Phel and Ex-4 dGln3 (table of Figure 19) exhibited faster recycling rates, whereas recycling rate was slower for the long residence time, extensively internalized Ex-4 Asp3. The same pattern of recycling differences was observed for GLP-1 -derived analogues with equivalent substitutions close to the agonist N-terminal.

Further, increased residual surface GLP-IR was observed in human islets treated overnight with Ex-4 Phel - versus exendin-4 (Fig. 21), consistent with the cumulative effect of reduced internalisation and faster recycling.

Example 3 - Desensitisation

GPCR recycling restores receptor responsiveness (Yu, S. S. et al, Journal of Biological Chemistry 268, 337-341 (1993)), so the effect of agonist pre-incubation on subsequent response to re-challenge with GLP-1 was measured.

Figure 8 shows cAMP response in INS-1 832/3 cells to ΙΟηΜ GLP-1 after prior exposure to ΙΟΟηΜ Ex-4, Ex-4 dHisl or Ex-4 Phe l or vehicle for 20 minutes (****p<0.001 vs Ex-4 using randomized block ANOVA + Sidak test.) The results in Figure 8 show an approximately 30% increase in GLP-IR desensitization for the analogues compared to Ex-4.

It was also found that exendin-phel induces less homologous desensitization than exendin-4 in human islets, as measured by calcium flux (Figs. 22-25). In keeping with these observations, beta cells exposed to Ex-4 Phe 1 continued to generate cAMP for longer than those exposed to exendin-4 (not shown).

Example 4 - insulin secretion β-arrestin recruitment and signaling from internalized receptor are reported to be critical for therapeutic GLP-IR stimulus-secretion coupling (Kuna, R. S. et al. Am J Physiol Endocrinol Metab 305, E161-70 (2013); Sonoda, N. et al., Proc Natl Acad Sci USA 105, 6614-6619 (2008)). Therefore, to determine the downstream consequences of alterations to GLP-IR binding kinetics, trafficking and signal bias, insulin secretion was measured using INS-1 832/3 cells, and intact human islets, with prolonged incubation to mimic in vivo drug exposure.

Figure 7 shows the insulin secretion response in INS-1 832/3 cells after 24 h incubation at 1 ImM glucose (Gl 1), n=6. Ex-4, Ex-4 dHisl or Ex-4 Phe l administered at ΙΟΟηΜ. The response is expressed as insulin stimulation index (ISI) relative to Gl 1. (*p<0.05, **p<0.01 vs Ex-4 using randomized block ANOVA + Friedman test.) The results in Figure 7 show that the analogues were approximately twice as insulinotropic as Ex-4 in vitro. Later experiments performed with the refined suspension cell protocol, summarized in the table of

Figure 26, confirmed the greater insulinotropic properties of Ex-4 dHisl and ex-4 Phel, also revealing notable differences with other exendin-4-derived agonists, in a manner indicating a consistent relationship between residence time, biased signaling, and GLP-1R trafficking. Specifically, agonists with short residence times, bias away from bARR2 responses, reduced internalisation (and fast recycling), exhibited the greatest insulin release, despite loss of potency for cAMP generation at the GLP-1R.

Dose responses were obtained for Ex-4 Phel and Ex-4 Asp3 in comparison to exendin-4 in INS-1 832/3 cells (figure 27). These results provide further evidence of the relationship between maximal insulin release and residence time, biased signaling and GLP-1R trafficking. Furthermore, Ex-4 Phel was also tested for insulin secretion versus exendin-4 in intact human islets to obtain verification of the observed effects in human tissue and increase potential for clinical application, and was again found to be more insulinotropic (see figure 28). Therefore, in contrast to a recently described, but poorly insulinotropic biased GLP-1R agonist

(Zhang, H. et al., Nat Commun 6, 8918 (2015), there was a clear inverse relationship between insulin secretion and either agonist residence time, internalization or β-arrestin recruitment (not shown), with exendin-phel being particularly effective (Figs. 27-28). Cross-reactivity with other receptors was excluded as insulin secretion effects could be blocked with exendin(9-39) (not shown). Conversely, the same pattern was not observed with shorter incubation times (not shown). A similar agonist rank order was noted for protection against apoptosis during both glucolipotoxicity (Buteau, J. et al., Diabetologia 47, 806-815 (2004)) and endoplasmic reticulum (ER) stress (Yusta, B. et al., Cell Metab 4, 391-406 (2006)) (not shown). These results suggest that the canonical effects of internalization and β-arrestin recruitment, namely signal termination by receptor sequestration within endosomes and desensitization, are important in determining GLP-1R agonist downstream responses. Example 5 - in vivo Studies

Single dose lowering of blood glucose

Female C57BL/6J mice, were fed a high fat diet for 3 months to induce dysglycaemia. On the morning of the experiment, mice were lightly fasted for 2 hours and baseline glucose measured by tail vein venesection. lOug/kg BW exendin-4, test analogue Ex-4 dHisl or Ex-4 Phel, or vehicle (0.9% NaCl) were then administered by intraperitoneal injection. Serial glucose measurements during ongoing fasting were taken over the subsequent 22 hours. Figures 9 to 12 show the results of this experiment. Figures 9 and 10 show the reduction in glucose level over time in mice for administered with vehicle, Ex-4, and Ex-4 dHisl . Figures 11 and 12 show the reduction in glucose level over time in mice for administered with vehicle, Ex-4, and Ex-4 Phe l . Area under curve for glucose over 22 hours with Ex-4 dHis 1 and Ex-4 Phe 1 was reduced by approximately 30% compared to exendin-4 at the same dose indicating that the analogues of the invention can achieve good in vivo reduction in glucose levels (*p<0.05, ** p<0.01, *** p<0.001 by one way ANOVA + Tukey test).

Similar experiments were performed in male C57B1/6 mice fed a high fat, high sucrose diet for 4 months, and are shown in Figures 29 and 30. Figure 29 shows change in blood glucose level (mM) over 8 hours in mice following administration of 0.24 or 2.4 nmol/kg Exendin-4 (Ex-4), Ex-4 Phe l or vehicle. Figure 30 shows the change in glucose AUC (mM.hr) calculated from the data in Figure 29 for Exendin-4, Ex-4 Phel, and vehicle. As can be seen from the Figures, administration of Ex-4 Phel provided a reduction in area under curve for glucose concentration compared to exendin-4. Single dose glucose tolerance tests:

To obtain further information on the potential therapeutic effects of ex-phe 1 on glucose metabolism, intraperitoneal glucose tolerance tests (IPGTTs) were performed in HFHS-fed mice. When these were performed at 0, 4 and 8 h after agonist administration at 2.4 nmol/kg a strikingly persistent anti- hyperglycemic effect of Ex-4 Phe 1 versus exendin-4 was revealed, associated with greater insulin release (Figs. 31-33). Similar findings were seen at 0.24 nmol/kg agonist, a dose comparable to that used clinically in humans after allometric scaling (Figs. 35-37). Other exendin-4 analogues were also tested for their effect on glucose tolerance 8 hours after agonist (2.4 nmol/kg) treatment, revealing large differences (Fig. 38), which correlate with in vitro measures of insulin secretion (Fig. 26) and other pharmacological properties including agonist residence time, signaling bias, and GLP-1R trafficking, as discussed earlier. Single dose effects on appetite and nausea:

As GLP-R agonists are known to suppress appetite and also induce nausea in humans, these effects were also tested. To determine the acute anorectic effect of ex-phel versus exendin-4, male C57B1/6 mice on a HFHS diet were fasted overnight and then injected with agonist or vehicle, with diet then returned and consumption serially monitored up to 8 hours. At a dose of 2.4 nmol/kg (Fig. 34) and 0.24 nmol/kg (Fig. 35), both exendin-4 and Ex-4 Phel reduced food intake compared to vehicle.

A conditioned taste aversion experiment in mice was carried out to assess nausea, showing the lack of aversive effect of Ex-4 Phe l at two doses (0.24 nmol/kg and 2.4 nmol/kg). Mice (n=8/group) were conditioned to associate the indicated treatment with Kool-Aid consumption, prior to being given free choice of Kool-Aid versus water. Two flavours of Kool-Aid were used, cherry and grape. The results are shown in Figures 39 and 40. As can be seen, compared with the positive control (lithium chloride), the analogues did not exhibit an aversive effect, suggesting that there is unlikely to be any difference between these two compounds in their propensity to induce nausea when used clinically at equimolar doses. Nevertheless, the greater gluco-modulatory effect of Ex-4 Phe 1 may allow lower dosing for the same beneficial effects on glucose metabolism, reducing the likelihood of nausea.

Pharmacokinetics

A pharmacokinetic study was also carried out to determine plasma concentrations of exendin-4 and Ex-4 Phe 1. Figure 47 shows in vivo plasma concentrations of exendin-4 and Ex-4 Phe 1 at 4 and 8 hrs following administration to mice. There was no difference in the apparent rate of disappearance from the circulation, excluding this as a facile explanation for the differential effects on glucose metabolism. Chronic administration study

Agonists were administered continuously to HFHS-fed mice for 2 weeks via subcutaneous minipumps, and it was found that the hypoglycemic effect was again greatest with Ex-4 Phel (Figs. 41-43) with no difference in cumulative food intake or weight loss (Figs. 44-45). In view of recent interest in GLP-1R agonism as a non-alcoholic fatty liver disease treatment (Armstrong, M. J. et al. Lancet 387, 679-690 (2016)), liver histology was evaluated which showed greater steatosis resolution with Ex-4 Phel (Fig. 46). Therefore, metabolic improvements from acute and chronic administration of Ex-4 Phel exceeded those of exendin-4 without increased nausea-related appetite inhibition.

In summary, this study identifies a set of linked agonist characteristics associated with improved GLP-1R agonist therapeutic efficacy, namely fast dissociation kinetics, reduced β-arrestin recruitment, reduced internalization propensity, and increased plasma membrane recycling. These appear contrary to the prevailing view that longer residence time and sustained signaling from internalized receptors are desirable for prolonged in vivo action (Guo, D. et al, ACS Med Chem Lett 7, 819-821 (2016); Copeland, R. A., Nat Rev Drug Discov 15, 87-95 (2016); Hothersall, J. D. et al, Drug Discov. Today 21, 90-96 (2016)). It is proposed that, for the GLP-1R, extensive receptor internalization, although initially able to generate enhanced responses (particularly in recombinant systems), may attenuate reestablishment of an adequate population of surface receptors to optimally respond to extracellular ligand. Importantly, the augmented action of Ex-4 Phel on beta cells did not extend to food intake inhibition, suggesting that this class of compound can achieve full glycemic improvement at a lower dose, thereby reducing or avoiding nausea. Without being bound by any theory, reduced GLP-1R endocytosis-mediated delivery of Ex-4 Phel to hypothalamic nausea and appetite centers (Secher, A. et al, J Clin Invest 124, 4473-4488 (2014)) may be involved.

Claims

1. A peptide hormone analogue, or a derivative of the peptide hormone analogue,

or a salt of the compound or the derivative, which is:

(i) an analogue of Exendin-4 which is a compound of formula (I):

(I)

wherein

Xaa¹ is selected from the group consisting of Phe, His, D-His, Asn, Gin, Tyr, and D-Tyr; Xaa² is selected from the group consisting of Gly, Ala, an a-aminoisobutyric acid residue (AIB), Ser, D-Ser, Thr and Pro;

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and

Xaa⁵ is selected from the group consisting of Thr and Ser;

Xaa^p is selected from the group consisting of Gin and Tyr,

(ii) an analogue of GLP-1 which is a compound of formula (II):

Xaa^Xaa^Xaa^Xaa^Xaa^Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa^a

(II)

wherein

(iii) an analogue of oxyntomodulin which is a compound of formula (III):

Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- Asp-Phe-Val-Gln-T -Leu-Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala

(III)

wherein

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and

Xaa⁵ is selected from the group consisting of Thr and Ser; or a variant thereof having up to three conservative amino acid substitutions at any one of positions 6 to 37;

(iv) an analogue of glucagon which is a compound of formula (IV)

Xaa^Xaa'-Xaa'-Xaa'-Xaa'-Phe-Thr-Ser-Asp-Xaa^-Ser-Xaa^-Xaa^-Leu-Xaa^-Xaa^-Xaa¹⁷- Xaa¹⁸-Ala-Xaa²⁰-Xaa²¹-Phe-Xaa² -Xaa²⁴-T -Leu-Leu-Asn-Xaa²⁹-V

(IV) wherein

Xaa¹⁰ is selected from the \ group consisting i of Tyr and Leu;

Xaa¹² is selected from the \ group consisting i of Lys, His and Arg;

Xaa¹³ is selected from the \ group consisting i of Tyr, Gin and His;

Xaa¹⁵ is selected from the ; group consisting ; of Asp and Glu;

Xaa¹⁶ is selected from the ; group consisting ; of Glu, Gin and Ser;

Xaa¹⁷ is selected from the ; group consisting ; of Arg, His and Lys;

Xaa¹⁸ is selected from the \ group consisting i of Arg and Lys;

Xaa²⁰ is selected from the \ group consisting i of His and Gin;

Xaa²¹ is selected from the \ group consisting i of Glu, His and Asp;

Xaa²³ is selected from the ; group consisting ; of lie and Val;

Xaa²⁴ is selected from the ; group consisting ; of Gin and Glu;

Xaa²⁹ is selected from the \ group consisting i of Thr and Gly;

V is selected from the group consisting of His, H1S-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, His-His-His-NH2, and a C-terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues; or V is absent; with the proviso that when the peptide hormone analogue is:

(ii) an analogue of GLP-1 which is a compound of formula (II), Xaa¹-Xaa²-Xaa -Xaa⁴-Xaa⁵ is not His-Ala-Glu-Gly-Thr;

(iii) an analogue of oxyntomodulin which is a compound of formula (III), Xaa¹-Xaa²-Xaa -Xaa⁴- Xaa⁵ is not His-Ser-Gln-Gly-Thr; and

2. A peptide hormone analogue as claimed in claim 1, wherein Xaa¹ is selected from the group consisting of Phe, Asn, Tyr, D-Tyr, and D-His.

3. A peptide hormone analogue as claimed in claim 2, wherein Xaa¹ is selected from the group consisting of Phe and D-His.

4. A peptide hormone analogue as claimed in claim 1, wherein Xaa¹ is His.

5. A peptide hormone analogue as claimed in any one of claims 1 to 4, wherein Xaa² is selected from the group consisting of Ala, Ser, D-Ser, Gly, Thr and Pro.

6. A peptide hormone analogue as claimed in any one of claims 1 to 4, wherein Xaa² is selected from the group consisting of Ala, AIB, D-Ser, Gly, Thr and Pro (preferably Ala, D-Ser, Gly, Thr and Pro, more preferably D-Ser, Thr and Pro).

7. A peptide hormone analogue as claimed in any one of claims 1 to 3, wherein the analogue is (i) an analogue of Exendin-4 which is a compound of formula (I) and Xaa² is Gly; (ii) an analogue of GLP-1 which is a compound of formula (II) and Xaa² is Ala; (iii) an analogue of oxyntomodulin which is a compound of formula (III) and Xaa² is Ser; or (iv) an analogue of glucagon which is a compound of formula (IV) and Xaa² is Ser.

8. A peptide hormone analogue as claimed in any one of claims 1 to 7, wherein Xaa³ is selected from the group consisting of D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, His, Ala, Tyr, Leu and Lys).

9. A peptide hormone analogue as claimed in any one of claims 1 to 3, wherein the analogue is (i) an analogue of Exendin-4 which is a compound of formula (I) and Xaa³ is Glu; (ii) an analogue of GLP-1 which is a compound of formula (II); or (iii) an analogue of oxyntomodulin which is a compound of formula (III) or (iv) an analogue of glucagon which is a compound of formula (IV) and Xaa³ is Gin.

10. A peptide hormone analogue as claimed in any one of claims 1 to 9, wherein Xaa⁴ is selected from the group consisting of Gly, Leu, Ala and Ser (preferably Leu, Ala and Ser).

11. A peptide hormone analogue as claimed in any one of claims 1 to 9, wherein Xaa⁴ is Gly.

12. A peptide hormone analogue as claimed in any one of claims 1 to 11, wherein Xaa⁵ is Thr.

13. A peptide hormone analogue as claimed in any one of claims 1 to 12, wherein Xaa¹ is selected from the group consisting of D-His and D-Tyr; and/or Xaa² is D-Ser and/or Xaa³ is D-Gln (for example, Xaa¹ is D-His and Xaa² is D-Ser).

14. A peptide hormone analogue as claimed in any one of claims 1 to 13, wherein the analogue is (i) an analogue of Exendin-4 which is a compound of formula (I) or a variant thereof having up to three conservative changes.

15. A peptide hormone analogue of formula (I) as claimed in claim 14, wherein

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Tyr, and D-Tyr (preferably His, Asn, Tyr or Phe);

Xaa² is Gly;

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln, Ala, Tyr, and Lys; more preferably Glu); Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and Xaa⁵ is Thr.

16. A peptide hormone analogue of formula (I) as claimed in claim 14, wherein

Xaa¹ is His;

Xaa² is Gly;

Xaa³ is selected from the group consisting of Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln, Ala, Tyr, and Lys);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

17. A peptide hormone analogue of formula (I) as claimed in claim 14, wherein

Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Tyr, and D-Tyr

(preferably Asn, Tyr or Phe);

Xaa² is Gly;

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

18. A peptide hormone analogue of formula (I) as claimed in claim 14, wherein

Xaa¹ is selected from the group consisting of His and Gin;

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and

Pro (preferably Gly, Ala and AIB);

Xaa³ is selected from the group consisting of Asp, Glu and Gin (preferably Glu and Gin, more preferably Glu);

Xaa⁴ is Gly; and Xaa⁵ is selected from the group consisting of Thr and Ser.

19. A peptide hormone analogue as claimed in any one of claims 1 to 13, wherein the analogue is (ii) an analogue of GLP-1 which is a compound of formula (II) or a variant thereof having up to three conservative changes.

20. A peptide hormone analogue of formula (II) as claimed in claim 19, wherein

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn and Tyr (preferably His, D-His and Phe);

Xaa² is Ala;

Xaa³ is selected from the group consisting of Glu, D-Gln, His, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln, Ala, Tyr and Lys);

Xaa⁴ is selected from the group consisting of Gly, Leu, Ala, Ser and AIB; and Xaa⁵ is Thr.

21. A peptide hormone analogue of formula (II) as claimed in claim 19, wherein

Xaa¹ is His,

Xaa² is Ala;

Xaa³ is selected from the group consisting of D-Gln, His, Ala, Tyr, Leu and Lys (preferably D-Gln, Ala, Tyr and Lys);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

22. A peptide hormone analogue of formula (II) as claimed in claim 19, wherein

Xaa¹ is selected from the group consisting of D-His, Phe Asn and Tyr (preferably

D-His and Phe),

Xaa² is Ala;

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

23. A peptide hormone analogue of formula (II) as claimed in claim 19, wherein

Xaa¹ is His;

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (for example Ala); Xaa³ is selected from the group consisting of Glu, Gin and Asp (for example Gin and Asp, or for example Glu);

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser.

24. A peptide hormone analogue as claimed any one of claims 1 to 13, wherein the analogue is (iii) an analogue of oxyntomodulin which is a compound of formula (III) or a variant thereof having up to three conservative changes.

25. A peptide hormone analogue of formula (III) as claimed in claim 24, wherein

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D-Tyr (preferably His, D-His, Gin, Phe and D-Tyr, more preferably His, D-His and Asn);

Xaa² is Ser of D-Ser (preferably Ser);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, Glu, D-Gln and His, more preferably Gin, D-Gln and His);

Xaa⁴ is selected from the group consisting of Gly Leu, Ala, Ser and AIB; and Xaa⁵ is Thr.

26. A peptide hormone analogue of formula (III) as claimed in claim 24, wherein

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is selected from the group consisting of Glu, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Glu, D-Gln and His, more preferably D-Gln and His);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

27. A peptide hormone analogue of formula (III) as claimed in claim 24, wherein

Xaa¹ is selected from the group consisting of D-His, Phe, Asn, Gin, Tyr, and D- Tyr (preferably D-His, Gin, Phe and D-Tyr, more preferably D-His and Asn); Xaa² is Ser;

Xaa³ is Glu;

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

28. A peptide hormone analogue of formula (III) as claimed in claim 24, wherein

Xaa¹ is selected from the group consisting of His, Gin, Phe and D-Tyr (preferably His and Gin);

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ser, D-Ser, Gly, Thr and Pro, most preferably Thr and Pro);

Xaa³ is selected from the group consisting of Asp, Glu and Gin (preferably Glu and Gin);

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Ser).

29. A peptide hormone analogue as claimed in any one of claims 1 to 13, wherein the analogue is (iv) an analogue of glucagon which is a compound of formula (IV).

30. A peptide hormone analogue of formula (IV) as claimed in claim 29, wherein

Xaa¹ is selected from the group consisting of His, D-His, Phe, Asn, Gin, Tyr, and D-Tyr (preferably His, D-His and Asn);

Xaa² is Ser or D-Ser (preferable Ser);

Xaa³ is selected from the group consisting of Glu, Gin, D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably Gin, D-Gln and His);

Xaa⁴ is selected from the group consisting of Gly Leu, Ala, Ser and AIB; and Xaa⁵ is selected from the group consisting of Thr.

31. A peptide hormone analogue of formula (IV) as claimed in claim 29, wherein

Xaa¹ is His;

Xaa² is Ser;

Xaa³ is selected from the group consisting of D-Gln, His, Asp, Ala, Tyr, Leu and Lys (preferably D-Gln and His);

Xaa⁴ is Gly; and

Xaa⁵ is Thr.

32. A peptide hormone analogue of formula (IV) as claimed in claim 29, wherein

Xaa¹ is selected from the group consisting of D-His, Asn, and Tyr (preferably D- His and Asn);

Xaa² is Ser;

Xaa³ is Glu;

Xaa⁴ is Gly; and Xaa⁵ is Thr.

33. A peptide hormone analogue of formula (IV) as claimed in claim 29, wherein

Xaa² is selected from the group consisting of Ala, AIB, Ser, D-Ser, Gly, Thr and Pro (preferably Ser, D-Ser, Gly, Thr and Pro, most preferably Ser, Thr and Pro); Xaa³ is selected from the group consisting of Asp, Glu and Gin (preferably Glu and Gin);

Xaa⁴ is Gly; and

Xaa⁵ is selected from the group consisting of Thr and Ser (preferably Ser).

34. A peptide hormone analogue as claimed in any one of claims 1 to 13 or 29 to 33, wherein V is selected from the group consisting of His, H1S-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; or a C- terminal extension amino acid sequence comprising at least four amino acid residues, at least three of said amino acid residues being His residues.

35. A peptide hormone analogue as claimed in any one of claims 1 to 13 or 29 to 34, wherein V comprises 3, 4 or 5 His residues.

36. A peptide hormone analogue as claimed in any of claims 1 to 13 or 29 to 35, wherein V is selected from the group consisting of His-His, His-His-NH2, Gly-His, Gly-His-NH2, Gly-His-His- His-His-Gln-CONH₂, Gly-His-His-His-His-His-Glu-CONH₂, Gly-His-His-His-His-Ser-CONH₂, Gly-His-His-His-His-Gln-Gln-CONH₂, His-His-His-His-Gly, His-His-His-His-Ala-Gly, Gly-His- His-His-CONH₂, His-Gly-His-His-CONH₂, His-His-His-His-CONH₂, His-His-His-His-His-Gln- NH₂, His-His-His-Gly and His-His-His-His-His-Gly.

37. A peptide hormone analogue as claimed in any of claims 1 to 13 or 29 to 36, wherein Xaa²⁰ is His and/or wherein Xaa¹⁰ is Tyr and/or Xaa¹⁶ is Ser or Glu.

38. A peptide hormone analogue as claimed in any of claims 1 to 13 or 29 to 37, wherein Xaa¹³ is Tyr and/or Xaa¹⁵ is Asp and/or Xaa¹⁷ is Arg or Lys and/or Xaa¹⁸ is Arg and/or Xaa²¹ is Glu and/or Xaa²³ is Val or lie.

39. A peptide hormone analogue as claimed in any one of claims in any of claims 1 to 13 or 29 to

38, wherein Xaa¹⁰ is Tyr, Xaa¹² is Lys, Xaa¹³ is Tyr, Xaa¹⁵ is Asp, Xaa¹⁶ is Glu or Gin (for example Glu), Xaa¹⁷ is Lys or Arg (for example Lys), Xaa¹⁸ is Arg, Xaa²⁰ is His, Xaa²¹ is Glu, and Xaa²³ is lie.

40. A peptide hormone analogue as claimed in any one of claims in any of claims 1 to 13 or 29 to

39, wherein Xaa²⁹ is Gly.

41. A peptide hormone analogue as claimed in any one of claims 1 to 40, wherein the compound consists of an amino acid sequence represented by formula (I), (II), (III) or (IV).

42. A peptide hormone analogue as claimed in any one of claims 1 to 41 which is a derivative that has been modified by one or more processes selected from amidation (for example amidation of the c-terminal amino acid), glycosylation, carbamylation, acylation, sulfation, phosphorylation, cyclization, lipidization, pegylation and fusion to another peptide or protein to form a fusion protein; preferably amidation (for example amidation of the c-terminal amino acid), lipidization, pegylation and fusion to another peptide or protein to form a fusion protein.

43. A peptide hormone analogue as claimed in any one of claims 1 to 41 which is not a derivative.

44. A peptide hormone analogue as claimed in any one of claims 1 to 28 and 41 to 43 which is not a variant.

45. A peptide hormone analogue as claimed in claim 1, wherein the analogue is selected from the group consisting of analogue number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of Figure 1.

46. A peptide hormone analogue as claimed in claim 1, wherein the analogue is selected from the group consisting of analogue number 12, 13, 14 ,15, 16, 17, 18, 19„ 20, 21, 22, 23 and 24 of Figure 1.

47. A peptide hormone analogue as claimed in claim 1, wherein the analogue is selected from the group consisting of analogue number 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42 of Figure 1.

48. A peptide hormone analogue as claimed in any one of claims 1 to 47, wherein the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to the corresponding native peptide.

49. A peptide hormone analogue as claimed in any one of claims 1 to 47, wherein the peptide hormone analogue is biased for the GLP-1 receptor G-protein-independent pathway compared to the corresponding native peptide.

50. A peptide hormone analogue as claimed in any one of claims 1 to 47, wherein the analogue is:

(i) an analogue of Exendin-4 which is a compound of formula (I) (for example a compound as claimed in any one of claims 15 to 17), and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to Exendin-4;

(ii) an analogue of GLP-1 which is a compound of formula (II) (for example a compound as claimed in any one of claims 20 to 22), and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to GLP-1;

(iii) an analogue of oxyntomodulin which is a compound of formula (III) (for example a compound as claimed in any one of claims 25 to 27), and the peptide hormone analogue is biased for the GLP- 1 receptor G-protein-dependent pathway compared to oxyntomodulin; or

(iv) an analogue of glucagon which is a compound of formula (IV) (for example a compound as claimed in any one of claims 30 to 32 or 34 to 40 when dependent on claims 30 to 32), and the peptide hormone analogue is biased for the GLP-1 receptor G-protein-dependent pathway compared to glucagon or a glucagon analogue having Xaal = His, Xaa2 = Ser; Xaa3 = Gin; Xaa4 = Gly and Xaa5 = Thr.

51. A peptide hormone analogue as claimed in any one of claims 1 to 47, wherein the analogue is:

(i) an analogue of Exendin-4 which is a compound of formula (I) (for example a compound as claimed in claim 18), and the peptide hormone analogue is biased for the GLP-1 receptor G- protein-independent pathway compared to Exendin-4; (ii) an analogue of GLP-1 which is a compound of formula (II) (for example a compound as claimed in claim 23), and the peptide hormone analogue is biased for the GLP-1 receptor G- protein-independent pathway compared to GLP-1;

(iii) an analogue of oxyntomodulin which is a compound of formula (III) (for example a compound as claimed in claim 28), and the peptide hormone analogue is biased for the GLP-1 receptor G- protein-independent pathway compared to oxyntomodulin; or

(iv) an analogue of glucagon which is a compound of formula (IV) (for example a compound as claimed in claim 33), and the peptide hormone analogue is biased for the GLP-1 receptor G- protein-independent pathway compared to glucagon or a glucagon analogue having Xaal = His, Xaa2 = Ser; Xaa3 = Gin; Xaa4 = Gly and Xaa5 = Thr.

52. A peptide hormone analogue as claimed in any one of claims 1 to 51 together with a further therapeutic agent, for simultaneous, sequential or separate administration.

53. A pharmaceutical composition comprising a peptide hormone analogue as claimed in any one of claims 1 to 51 together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

54. A pharmaceutical composition as claimed in claim 53, present in a syringe or other administration device for subcutaneous administration to humans.

55. A pharmaceutical composition as claimed in claim 53 or 54 wherein the composition has a pH of less than 5 prior to administration and wherein the composition comprises zinc ions.

56. A peptide hormone analogue as claimed in any one of claims 1 to 52, or a pharmaceutical composition as claimed in any one of claims 53 to 55, for use as a medicament.

57. 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 peptide hormone analogue as claimed in any one of claims 1 to 52, or a pharmaceutical composition as claimed in any one of claims 53 to 55.

58. A peptide hormone analogue as claimed in any one of claims 1 to 52, or a pharmaceutical composition as claimed in any one of claims 53 to 55, for use in the prevention or treatment of obesity and/or diabetes (for example type 2 diabetes or type 1 diabetes (especially type 2 diabetes)), for use in the prevention or treatment of obesity related diseases and/or diabetes related diseases (for example diabetic peripheral neuropathy, diabetic retinopathy and other forms of diabetic eye disease, diabetic nephropathy, fatty liver disease, non-alcoholic steatosis, nonalcoholic steatohepatitis, obstructive sleep apnoea and/or polycystic ovarian syndrome), increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject; restoring insulin responsiveness in a subject, for providing long-term glycemic control in a subject and/or for use as a cytoprotective agent.

59. A peptide hormone analogue as claimed in claim 58, for use in the prevention or treatment of diabetes (for example type 2 diabetes or type 1 diabetes (especially type 2 diabetes)), improving insulin release in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de- sensitization in a subject; restoring insulin responsiveness in a subject and/or for providing long- term glycemic control in a subject.

60. A peptide hormone analogue as claimed in claim 59, for use in the prevention or treatment of diabetes, type 2 diabetes, type 1 diabetes (especially type 2 diabetes), type 2 diabetes in subjects with insulin resistance, and type 2 diabetes in subjects with reduced beta cell function.

61. A method of treating or preventing obesity and/or diabetes (for example type 2 diabetes or type 1 diabetes (especially type 2 diabetes)), obesity related diseases and/or diabetes related diseases (for example diabetic peripheral neuropathy, diabetic retinopathy and other forms of diabetic eye disease, diabetic nephropathy, fatty liver disease, non-alcoholic steatosis, non-alcoholic steatohepatitis, obstructive sleep apnoea and/or polycystic ovarian syndrome), increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject; restoring insulin

responsiveness in a subject, for providing long-term glycemic control in a subject and/or for use as a cytoprotective agent, comprising administration of a therapeutically effective amount of a peptide hormone analogue as claimed in any one of claims 1 to 52, or a pharmaceutical composition as claimed in any one of claims 53 to 55.

62. Use of a peptide hormone analogue as claimed in any one of claims 1 to 52 for the manufacture of a medicament for the prevention or treatment of obesity and/or diabetes (for example type 2 diabetes or type 1 diabetes (especially type 2 diabetes)), obesity related diseases and/or diabetes related diseases (for example diabetic peripheral neuropathy, diabetic retinopathy and other forms of diabetic eye disease, diabetic nephropathy, fatty liver disease, non-alcoholic steatosis, nonalcoholic steatohepatitis, obstructive sleep apnoea and/or polycystic ovarian syndrome), increasing the energy expenditure of a subject, improving insulin release in a subject, improving carbohydrate metabolism in a subject, improving the lipid profile of a subject, reducing appetite in a subject, reducing food intake in a subject, reducing calorie intake in a subject, improving carbohydrate tolerance in a subject, decreasing rate of breakdown of insulin in a subject, improving insulin signaling in a subject, reducing insulin de-sensitization in a subject; restoring insulin

responsiveness in a subject, for providing long-term glycemic control in a subject and/or for use as a cytoprotective agent.

63. An analogue or pharmaceutical composition for use as claimed in any one of claims 58 to 60, a method as claimed in claim 61, or a use as claimed in claim 62, wherein the subject is overweight and/or obese and/or diabetic.

64. A method of causing weight loss or preventing weight gain in a subject for cosmetic purposes comprising administration of an effective amount of a peptide hormone analogue as claimed in any one of claims 1 to 52, or a composition as claimed in any one of claim 53 to 55.

65. Use of a peptide hormone analogue as claimed in any one of claims 1 to 51 in labelled form as a diagnostic agent for the diagnosis of conditions associated with a disease or disorder associated with the GLP-1 receptor, for example as a diagnostic agent for the diagnosis of conditions selected from the group consisting of obesity, diabetes, a disease or disorder associated with the GLP-1 receptor G-protein-dependent pathway, and a disease or disorder associated with the GLP-1 receptor G-protein-independent pathway.

66. Use of a peptide hormone analogue as claimed in any one of claims 1 to 51 or a labelled form of such a peptide hormone analogue as a reference compound in a method of identifying ligands for the GLP-1 receptor.

67. Use of a peptide hormone analogue or a labelled form of such a peptide hormone analogue as claimed in any one of claims 1 to 13, 15 to 17, 20 to 22, 25 to 27, 30 to 32, and claims 34 to 51 when dependent on any one of claims 1 to 13, 15 to 17, 20 to 22, 25 to 27, 30 to 32, as a reference compound in a method of identifying ligands for the GLP-1 receptor that are biased for the GLP-1 receptor G-protein-dependent pathway.

68. Use of a peptide hormone analogue or a labelled form of such a peptide hormone analogue as claimed in any one of claims 1 to 13, 18, 23, 28 and 33, and claims 34 to 51 when dependent on any one of claims 1 to 13, 18, 23, 28 and 33, as a reference compound in a method of identifying ligands for the GLP-1 receptor that are biased for the GLP-1 receptor G-protein-independent pathway.