PISCINE-DERIVED PYY PEPTIDES FOR USE IN TREATING A METABOLIC DISORDER

Abstract

The present invention relates to peptides for use in the treatment of metabolic disorders. In particular, the present invention relates to peptides for use in the treatment of diabetes. According to the present invention, there is provided a peptide for use in the treatment of a metabolic disorder; wherein the peptide is a piscine-derived PYY or fragment or analogue thereof.

Claims

1. A piscine-derived PYY peptide for use in the treatment of a metabolic disorder.

2. The peptide for use according to claim 1; wherein the peptide comprises the amino acid sequence defined by SEQ ID NO: 2.

3. The peptide for use according to claim 1 or 2; wherein the peptide comprises the amino acid sequence defined by SEQ ID NO: 3.

4. The peptide for use according to claim 1 or 2; wherein the N-terminus of the peptide comprises an amino acid sequence defined by a sequence selected from the group comprising: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

5. The peptide for use according to any preceding claim; wherein the peptide comprises an amino acid sequence having at least 60% identity to a sequence selected from the group comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

6. The peptide for use according to any preceding claim; wherein the peptide comprises the amino acid sequence defined by a sequence selected from the group comprising: SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

7. The peptide for use according to any preceding claim; wherein the use of the peptide in the treatment of a metabolic disorder has no significant impact on patient appetite.

8. The peptide for use according to any preceding claim; wherein the use of the peptide in the treatment of a metabolic disorder has no significant impact on patient appetite, compared to a comparable dose of human PYY(1-36).

9. The peptide for use according to any preceding claim; wherein the peptide has a half life of greater than 2 hours under physiological conditions.

10. The peptide for use according to any preceding claim; wherein the peptide has a half life of greater than 2 hours against cleavage and/or degradation by DPP-4 or C-terminally directed protease enzymes.

11. The peptide for use according to any preceding claim; wherein the use comprises administration of the peptide during a pre-determined first period during every 24 hours.

12. The peptide for use according to any preceding claim; wherein the use comprises administration of the peptide within a pre-determined period of time before or while the patient is asleep.

13. The peptide for use according to any preceding claim; wherein the use comprises administration of the peptide in alternating combination with at least one GLP-1 receptor agonist.

14. The peptide for use according to claim 13; wherein at least one GLP-1 receptor agonist is liraglutide or exenatide.

15. The peptide for use according to claim 13 or 14, wherein the use comprises administration of the at least one GLP-1 receptor agonist within a pre-determined period of time while the patient is awake.

16. The peptide for use according to any preceding claim; wherein the metabolic disorder is a pancreatic disorder.

17. The peptide for use according to any preceding claim; wherein the metabolic disorder comprises diabetes mellitus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0168] FIG. 1 shows a chart illustrating insulin release (ng/10.sup.6 cells/20 minutes) from rodent BRIN-BD11 beta-cells on the Y-axis against the concentration of various peptides on the X-axis. The peptides include Human PYY(1-36), Bowfin PYY, Trout PYY, Sea Lamprey PYY, Sturgeon PYY, as shown by the key. Values are mean±SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 compared to 16.7 mM glucose control.

[0169] FIG. 2 includes two charts. FIG. 2A shows a chart illustrating proliferation frequency of human 1.164 beta-cells (percent of total cells analysed) on the Y-axis against various treatments including human PYY(1-36) and four piscine-derived PYY variants. As shown by the key, each treatment was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=4). *p<0.05, **p<0.01, ***p<0.001 compared to untreated media control. .sup.Δp<0.05 compared to respective concentration of human PYY(1-36). FIG. 2B shows a chart illustrating TUNEL positive apoptotic cells of human 1.1B4 beta-cells (percent of total cells analysed) on the Y-axis against various treatments including human PYY(1-36) and four piscine-derived PYY variants on the X-axis. As shown by the key, each PYY peptide was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=4). *p<0.05, **p<0.01, ***p<0.001 compared to untreated media control.

[0170] FIG. 3 shows a chart illustrating cumulative food intake (g) in overnight fasted mice on the Y-axis against time (minutes). As shown by the key, cumulative food intake was assessed following intraperitoneal administration of vehicle (0.9% NaCl) alone or in combination with test peptide (each at 25 nmol/kg bw). As shown by the key, test peptides included human PYY(1-36), Bowfin PYY, Trout PYY, Sea Lamprey PYY, Sturgeon PYY. Values are mean±SEM (n=6). *p<0.05, **p<0.01 compared to vehicle treated control. .sup.Δp<0.05, .sup.ΔΔp<0.01 .sup.ΔΔΔp<0.001 compared to human PYY(1-36).

[0171] FIG. 4 shows a chart illustrating insulin release (ng/10.sup.6 cells/20 minutes) from immortalised rodent BRIN-BD11 beta-cells at basal, 5.6 mM, glucose concentration on the Y-axis, against the concentration of several human PYY metabolites on the X-axis. The human PYY metabolites include PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34, as shown by the key. Values are mean±SEM (n=8). **p<0.01, ***p<0.001 decreases compared to 5.6 mM glucose control. .sup.σp<0.05, .sup.σσp<0.01 increases compared to human PYY(1-36); .sup.σσσp<0.001 increases compared to 5.6 mM glucose control.

[0172] FIG. 5 shows a chart illustrating insulin release (ng/10.sup.6 cells/20 minutes) from immortalised rodent BRIN-BD11 beta-cells at elevated, 16.7 mM, glucose concentration on the Y-axis, against the concentration of several human PYY metabolites on the X-axis. The human PYY metabolites include PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34, as shown by the key. Values are mean±SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 decreases compared to 16.7 mM glucose control. .sup.σσσp<0.001 increases compared to 16.7 mM glucose control.

[0173] FIG. 6 includes two charts. FIG. 6A shows a chart illustrating proliferation frequency in immortalised rodent BRIN-BD11 cells (percent of total cells analysed) on the Y-axis against various treatments including GLP-1, PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34 As shown by the key, each treatment was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=3). .sup.++p<0.01, .sup.+++p<0.001 compared to untreated, media control. *p<0.05, ***p<0.001 compared to cytokine mixture alone. FIG. 6B shows a chart illustrating TUNEL positive apoptotic cells of human 1.164 cells (percent of total cells analysed) on the Y-axis against various treatments including GLP-1, PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34 on the X-axis. As shown by the key, each PYY peptide was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=4). *p<0.05, ***p<0.001 compared to cytokine mixture alone. .sup.++p<0.01 compared to untreated, media control.

[0174] FIG. 7 includes two charts. FIG. 7A shows a chart illustrating proliferation frequency in immortalised rodent BRIN-BD11 cells (percent of total cells analysed) on the Y-axis against various treatments including GLP-1, PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34 As shown by the key, each treatment was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 compared to control. FIG. 7B shows a chart illustrating proliferation frequency in 1.164 cells (percent of total cells analysed) on the Y-axis against various treatments including GLP-1, PYY.sub.1-36, PYY.sub.3-36, PYY.sub.1-34, PYY.sub.3-34 As shown by the key, each treatment was tested separately at 10.sup.−8 and 10.sup.−6 M. Values are mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 compared to control.

[0175] FIG. 8 shows a chart illustrating effects of twice-daily administration of test-peptide on body weight. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on bodyweight (FIG. 8A). Measurements were taken three days prior to and throughout the treatment period, at regular intervals. (Treatment period demonstrated via the horizontal bar parallel to x-axis) The % bodyweight change, from first to final treatment day, is also provided (FIG. 8B). Values are mean±SEM (n=6). .sup.Δp<0.05 compared to lean, saline-only control.

[0176] FIG. 9 shows a chart illustrating effects of twice-daily administration of test-peptide on terminal body composition. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on body composition, with total lean mass and total fat mass provided for each group. Values are mean SEM (n=6). *p<0.05 compared to STZ+saline control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to lean, saline-only control.

[0177] FIG. 10 shows a chart illustrating effects of twice-daily administration of test-peptide on food and fluid intake. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on cumulative food (FIG. 10A) and cumulative fluid (FIG. 10B) intake, over the treatment period. Values are mean±SEM (n=6). *p<0.05, **p<0.01 compared to stz+saline control. .sup.Δp<0.05, .sup.ΔΔp<0.01, .sup.ΔΔΔp<0.001 compared to lean, saline-only control.

[0178] FIG. 11 shows a chart illustrating effects of twice-daily administration of test-peptide on blood glucose and circulating plasma. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on blood glucose (FIG. 11A) and circulating plasma insulin (FIG. 11C). Measurements were taken three days prior to and throughout the treatment period, at regular intervals. (Treatment period demonstrated via the horizontal bar parallel to x-axis) Respective areas under the curve are provided (FIG. 11B, FIG. 11D). Values are mean±SEM (n=6). Values are mean±SEM (n=6). *p<0.05, **p<0.01, ***p<0.001 compared to stz+saline control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to lean, saline-only control.

[0179] FIG. 12 shows a chart illustrating effects of twice-daily administration of test-peptide on terminal glucose tolerance and plasma insulin over a two hour period. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on glucose tolerance (FIG. 12A) and circulating plasma insulin (FIG. 12C). Glucose was administered following an overnight (16 hour) fast via i.p. injection at an 18 mmol/kg bw dose. Blood glucose and plasma insulin were measured prior to, and after i.p. injection at regular intervals. Experiment was performed following 21 days of treatment with test peptide. Respective areas under the curve are provided (FIG. 12B, FIG. 12D). Values are mean±SEM (n=6). *p<0.05, **p<0.01, ***p<0.001 compared to STZ+saline control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to lean, saline-only control.

[0180] FIG. 13 shows a chart illustrating effects of twice-daily administration of test-peptide on terminal insulin sensitivity. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on insulin sensitivity, as demonstrated via percentage fall in blood glucose over 60 minutes (FIG. 13A). Insulin was administered at a 25 U/kg/bw dose in non-fasting mice. Experiment was performed following 21 days of treatment with test peptide. Respective areas under the curve are provided (FIG. 13B). Values are mean±SEM (n=6). *p<0.05 compared to stz+saline control. .sup.ΔΔΔp<0.001 compared to lean, saline-only control.

[0181] FIG. 14 shows a chart illustrating effects of twice-daily administration of test-peptide on pancreatic histology. Effects of twice-daily administration of saline control or test peptide in combination with saline (Exendin-4, Sturgeon PYY(1-36) and Sea Lamprey PYY(1-36) at 25 nmol/kg bw dose) on pancreatic morphology. Variables were assessed using CelIF image analysis software after 21 days of twice-daily i.p injections. Values are means±SEM of 6-8 mice per group, with approximately 50 islets being analysed per group (A-D). *p<0.05, **p<0.01, ***p<0.001 compared to STZ+saline control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to lean, saline-only control.

[0182] FIG. 15 shows representative images, demonstrating the effects of twice daily administration of test peptide on pancreatic morphology. Representative images of islets showing insulin (red) and glucagon (green) immunoreactivity from pancreatic tissues extracted from lean Swiss TO mice following streptozotocin pretreatment. Animals received twice daily doses of saline control (FIG. 15A) or test peptide in combination with saline (Exendin-4 (FIG. 15B), Sturgeon PYY(1-36) (FIG. 15C) and Sea Lamprey PYY(1-36) (FIG. 15D) at 25 nmol/kg bw dose). Images were obtained at 40× magnification using CelIF software.

[0183] FIG. 16 shows a chart illustrating insulin secretory activity of Sea Lamprey PYY, (Ile3)Sea Lamprey PYY, (D-Arg35)Sea Lamprey PYY and (Ile3, D-Arg35)Sea Lamprey PYY in BRIN BD11 beta-cells. Effects of PYY(1-36), Sea Lamprey PYY(1-36) and related analogues on insulin release from rodent BRIN-BD11 beta-cells at 16.7 mM, glucose concentration. Values are mean±SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 decreases compared to 5.6 mM glucose control.

[0184] FIG. 17 shows a chart illustrating proliferative activity of Sea Lamprey PYY, (Ile3)Sea Lamprey PYY, (D-Arg35)Sea Lamprey PYY and (Ile3, D-Arg35)Sea Lamprey PYY in rodent BRIN BD11 and human 1.1B4 clonal beta-cells. Effects of PYY(1-36), Sea Lamprey PYY(1-36) and related analogues on proliferation of BRIN-BD11 (FIG. 17A) and 1.1 B4 cells (FIG. 17B) at 10-6 and 10-8 M concentrations. Values are mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 compared to control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to native PYY(1-36) at the same concentration.

[0185] FIG. 18 shows a chart illustrating effects of Sea Lamprey PYY, (Ile3)Sea Lamprey PYY, (D-Arg35)Sea Lamprey PYY and (Ile3, D-Arg35)Sea Lamprey PYY on protection against cytokine-induced apoptosis in rodent BRIN BD11 and human 1.1B4 clonal beta-cells. Effects of PYY(1-36), Sea Lamprey PYY(1-36) and related analogues on protection against cytokine-induced (IL-1β (100 U/mL), IFNγ (20 U/mL), TNFα (200 U/mL)) apoptosis in BRIN-BD11 (FIG. 18A) and 1.1 B4 cells (FIG. 18B) at 10-6 and 10-8 M concentrations. Values are mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 compared to control. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to native PYY(1-36) at the same concentration.

[0186] FIG. 19 shows a chart illustrating effects of Sea Lamprey PYY, (Ile3)Sea Lamprey PYY, (D-Arg35)Sea Lamprey PYY and (Ile3, D-Arg35)Sea Lamprey PYY on food intake in overnight fasted mice. Acute effects of PYY(3-36), Sea Lamprey PYY(1-36) and related analogues on food intake in 18 h fasted mice. PYY(3-36) was also employed as a positive control. Cumulative food intake was assessed following intraperitoneal administration of dissolution vehicle (0.9% NaCl) alone or in combination with test peptide at a dose of 25 nmol/kg bw. Values are mean±SEM (n=6). *p<0.05, **p<0.01, ***p<0.001 compared to saline alone. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to PYY(3-36) control.

DETAILED DESCRIPTION OF THE INVENTION

[0187] Beta-cell rest: Previous work by the inventors has already shown that sustained periods of beta-cell rest using a GIP receptor inhibitor, combined with suitable intervals of beta-cell stimulation with a GLP-1 agonist, has significant therapeutic potential for diabetes. In agreement, there is known to be a preservation of residual beta-cells in various diabetes conditions and, without being bound by theory, it is believed that inducing beta-cell rest offers a route for reactivating these cells, and preserving beta-cell function. As such, prolonged periods of rest are thought to allow chronically overstimulated beta-cells to replenish the immediately secretable insulin granule pool. Other positive actions known to occur during extended periods of beta-cell rest include enhanced beta-cell glucokinase activity and improved hepatic glucose handling. There is also a suggestion that beta-cell rest can directly impede beta-cell apoptosis. Moreover, the undetermined mechanism whereby thiazolidinediones exert a protective effect on beta-cell function has now been directly linked to beta-cell resting actions. Finally, a recent study has highlighted the plasticity of pancreatic beta-cells and their ability to correct inherent insulin secretory dysfunction through sustained periods of beta-cell rest. This gives further credence to the idea of sequential periods of beta-cell rest as a treatment strategy for diabetes.

[0188] GLP-1 and PYY: Like many gut peptides, both native GLP-1 and PYY are rapidly degraded by plasma enzymes, particularly dipeptidylpeptidase-4 (DPP-4). For GLP-1, this degradation annuls insulin secretory and protective beta-cell effects. As such, numerous enzymatically stable long-acting forms of GLP-1 are now clinically available. For PYY, the story is only just beginning to emerge. Until recently, the principal biological action of PYY was believed to be linked to induction of satiety and regulation of energy balance. This action is initiated by the enzymatic action of DPP-4 on PYY, cleaving native PYY(1-36) to PYY(3-36). Interestingly, these energy-regulating effects are believed to be exerted through interaction of PYY(3-36) with the neuropeptide Y (Y) receptors, specifically hypothalamic Y.sub.2 receptors. However, more recent investigations are beginning to reveal a critical role for PYY(1-36) in the control of beta-cell function and survival, through activation of Y.sub.1 receptors. Thus, PYY(3-36) is a more specific Y.sub.2 receptor agonist, whereas PYY(1-36) is known to interact mainly with Y.sub.1 receptors. Indeed, the Y.sub.1 receptor is recognised as the most prominent NPY receptor form in the endocrine pancreas. This knowledge is further strengthened through demonstration that selective destruction of PYY-expressing cells in adult mice induces dysfunction and loss of beta-cells, and that transgenic mice selectively overexpressing PYY in pancreatic beta-cells present with increased beta-cell mass. Studies by the inventors, along with the recent evidence of PYY-mediated beneficial effects of Roux-en-Y gastric bypass surgery, suggest that sustained activation of Y.sub.1 receptors by enzymatically stable PYY(1-36) analogues will exert clear benefits for diabetes treatment, linked to both induction of beta-cell rest and direct promotion of beta-cell survival.

[0189] Stable PYY and GLP-1 peptides: Numerous well-characterised GLP-1 receptor agonists are now available, including liraglutide. The major challenge for the current study in relation to GLP-1 involves timing of injections, relative to sequential PYY injections, to evoke maximum possible benefits. Harnessing PYY therapeutically is more challenging. As noted above, activation of Y.sub.1 receptors by PYY(1-36) is key for direct beneficial beta-cell effects. Based on the origin and evolution of PYY, and therefore most prolonged conservation of sequence, the inventors have screened a series of piscine-derived PYY peptides for potential therapeutic exploitation. Thus, just as was the case for the clinically utilised GLP-1 receptor agonists, the inventors believe that nature could hold the key to uncovering the first-in-class, stable PYY peptide. The inventors were initially able to narrow down potential lead candidates based on computation structure/function analyses to yield stable, Y.sub.1 receptor specific, PYY peptides. Thus, trout, bowfin, sturgeon and sea lamprey PYY forms were chosen for further analysis. Subsequent detailed pilot studies by the inventors revealed, as expected from structural analyses, all four peptides were enzymatically stable (Table 1). In addition, the piscine-derived PYY peptides also induced similar insulinostatic (FIG. 1), beta-cell proliferative and anti-apoptotic (FIG. 2) effects as human PYY, confirming retention of bioactivity, presumably through activation of pancreatic Y.sub.1 receptors. It might be expected that activation of hypothalamic Y.sub.1 receptors would stimulate hunger, whilst this was true for trout, bowfin and sturgeon PYY, sea lamprey PYY had no significant impact on appetite (FIG. 3). These observations make sea lamprey PYY the preferred lead candidate to have taken forward to efficacy testing in diabetes, alongside treatment with an established GLP-1 mimetic.

[0190] Exploiting sequential beta-cell rest and stimulation: GLP-1 and PYY possess complementary beneficial effects on the preservation of beta-cell mass. Since PYY evokes beta-cell rest, it is logical that treatment with PYY-based agents should be introduced at a time when the beta-cell is least active, that is, during sleeping hours. Indeed, it would be imagined that any potential stimulation of hypothalamic Y.sub.1 receptors would have less potential impact on appetite at this time. In any case, the inventors do not see this as a major issue for sea lamprey PYY, based on the pilot observations (FIG. 3). Following on from this, activation of GLP-1 receptors during awake hours, when the beta-cell is more likely to be stimulated, is also contemplated. The inventors already have experience in this type of scenario in mice. Since a 12 hour dark:light cycle is standard procedure in rodent research facilities worldwide, the inventors have created a precise dosing schedule where beta-cells will be rested for 8-12 hours and then subsequently stimulated for 8-12 hours. In the human setting, the inventors recognise that these timings might need to be adapted, but the same theory applies. The staggered treatment scenario should also avoid any potential receptor downregulation and encourage preservation of beta-cell mass during the entire day, without adversely affecting insulin secretory function. Taken together, the potential interplay that exists between PYY and GLP-1 receptor signalling, enabling preservation and possible replenishment of beta-cell mass, promotes this approach as a new and effective treatment paradigm for diabetes.

EXAMPLES

[0191] Insulin Secretion Methodology

[0192] Effects of peptides on insulin secretion were examined using immortalised rodent BRIN-BD11 and human 1.1B4 beta-cells. Cells were seeded (150,000/well) into 24-well plates (Nunc, Roskilde, Denmark) and allowed to attach overnight at 37° C. Following 40 min pre-incubation (1.1 mmol/l glucose; 37° C.), cells were incubated (20 min; 37° C.) in the presence of 5.6 or 16.7 mmol/L glucose, as appropriate, with a range of test peptide concentrations as appropriate. After 20 min incubation, buffer was removed from each well and aliquots stored at −20° C. prior to determination of insulin by radioimmunoassay.

[0193] Beta-Cell Proliferation and Apoptosis Studies Methodology

[0194] To assess the effects of peptides on rodent BRIN-BD11 and human 1.164 cell proliferation, cells were seeded at a density of 150,000 cells per well and cultured overnight in the presence of peptides. Cells were rinsed with PBS and fixed using 4% paraformaldehyde. After antigen retrieval with citrate buffer at 95° C. for 20 min, tissue was blocked using 2% BSA for 45 min. The slides were then incubated with rabbit anti-Ki-67 primary antibody, and subsequently with Alexa Fluor® 594 secondary antibody. Slides were viewed using fluorescent microscope (Olympus System Microscope, model BX51; Southend-on-Sea, UK) and photographed by DP70 camera adapter system. Proliferation frequency was determined in a blinded fashion and expressed as % of total cells analysed. Approximately 150 cells per replicate were analysed. For analysis of ability to protect against apoptosis, BRIN-BD11 and 1.1B4 cells were seeded as above. Cells were then exposed to cytokine mixture, as described, in the presence or absence of test peptides for 2 h. Cells were then harvested and a TUNEL assay was performed. Slides were viewed under appropriate filter using an Olympus™ fluorescent microscope.

[0195] Food Intake Methodology

[0196] Studies were carried out using adult male NIH Swiss mice (12 weeks of age, Envigo Ltd, UK), housed individually in air conditioned room at 22±2° C. with 12 h light and dark cycle and ad libitum access to standard rodent diet (10% fat, 30% protein and 60% carbohydrate; Trouw Nutrition, Northwich, UK) and drinking water. All experiments were carried out in accordance with the UK Animal Scientific Procedures Act 1986. Mice were fasted (18 h) and then received an i.p. injection of saline alone (0.9% (w/v) NaCl) or in combination with test peptides (25 nmol/kg body weight) and food intake measured at 30 min intervals.

Example 1 Identification of Stable, Y1 Receptor Specific, PYY Peptides; and DPP-4 and Murine Plasma Degradation Analyses

[0197] The inventors were initially able to narrow down potential lead candidates based on computation structure/function analyses to yield stable, Y.sub.1 receptor specific, PYY peptides. Thus, trout, bowfin, sturgeon and sea lamprey PYY forms were chosen for further analysis.

[0198] Following incubation of peptides with purified DPP-4 or murine plasma and HPLC analysis, collected fractions for each time-point (0, 2 and 8 h) were subsequently mixed with α-cyano-4-hydroxycinnamic acid and applied to a Voyager-DE BioSpectrometry Workstation and mass-to-charge (m/z) ratio verses peak intensity recorded. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Amino acid sequence of PYY peptides, as well as DPP-4 and murine plasma degradation analyses SEQ DPP-4 Plasma ID half- half- Amino acid sequence NO Peptide life life YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY- 8 Human <2 h <2 h NH.sub.2 PYY(1-36) YPPKPENPGEDAPPEELARYYSALRHYINLITRQRY- 5 Bowfin PYY >8 h >8 h NH.sub.2 YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY-NH.sub.2 6 Trout PYY >8 h >8 h MPPKPDNPSPDASPEELSKYMLAVRNYINLITRQRY- 4 Sea >8 h >8 h NH.sub.2 lamprey PYY YFPPKPEHPGDDAPAEDVVKYYTALRHYINLITRQRY- 7 Sturgeon >8 h >8 h NH.sub.2 PYY

Example 2 Insulinostatic Effects of Human PYY and Related Piscine-Derived PYY Species

[0199] The effects of human PYY(1-36) and four piscine-derived PYY variants on insulin release (20 min) from rodent BRIN-BD11 beta-cells at 16.7 mM glucose were investigated; as described in the insulin secretion methodology above. The results are shown in FIG. 1. Values are mean±SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 compared to 16.7 mM glucose control.

Example 3 Effects of Human PYY and Related Piscine-Derived PYY Species on Human Beta-Cell Proliferation and Apoptosis

[0200] The effects of human PYY(1-36) and four piscine-derived PYY variants (each at 10.sup.−8 and 10.sup.−6 M) on proliferation and protection against apoptosis in human 1.1B4 beta-cells were investigated; as described in the beta-cell proliferation and apoptosis studies methodology above. Proliferation was measured by Ki-67 staining and apoptosis by TUNEL assay following incubation of peptides in the presence of a cytokine cocktail (TNF-α (200 U/mL), IFN-γ (20 U/mL) and IL 1B (100 U/mL)). The results are shown in FIG. 2. FIG. 2A shows the effects of human PYY(1-36) and four piscine-derived PYY variants (each at 10.sup.−8 and 10.sup.−6 M) on proliferation in human 1.1B4 beta-cells. FIG. 2B shows the effects of human PYY(1-36) and four piscine-derived PYY variants (each at 10.sup.−8 and 10.sup.−6 M) on protection against apoptosis in human 1.1B4 beta-cells. Values are mean±SEM (n=4). *p<0.05, **p<0.01, ***p<0.001 compared to untreated media control. .sup.Δp<0.05 compared to respective concentration of human PYY(1-36).

Example 4 Effects of Human PYY and Related Piscine-Derived PYY Species on Feeding

[0201] The effects of human PYY(1-36) and four piscine-derived PYY variants on food intake in overnight fasted mice were investigated; as described in the food intake methodology above. Cumulative food intake was assessed following intraperitoneal administration of vehicle (0.9% NaCl) alone or in combination with test peptide (each at 25 nmol/kg bw). The results are shown in FIG. 3. Values are mean±SEM (n=6). *p<0.05, **p<0.01 compared to vehicle treated control. .sup.Δp<0.05, .sup.ΔΔp<0.01 .sup.ΔΔΔp<0.001 compared to human PYY(1-36).

Example 5 Insulin Secretion

[0202] The effects of PYY.sub.1-36 and its N- and C-terminal metabolites on insulin release from immortalised rodent BRIN-BD11 beta-cells at basal (5.6 mM) and elevated (16.7 mM) glucose concentrations were investigated; as described in the insulin secretion methodology above. The results for basal, 5.6 mM, gluclose concentration are shown in FIG. 4, while the results for elevated, 16.7 mM, glucose concentration are shown in FIG. 5. Values are mean±SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 decreases compared to 5.6 mM glucose control. .sup.σp<0.05, .sup.σσp<0.01, .sup.σσσp<0.001 increases compared to the glucose control (5.6 mM glucose in FIG. 4, 16.7 mM glucose in FIG. 5).

Example 6 Protection from Apoptosis

[0203] The effects of PYY.sub.1-36 and its N- and C-terminal metabolites (3-36), (1-34) and (3-34) on apoptosis in immortalised rodent BRIN-BD11 cells and 1.1B4 cells at 10.sup.−6M and 10.sup.−8 M concentrations were investigated; as described in the beta-cell proliferation and apoptosis studies methodology above.

[0204] Peptides were incubated with cells in the presence of a cytokine cocktail mixture (TNF-a (200 U/mL, IFN-y (20 U/mL and IL 1B (100 U/mL))). The results are shown in FIG. 6. FIG. 6A shows the effects of PYY.sub.1-36 and its metabolites (3-36), (1-34) and (3-34), each at 10.sup.−8 and 10.sup.−6 M, on apoptosis in immortalised rodent BRIN-BD11 cells. FIG. 6B shows the effects of PYY.sub.1-36 and its metabolites (3-36), (1-34) and (3-34), each at 10.sup.−8 and 10.sup.−6 M, on apoptosis in 1.1B4 cells. Values are mean±SEM (n=3). .sup.+p<0.05, .sup.++p<0.01, .sup.+++p<0.001 compared to untreated, media control. *p<0.05, **p<0.01, ***p<0.001 compared to cytokine mixture alone.

Example 7 Proliferation

[0205] The effects of PYY.sub.1-36 and its N- and C-terminal metabolites (3-36), (1-34) and (3-34) on proliferation of immortalised rodent BRIN-BD11 cells and 1.164 cells at 10.sup.−6 M and 10.sup.−8 M concentrations were investigated; as described in the beta-cell proliferation and apoptosis studies methodology above. The results are shown in FIG. 7. FIG. 7A shows the effects of PYY.sub.1-36 and its metabolites (3-36), (1-34) and (3-34), each at 10.sup.−8 and 10.sup.−6 M, on proliferation of immortalised rodent BRIN-BD11 cells. FIG. 7B shows the effects of PYY.sub.1-36 and its metabolites (3-36), (1-34) and (3-34), each at 10.sup.−8 and 10.sup.−6 M, on proliferation of immortalised rodent 1.1B4 cells. Values are mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 compared to control.

Example 8 Effects of Sea Lamprey PYY and Sturgeon PYY on Body Weight, Terminal Body Composition, Food and Fluid Intake, Blood Glucose and Circulating Plasma, Terminal Glucose Tolerance and Plasma Insulin, Terminal Insulin Sensitivity, Pancreatic Histology, and Pancreatic Morphology

[0206] The effects of sea lamprey PYY and sturgeon PYY on body weight, terminal body composition, food and fluid intake, blood glucose and circulating plasma, terminal glucose tolerance and plasma insulin, terminal insulin sensitivity, pancreatic histology, and pancreatic morphology in overnight fasted mice were investigated; as described in the food intake methodology above. Each of body weight, terminal body composition, food and fluid intake, blood glucose and circulating plasma, terminal glucose tolerance and plasma insulin, terminal insulin sensitivity, pancreatic histology, and pancreatic morphology was assessed following intraperitoneal administration of vehicle (0.9% NaCl) alone or in combination with test peptide (each at 25 nmol/kg bw) twice daily for 21 days to a multiple low-dose streptozotocin (50 mg/kg for five consecutive days) mouse model of diabetes. The results are shown in FIGS. 8-15. These data provide evidence for the beneficial metabolic and islet architecture effects of sturgeon, and especially sea lamprey, PYY.

Example 9 Generation of Stable PYY Peptide Analogues

[0207] Further to the recognised importance of maintenance of C-terminal integrity for PYY peptide bioactivity as shown in FIGS. 4-7, the inventors have generated novel analogues of Sea Lamprey PYY (Table 2). Essentially, the inventors have generated a combination of Sea Lamprey PYY peptides that are enzymatically stable or liable at both the N- and C-termini. As such, native Sea Lamprey PYY is N-terminally stable but C-terminally liable. (Ile3)Sea Lamprey PYY is enzymatically liable at both the N- and C-termini. (D-Arg35)Sea Lamprey PYY is enzymatically stable at both the N- and C-termini. Finally, (Ile3, D-Arg35)Sea Lamprey PYY is enzymatically liable at the N-terminus but stable at the C-terminus. The data contained below confirm N- and C-terminal stability of the Sea Lamprey PYY peptides (Table 3). In addition to this, the example also show insulin secretory actions (FIG. 16) together with effects on pancreatic beta-cell proliferation (FIG. 17) and protection against apoptosis (FIG. 18), of all peptides. Finally, the effects of Sea Lamprey PYY, and related analogues, on re-feeding in overnight fasted mice is also presented (FIG. 19).

TABLE-US-00002 TABLE 2 Amino acid sequence of PYY peptide analogues SEQ ID Amino acid sequence NO Peptide MPPKPDNPSPDASPEELSKYMLAVRNYINLITRQRY-NH.sub.2  4 Sea Lamprey PYY(1-36) MPIKPDNPSPDASPEELSKYMLAVRNYINLITRQRY-NH.sub.2  9 [Iso-3]Sea Lamprey PYY (1-36) MPPKPDNPSPDASPEELSKYMLAVRNYINLITRQ(d-R)Y- 10 (D-Arg-35)Sea Lamprey NH.sub.2 PYY(1-36) MPIKPDNPSPDASPEELSKYMLAVRNYINLITRQ(d-R)Y- 11 [Iso-3](d-Arg-35)Sea NH.sub.2 Lamprey PYY(1-36)

TABLE-US-00003 Enzymatic stability of PYY peptide analogues SEQ ID % Identified Peptide NO Peptide Name Degradation Fragment(s) 8 Native PYY (1-36) 49 PYY (3-36), PYY (1-34) Native PYY (3-36) 36.4 PYY (3-34) 4 Sea Lamprey PYY(1-36) 47.4 Sea Lamprey PYY (1-34) 9 [Iso-3] Sea Lamprey 82.1 [Iso-3] Sea Lamprey PYY (1-36) PYY (3-36) [Iso-3] Sea Lamprey 20.1 [Iso-3] Sea Lamprey PYY (1-36) + Sitagliptin PYY (1-34) 10 (d-Arg-35) Sea Lamprey 0 None PYY (1-36) 11 [Iso-3] (d-Arg-35) 28.9 [Iso-3](d-Arg-35) Sea Lamprey PYY (1-36) Sea Lamprey PYY (3-36)

[0208] A summary of plasma degradation of human PYY(1-36) and PYY(3-36) as well as Sea Lamprey PYY(1-36) and its three related analogues is given in Table 3. Peptides were incubated at 37° C. in 50 mM TEA buffer containing plasma extracted from lean, overnight fasted, NIH, Swiss mice and reactions stopped at 4 h with 10% TFA. Degradation mixtures were then separated via RP-HPLC, with any relevant peaks being collected for identification via MALDI-TOF. Percentage of degradation is calculated via the peak area functionality in Chromquest Version 4.